Citation
A stability and solubility study of riboflavin and some derivatives

Material Information

Title:
A stability and solubility study of riboflavin and some derivatives
Creator:
Hertz, Joel John, 1926-
Publication Date:
Language:
English
Physical Description:
xi, 145 leaves : ill. ; 29 cm.

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D)
Degree Grantor:
University of Florida
Degree Disciplines:
Pharmacy
Committee Chair:
Becker, Charles H.
Committee Members:
Husa, William J.
Johnson, Carl H.
Stearns, Thomas W.
Lauter, Werner M.

Subjects

Subjects / Keywords:
Aqueous solutions ( jstor )
Bottles ( jstor )
Flint ( jstor )
Fluorescence ( jstor )
pH ( jstor )
Potassium ( jstor )
Sodium ( jstor )
Solubility ( jstor )
Solvents ( jstor )
Water distillation ( jstor )
Dissertations, Academic -- Pharmacy -- UF ( mesh )
Pharmacy thesis Ph.D ( mesh )
Riboflavin ( mesh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1954.
Bibliography:
Includes bibliographical references (leaves 139-143).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Joel John Hertz.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright Joel John Hertz. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
024250310 ( ALEPH )
25743313 ( OCLC )
AEK7303 ( NOTIS )
AA00004956_00001 ( sobekcm )

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A STABILITY AND SOLUBILITY STUDY OF

RIBOFLAVIN AND SOME DERIVATIVES

A '


By
JOEL JOHN HERTZ











A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY









UNIVERSITY OF FLORIDA
June, 1954























ACKNOWLEDGMENT


The author wishes to express his sincere appreciation to

Dr. Charles H. Becker, Chairman of the Supervisory Committee, for

his untiring patience and encouraging guidance in the direction

of this work.

The valuable assistance of Dr. Werner M. Later is also

gratefully aaknowedged.

The author would also like to express his appreciation to

Ell Iy and Company for their financial assistance to his and for

purchasing equipment and supplies.













TABLE OF CONTENTS


Page
LIST OF TABLES

INTRODUCTION 1

REVIEW OF SE LITERATURE 3

Historical Sketch 3

Isolation 7

Occurrence 9

Physical and Chemical Properties 1

Assay Determinations 15

Increasing Solubility 19

EXPERIMENTAL 26

Materials Used 26

Preparation of Riboflavin Derivatives 29

Pyruvie Acid, Levulinic Acid and Citraconie
Anhydride Derivatives 29

Scope of the Fluorophotometer 31

Standardization of the Lumetron 34

Preparation of Solutions 34
Adjusting the Iumstron for Analysis 34

Stability Study Methods 37

Solutions Used 37
Procedure 40

Solubility Study Methods 43

Solvents Used 43
Procedure 43


iII











Page
Aseay Method of Riboflavin Derivatives 45

Results with Riboflavin 47

Results with Riboflavin-5-Phosphate Sodium 63

Results with Flawaxin Soluble 79

Results with a Pyruvic Acid Derivative of
Riboflavin 95

RIeslta with a Levulinic Acid Derivative of
Riboflavin i

Results with a Citraeonia Anhydride Derivative
of Riboflavin 127
DISCUSSION OF RESULTS 129

SU(MIAR AIN COINCLUSIONS 13

BIBBLOGRAB 139

BIOGRAPHICAL ITEMS 14

CGOMtTTEE REPOT 145












LIST OF TABLES


Table Page

1. Description of Drugs and Chemicals Used 27

2. Lumetron Readings of Various Dilutions of a Standard
Riboflavin Solution Containing 2 Mg./Liter 36

3. The Stability of Riboflavin in Distilled Water Stored
Under Various Conditions in Flint and Amber Bottles 48

4. The Stability of Riboflavin in Distilled Water Buffered
at pH 6 Stored Under Various Conditions in Flint and
Amber Bottles 49

5, The Stability of Riboflavin in Distilled Water Buffered
at pH 5 Stored Under Various Conditions in Flint and
Amber Bottles 50

6. The Stability of Riboflavin in Distilled Water Buffered
at pH 4 Stored Under Various Conditions in Flint and
Ambor Bottles 51

7. The Stability of Riboflavin in 25 Per Cent Glycerin in
Distilled Water Stored Under Various Conditions in Flint
and Ambor Bottles 52

8. The Stability of Riboflavin in 50 Per Cent Glycerin in
Distilled WJater Stored Under Various Conditions in Flint
and Amber Bottles 53

9, The Stability of Riboflavin in 25 Per Cent Propylene
Glycol in Distilled Water Stored Under Various Condi-
tions in Flint and Amber Bottles 54

10. The Stability of Riboflavin in 50 Per Cent Propylene
Glyool in Distilled Water Stored Under Various Condi-
tions in Flint and Amber Bottles 55

U1 The Stability of Riboflavin in a Saturated Solution of
Ethyl Aminobensoate in Distilled Water Stored Under
Various Conditions in Flint and Amber Bottles 56

12. The Stability of Riboflavin in 0.01 Per Cent Quinine
Bisulfate in Distilled Water Stored Under Various
Conditions in Flint and Amber Bottles 57











Table Pag

13% The Stability of Riboflavin in a Saturated Solution of
Beta-Methyl Umbelliferone in Distilled Water Stored
Under Various Conditions in Flint and Amber Bottles 58

14 The Stability of Riboflavin in 1*0 Per Cent Urea In
Distilled Water Stored Under Various Conditions in
Flint and Amber Bottles 99

15. The Stability of Riboflavin in 0.1 Per Cent Teen 80
in Distilled Water Stored Under Various Conditions in
Flint and Amber Bottles 60

16, The Stability of Riboflavin in 0.5 Per Cent Niacin in
Distilled Water Stored Under Various Conditions in
Flint and Amber Bottles 61

17. The Solubility of Riboflavin in Some Aqueous Solutions
and Other Solvents 62

I,. The Stability of Riboflavin-5'-Phosphate Sodium in
Distilled Water Stored Under Various Conditions in
Flint and Amber Bottles 64

19. The Stability of Riboflavin-5'-Phosphate Sodium in
Distilled Water Buffered at pH 6 Stored Under Various
Conditions in Flint and Amber Bottles 65

20. The Stability of Riboflavin-5'-Phosphate Sodium in
Distilled Water Buffered at pH 5 Stored Under Various
Conditions in Flint and Amber Bottles 66

21. The Stability of Riboflavin-5'-Phosphate Sodium in
Distilled Water Buffered at pH 4 Stored Under Various
Conditions in Flint and Amber Bottles 67

22. The Stability of Riboflavin-5'-Phosphate Sodium in 25
Per Cent Glycerin in Distilled Water Stored Under Var-
ious Conditions in Flint and Amber Bottles 68

23. The Stability of Riboflavin-5'-Phosphate Sodium in 50
Per Cent Glycerin in Distilled Water Stored Under Var-
ious Conditions in Flint and Amber Bottles 69

24. The Stability of Riboflavin-5'-Phosphate Sodium in 25
er Cent Propylene Glycol in Distilled Water Stored
Under Various Conditions in Flint and Amber Bottles 70









vii


Table Page

25. The Stability of Riboflavin-51-Phosphate Sodium in 50
Per Cent Propylene Glycol in Distilled Water Stored
Under Various Conditions in Flint and Amber Bottles 71

26. The Stability of Riboflavin-5'-Phosphate Sodium in a
Saturated Solution of Ethyl Aminobensoate in Distilled
Water Stored Under Various Conditions in Flint and
Amber Bottles 72

27. The Stability of Riboflavin-51-Phosphate Sodium in 0.01
Per Cent Quinine Bisulfate in Distilled Water Stored
Under Various Conditions in Flint and Amber Bottles 73

28. The Stability of Riboflavin-5'-Phosphate Sodium in a
Saturated Solution of Beta-Methyl Umbelliferone in Dis-
tilled Water Stored Under Various Conditions in Flint
and Amber Bottles 74

29, The Stability of Riboflavin-5'-Phosphate Sodium in 1.0
Per Cent Urea in Distilled Water Stored Under Various
Conditions in Flint and Amber Bottles 75

30. The Stability of Riboflavin-55-Phosphate Sodium in 0.1
Per Cent Tueen 80 in Distilled Water Stored Under Various
Conditions in Flint and Amber Bottles 76

319 The Stability of Riboflavin-5 -Phosphate Sodium in 0.5
Per Cent Niacin in Distilled Water Stored Under Various
Conditions in Flint and Amber Bottles 77

32. The Solubility of Riboflavin-5'-Phosphate Sodium in Some
Aqueous Solutions and Other Solvents 78

33. The Stability of Flavazin Soluble in Distilled Water
Stored Under Various Conditions in Flint and Amber Bottles 80

34. The Stability of Flavaxin Soluble in Distilled Water
Buffered at pH 6 Stored Under Various Conditions in
Flint and Amber Bottles 81

35, The Stability of Flavaxin Soluble in Distilled Water
Buffered at pH 5 Stored Under Various Conditions in
Flint and Amber Bottles 82

36. The Stability of Flavexi Soluble in Distilled Water
Buffered at pH 4 Stored Under Various Conditions in
Flint and Amber Bottles 83









vini


Table Page

37. The Stability of Flavaxin Soluble in 25 Per Cent Glycerin
in Distilled Water Stored Under Various Conditions in
Flint and Amber Bottles 84

38. The Stability of Flavaxin Soluble in 50 Per Cent Glycerin
in Distilled Water Stored Under Various Conditions in
Flint and Amber Bottles 85

39. The Stability of Flavaxin Soluble in 25 Per Cent Pro-
pylene Glyool in Distilled Water Stored Under Various
Conditions in Flint and Amber Bottles 86

40. The Stability of Flavaxin Soluble in 50 Per Cent Pro-
pylene Glycol in Distilled Water Stored Under Various
Conditions in Flint and Amber Bottles 87

41. The Stability of Flavaxin Soluble in a Saturated
Solution of Ethyl Aminobensoate Stored Under Various
Conditions in Flint and Amber Bottles 88

42. The Stability of Flavaxin Soluble in 0.01 Per Cent
Quinine Bisulfate in Distilled Water Stored Under
Various Conditions in Fliv.t and Amber Bottles 89

43. The Stability of Flavaxin Soluble in a Saturated
Solution of Beta-Methyl Umbelliferone Stored Under
Various Conditions in Flint and Amber Bottles 90

44. The Stability of Flavaxin Soluble in 1.0 Per Cent Urea
in Distilled Water Stored Under Various Conditions In
Flint and Amber Bottles 91

45. The Stability of Flavaxin Soluble in 0.1 Per Cent Tueen
80 in Distilled Water Stored Under Various Conditions
in Flint and Amber Bottles 92

46. The Stability of Flavaxin Soluble in 0.5 Per Cent Niaein
in Distilled Water Stored Under Various Conditions in
Flint and Amber Bottles 93

47. The Solubility of Flavaxin Soluble in Some Aqueous
Solutions and Other Solvents 94

48. The Stability of a Pyruvic Acid Derivative of Riboflavin
in Distilled Water Stored Under Various Conditions in
Flint and Amber Bottles 96











VUame page
49 The Stability of a Pyruvic Acid Derivative of Riboflavin
nl Distilled Water Buffered at pH 6 Stored Under Various
Conditions in Flint and Amber Bottles 97

50. The Stabilit of a Pyruvic Acid Derivative of Riboflavin
in Distilled Water Buffered at pB 5 Stored Under Various
Conditions in Flint and Amber Bottles 98

51. The Stability of a Pyruvic Acid Derivative of Riboflavin
in Distilled Water Buffered at pH 4 Stored Under Various
Conditions in Flint and Amber Bottles 99

52. The Stability of a Pyruvic Acid Derivative of Riboflavin
in 25 Per Cent Glyoerin in Distilled Water Stored Under
Various Conditions in Flint and Amber Bottles 100

53. The Stability of a Pyruvic Acid Derivative of Riboflavin
in 50 Per Cent Glycerin in Distilled Water Stored Under
Various Conditions in Flint and Amber Bottles 101

54. The Stability of a Pyruvic Acid Derivative of Riboflavin
in 25 Per Cent Propylene Glycol in Distilled Water Stored
Under Various Conditions in Flint and Amber Bottles 102

55. The Stability of a Pyruvic Acid Derivative of Riboflavin
in 50 Per Cent Propylene Glyool in Distilled Water Stored
Under Various Conditions in Flint and Amber Bottles 103

56. The Stability of a Pyruvic Acid Derivative of Riboflavin
in a Saturated Solution of Ethyl Aminobensoate in Dis-
tilled Water Stored Under Various Conditions in Flint
and Amber Bottles 104

57. The Stability of a Pyru-ic Acid Derivative of Riboflavin
in 0.01 Per Cent Quinine Bisulfate in Distilled Water
Stored Under Various Conditions in Flint and Amber Bottles 105

58. he Stability of a Pyruvic Acid Derivative of Riboflavin
in a Saturated Solution of Beta-Methyl Umbelliferone in
Distilled Water Stored Under Various Conditions in Flint
and Amber Bottles 106

59. The Stability of a Pyruvic Acid Derivative of Riboflavin
in 1.0 Per Cent Urea in Distilled Water Stored Under Var-
ious Conditions in Flint and Amber Bottles 107











Table Fags

60. The Stability of a Pyruvic Acid Derivative of Riboflavin
in 0.1 Per Cent Tween 80 in Distilled Water Stored Under
Various Conditions in Flint and Amber Bottles 108

61 The Stability of a Pyruvic Acid Derivative of Riboflavin
in 0.5 Per Cent Niacin in Distilled Water Stored Under
Various Conditions in Flint and Amber Bottles 109

62. The Solubility of a Pyruvic Acid Derivative of Riboflavin
in Some Aqueous Solutions and Other Solvents 110

63, The Stability of a Levulinic Acid Derivative of Riboflavin
in Distilled Water Stored Under Various Conditions in
Flint and Amber Bottles 112

64 The Stability of a Levulinic Acid Derivative of Riboflavin
in Distilled Water Buffered at pH 6 Stored Under Various
Conditions in Flint and Amber Bottles 113

65. The Stability of a Levulinic Acid Derivative of Riboflavin
in Distilled Water Buffered at pH 5 stored Under Various
Conditions in Flint and Amber Bottles 114

66. The Stability of a Levulinic Acid Derivative of Riboflavin
in Distilled Water Buffered at pH 4 Stored Under Various
Conditions in Flint and Amber Bottles 115

67. The Stability of a Levulinic Acid Derivative of Riboflavin
in 25 Per Cent Glycerin in Distilled Water Stored Under
Various Conditions in Flint and Amber Bottles 116

68. The Stability of a Levulinic Acid Derivative of Riboflavin
in 50 Per Cent Glycerin in Distilled Water Stored Under
Various Conditions in Flint and Amber Bottles 117

69. he Stability of a Levulinic Acid Derivative of Riboflavin
in 25 Per Cent Propylene Glycol in Distilled Water Stored
Under Various Conditions in Flint and Amber Bottles 118

70. The Stability of a Levulin i Acid Derivative of Riboflavin
in 50 Per Cent Propylene Glyool in Distilled Water Stored
Under Various Conditions in Flint and Amber Bottles 119

71, The Stability of a Levulinic Acid Derivative of Riboflavin
in a Saturated Solution of Ethyl Aminobensoate in Distilled
Water Stored Under Various Conditions in Flint and Amber
Bottles 120











Table Page

72. The Stability of a Levulinic Acid Derivative of Riboflavin
in 0.01 Per Cent Quinine Bisulfate in Distilled Water
Stored Under Various Conditions in Flint and Amber Bottles 12

73, The Stability of a Levulinic Acid Derivative of Riboflavin
in a Saturated Solution of Beta-Methyl Umbelliferone in
Distilled Water Stored Under Various Conditions in Flint
and Amber Bottles 122

74 The Stability of a Levulinic Acid Derivative of Riboflavin
in 1.0 Per Cent Urea in Distilled Water Stored Under VTa-
ious Conditions in Flint and Amber Bottles 123

75. The Stability ef a Levulinic Acid Derivative of Riboflavin
in 0.1 Per Cent Tveen 80 in Distilled Water Stored Under
Various Conditions in Flint and Amber Bottles 124

76. The Stability of a Levulinic Acid Derivative of Riboflavin
in 0.5 Per Cent Niacin in Distilled Water Stored Under
Various Conditions in Flint and Amber Bottles 125

77. The Solubility of a Levulinic Acid Derivative of Riboflavin
in Same Aqueous Solutions and Other Solvents 126

78. The Solubility of a Citraconic Anhydride Derivative of
Riboflavin in Some Aqueous Solutions and Other Solvents 128













INTRODUCTION


Almost since the discovery and isolation of vitamin 2 or

riboflavin, the problem of solubility and stability in solution has

been the abuse of much concern.

Numerous methods have been suggested for preparing solutions

containing a relatively high concentration of riboflavin. Most of

these suggested methods, however, do not show any increase in stability

greater than that of the pure vitamin itself. One of the purposes of

this investigation was to prepare a more soluble form or derivative of

this vitamin. Another aai was to prepare solutions of riboflavin, or

a derivative thereof, which would be more stable to light and yet re

tain active physiological activity. An evaluation of riboflavin and

same derivatives was made with regard to solubility and stability in

various solvents and under different storage conditions,

It is often desirable to administer riboflavin parenterally,

and to do so, it is necessary that the vitamin be present in a there.

peutically effective amount and in a reasonable quantity of harmless

diluent. It is likewise desirable to administer such riboflavin solu-

tions bV oral route. Such a form of vitamin B2 oould also be used for

the enrichment of foodstuffs, for infant preparations and in many other

pharmaceuticals.

Riboflavin is only very sparingly soluble in both water and in

aqueous acidic solution. At 200 C. (1) only 0.12 mg. per ml. of water

1










2
will dissolve. Although more riboflavin is soluble in alkaline aqueous

solutions, such solutions are extremely unstable and the riboflavin

soon loses its physiological activity. It is of advantage to have a

salt producing an acid pH present in the preparation of such solutions.

This type of salt could serve to maintain the pH of the solution near

the isoelectric point of riboflavin, thereby increasing its stability.

In human beings (2) natural as well as artificially induced

riboflavin deficiencies have been observed. Lesions on the lips, and

fissures at the angles of the mouth (cheilosis) are characteristic symp-

tons which are promptly relieved by the administration of pure ribo-

flavin. Human pellagra is often accompanied by definite symptoms of

riboflavin deficiency, and in general, some riboflavin deficiency prob-

ably exists whether the outward symptoms are detectable or not.

Thus, it is ap aren't that the value of a concentrated and stable

solution of vita:nin B2 is of prim- importance. An attempt has been made

in this investigation to prepare such types of solutions.













A REVIEW OF T:E LITERATURE


Historical Sketch

The chemical nature of the yellow-green fluorescent pigment of

whey, now referred to as riboflavin (synonymous with! lactoflavin, vita-

min G and vitamin B2) commanded the attention of chemists (3) as early

as 1879. A considerable concentration of this pigment was effected and

certain of its more obvious chemical properties were clearly set forth

by Bleyer and Kallmann (4) in 1925. No unusual significance was asso-

ciated with this pigment by these early workers, who apparently regarded

it only as one of the minor constituents of milk. The chemical nature

of the pigment was still quite obscure.

In the course of an investigation into the nature of pellagra,

Goldberger and Lillie (5) produced a deficiency disease in rats, char-

acterized by ophthalmic and bilaterally symmetrical denuded areas. The

factor that prevented these lesions was heat-stable, in contrast to

vitamin B1 which was heat-labile. It was termed by Goldberger, the

P. P. (pellagra-preventing) factor but was later designated vitamin B2

in Great Britain and vitamin G in the United States (6). It is now

known that vitamin B2 or riboflavin is not the rat pellagra-preventative

factor but owing to the lack of knowledge at that ti-e of the existence

of other members of the B complex, this -isconception was widely preva-

lent.











In 1932 Warburg and Christian (7) described a new oxidation

en"yme obtained from aqueous extracts of yeast. The enzyme in water

solutions was yellow and exhibited a green fluorescence. It has nw

been established (32) tVat this "yellow enayme" is present in every

living cell or at least in the cells of all the higher forms of life.

The symptoms reported by oth-r workers as characteristic of

vitamin B2 deficiency varied considerably, howvowr, and frequently dif-

fered markedly from those observed by Goldberger and Lllie (5). In

particular, some workers reported only an absence of growth, while

others noted te appearance of a dermatitis in some of the experimental

animals.

In 1934 Oyorgy (8) (9) showed the fallacy of Goldberger and

Lillie's experiments. This observer showed that rats ma. ntained on a

vitamin B free diet, with Bj concentrate and lactoflavin added, devel-

oped a number of pellagra-like changes which were not only unrelieved,

but even made worse by the addition of more vitamin B2. These lesions,

so produced, which were of a somewhat different character from those

produced by Goldberger and Lillie, were cured by an unknown factor,

tentatively named by (yorgy vitamin B6. It was contained in the "Peter's

Eluate" from charcoal as prepared from yeast extract. This author ad-

mitted that certain skin lesions can be produced by deprivation of Vita-

min B2 but made a sharp distinction between the manifestations so pro-

duced and those due to deprivation of vitamin Bg.

Thus, ir.tial1y, the term vitamin B2 was intended to describe

the factor that caused pellagra, now known to be identical with nicotinic












acid. Subsequently, it came to be used to denote the rat growth

factor, riboflavin.

The first step towards an understanding of the nature of vita-

min B2 was taken by Kuhn, Qyorg and ;a.-ner-Jauregg (10), who isolated

from egg-white a compound with a strong yellowish-Sreen fluorescence.

They called this substance ovoflavinn" and shoved that it stimulated

the growth of rats.

In the same journal containing the paper by Kuhn e al., there

appeared a paper by -llinger and Koschara (11). They reported the

presence of similar fluorescent substances in milk, liver, kidney,

urine, muscle, yeast and in certain plant materials. They described

the isolation of a crystalline fluorescent substance from whey. This

substance obtained from whey they called lactoflavinn" and they pro-

posed the neme "lyochromes" for the group to which all these substances

belonged. This term was in contradistinction to a group of naturally

occurring fat-soluble pigments called "lypoohromes." Both Kuhn get g

and Koschara (11) suggested that the pipgmonts might be related to the

"yellow enzyme" discovered in yeast. In fact, Kuha showed that one and

the same substance, lumiflavin, was produced by irradiation of the yel-

low enayme and of riboflavin.

Shortly after the publication of these papers, Booher (12) re-

ported the preparation of a concentrate froth whey powder that shoved a

strong yellow fluorescence and had growth-pro'moting properties for the

rat.

At first, these pigments isolated from various substances (13)










6
were given specific names according to their origin, for examples ovo-

flavin, lactoflavin, uroflavin and hepatoflavin. It was later realized

that they were all probably identical with one another. This was con-

firmed by direct comparison of some of the compounds, but several were

isolated in such small amounts that rigid proof of identity was not

possible.

In 1936, at a conference group of tle American Chemical Society

meeting at Pittaburgh (14), the opinion was unanimous that the term

flavin should be used to designate the water-soluble pigment that has

been demonstrated to be necessary for the normal nutrition of the rat

and for growing chicks. It was also determined at this meeting that

the terms lactoflavin, vitamin G and vitamin B2 should not be used.

Riboflavin was to be the accepted name for vitamin B2.











Isolation

aiboflavin has been isolated from a wide variety of animal and

plant products (15) including egg-white, milk, liver, kidney, urine,

barley malt, dandelion blossoms, raises, egg yolk and retinas of fish

eyes. It can be stated with absolute certainty that the crystalline

flavin obtained from each of these various sources was chemically iden-

tical with riboflavin. At least such is the case for those to which

adequate determinative tests have been applied.

The methods of isolation (16) varied somewhat in different lab-

oratories and with the raw materials employed, but nearly all the workers

used adsorption on fuller's earth (or in some instances lead sulfide)

from a slightly acid-aqueous or aqueous-alcoholic extract. The result-

ing adsorbate was eluted with pyridine, or pyridine-methanol-water mix-

ture or dilute ammonia, and the eluate, after being concentrated, was

treated with a heavy metal, such as silver or thallium, to precipitate

the flavin in the form of a salt. The free flavin was recovered from

the precipitate by suitable treatment and recrystallized from water,

dilute alcohol or dilute acetic acid.

In their earlier work, Kuhn and his co-workers (10) obtained

from 100 Kg. of dried egC albumin, corresponding to about 33,000 eggs,

10 mg. of thrice recrystallized flavins. According to subsequent meas-

urements (17) of the quantities of flavin normally present in dried egg

albumin, this yield would correspond to about 7 per cent of the total

flavin present in the egg albumin. Similarly the yield of crystalline









8
flavin from milk, as reported in the earlier work (18), was not greater

than 5 pe cent of the total quantity present. The use of heavy metal
precipitation increased the yield to about 18 to 20 per cent of the

quantity reported (17) to be normally present in milk,

Synthetic flavin was first prepared la 1934 by Karrer and Kuhn

(19) and also by pRenemund and .eygand (20). This synthetic flavin was

shown in both eases to be chemically identical with the flavin isolated

from milk and to have the same biological value for rats. Karrer (21)

first used the term riboflavin and its synthesis proved it to be an

alloxasine structure combined with ribose.











Occurrence

Riboflavin (1) is widely distributed in nature in both plants

and animals, being found as a free pigment or combined with a protein.

It is an essential constituent of all living cells.

In the plant world, analysis (22) has shown that riboflavin

occurs naturally in the green actively growing leaves and that it per-

sists there in higher concentration than in other parts of the plant.

Consequently, green stems and leaves are a much richer source of the

vitamin than the flower or root. However, the vitamin is present in

small amounts in practically all root vegetables and tubers.

There is reason to believe that as the leaves mature and dry,

the riboflavin content may be correspondingly diminished (23). This

may have a bearing on the vitamin content of milk since it has been

found that cowv fed on fresh young grass yield milk richer in ribofla-

vin than animals receiving a more mature and drier grass (24). However,

milk, either fresh or processed, seems to be a relatively rich and con-

stant source of riboflavin.

The concentration of the vitamin in seeds (23) is subjected to

considerable variation and reaches its maximum in the gem portion.

Legumes, peas and beans provide a moderately rich source while nuts and

cereal grains are somewhat poorer in their content. Fruits generally,

and particularly citrus, have been proved to provide only a trifling

amount of this substance.

The glandular organs of animals (24) constitute the richest of

all foodstuffs in their riboflavin content. The lean musle flesh











contains very considerable quantities.

Many species of microorganisms (25) are capable of syntheaising

riboflavin, and because of the extensive bacterial growth in the human

intestinal tract, this may form an important and constant source of

supply.

The retinas of the eyes of many species of animals have been

reported (26) to contain relatively high concentrations of flavin. It

was supposed that the flavins are involved in some light sensitized re-

actions concerned with dim vision.

Riboflavin is synthesized commercially (1) on a large scale for

addition to bread, flour and other dietary and pharmaceutical prepara-

tions.











Physical and Chemical Properties


Riboflavin (27) is a yellow to orange-yellow crystalline pow-

der having a slight odor. It melts at about 2800 C. and its saturated

solution is neutral to litmus. Riboflavin is quite stable in strong

mineral acids. When dry it is not appreciably affected by diffused

light, but in solution, especially in the presence of alkali, it dete-

riorates quite rapidly, the deterioration being accelerated by light.

Riboflavir. is so sensitive to light that on irradiation with ultravio-

let rays or visible light (1) it undergoes irreversible decomposition.

Riboflavin is 6,7-dimethyl-9-D-l'-ribitylisoalloxazine. It is

thus a nitrogenous polyhydroxy alcohol (1).

At least one of the methyl groups in position 6 or 7 is essen-

tial in order that the flavin molecule shall possess vitamin activity.

The absence of both the 6 and 7 methyl groups actually appears to be

accompanied with toxicity (28). With regard to the side-chain, only

the D-ribose or D-arabinose residue attached to the nitrogen atom in

position 9 has thus far proved to be compatible with vitamin activity

of the flavins. Exceedingly small variations in the side-chain often

cause complete lack of vitamin activity.

The flavins, as a group, all share the tricyclic chromophoric

nucleus that confers on them the yellow color to which they owe their

group name. The vitamin activity (29) of the various members of this

group of yellow pigments is profoundly influenced by the position and

nature of the subatituent groups in the benzene nucleus and by the na-

ture of the side-chain attached to the pyrasine ring,









12
Riboflavin, the empirical formula of which is C17W$20406, ham

a solubility in water of 12 mg. per 200 ml. at 27.50 C. Some variations

in solubility have been noted and this is due to differences in the in-

ternl crystalline structure of the vitamin. The aqueous solution has

a strong yellowish-greon fluorescence which is discharged by acid or

alkali. This is used as a basis for identification by the U. S. P. (27).

Riboflavin (25) (27) is sparingly soluble in ethyl alcohol (4.5

mg. per 100 al. at 27.50 C.) anyl alcohol, cyclohexanol, phenol or aaml

acetate, but insoluble in acetone, ether, benzene or chloroform. It

is more soluble in iaotonic sodium chloride solution and very soluble

in dilute alkali. By splitting off the D-ribityl side-chain (25) th

resultant molecule becomes soluble in chloroform.

To increase the solubility of riboflavin in water (for injec-

tion use) the U. S. P. allows suc, preparations to contain nicotinamide,

urea or other suitable harmless solubilizing agents.

In neutral or acid-aqueous solution, riboflavin (30) shows no

rotation, but in alkaline solution it is strongly 1-rotatory,

Riboflavin is amphotoric in nature, with an isoelectric point

at pH 6 (31). The dissociation constants areas


Ku a 63 x 10-12 and Kb a 05 x 10-5.

On acetylation (10), a tetraacetate with a melting point of

2420 C, is formed.

On irradiation in alkaline solution (32), riboflavin yields

luniflavin, C1312N402, and this being sparingly soluble in water,










13
separates fro- the irradiated solution. Irradiation of neutral or acid

solutions of riboflavin (33) is attended witi the formetion of 6,7-

dimethyl-alloxaaine or lumichrome which exhibits an intense blue fluores-

cence.

Goldblith and Proctor (34) showed that elootro a or X-rays (3

magavolts from a Trump generator), which were used to irradiate solu-

tions of pure riboflavin in metallic petri dishes, caused destruction

of this fluorescent substance according to accepted methods of assay.

The higher the concentration of irradiated vitamin, the less was the

percentage of destruction. The products of irradiated riboflavin wer

lumichrome plus fragments.

Ellinger and Koschara (11) found vitamin B2 to be reversibly

reduced by sodium dithionite solution, by since in acid solution, by by-

drogen sulfide in alkaline solution, by hydrogen in the presence of a

catalyst, or by titanous chloride to a leuoo-compound which was reoc-

idised to riboflavin and the color and fluorescence restored on exposure

to air,

By maintaining riboflavin (35), both synthetic and natural, in

a reduced state vith sodium hydrosulfite, it can be protected ftro sun.

light destruction, The reduced state can be reoxidised by vigorous

shaking with excess air. Conclusions were that unreduced controls

shoved a 90 per cent destruction in a thirty minute exposure.

Riboflavin was said to be rendered more stable to light by the

presence of sodium dithionite (36) or by heating with boric acid (37).

Solutions containing boric acid are recommended for injection and are











said to be self-sterilizing as well as photo-stable.

Riboflavin (25) has a characteristic absorption spectrum, the

peaks of the absorption bands being 221, 266, 359, and 445 Im.

Crystalline riboflavin is stable in the dark at ordinary tea

peretures but decomposes on exposure to light. Vitamin B2 (38) is rel-

atively heat-stable in acid solution, and the rate of destruction is

rapidly increased with increasing alkalinity. In alkaline solutions

it is unstable, especially when these solutions are exposed to light.

Ellinger and Holden (39) showed that at high concentrations of

riboflavin in solution, the effect of "quenching" comes into play. It

was considerably affected by certain anions, such as halides, cyanide,

thiocyanide, sulfite and nitrite. Ferrous and ferric salts (am

oxidation-reduction process) has a similar "quenching" effect.

Epley and Hall (40) experimented with several of the F. D. and

C. colors and showed that riboflavin was unstable to F. D. and C. green

number 3. However, F. D. and C. red number 3 and F. D. and C. orange

number 1 seemed to protect vitamin B2 from photochemical destruction.

Common cork, unless previously soaked in a large volume of water

(41), contains a substance which strongly inhibits the fluorescence of

riboflavin.

No appreciable destruction occurred when milk was incubated by

Sure and Ford (42) for twenty-two hours at 310 to 370 C. or during the

cooking of foods (43). When, on the other hand, milk in bottles was

exposed to sunlight by Peterson gji ,. (44), more than half the ribo-

flavin was destroyed within two hours,











Assay DLtertinations

The U. S. P. (27) gives both a microbiological assay procedure

and a fluorophotometric method for determining riboflavin. The fluoro-

photometric method is based on the measure of fluorescence in acid solu-

tion. By comparing the concentration of an unknown solution with that

of a prepared standard using a fluorophotometer that can accurately

measure riboflavin activity in approximately 0.1 to 0.2 mag. per ml.,

the amount present can accordingly be calculated. The microbiological

method is carried out in acid media. It is also based on a comparison

of an unknown with a standard solution using a pure culture of lt4



Visual methods for the determination of fluorescence have been

employed, but photoelectric techniques (45) have been almost universally

adopted in more recent years.

Loy (46) made a study of the fluorometric method and the micro-

biological method of assay of riboflavin. He found that there were no

statistical differences between the results of the two methods,

When an amber transparent shade of the type commonly used in

department store display windows for filtering out ultraviolet rays was

placed over laboratory windows, it was found to minimize the destruction

of riboflavin (47) by light rays during the course of assay. The use

of added artificial light resulted in appreciable destruction of ribo-

flavin,

So sensitive is riboflavin to the action of light that ribo-

flavin assays should be carried out in dim light and preferably in red











light. De Merre and Brown (48) recommended a 150-watt lamp screened

with a red oellophane filter. The light from the lamp normally ema

played in a Coleman spectrophotometer, however, does not cause appre-

ciable destruction.

Various standards have been used for comparison with the in-

tensity of fluorescence of the unknown, for example, pure riboflavin

(49), potassium dichromate (50), fluorescein (51) and uranium glass

(52) have been used as such standards.

Cohen (53) used a Kleinmann nephelometer with light from a

mercury lap filtered through a screen of nickel oxide for the deter-

mination of vitamin B2 by means of its fluorescence.

To prepare a solution for fluorescent analysis, Weiaberg and

Levin (50) recommended the use of a clear solution. Various dilutions

of this solution were made, up to 50 al., in square 60 al. bottles and

compared under ultraviolet light with standards of sodium fluorescein.

The standards were made up in terms of 0.1-1.0 meg. of riboflavin per

al.

Riboflavin can be determined in concentrations of 0.005-2.0 mg,

per liter as shown by Kavanagh (54). He used a two photocell balanced

circuit with a galvanometer as a null point indicator to measure the

ratio of the fluorescence of the unknown to that of a standard glass or

quinine solution. With the use of proper filters, small amounts of

suspended material did not interfere with the experiment.

For determining riboflavin content, Hodson and Norris (45)
based their methods on the utilization of certain properties of ribo-









17
flavin, Basing their work on fluorometric methods their claims were

1. Riboflavin fluoresced gre.:-n when radiated with a blue

light.

2. It was not destroyed by mild oxidation or reduction.

3. It could be reduced to a non-fluorescing form with sodium

hydrosulfite and reoxidised readily by shaking with air.

4, It was not reduced by stannous chloride.

5. The intensity of the fluorescence could be measured with a

photoelectric cell

In further experimental work, Kuhn and Moruszi (31), took meas-

urements of the fluorescence of riboflavin solutions with graded pH

values and shoved that at a pH of 1.7 on the acid aide and pH 10.2 on

the alkaline side, the fluorescence brightness seemed to be propor-

tional to the riboflavin concentration.

Jones and Christiansen (55) reported that riboflavin gave a

maxima fluorescence at a pH of 6 to 7 whereas Karrer and Fritsche (56)

pointed out that a maximum fluorescence was exhibited by a 0.003 per

cent solution of riboflavin at pH 7.0.

Conner and Straub (57) shoved that a linear relationship between

fluorescence and concentration of riboflavin exists between the limits

of 0.013 to 0.13 meg. per ml.

Hanson and Weiss (58) recommended that in determining ribo-

flavin concentrations, a standard solution containing 50 mag. per ml.

(50 parts per million) should be used. Although they found that it was

difficult to get that amch riboflavin into perfect solution, they









18
observed that fluorescence was proportional up to about 30 parts per

million. In some cases the straight-line relationship between concen-

tration and fluorescence would possibly co-tinue up to 50 parts per

million. It was said to be safor to work at a concentration not greater

than 30 parts per million.

At the University of Witvatersrand in Johannosburg, Alper (59)

claimed that substances which' fluoresce exhibit fatigue when exposed

continuously to the radiation which causes fluorescence. A dilute

aqueous solution of riboflavin gave a straight line when the logarithm

of the intensity of the fluorescent light was plotted against t-me.

Every trial ended, not with an equilibrium state, but with a rate of

fading which could no long-*r be measured. This photofatigue was impor-

tant in the fluorometric assay of substances llke riboflavin.

Slater and Morell (60), who worked with the Klett photoelectric

fluoroneter-colorimeter and the Klett photoelectric fluorometer, gave

detailed procedures for making thiochrome and riboflavin solutions. It

was shown that many precise measurements of the fluorescent intensity

of solutions were possible with a fluorometer,











Increasing Solubility

In view of the low solubility of riboflavin, numerous methods

have been suggested and many derivatives made for the preparation of

solutions containing a relatively high concentration of the vitamin.

One of the earliest patents obtained to increase the solubility

of riboflavin was received by Auhagen (61) in 1941. He claimed to have

gotten up to 0.25 Gm. of riboflavin in 100 ml. of a 10 per cent nilo-

tinamide or salts of nicotinic acid in water.

Frost (62) showed that riboflavin-nicotinamide solutions may be

physiologically stabilized by adjusting the pH value of the solutions

to 2.6-6.6 and preferably from 4.4-6.6. In a later work, Frost (63)

proved that the solubility of riboflavin in nicotinamide solutions de-

creases progressively at pH values more acid than 5.0. As the nico-

tinamide concentration was increased from 5 to 50 per cent, the solu-

bility of riboflavin increased at pH 5 from about 0.1 to about 2.5 per

cent. The observed strong solvent effect of niacin on riboflavin ap-

peared to be related to its chemical constitution with both CoH4N and

CONE2 groups being involved. An acid which formed an addition salt

reduced the solvent action of nicotinamide but did not eliminate it.

Various details vcre given for the production of double salts

of riboflavin, such as sodium riboflavin and an alkali metal borate by

Auerbach (64). He claimed that the use of borax with an alkali was

said to give the complex:


C17H1906N4Na-Na2B4. lO10H20.










20
In 1942, Frost (37) chowed that a riboflavin-boron solution of

good stability, containing up to 0.3 per cent riboflavin, can be ob-

tained by heating an aqueous solution of riboflavin and up to 5 per

cent boric acid at pH 6.5 for three hours at 95P C. With aetaboric

acid less heating was required, The specific rotation of riboflavin

below pH 6 was enIL. ed in a positive direction by boron but the sol-

vent effects of boron compounds were small below pH 6.0 and were in-

creased above pH 6.0. It was found that the ribityl group was involved

in the solvent reaction with boric acid and that the effect was inde-

pendent of the very insoluble isoalloxanine group. Any attempt to

bensoylate riboflavin in the presence of borates gave no reaction.

Riboflavin tetrabenzoate and riboflavin monoborate were prepared. Iao-

tonic preparations of the riboflavin-boron complex were self-sterilising

toward molds and bacteria and were suitable for injection.

Moran and Stein (65) experimented with the sodium salt of ribo-

flavin and a polybasic carboxylic acid such as phthalic or succinic

acid or their anhydrides refluxed in pyridine. After the reaction was

completed the pyridine was evaporated, the residue dissolved in water

and acidified. Crystals that separated were recrystallized from boil-

ing water. These derivatives, particularly the succinate, were shown

to have increased the solubility in water very favorably as compared

to that of pure riboflavin,

Hoffer (66) showed that in 180 ml. of a 2 per cent solution of

a lower alkylolamide of gentisic acid in water, he was able to get up

to 0.33 Cm. of riboflavin soluble.









21
Hoffer in collaboration vith Purter (67) showed that in aqueous

solution containing 5 p .r cent gontisic acid and 5 per cent sodium

gentisate at pH 5.0, riboflavin, up to 16 GO was soluble in each 100 al.

Jurist (68) showed that concentrated riboflavin solutions were

obtained by dissolving the vitamin in an aqueou solution of a pharmsoao

dynamically unobjectionable aliphatic amidine acid addition product,

such as acetamidine hydrochloride. A 20 per cent aqueous solution of

aoetamidine hydrochloride will carry 1900-2000 meg. per il. Solutions

did not deposit riboflavin when chilled to 80 C. and they were heat-

sterilised and stored without changes in color or clarity,

By using liver extract as a solvent, having 250 to 350 mg. per

al. of liver solids, Shelton (69) showed that it was possible to get up

to 0.2 Qu of riboflavin soluble por 100 ml.

Bird and Kuna (70) demonstrated that riboflavin was readily

brought into solution with gallic acid or its alkali salts. Ten milli-

liters of a 10 per cent solution of gallic acid in 50 per cent aqueous

ethyl alcohol dissolved 14 nmg. of riboflavin. Sodium gallate in a 10

per cent aqueous solution at pH 6,7 dissolved 58 mg. of riboflavin in

30 al. at 24.50 C. Dry mixtures of the vitamin and the salts of gallio

acid dissolved readily in water.

Riboflavin was converted into a soluble complex by treatment
with gallic acid in the presence of water and an inorganic acid by

Bentner (71).

Preisverk (72) reported a solubility of 4 Gm of riboflavin
per 100 ml. of an aqueous solution containing 25 per cent or more of a









22
water soluble salt of 2,4-dihydroxybensoic acid or its lower monoaliyl

ethers. The ortho, meta and para compounds of the latter were specified.

Water-soluble salts of bensoic acid and its amino or hydroxy-

substituted derivatives were used as solubilizing agents in aqueous

solutions by Miller (73). He showed that alkali benzoates (including

sodium p-hydroxybensoate and sodium p-aminobenzoate), magnesium or

sodium salicylate and monoethanolamine salicylate all helped to increase

the solubility of riboflavin.

Haas (74) claimed to have gotten up to 8.0 Cm. per ml. soluble

as the citrate of diothylaminoacetyl riboflavin.

Upham (75) prepared stable, sterile and clear solutions of ribo-

flavin citrate in propylene glycol. He reported up to 40 mg. per al.

as the possible solubility. Solutions containing additional substances

in the same solvent were also given.

Moos and Upham (76) prepared citric, malic and tartaric acid

esters of riboflavin by heating the acid and vitamin in phenol at 1200

to 1400 C. The esters were separated by pouring the cooled mixture into

ether. The esters were water soluble and stable at pH 5.5-7.5 which

were suitable for solutions for parenteral administration.

Knauf and Kirchmeyer (77) prepared solutions of riboflavin con-

taining 0.1 to 0.3 per cent using water and 1 to 4 per cent veratyl al-

cohol as the solubilizer.

Solutions suitable for oral or parenteral administration and

containing 0.15 to 0.3 per cent of riboflavin were reported by Charney

(78). These solutions of riboflavin, alone or with other substances,











were prepared by usinC 1 per cent vanillin as the solubilising agent

in water or propylene glycol.

Charney (79) also reported the solubility of riboflavin in 4

per cent aqueous L-tyrosine amide at pH 5.0 and in 4 per cent aqueous

L-tyrosine amide plus 10 per cent nicotinamide at pH 5.0.

The effect of sodium chloride, glycerin, urea, boric acid and

sodium sallicylte as solubilizers and stabilizers for concentrated

solutions of riboflavin were studied by Gupta and Qupta (80). Boric

acid was found to be the most efficient stabilizer but was painful when

administered intramuscularly. Sodium sallcylate (5 per cent) in a con-

centration of 2.5 ag. of riboflavin per ml. was found effective.

Gerlough and Smith (81) found that acetyltryptophan had a solu-

bilising action on riboflavin and could be used in the preparation of

solutions for parenteral administration.

The solubility of riboflavin in aqueous media was increased by

the addition of 20 parts of pyridylcarbinol (82) to 80 parts of water.

Schoen and Gordon (83) worked with -ater soluble methylol de-

rivatives of riboflavin. By reacting formaldehyde with vitamin B2, the

monomethylol and dimethylol derivatives were obtained. Such compounds

were found to be stable to potassium permanganate at room temperature

but not at 500 C.

When riboflavin was fused with an amide, such as urea, urethane,

acetanide or niacin, a product was obtained which yielded water solu-

tions containing up to 6 per cent riboflavin. Stecher (84) showed that

an acid salt, such as NaH2PO4.H20 may be incorporated in the melt or










24
added to the dry material. The composition of the used product was

not determined but it was assumed to contain equimolecular amounts of

riboflavin and amide.

Stone (85) made a water-soluble derivative of riboflavin by

dissolving it first in concentrated sulfuric acid, neutralizing with

calcium hydroxide and freese-drying. This calcium salt of a sulfate

ester of riboflavin in aqueous solution was found to be stable in air

and 1000 times as soluble as U. S. P. Riboflavin. It wae soluble in

methyl alcohol, glycerol and propylene glycol and slightly soluble in

ethyl alcohol. Analysis indicated an empirical formulas


C17H18N4015S3Ca.

A portion of the sulfur was present as the sulfate. Fluorometric assay

gave a riboflavin content of 57.2 per cent.

Riboflavin-5'-phosphate ester monosodium salt (86) was prepared

by phosphorylation of riboflavin with chlorophosphoric acid. The solu-

bility in water was claimed to be more than 100 timas that of riboflavin.

The empirical formula was reported as:


C17.I204O0 Pa. 2H20.

It was fully active biologically, microbiologically ard ensynatically.

However, the greater sensitivity of the phosphate ester to destruction

by ultraviolet light necessitated careful protection of dilute solutions

from exposure. The monodiethanolamine salt, with a water solubility of

more than 200 times that of riboflavin, was also prepared. This salt










25
was slightly acid in aqueous solutions.

A method of solubilizing riboflavin with sodium 3-hydroxy-2-

naphthoate was developed by Arnold and Auerback Jet #. (87). In the

procedure, the riboflavin itself was not treated. An aqueous solution

with the naphthoate salt was prepared with the concentration being bout

double that of the desired concentration of riboflavin. The vitamin was

then added and after a little stirring it went into solution. An exact

mechanism of the solubilisation effect was not proposed but it was be-

lieved that some sort of complex was formed.













EXP K.IT QITAL


Materials Used


The drugs and chemicals used in this investigation together

with the source, grade and lot number are shown in Table 1. The manu-

facturers of these materials are indicated by letters as follows


t Eli Lilly and Co.

M Merck and Co., Inc.

W-S 'inthrop-Stearns, Inc.

H-LR Hoffmann-La Roche, Inc.

MA Matheson Co., Inc.

0 General Chemical Division

SA Sargent Chemical Co.

E Eastern Chemical Co.

S Swift and Co.

D Dow Chemical Co.

F Fisher Scientific Co.

K Koppers Co., Inc.

ML Mallinckrodt Chemical Works

SNr Smith New York

C Carbide and Carbon Chemicals

B Baker Chemical Co.

A Atlas Powder Co.

26











TABLE 1

DESCRIPTION OF DRUGS AND CHEMICALS USED


Material Manufacturer Grade Lot Number
,.. _._. ____ IL I~ II.. / III-llU l I I If I Jll I IIII 1 I .L ll l .


Riboflavin


Flavain Soluble
(Riboflavin Sodium-
Sodium Tetraborate)

Riboflavin-5'-Phosphate
Sodium

Pyruvic Acid

Sodium Hydroxide

Barium Hydroxide

Potassium Acid Phthalate

Fluoreseein Sodium

Ethyl Ether

Glycerin

Propylene Glycol

Ethyl Aminobensoate

Quinine Bisulfate

Beta-Methyl Umbelliferone

Nicotinic Acid (Niacin)

Citraconic Anhydride

Liquefied Phenol

LeTllnic Acid (Liquid)

Ethyl Alcohol


L
H

8




H-LR

MA

0

SA



M

M

S

D

F

M

I

ML

SNY

ML

MA

C


USP
USP








CP

Reagent

Reagent

Reagent



USP
USP


US?

USP

USP



USP



USP

Op
UCP

USP


Sample
42989

R-023-IN




510123

5300

J089

24392

92353

50223

0638

800

1855550

13847

43587

C5P-2132

2697

Investigational

0024

2404











TABLE 1-Continued


Material Manufacturer Grade Lot Number

Sodium Chloride M Reagent 41439

Potassium Chloride M Reagent 42378

Sodium Acid Phosphate ML USP 7868

Potassium Acid Phosphate B USP 2251

Nicotinamide N USP 50180

Urea B USP 1490005

Polyoxyethylene Sorbitan
Honooleate (Tween 80) A USP 299











Preparation of Riboflavin Derivatives


Pymruc Acid. Imulino Acid and
Citraconic Anb drlde Derivatives

To 5 Qm. of riboflavin, placed in a 250-ml. red glass flask,

were added 5 ml. of pyruvic acid, levulinic acid or citraconia anhy

dride (whichever was indicated), and 50 ml. of liquefied phenol. The

flask was set in an oil bath and refluxed from four to six hours at a

temperature range fron 100-11&0 C, The reaction mixture was allowed

to eool to room temperature and then poured, with constant stirring,

into 500 al. of ether in which the riboflavin derivative precipitated.

The colored, crystalline precipitate was separated from the ether mix-

ture ty filtration on a Buchner funnel, and the product was washed

with several portions of ether and dried between sheets of filter paper.

The dried derivative was further purified by placing it in a mortar and

triturating with a 100 ml. portion of ether and filtering. This pro-

cedure was repeated three times after which the preparation was again

dried between sheets of filter paper. It was dried in an oven at 600 C.

for four hours and then placed in the dark in a desiccator over phos-

phorous pentoxide. A working yield of derivative was obtained by this

method.

Another method of synthesis, which did not prove as successful

as that just mentioned, involved the refluxing of 2 Gn. of riboflavin

and 8 al. of the organic acid or anhydride in media made up of 2 al.

of concentrated sulfuric acid in 30 ml. of distilled water. This m1.0

ture was also refluxed in an oil bath at a temperature range fro











100-1100 C for four to six hours. After the reaction mixture was

cooled to room temperature, the sulfuric acid was carefully neutralized

with a slurry of calcium oxide in water. The precipitated calcium sul-

fate was removed by filtration and the aqueous solution containing the

derivative was evaporated to dryness in an oven at 600 C. This method

was not generally employed in the preparation of soluble derivatives

due to the apparently low riboflavin yields and the difficulty encoun-

tered with the isolation.

Other methods tried for isolating the riboflavin derivative

from the reaction mixture were vacuum distillation and steam distillam

tion. Although the compound was isolated with vacuum distillation,

this procedure was found too time consuming. Steam distillation de-

stroyed most of the vitamin.











Scope of the Fluorophotometer


The instrument used to determine the stability and solubility

of riboflavin and some of its derivatives was the lumetron photo-

electric fluorescence meter model 402-EF.

The operation of this type of fluorophotometer (88) is based

on the light of a mercury vapor lamp which is condensed by an optical

system to form a parallel beam. This beam is passed through a narrow-

band filter which isolates the exciting light of the proper wave length.

The exciting beai is split into two parts. One part enters the sample

holder whict is provided with a thin front window of low ultraviolet

absorption. The fluorescence of the liquid is registered by two large

barrier-layer photoells which are arranged laterally on both sides of

the sample holder in the fluorescence pick-up unit, Filters between

sample holder and photocells serve to isolate the specific fluorescence

band and to eliminate the influence of primary light which may be scat-

tered by particles suspended in the liquid. The other part of the beam

is deflected by a front surface mirror and acts upon the balance which

is mounted so that it can be turned through an angle of 900. The two

measuring photocells and the balance photocell are connected in a

bridge circuit with a slide wire and with a galvanometer as the sero

indicator, The purpose of the bridge circuit is to eliminate the in-

fluence of light intensity variations of the mercury vapor lamp.

The galvanometer was set to the sero mark in the center of the

acale by means of the galvanometer zero adjustment knob. This setting

was checked from time to time and readjusted when necessary. However,











it was not found necessary to have the light spot always exactly on

the Mero mark of the scale. The high sensitivity of the multiple re-

flection galvanometer made it necessary to treat the balancing opera-

tion in a manner differing slightly from the balancing of circuits ea*

played in less sensitive galvanozmters. A very convenient electrical

method to accomplish this is used in the instrument. The middle posi-

tion of the galvanometer key switch (off position) is made not to dis-

connect the galvanometer, but to connect the galvanometer and the other

parts of the circuit into a substitute circuit in which all interfer-

ences but no photocurrents register on the galvanometer.

The lumetron is furnished with 10 bakelite plates numbered from

1 through 10. Plate 1 has no aperture whereas plates 2 through 10 ae

provided with apertures ranging in diameter from 1/16 of an inch to 3/4

of an inch. These plates fit into a slot on the right of the filter

holder compartment and serve the purpose of reducing or blocking out

the light on the balance photooell or on the measuring photocell. The

most suitable reduction plate is selected by trying out the various ap-

ertures starting with the larger apertures and proceeding to the smaller

ones. Finally, a very small aperture is found which no longer permits

balancing even though the balance control is turned all the way counter-

clockwise. The reason for this is that with this reduction plate the

balance beam has been reduced so much that the balance cell, even if

turned to face the balance beam squarely, can no longer balance the eur-

rent of the measuring cells. The smallest aperture with which it is

still possible to balance the circuit or the next larger aperture should









33
be used. The selection of the aperture has no effect upon the fluores-

cenoe reading obtained but only upon the convenience in balancing with

the balance cell controls.

In order to measure, by fluorescence, the concentration of an

ingredient in a solution, it is necessary to have samples of the sol-

vent alone as well as of the ingredient alone. A known amount of the

ingredient is dissolved in the solvent and serves as the standard for

which the instrument is balanced with the slide wire on 100. The sol-

vent alone serves as the standard blank. If this blank show a reading

on the instrument (either due to inherent fluorescence of the solvent

or due to scattered primary light), this reading is suppressed by mans

of a sero suppressor knob so as to make the blank read sero on the

slide wire dial. The length of the slide wire scale from 0 to 100,

then, covers the range of concentration fro o to the known concen-

tration of the standard.











Standardization of the Lunetron


Preparation of Solutions

To prepare the lunetron for stability studies of riboflavin

and some of its derivatives, it was necessary to adjust the instrument

in such a manner so that there would be a linear relationship between

concentration and fluorescence. Such a relationship exists in concen-

trations up to 2 mg. per liter (54). The plotting of a calibration

curve is not necessary in this range, since the amount of fluorescence

is directly proportional to the concentration.

The preparation of a standard stock solution of riboflavin was

made by taking exactly 50 mg. of riboflavin, previously dried at 105 C.

for two hours, and dissolving it in enough distilled water (acidified

with 1 al. of glacial acetic acid per liter) to make 1000 al. To pre-

pare a solution dilute enough for fluorophotometric analysis, this so.

lution had to be further diluted by taking a 40 ml. aliquot and diluting

with a sufficient amount of distilled water to make 1000 ml. The re-

sulting concentration of this standard solution was 2 ag. per liter o

2 mag. per al.


Adlusting the uIamtron for Analvsia
After setting the galvanometer to the ero mark, the primary

and secondary filters were inserted into the lumetron. The sample

holder, with 25 ml. of the riboflavin standard solution containing 2 meg.

per ml., was placed into the fluorescence pick-up unit. With the slide

wire dial set at 100 and after the insertion of the proper reduction










35
plate, the lumetron balance cell was adjusted. This gave a reading of

100 with a solution containing 2 mog. per ml.

The slide wire dial was then set at sero and a blank solution

(solvent only) was placed in the pick-up unit. Here, the lumetron was

adjusted to give a reading of sero by turning the suppressor knob

counter-olockwise,

A solution containing 1 meg. per ml. of fluorescent sodium was

similarly prepared using distilled water. The fluorescence reading was

taken after adjustment of the lumetron with the riboflavin solution and

the blank. This fluorescence reading was used as a standard so that

the instrument could be properly checked and adjusted each day before

use.

After the lumstron was set for the maximum and minimum values,

various dilutions were made of the standard riboflavin solution and

fluorophotoetrio readings were taken as described in Table 2*











TABIE 2
LUMETRON READINGS OF VARIOUS DILUTIONS OF A STANDARD
RIBOFLAVIN SOLUTION CONTAINING 2 MO./LITER


M1. of Standard Ml. of Distilled
iboflavin Solution Water Added


Mea./l1.


- fhi6aon-


ReadinE


0.50 24.50 0.04 1.5

1.25 23.75 0,1 4.7
2.50 22.50 0.2 9.8

3.75 21.25 0.3 15.0
5.00 20.00 0.4 20.5
6.25 18.75 0.5 25.3

7.50 17.50 0.6 30.5
8.75 16.25 0.7 35.5

10.00 15.00 0.8 40.5

11.25 13.75 0.9 45.3

12.50 12.50 1.0 50.1

13.75 11.25 1.1 55.3

15*00 10.00 1.2 60.5
16.25 8.75 1.3 65.3

17.50 7.50 1.4 70.2

18.75 6.25 1.5 75.2
20.00 5.00 1.6 80.4
21.25 3.75 1.7 84.8

22.50 2.50 1.8 90.0
23.75 1.25 1.9 95.0
25.00 0.00 2.0 100.0











Stability Study Methods


Solution Used
The solutions used as solvents for the investigation of the

stability of riboflavin and some of its derivatives were as follows


Buffer Solution at pH 6.0

Buffer Solution at pH 5,0

Buffer Solution at pH 4.0

25 Per Cent Glycerin in Distilled Water

50 Per Cent Glycerin in Distilled Water

25 Per Cent Propylene Glycol in Distilled Water

50 Per Cent Propylene Glycol in Distilled Water

Saturated Solution of Ethyl Aminobensoate in Distilled Water

0,01 Per Cent Quinine Bisulfate in Distilled Water

Saturated Solution of Beta-Methyl Umbelliferone in Distilled Water

1,0 Per Cent Urea in Distilled Water

0.1 Per Cent Tween 80 in Distilled Water

0.5 Per Cent Nicotinio Acid in Distilled Water


Buffer solutions from pH 4 to 6 were used in view of the lit-

erature reports that this range was most favorable to the stability of

riboflavin solutions. Glycerin and propylene glycol in distilled water

were selected because these are widely used vehicles in pharmaqy* Aque-

ous solutions of ethyl aminobensoate, quinine bisulfate and beta- tthyl

umbelliferone were used as solvents since it was thought that they might

delay destruction of the vitamin in the presence of light due to their











sun screening properties. Both urea and nicotinic acid are used Iy

some pharmaceutical houses as solubilisers for riboflavin, and it was

thought these might be effective for stabilizing solutions of the ribo-

flavin derivatives prepared in this investigation. Teen 80 was se-

lected because it is conmonly used in oral vitamin drops.

The distilled water used to make up all solutions throughout

this investigation was laboratory distilled water. The pH varied from

6.1 to 6.4.

The solutions used in the preparation of buffer mixtures were

prepared according to the Clark and Lube procedure (27) as follow i

Bariuma Idroxide Test Solution. A saturated solution of bariua

hydroxide was prepared by adding an excess amount to recently boiled

distilled water, shaking thoroughly and then filtering. The test solu-

tion was freshly prepared each time it was needed.

0.2 M Sodium Rvdroxide Solution. This solution was prepared

by dissolving 9,00 Oa. of sodium hydroxide in 950 al. of distilled water.

A freshly prepared saturated solution of reagent barium hydroxide was

added drop by drop until no more precipitate was formed. The mixture

was thoroughly shaken and allowed to stand overnight in a stoppered

bottle. The next day, the precipitate was filtered off and the resulting

product gave a carbonate free solution of sodium hydroxide. To standard-

ise the product, 10 al. of normal sulfuric acid was diluted with 50 1.

of carbon dioxide free distilled water and two drops of phenolphthalein,

T. S. was added. This solution was titrated with the sodium hydroxide

solution until a permanent pink color was produced. The normality of










39
the sodium hydroxide solution was calculated and adjusted to exactly

0.2 M with freshly boiled and cooled distilled water.

0.2 M Potassium Biphthalate Solution. Exactly 40.843 Bno of

potassium biphthalate was dissolved in 900 Al. of distilled water and

then sufficient distilled water added to make 1000 al. The polarity

was then determined and adjusted to exactly 0.2 M by titration with the

prepared 0.2 M sodium hydroxde solution using phenolphthalein as the

indicator,

0.2 M Monobasia Potassium Phosphate Solution. This preparation

was made by dissolving exactly 27.218 Gm. of monobasic potassium phos-

phate in distilled water and diluting with sufficient distilled water

to make 1000 al. The polarity was determined and adjusted by titrating

against 0.2 X sodium hydroxide solution.

The buffer solution at pH 6,0 was prepared by adding 50 ml. of

0.2 M monobasio potassium phosphate to 5.64 ml. of 0.2 X sodium hydrox-

ide solution and diluting to 200 ml. with distilled water.

The buffer solution at pH 5.0 was prepared by taking 23.65 m.l

of 0.2 M sodium hydroxide and adding it to 50 al. of potassium htphthal-

ate. This was diluted to 200 al. with distilled water.

The buffer solution at pH 4.0 was prepared by adding 0.40 al,

of 0.2 M sodium hydroxide to 50 ml. of potassium biphthalate and diluting

to 200 al. with distilled water.

Solutions of glycerin, propylene Clyool and polyoaethylene
sorbitan monooleate (Tween 80) in distilled water were prepared on a

volume to volume basis whereas solutions of quinine bisulfate, urea and










40
nicotinic acid in distilled water were prepared on a weight to volume

basis.

The saturated solution of ethyl aminobonsoate in distilled wa

ter was prepared by taking an amount of ethyl aminobensoate that would

normally dissolve in the desired quantity of water and dissolving it

first in the smallest amount of ethyl alcohol. This alcoholic solution

was then added to the distilled water a little at a time with thorough

agitation. The preparation was allowed to stand overnight in a well

stoppered bottle and then filtered the next day,

The saturated solution of beta-methyl umbelliferone in distilled

water was prepared bl adding 1 nG. of beta-methyl umbelliferone to a

liter of boiling distilled water with constant agitation, allowing it

to cool to room temperature and setting it aside overnight in a well

stoppered bottle, The next day the needle-like crystals which precip-

itated out of solution were removed by filtration.



Stability studies were evaluated for the following preparations


Riboflavin

Pyruvic Acid Derivative of Riboflavin

Levulinic Aaid Derivative of Riboflavin

Flavaxin Soluble

Riboflavin-5 -Phosphate Sodium


Solutions of the above were prepared in different conoentrations

in 250-ml. red volumetric flasks. Each solution was kept in the dark










41
overnight in red colored and well stoppered bottles. An initial reading

was taken just before the start of storage under various conditions of

light.

Flint and amber bottles, commonly employed in the storage of

pharmaceutical products, were used as the containers for the solutions.

Three sets of each of the vitamin solutions were prepared for each sol-

vent. One set of solutions was kept in direct sunlight by placing the

containers on the flat roof of the building. Another set was kept in

the diffused light of the laboratory by placing the containers on a ta*

ble. The third set was kept in total darkness by setting the bottles

in closed cardboard boxes in a laboratory desk locker.

Twenty-five milliliters were stored in each type of container.

Approximately 1 ml. of toluene, enough to produce a layer on the top

of the solution, was added to each container to prevent mold growth.

The lumetron was set to give a reading of 100 with a standard

riboflavin solution containing 2 m g. per al. and a reading of zero

with the particular solvent used. A certain quantity of the more con-

centrated solutions had to be diluted with enough distilled water to

fall into the range of the lumetron.

Along with the storage of the solutions containing riboflavin

or its derivatives, a set of blanks (the solvent only) was also stored

under the same conditions. The blanks also were stored in amber and

flint bottles with a layer of toluene on the top. The use of blanks

was deemed necessary especially with solvents of 0.01 per cent quinine

bisulfate in distilled water and with a saturated solution of beta-metlyl










42
umbelliferone in distilled water which normally have their own inherent

fluorescence. Accordingly, before a fluorophotometric reading was de-

termined, the lumetron was adjusted to read sero with the insertion of

the blank.

The vitamin content of all solutions in both flint and amber

bottles vas evaluated fluorophotometrically at the end of the following

time intervals: one day, three days, five days, seven days, ten days,

fifteen days, twenty days, thirty days and sixty days.

Riboflavin was used as a control in the stability study. Both

Flavazin Soluble and riboflavin-5'-phosphate sodium wee selected be-

cause they are recognized as fairly soluble riboflavin derivatives and

are available on the market. These were compared for stability with

the riboflavin derivatives prepared in this investigation.











Solubility Study Methods



The solvents and solutions used for the investigation of the

solubility of riboflavin and some of its derivatives were as follows


Distilled Water

Glycerin

Propylene Glycol

0.9 Per Cent Sodium Chloride in Distilled Water

0.9 Per Cent Potassium Chloride in Distilled Water

1.0 Per Cent Sodium Acid Phosphate in Distilled Water

1.0 Per Cent Potassium Acid Phosphate in Distilled Water

Ethyl Aloohol

1.0 Per Cent Niacinamide in Distilled Water

1.0 Per Cent Urea in Distilled Water


The above percentage solutions were prepared on a weight to

volume basis. A sufficient quantity of salt was weighed out, dissolved

in a portion of distilled water and then made up to volume with more

distilled water.


Procedure

The method used for determining the solubility of riboflavin

and some of its derivatives consisted of preparing saturated solutions

in the solvents and solutions listed. Saturation was attained ty placing

an excess amount of riboflavin or a derivative thereof in the solvent,









44
heating to 600 C. for several minutes with constant agitation and then

cooling to room temperature. Care wa taken to avoid any unnecessary

exposure to light in this procedure. After cooling, the preparations

ware well stoppered and stored overnight in total darkness. The next

day, the excess amount of crystals was removed by oentrifuging.

One milliliter of the solution was carefully pipetted into

950 al. of distilled water and then further diluted to 1000 ml. with

more distilled water. A 25-ml. aliquot of this solution was used to

obtain a fluorophotometric reading. With the more soluble derivatives,

it was found necessary to further dilute 1 ml. of the above solutions

to 250 al. in a red volumetric flask. This was sometimes found neces-

sar because the lumetron was adjusted to give a reading of 100 with

the standard riboflavin solution and a reading of sero with a distilled

water blank. The preparation of the standard riboflavin solution ws

the sau as that described under assy procedure.

Solubilities were determined for each of the following


Riboflavin

Pyruic Acid Derivative of Riboflavin

Levulinic Aaid Derivative of Riboflavin

Citraconic Anhydride Derivative of Riboflavin

Flavaxin Soluble

Riboflavn-5 '-Phosphate Sodium











Assay Method of Riboflavin Derivatives

The determination of the amount of riboflavin equivalent to a

specific quantity of derivative was determined fluorophotometrically,

A standard riboflavin stock solution was first prepared. Riboe

flavin, U. S. P., was dried at 1050 C. for two hours and stored in the

dark in a desiccator over phosphorous pentoxide. Exactly 50 mg. were

carefully weighed and dissolved in distilled water to make one liter.

This solution was stored in a red glass bottle under toluene and placed

in a refrigerator which was set at !0 C. Each ml. represented 50 meg.

of U. S. P. Riboflavin.

A standard riboflavin solution was prepared from the above stock

solution by placing 10 ml. of the above preparation in a red glass vol.

metric flask and diluting to 250 al. with distilled water. Each ml.

represented 2 mag. of riboflavin. A 25-ml. aliquot of this solution

was placed in a sample container and the lumetron adjusted to read 100

with this concentration. The same amount of distilled water was used

as the blank and the sero suppressor knob was adjusted to read sero.

Accordingly, the preparation of solutions of riboflavin deriv-

atives were made in a similar manner as that described for riboflavin

solutions. The resulting concentrations were also 2 meg, per al, After

adjusting the lumetron with the standard riboflavin solution and the

blank, a 25-al. aliquot of the derivative solution was placed in a saa-

ple container. The percentage of riboflavin was determined directly

by the amount of fluorescence registered on the lumetron.










46
The following derivatives of riboflavin were assayed in this

nmnneri


Pyruvic Acid Derivative

Citraconic Anhydride Derivative

Levulinic Acid Derivative

Flavaxin Soluble

Riboflavin-5'-Phosphate Sodium











Results with Riboflavin

Stability studies were evaluated for riboflavin in various sol-

vents and the results were used as a control for the derivatives an-

thesised in this investigation.

Fifty milligrams of riboflavin were added to a liter of each

of the solvents listed under solubility studies. Since the luetron

was set for determinations up to 2 mew. per ml., the riboflavin solu-

tions were diluted by adding 1 al. of the vitamin solutions to 24 Ait

of distilled water. An initial lumetron reading was determined before

the onset of storage.

Riboflavin was used as the basis for assay of the other deriv-

atives studied in this investigation.

The solubility of riboflavin in various solvents vas determined

and the amount of riboflavin present in each al. of saturated solution

was evaluated fluorophotometricallyo











TABLE 3

THE STABILITY OF RIBOFLAVIN IN DISTILLED WATER STORED
UNDER VARIOUS CO;DITIOS IN FLINT AND AMBER BOTTLES


Ditn ed LAron
Lumetron


daeR ina Me M anding Mon.


Lumetron
Reading M?4ciMl.


Lmetron Reading of


One Day
Amber
Flint

Three Days
Amber
Flint

Five Days
Amber
Flint

Seven Days
Amber
Flint

Ten Days
Amber
Flint

Fifteen Days
Aaber
Flint

Twenty Days
Amber
Flint

Thirty Days
Amber
Flint

Sixty Days
Amber


67.9
2.5


43.0
2.0


37.1
1.8


19.2
0.7


14.8
0.1


9.8
0.0


3.5
e*0


2.0
* *


Freshly Prepared Samples 95.4 or 1.908 mag./m1.


1.358
0.050


0.860
0.040


0.742
0.036


0.384
0.014


0.296
0.002


0.196
0.000


0.090



0.040
* o


92,0
46.5


90.5
40.0


89.8
23.0


88.4
10.5


87.8
4.5


86.5
2.2


76.2
1.1


71.5
0.8


1.840
0.930


1.810
0.800


1.796
0.460


1.768
0.210


1.756
0.090


1.730
0.044


1.524
0.022


1.430
0.016


95.4



95.4


95.4



95.4



95.4



95.0



95.0



94.5


1.908



1.908


1.908



1.908



1.908



1.900



1.900



1.890


1.086 93.4 1.868


Sunll.n f
Lumetron


--- --- -- ----


.-3imn .v -- M--a-


0.0


0.000 54,3












TABI 4

THE STABILITY OF RIBOFLAVI' IN DISTILL D WATER BUFFER'ID AT pH 6
STORF;D UNDLR VARIOUS CONDITIONS IN FLINT AND AMBER BOTTLES

engght Difmused Liht Darkness
Lumetron Luaetron Luametron
eadin- Wha-.lj Reading Meag./M. Reading Mcg./uL.


Lumetron leading of Freshly Prepared Sample: 91.5 or 1.830 mcg./ml,

One Day
AAbr 66.1 1.322 91.0 1.820 91.5 1.83
Flint 2.0 0.040 42.4 0.848

Three Days
Amber 53.2 1.064 90.1 1.802 91.5 1.83
Flint 1.4 0.028 38.9 0.778

Five Days
Amber 38.0 0,760 89.2 1.784 91.5 1.83
Flint 0.8 0.016 22.1 0.442

Seven Days
Amber 19.1 0.382 87,7 1,754 91.5 1.83
Flint 0.2 0.004 8.9 0.178

Ten Days
Ambew 12.9 0.258 85.0 1,700 91.3 1.82
Flint 0.0 0.000 2.1 0.042

Fifteen Days
Amber 7.6 0.152 84.2 1.684 91.0 1.821
Flint ... .**.. 0.6 0.012


0



0



0





S


Twenty Days
Amber
Flint

Thirty Days
Amber
Flint

Sixty Days
Amber


1.4
0*5


0*5
* **


0.028



0.010
000.*


75.4
0,3


68.2
0.0


1.508
0.006


1.364
0.000


90.8



90.4


1.816



1.808


0,000 54.5 1.090


0.0


89#2 1.784











TABIX 5
THE STABILITY OF RIBOFLAVIN IN DISTILLED WATER BUFFERED AT pH 5
STORED UNDER VARIOUS CONDITIONS IN FLI7T AND AMBE BOTTLES

aggMl8ii Diftfed iUUah< Darss
Lumetrom Lumetron lumetron
R&adin=f E1mz./A. Reatnt HCMi2/H1. Raad1in=7 /Ml
Lametron Reading of Freshly Prepared Sample: 91,2 or 1.824 mog./ml.

One Day
Amber 66.4 1.328 91.0 1.820 91.2 1.824
Flint 2,2 0.044 40.2 0.804

Three Days
Amber 540 1.082 90.4 1.808 91.2 1.824
Flint 1.? 0.034 3492 0.684

Five Days
Amber 40.1 0.802 90.0 1.800 91.2 1.824
Flint 0.9 0.018 23.2 0.464

Seven Days
Amber 19.1 0.382 88.7 1,774 91.2 1.824
Flint .O 0.000 9.0 0.180

Ten Days
Amber 148 0.296 86.1 1.722 91.2 1.824
Flint .*, *...* 3.4 0.068

Fifteen Day
Amber 9.1 0.182 85.4 1.708 90.7 1.814
Flint .*0 ....* 1.1 0.022

Twenty Days
Amber 1,2 0.024 76.2 1.524 90.4 1.808
Flint ... ..... 0.4 0.008

Thirty Days
Amber 0.4 0.008 69.9 1,398 90.0 1.800
Flint *.. ..... 0.0 0.000

Sixty Days
Amber 0.0 0.000 55.8 1.116 89.1 1.782












TABLE 6

THE STABILITY OF RIBOFIAVIN IN DISTILLED WATER BUFFERED AT pH 4
STORED UNDER VARIOUS CONDITIONS IN FLINT AND AMBER BOTTLES
I~~~~~ ---if __.i liiIII I = I .. .. I ,


& Diffused Maht
Lumatron Luaetron
Readin Mea./ML. Randing Mano. A.


Lametron Reading of Freshly Prepared Sample:


aumetron
Reading Mao. Al.


93.3 or 1.866 mcg./m1.


One Day
Amber
Flint

Three Days
Amber
Flint

Five Days
Amber
Flint

Seven Days
Amber
Flint

Ten Days
Amber
Flint

Fifteen Days
Amber
Flint

Twenty Days
Amber
Flint

Thirty Days
Amber
Flint

Sixty Days
Amber


68.2
2.0


55.4
1.5


40.1
1.0


20.0
0,2


15,2
0.0


10.9
2.*


2.5
e**


1.1
..*


1.364
0.040


1.108
0.030


0.802
0.020


0.400
0.004


0.304
0.000


0.218



0.050



0.022
*. *..


93.3
43.1


92.6
36,0


91.8
24.2


90.9
9.2


90.1
2.2


89.4
1.4


79.8
0.5


75.0
0.0


1.866
0.862


1.852
0.720


1.836
0.484


1.818
0.182


1.802
0.044


1.788
0.028


1.596
0.010


1.500
0.000


93.3



93.3


93.3



93.3



93.3



92.9



92.5



92.0


1.866



1.866


1.866



1.866



1.866



1.858



1.850



1.840


0.000 60.1 1.202


91,3 1,826


0.0


~'T~~ ""~"'"I "R~-~'~ -"----- ---












TABLE 7

THE STABILITY OF RIBOFLAVIN IN 25 PER CENT GLYCERIN III DISTILLED WATER
STORED UNDER VARIOUS CONDITIONS IN FLT AN AMBER BOTTLES

uadi ts sDifused rLi.At bas aIJ
Lumetron Lumetron Lumetron
...e ading eas.AL.. Reading ej./Y ead~m Mis /MLz,


Lumetron Reading of Freshly Prepared Samples


98.0 or 1.960 mcg.,/l.


One Day
Amber
Flint

Three Days
Amber
Flint

Five Days
Amber
Flint

Seven Days
Amber
Flint

Ten Days
Amber
Flint

Fifteen Days
Amber
Flint

Twenty Days
Amber
lint

Thirty Days
Amber
Flint

Sixty Days
Amber


70.5
3.0


50.4
1,0


41.2
0.8


28.0
0.3


12.8
0.0


7.4



4.6



3.0
* *


1.410
0.060


1.008
0.020


0.824
0.016


0.560
0,006


0,256
0.000


0.148



0.092



0.060
*00* *


98.0
4645


94.5
40.1


93,.0
29.0


88.9
18.2


88.3
9.2


87.3
5.0


77,1
3.0


1.4


1.960
0.930


1.890
0.802


1.860
0.560


1.778
0.364


1.766
0.184


1.746
0.100


1.542
0.060


1.430
0.028


98.0


1.960


1.960


98.0


98,0



98.0



97.7


97.2


1.960



1.960



1.960



1.960



1.954



1.944


1.204 96.0 1.920


0.0


0.000 60.2











TABLE 8

THE STABILITY OF RIBOFLAVIN I 50 PER CENT GLYCEilIN IN DISTILLED WATER
STORED UND~R VARIOUS CONDITIONS IN FLIIT AND AMBER BOTTLES

Slight Diffused iaht Derkness
Lumetron Lumetron Lunetren
Beadina M0g./. ReadinE MHO./M1. Reading MaE.A1.

Lumetron Reading of Freshly Prepared Sampler 92.0 or 1.840 mcg./ml.

One Day
Amber 80,5 1.610 91.0 1.820 92.0 1.840
Flint 2.5 0.050 53.5 1.070

Three Days
Amber 99.0 1.180 90.2 1.804 92.0 1.840
Flint 1.0 0.020 46.0 0.920

Five Days
Amber 49.6 0.992 89.1 1.782 92.0 1.840
Flint 0.7 0.140 35.2 0.704

Seven Days
Amber 38.4 0.768 88.3 1,766 92.0 1.840
Flint 0.1 0.002 24.2 0.484

Ten Days
Amber 34.6 0.692 86.1 1.722 92.0 1.840
Flint 0.0 0.000 13.2 0.264

Fifteen Days
Amber 29.8 0.596 84.5 1.690 91.8 1.836
Flint ... ..... 9.4 0.188


Twenty Days
Amber
Flint

Thirty Days
Amber
Flint

Sixty Days
Amber


21.5



15.2
* *


0.430



0.304
.....


76.1
7.4


72.3
5.0


1.522
0.148


1.446
0.100


91.6



91.3


1.832


1.826


0.000 59.8 1.196


91.0 1.820


0.0











TASIE 9
THE STABILITY OF RIBOFLAVIN IN 25 PER CEIlT PROFPYL GLYCOL IN
DISTILLED WATER STORED UDER VARIOUS CONDITIONS
IN FLINT AND AMBR BOTTLES

Sa&i=sh Diffused laht sa
Lumetron lumetron Lumetron
Reading Mcg./MLt. Reading Mcg./M1. Reading Moa./M.

Lumetron Reading of Freshly Prepared Samplel 94.8 or 1.896 mag./Mal

One Day
Amber 63.5 1.270 94.0 1,880 94.8 1.896
Flint 1.5 0.030 42.1 0.842

Three Days
Amber 42.8 0.856 92.0 1.840 94.8 1.896
Flint 0.8 0.016 33.5 0.670

Five Days
Amber 37.6 0.752 90.6 1.812 94.8 1.896
Flint 0.1 0,002 24.5 0.490

Seven Days
Amber 23.1 0.462 89.2 1,784 94.8 1.896
Flint 0.0 0000 16.1 0.322

Ten Days
Amber 9.4 0.188 88,1 1.762 94.8 1,896
Flint .., ...,, 12.0 0.240

Fifteen Days
Amber 5.8 0.116 88.0 1.760 94.4 1.888
Flint ... ..... 6.3 0.126

Twenty Days
Amber 4.1 0.082 80.2 1.604 94.0 1.840
Flint ... ..... 5.2 0.104

Thirty Days
Amber 2.0 0.040 77,4 1.548 93.7 1.874
Flint ... ..... 3.8 0.076

Sixty Days
Amber 0.0 0.000 56.8 1.136 93.0 1.860











TABIE 10

THE STABILITY OF RIBOFLAVIN IN 50 PER WT PROPYIZNE GICOL IN
DISTILED WATER STORED UNDER VARIOUS CONDITIONS
IN FLINT AND AMBER BOTTLES

Sun lsht DiffuLsed Lbht Djnm
Lumetron Lumetron Lumetron
Reading MNa. Reading Mcg./Ml Reading Mo.ag/M

Lunetron Reading of Freshly Prepared Samples 89.5 or 1.790 mcg./ml.

One Day
Amber 82.5 1.650 88.5 1.770 89.5 1.790
Flint 1,5 0.030 49.0 0.980

Three Days
Amber 62.3 1.246 86.1 1.722 89.5 1.790
Flint 1.1 0.022 412 0.824

Five Days
Amber 48.9 0.978 84.0 1.680 89.5 1,790
Flint 0.3 0.006 29.8 0.598

Seven Days
Amber 40.2 0.804 83.2 1.664 89.5 1*790
Flint 0.0 0.000 16.4 0.328

Ten Daye
Amber 33.2 0.664 81.8 1.636 89.5 1.790
Flint ,. ,.... 10.1 0.202

Fifteen Days
Amber 25.8 0.516 80.1 1.602 89.1 1.782
Flint .. *..... 4.9 0.098

Twenty Days
Amber 19.0 0.380 73.8 1.576 89.0 1.780
Flint ... ..... 4.0 0.080

Thirty Days
Amber 12.1 0.242 76.8 1,536 88.7 1.774
Flint *.. ..... 3.0 0.060

Sixty Days
Aaber 0.0 0.000 54.3 1.086 88.0 1 760


v -Tvf


--1- ....











TABIE 11
THE STABILITY OF RIBOFLAVI N IN A SATURAT D SOLUTION OF rTHYL
AMINOBENZOATE IN DISTILLED WATER STORED UNDER VARIOUS
CONDITIONS IN FLINT AND AMBTR BOTTLES

Suad.l1f, manned Liht Diiraegssr
Luamtron Laxtron Lumetron
Reading HoSn.A1. Reading Mc,.flM. Readina WHar/M.
Lumetron Reading of Freshly Prepared Sample: 90.0 or 1.800 aog,/ml.

One Dan
Amber 75.5 1.510 90.0 1.800 90.0 1.800
Flint 4.0 0.080 67,0 1.340

Three Days
Amber 45.0 0.900 88.1 1,762 90.0 1.800
Flint 2.8 0.056 61.5 1.230

Five Days
Amber 34.1 0.682 87.5 1,750 90.0 1.800
Flint 0.7 0.014 54.8 1.096

Seven Days
Amber 28.2 0.564 87*2 1.744 90.0 1.800
Flint 0.2 0.004 40.6 0.812

Ten Days
Amber 23.7 0.474 87.0 1.740 90.0 1.800
Flint 0.0 0.000 40.1 0.802

Fifteen Days
Amber 12.1 0.242 84.6 1.692 90.0 1.800
Flint ... *.... 20.3 0.406

Twenty Days
Amber 6.4 0.128 80.5 1.610 90.0 1.800
Flint ..* ..... 73 0.146

Thirty Days
Amber 0.5 0.010 77.2 1,544 89.5 1.790
Flint ... ..... 2.5 0.050

Sixty Days
Arber 0.0 0.000 69.2 1.384 88.5 1.770











TABIE 12

THE STABILITY OF RIBOFLAVIN IN 0.01 PER CENT QUININE BISULFATE
IN DISTILLED WATER STORED UND R VARIOUS CONDITIONS
IN FLINT AND AMBER BOTTLES

Snali1rt Diffaeed Liaht Darimeay
lumetumetLumetron Lmetron
Reading MegH./M. Reading Macg/M. Reading Mag./M1.


Lumetron Reading of Freshly Prepared Samplet


90.0 or 1.800 mag./61.


One Day
Amber
Flint

Three Days
Amber
Flint

Five Days
Amber
Flint

Seven Days
Amber
Flint

Ten Days
Amber
Flint

Fifteen Days
Amber
Flint

Twenty Days
Amber
Flint

Thirty Days
Amber
Flint

Sixty Days
Amber


68,0
17.1


65.0
10.1


53.6
1.5


1.0


33.4
0.3


30.2
0.0


21.0



10.6



0.0


1.360
0.342


1,300
0,202


1,072
0.030


0*756
0.020


0.668
0.006


0.604
0.000


0.420


0.212
*0*.*


90,0
47.0


85.4
442


84.2
36.3


84.0
26.8


83.0
10.8


82.1
3.1


81.4
1.5


76.2
0.5


1,800
0.940


1.708
0.884


1,684
0.726


1.680
0.536


1.660
0.216


1.642
0.062


1.628
0.030


1.524
0.010


0.000 63.4 1.268


90.0



90,0


90,0



90.0



90.0



90.0



89.4


89.2


1,800



1,800


1.800



1,800


1.800



1.800



1.788


1,784


88,0 1,760











TABI~ 13

TE STABILITY OF RIBOFUIAVI IN A SATURATED SOLUTION OF BETA-MBTHYL
UMBELLIFERONE IN DISTILLED WATER STOED UNDER VARIOUS
CONDITIONS IN FLINT AND AER BOTTLES

iSu n "lt Diffued Jialt DrkaLes
Lumetron Lumetron Lummtron
Readin" Mgn.M1. Reading Mcg./KL. Reading- Moa./M

Lumetron Reading of Freshly Prepared Sample: 90.5 or 1.810 mg./ml.

one Day
Amber 69.0 1,380 90.5 1.810 90.5 1.810
Flint 2.2 0.0 67.5 1.350

Three Days
Amber 43.2 0.864 90.0 1.800 90.5 1.810
Fint 1.2 0.024 63.7 1.274

Pive Day
Amber 32.1 0.642 87.1 1.742 90.5 1.810
Flint 0.8 0.016 54.0 1.080

Seven Days
Amber 28.5 0.570 85,8 1,716 90.5 1.810
Flint 0.1 0.002 43.2 0.864

Ten Days
Amber 1.1 0.282 84.5 1.690 90.5 1.810
Flint 0.0 0.000 22.9 0.458

Fifteen Days
Amber 6.1 0.122 82.0 1.640 90.5 1.810
Flint ..... 15.4 0.308

Twenty Days
Amber 4.2 0.084 75.0 1.500 90.0 1.800
Flint *... .... 4.2 0.084

Thirty Days
Amber 0.2 0.040 71.0 1.420 89.8 1.796
Flint ... ,.... 0.1 0.002

Sixty Days
Amber 0.0 0.000 63.1 1.262 88.6 1.772


r











TABIE 14

TE STABILITY OF RIBOFLAVIN IN 1.0 PER GEIT UREA In DISTILLED WATER
STORED UND-R VARIOUS CONDITIONS IN FLIPT AlD AMBER BOTTLES

unliht Diffused Light Darkness
Lranmeron lametron lmatron
.ead4in MB../MN. Reading Mr./Mil. leadina dan.aI l


Lametron Reading of Freshly Prepared Samplet


92.0 or 1.840 mcg./al.


One Day
Amber
Flint

Three Days
Amber
Flint

Five Days
Amber
Flint

Seven Days
Amber
Flint

Ten Days
Amber
Flint

Fifteen Days
Amber
Flint

Twenty Days
Amber
Flint

Thirty Days
Amber
Flint

Sixty Day
Amber


64*5
1.5


448
0.8


33.0
0.0


17.0
0.0


4.5



2*4
9,


0.8



0.1
0e0


1.290
0.030


0.896
0.016


0.660
0,000


0.340
0,000


0.090



0.048



0.016
.9,.i


0.002
.**U*


92.0
40,1


90.0
32.1


85.2
14,8


79.4
6,5


75.2
2,1


69.8
1,2


62.5
0.8


53.1
0,0


1.840
0.802


1.800
0.642


1,704
0.296


1,588
0.130


1.504
0.042


1.396
0.024


1.250
0,016


1.062
0.000


92.0



92.0


92.0
92,0



92.0



91.5



91.3



91.0



91.0


1.840



1.840


1.840



1.840



1.830



1.826



1.820



1.820


42.8 0.856


90,4 1.808


0.0











TABIE 15
THE STABILITY OF RIBOFLAVIN IN 0.1 PER CENT 80 IN DISTILLED
WATER STORED UNDER VARIOUS CONDITIONS IN FLINT AND ABER BOTTLES


Lumetron Lumstron Lumetron
_,sdiai&-, oa./K.. R eadin MeM./Ml. Readin, Ma ./Kl.
Lumetron Reading of Freshly Prepared Samples 91.0 or 1.820 mag./l,.


ne Day
Amber
Flint

Three Days
Amber
Flint

Five Days
Amber
Flint

Seven Dys
Amber
Flint

Ten Days
Amber
Flint

Fifteen Days
Amber
Flint

entry Days
Amber
Flint

Thirty Day
Amber
Flint


62.8
4.0


41.9
2.8


32.0
1.4


15.8
0.8


2.7
0.4


1.8
0.0


0*4
too


0.1
'..


1.256
0.080


0.838
0.056


0.640
0.028


0.316
0.016


88,0
40.5

86.1
35.0


85.2
20.1


83.8
8.5


0.054 82.5
0.008 4.1


0.036
0.000


0.008
,0**0


80,2
0.8


71.1
0.3


0.002 65.1
**,.* 0.0


0.000 51,7 1.034


1,.760
0.810


1,722
0.700


1.704
0.402


1,676
0.170


1.650
0.082


1.604
0.016


1.422
0.006


1.302
0.000


91.0



91.0



91.0



91.0



91.0


90.7



90.4


89.8


1,820


1.820



1.820



1.820


1.820


1.814


1.808


1.796


Sixty Days 0.0


88,9 1,78











TABLE 16
THE STABILITY OF RIBOFLAVIN IN 0.5 PER CENT NIACIN II1 DISTILLED WATER
STORED UNDER VARIOUS CODITI0NS IN FINT A1D AMER BOTTLES

Sml&dht Diffnaed Liaht D m
Lumetron Lumetron Imetron
Readain )ri./fha. Readiena -./M. Refadlna Ha./X.


Luretron Reading of Freshly Prepared Samples 94.2 or 1.884 mag./ml.

One Day
Amber 68.8 1.376 94.0 1.880 94.2 1.88
Flint 1.0 0.020 40.0 0.800

Three Days
Amber 53.8 1.076 93.1 1.862 94.2 1.88
Flint 0.6 0.012 34.2 0.684

Five Days
Amber 39.2 0.784 92.4 1.848 94.2 1.88
Flint 0.2 0.004 22,0 0.440

Seven Days
Amber 17.9 0.358 91.1 1.822 94.2 1.88
Flint 0.0 0.000 7.2 0.144

Ten Days
Amber 13.1 0.262 89,4 1*788 94.2 1.88
Flint ... ..... 1.0 0.020

Fifteen Days
Amber 8.9 0.178 88.2 1.764 93.9 1l8g
Flint ... *...* 0.4 0.008


4


4


4


4


4


S


Twenty Days
Amber
Flnt

Thirty Days
Amber
Flint

Sixty Days
Amber


0,8


0.042


0.016


79.0
0.0

7442


1.580
0.000


93.4


1.484 93.0


1,868


1.860


0.000 59.2 1.184


0.0


92,1 1.842


















TABLE 17
THE SOLUBILITY OF RIBOFLATV IN I S0M AQUEOUS
SOLUTIONS AND OTH R SOLVENTS

Average
Lumetron Mg./Mt.
Reading
Distilled Water 7,2 0.14

0.9% Sodium Chloride 10.0 0.20

0.9% Potassium Chloride 9.5 0.19

1,0% Sodium Acid Phosphate 5,5 0.11

1.0% Potassium Acid Phosphate 5.5 0.11

1.0% Niacinamide 20.0 O.40

1.0% Urea 17.0 0.34

Propylene Glyool 26.4 0.52

Glycerin 42.0 0.84

Alcohol 2.2 0.04











Results with Riboflavin-5'-Phosphate Sodium

Hoffmann-La Roche's riboflavin-5'-phosphate sodium salt was

selected for study because of its unusually high solubility and its

availability on the market.

The assay of this salt by the fluorophotometric procedure

showed a 70.0 per cent riboflavin content. thua, 1 Gm. of riboflavin-

5'-phosphate sodium was equivalent to 0.7 Cn. of riboflavin.

Five milligrams of riboflavin-5'-phosphate sodium were added

to each ml. of the solvents used for stability study. Since the lume-

tro was set for determinations up to 2 mag. per ml., each solution

had to be further diluted by adding 0.4 ml. to a sufficient quantity

of distilled water to make 1000 ml. Twenty-five milliliters of this

dilution were used for fluorophotometric analysis,

The solubility of Hoffman-La Roche's product in some aqueous

solutions and other solvents vas determined. Due to its unusually high

solubility further dilutions with distilled water were necessary for

evaluation with the lumetron.












TABLE 18

THE STABILITY OF RIBOFLAVIN-5'-PHOSPHATE SODIUM IN DISTILLED WATER
STORED UNIDR VARIOUS CONDITIONS IN FIN AND AMBER BOTTIZS

Luta- nasa u-tZn
& 5 Baff Diffused LLght 50SS5e5s5
Lumetron Lumetron ZumXetron
Reading Meo./Ml. Reading MeC./m. Readin Mca./ILa


Lumetron Reading of Freshly Prepared Samples 70.0 or 1.400 mcg./.l,

One Day
Amber 15.2 0.304 66.2 1.324 70.0 1,40
Flint 1.0 0.020 33.0 0.660

Three Days
Amber 3.5 0.070 64.1 1.282 70.0 1.40
Flint 0.1 0.002 16.1 0.322

Five Days
Amber 2.0 0.040 60.4 1.208 70.0 1.40
Flint C.O 0.000 1,8 0.036

Seven Days
Amber 1,5 0.030 59.8 1.196 70.0 1.40
Flint *.. ..... 1.0 0.020

Ten Days
Amber 1.0 0.020 57,8 1.156 70.0 1.40
Flint ,,. .,.., 0.6 0.012

Fifteen Days
Amber 0,4 0.008 54.5 1.090 69.7 1.39
Flint ... .*,,, 0.1 0.002


Q



D



D



D



O


4


Twenty Days
Amber
Flint

Thirty Days
Amber
Flint

Sixty Days
Amber


0.0
999


*. 9


0.000

****


52.8
0.0


49.4



38.6


1.056
0.000


0.988
.9** ,


68.5



67.7


0.772 65.5


1,370



1.354


1.310











TABLE 19
THE STABILITY OF RIBOFLAVIN-5'-PHOSPHATE SODIUM IN DISTILLED WATER
BUFFERED AT pH 6 STORED UNDM R VARIOUS CONDITIONS
IN FLINT AND AMBER BOTTLES

BiA tg Diffunsd "At arknesa
Lunetron Luetron Lumetron
Reading Mc)g./l. Reading cg,./M1. Reading tg,./l.1

Lumtron Reading of Freshly Prepared Sampler 72.5 or 1.450 mcg./ml.

One Day
Amber 17.5 0.350 67.1 1.342 72,5 1.450
Flint 1.8 0.036 34.1 0.682

Three Dqe
Amber 4.8 0.096 63.8 1.276 72.5 1.450
Flint 0.6 0.012 17.1 0.342

Five Daya
Amber 2.5 0.050 62.1 1.242 72.5 1.450
Flint 0.0 0.000 2.8 0.056

Seven Days
Aaber 18 0.036 99,8 1.196 72.5 1.450
Flint *... .... 1.5 0.030

Ten Day
Amber 1.1 0.022 57., 1.48 72.0 1.440
Flint ... ,.... 1.0 0.020

Fifteen Days
Amber 0.6 0.012 56,0 1.120 71.6 1.432
Flint **. ...,* 0.4 0.008

Twenty Days
Amber 0.0 0.000 54.0 1.088 70.9 1.418
Flint ... ...** 0.0 0.000

Thirty Days
Amber .., .,,.. 51.6 1.032 70.0 1,400
Flint **o** *,to o***

Sixty Day.
Amber ., #..0. 40.3 0.806 67.8 1.356











TABLE 20
THE STABILITY OF RIBOFLATIN-5'-PHOSPHATE SODIUM IN DISTILLED WATER
BUFFERED AT pH 5 STOPRD UNDER VARIOUS CONDITIONS
IN FLI1T AND AMBER BOTTLES

Sgim t sterdo Ltht Mfffnd
Lumetron Iuattron Lumetron
Reading Mog,.al Reading Mog./kl, Beading Mcg.&.

Lumetron Reading of Freshly Prepared Samples 74.2 or 1.484 mg./al.

One Day
Amber 18*7 0.374 73,0 1.46 742 1.482
Flint 2.0 0.040 35.0 0.700

Three Days
Amber 5.0 0.100 71.4 1.428 74,2 1.482
Flint 0.8 0.016 18.5 0.370

Five Days
Amber 3.1 0,062 69.5 1.390 742 1.482
Flint 0.2 0.004 3.3 0.066

Seven Days
Amber 1.5 0.030 67.6 1.352 74.2 1.482
Flint 0.0 0.000 2.3 0.046

Ten Days
Amber 1.0 0.020 63.2 1.264 73.8 1.476
Flint .. ,..* 1.8 0.036

Fifteen Days
Amber 0.7 0.014 58.5 1.170 73.3 1.466
Flint ... *.... 0.6 0.012

Twenty Days
Amber 0.0 0.000 55.5 1.116 72.6 1.452
plint ,.. ... 0.2 0.004

Thirty Days
Amber ** *,*,, 53.4 1.068 72.0 1.440
Flint .** ..... 0,0 0.000

Sixty Days
Amber ... ...., 42.5 0.850 70.1 1.402











TABIE 21
TE STABILITY OF BIBOFLAVIN-5 -PHOSPHATE SODIUM IN DISTILLED WATER
BUFFERED AT pH 4 STCED UNDER VARIOUS CONDITIONS
IN FLINT AD AMBER BOTTLES

Rediffused Lght D-k eAA
Lutltron IlamtFon uLm rtroa
Reading Megl Reading Mo.yM1. Reading MN./t.


liuatron Reading of Freshly Prepared Samples


73.6 or 1.472 mog./al,


One Day
Amber
Flint

Thre Days
Amber
Flint

Five Days
Amber
Flint

Seven Days
Amber
Flint

Fn Days
Amber
Flint

Fifteen Days
Amber


Twenty Da
Amber
Flint

Thirty Days
Amber
Flint

Sixty Days
Amber


18.0
1.8


4.7
1.0


2,8
0.4


1,3
0,0


0,8



0.2


0,0
0.10



*to
'99


0.360
0.036


0.094
0.020


0.056
0.008


0,026
0,000


0.016
****<

0.004



0,000
,*4# V


**. *


72.0
42.5


70.4
17.1


69,5
5,6


68,0
3,8


64.2
1,5

62.2
1,0


60.5
0.0


55.0
0#0


1.440
0.850


1.408
0.342


1.390
0.112


1.360
0,076


1.284
0.030


1.244
0,020


1.210
0,000


1.100
#*Off


44.0 0.880


73,6



73.6


73.6



73.6



73.0


1.472



1.472


1.472



1,472



1.460


72,4


71,9



71.5


1.438



1.430


69,8


1.398


n*0











TABLE 22
THE STABILITY! OF RIBOFIAVIN-5'-PHOSPHATE SODIUM IN 25 PER CENT
GLICERIN IN DISTILLED WATER STORED UNDER VARIOUS
CONDITIONS IN FLINT AND AMBER BOTTLES

8g=afght Diffused Lift tBEsrAr
Luma tron Iumtroan lametrom
Reading M.g./Z.. Reading M~g./M,. Reading Nog./_ .

Lumtron Reading of Freshly Prepared Samples 73.9 or 1.478 mcg./al.

One Day
Amber 23*1 0.462 73.0 1.460 73.9 1.478
Flint 2.1 0.042 57,5 1.150

Three Days
Amber 10.1 0.202 72.8 1.456 73.9 1.478
Flint 1.4 0.028 40.7 0.814

Five Days
Amber 2.1 0.042 72,1 1.442 73.9 1.478
Flint 0.7 0.014 26.6 0.532

Seven Days
Amber 1.8 0.036 71.0 1.420 73.9 1.478
Flint 0.1 0.002 18.8 0.376

Ten Days
Amber 1.5 0.030 67.0 1.340 73#3 1.476
Flint CC 0C.00 8.0 0.160

Fifteen Days
Amber 0.9 0.018 63,5 1.270 72,8 1.456
Flint ... ..... 2.3 0.026

Twenty Days
Amber 0.3 0.006 60.8 1.216 72.6 1.452
Flint ,*. .*,,. 0.9 0.018

Thirty Days
Amber C.C C.GCC 54.1 1.082 72.4 1.448
Flint ... ..... C.C C,000

Sixty Days
Amber ,*. *,., 44.8 0.896 70.1 1.402











TABLE 23

THE STABILITY OF RIB LAVIN-5'-PHOSPHATE SODIUM IN 50 PER CENT
GLYCERIN IN DISTILLED WATER STORED UNDER VARIOUS
CONDITIONS IN FLINT AND AMNER BOTTLES


Iumtron TastML n Luatrac
Beading cg. eang dig g./. Reading Mog/k.

Iumatron Reading of Freshly Prepared Samples 69.3 or 1.386 macg/al.

One Day
Amber 23.8 0.476 69.0 1.380 69.3 1.386
Flint 2.2 0.044 46.1 0.922

Three Day
Amber 16,1 0.322 68.3 1.366 69.3 1.386
Flint 1.3 0.016 29.1 0.582

Five Days
Aaber 8.9 0.178 67.8 1.356 69.3 1.386
Flint 0.6 0.012 20.6 0.412

Seven Days
iAber 4.6 0.092 66.0 1.320 69.3 16386
Flint 0.2 0.004 18.2 0.364

Ten Days
Amber 3.0 0.060 62,1 1.242 69.3 1.386
Flint 0.0 0.000 12.5 0.250

Fifteen Days
Amber 2.1 0.042 61.1 1,222 68,9 1.378
Flint 9., *..., 9.1 0.182

Twenty Days
Amber 1.1 0.022 59.6 1.192 68.5 1.370
Flint ,., .,.6o 6.6 0.132

Thirty Days
Amber0.0 0.000 56.3 1.126 67.8 1.356
Flint ,** *...* 3.2 0.064

Sixty Days
Amber *,, ,.,, 48.5 0.970 66.4 1.328











TABIE 24
ME STABILITY OF RIBOFIAVIN-51-PIOSPHATE SODIUM IN 25 PER CENT
ROPLEE GLCOL IN DISTILLED WATER STORED UNDER VARIOUS
CONDITIONS ITI FLINT AND AMBR BOTTLES


MItroM I ,tran Lletron
Reading -eg./l1. Reading Mog./Ml, Reading Ncgy/l1.

Lumetron Reading of Freshly Prepared Samplez 72.0 or 1.44O mog,/Al.

One Day
Amber 19.0 0.380 67,7 1.354 72.0 1.440
Flint 1.9 0.038 52.3 1.046

Three Days
Amber 9.2 0.184 65,7 1.314 72.0 1.440
Flint 1,0 0,020 28.6 0.572

Five Day
Amber 2.0 0.040 64.2 1.284 72.0 1.440
Flint 0.6 0.012 14.7 0.294

Sewn Days
Amber 1.6 0.032 62.2 1.244 72.0 1.440
Flint 0.2 0.004 10.8 0.216

Ten DIy"
Amber 1.0 0.020 57,9 1.158 71.6 1.432
Flint 0.0 0.000 4.3 0.086

Fifteen Days
Amber 0.3 0.006 56.0 1.120 71.2 1.432
Flint ... .*... 3.4 0.068

Twenty Days
Amber 0,0 0.000 55.3 1.106 70.9 1.418
Flint ... *** 1.8 0.036

Thirty Daym
Amber ... *.... 53.8 1.076 70.7 1.414
Flint ... ..*** 0.0 0.000

Sixty DoOa
Amber ... ..... 44.2 0.884 69.8 1.396











TABIE 25
THE STABILITY OF RIBOFLAVIN-5 '-PHOSPHATE SODIUM IN 50 PER CT
PROFILENE GLYCOL IN DISTILLED WATER STORED UNDER VARIOUS
CONDITIONS IN FLINT AND AMBER BOTTLES

a2gt manfed ht aam
Lnastron L tIraon Immetrou
Reading Mc,1 ,t .Reading .Ng./M. Roadlng Mg.s,

Luamtron Reading of Freshly Prepared Scaples 72.4 or 1.448 ng./fl.

One Day
Amber 20.2 0.404 72.0 1.,40 72.4 1.448
Flint 2.0 0.040 55.0 1.100

Three Days
Amber 8.0 0.160 71.5 1.430 72.4 1.448
Flint 0.9 0.008 29.3 0.586

Five Days
Amber 1.6 0.032 69.0 1.380 72.4 1.448
Flint 0.3 0.006 16.1 0322

Seven Days
haber 1.0 0.020 67.7 1.354 72.4 1.448
Flint 0.0 0.000 12.1 0.242

Ten Days
Amber 0.9 0.018 65.2 1.304 72.0 1.440
Flint 9,. *..* 5.0 0.100

Fifteen Days
Amber 0.1 0.002 62,4 1.248 71.8 1436
Flint .,* ..... 4*4 0.088

Twenty Da"s
Amber 0.0 0.000 58.5 1.170 71,6 1.432
Flint *.. ..... 2.5 0.050

Thirty Days
Amber *,, *,.** 56.3 1.126 71.0 1.420
Flint ... ...., 0.0 0.000
Sixty D ay
Amher ... *.... 47.3 0,946 69.4 1.388











TABlE 26
THE STABILITY OF RIBOFIAVIN-5'-PHOSPHATE SODIUM IN A SATURATED
SOLUTION OF ETHYL AMINOBENZOATE IN DISTIL CID WATER STORED
UNDER VARIOUS CONDITIONS IN FLINT AND AMBER BATTLES

Ililfl PItfflg IBM- I11II I II 1
Lumetron LIaet-r n Iaumatron
Reading Mcg./MI. Reading Mcg./M1. Reading Mcg./M1.

Lnmetron Reading of Freshly Prepared Samples 71.2 or 1.44 mog./ml.

One Day
Amber 40.1 0.802 70.9 1.418 71.2 1.42
Flint 4.1 0.082 51.2 1.024

Three Days
Amber 27.2 0.54 70.4 1.408 71.2 1.424
Flint 3.2 0.064 43.3 0.866

Five Days
Amber 11 0.222 69.3 1,386 71.2 1.424
Flint 1.8 0.036 35.1 0,702

Seven Days
Amber 6.4 0.128 68.0 1,360 71.2 1.424
Flint 1.0 0.020 31.0 0.620

Ten Days
Amber 3.9 0.078 67,0 1.340 70.8 1.416
Flint 0.6 0.012 21.0 0.420

Fifteen Days
Amber 1.2 0.034 65,5 1.310 70,4 1.408
Flint 0.2 0.004 12.3 0.246

Twenty Days
Amber 0.5 0.010 63.5 1,270 70.0 1.400
Flint 0.0 0.000 5.8 0,116

Thirty Days
Amber 0,0 0.000 60.5 1.210 69.5 1,390
Flint ,.. *.,*2 2.9 0.058

Sixty Days
Amber ... ..... 55.2 1.044 68.7 1.374












TABIE 27

THE STABILITY OF RIBOFLAIf-5 '-PROSPHATE SODIUM IN 0.01 PER CEMT
QUININE BISULFATE IN DISTILLED lWTER STORED UNDER VARIOUS
CONDITIONS IN FLINT AND AMER BOTTLES


Lumetrom
Readinga NMe.1..


Luamtron umeTtron
Readin Mar./M1. Reading Moei./L.


Lmatron Reading of Freshly Prepared Ssuples 76,2 or 1.524 ag./al.

One Day
Amber 23.4 0.468 71.1 1.422 76.2 1.52
Flint 3.8 0.076 48.8 0.976

Three Days
Amber 19.2 0.384 70.1 1.402 76.2 1.52
Flint 2.2 0.044 25.2 0.504

Five Dayo
Amber 12,8 0,256 69.6 1,392 76.2 1.52
Flint 1.0 0.020 16.2 0.324

Seven Day
Amber 10.6 0.212 68.9 1.378 76.0 1,52
Flint 0.5 0.010 1.4 0.288

Ton Days
Amber 5.0 0.100 64.1 1.282 75.9 1.51
Flint 0.0 0.000 10,1 0.202

Fifteen Days
Amber 34 0.068 62.5 1.250 75.7 1.51
Flint ... **... 8.0 0.160


4



4



4.



0



S



4.


Twenty Day
Amber
Flint

Thirty Days
Amber
Flint

Sixty Day
Amber


1.9
.*.


0.2
0*0


0.0


0.038



0.004
*#**O


61,8
6.8


58.7
3.2


1.236
0.136


1.174
0.064


0.000 51.5


75.2


1.510



1.504


74.1 1.482


m


-- --


- ---- --- ----











TABLE 28
THE STABILITY OF RIBOFLAVIN-5 -PHOSPHATE SODIUM IN A SATURATED
SOLUTION OF BETA-METIffL UMBELUIFERONT IN DISTILLED WATER
STORKD UNDER VARIOUS C0aDITIONS
IN FLINT AND AMBER BOTTLES

Munligh Diffnaed Liaht DarkneM
Luetron LZmtron umetron
Reading Mog./M1. Reading Mag./l. Beading nog./KL.

uIetron Reading of reshly Prepared Samples 69.7 or 1.394 mag./al.

One Dar
Amber 29.1 0.582 64.8 1.296 69.7 1.394
Flint 3.2 0.064 43.8 0,876

Three Days
Amber 11. 0,220 64, 5 1,290 69.7 1.394
Flint 2.0 0.040 34.5 0.690

Five Days
Amber 6.5 0.130 640 1.280 69.7 1.394
Flint 1.1 0.022 25.2 0.504

Seven Days
Amber 4,8 0.096 63.0 1.260 69.7 1.394
Flint 04 0.008 20.0 0.400

Tea Days
Amber 1.5 0.030 59.3 1.186 69.5 1.390
Flint 0.0 0.000 9.5 0.190

Fifteen Days
Amber 0.8 0.016 57.0 1.140 68.9 1.378
Flint **... ... 2,0 0.040

Twenty Days
Aaber 0.2 0.004 541 1.082 68.9 1.378
Flint *... .... 1.4 0.028

Thirty Days
Amber 0.0 0.000 50.2 1.004 68.5 1.370
Flint ... .*..0 0.3 0.006

Sixty DeVs
Amber ... ..... 47.2 0.944 67.2 1.344












TABIE 29
THE STABILITY OF RIBOFIATIN-5'-PIOSPH.TE SODIUM IN 1.0 PER CENT UREA
IN DISTILLED WATER STORED U:i.l R VARIOUS CONDITIONS
IN FLIIT AND AMBER BOTTLES

Su s Digffused ALiht Darkness
laumtron Lumetron Lumetron
Reading Mog./m. Reading Miog a Reading Ncg./.l,

LuAmetron Reading of Freshly, Prepared Samples 70.8 or 1,416 mcg./ml,

one Day
Amber 6.1 0.122 68.8 1,376 70.8 1.416
Flint 1.1 0.022 31.1 0.622

three Das
Amber 58 0.116 67,8 1.356 70.8 1.416
Flint 0.8 0.016 11.0 0.220

Five Days
Amber 5.2 0.104 65,6 1.312 70.8 1.416
Flint 0.5 0.010 5.2 0.104

Seven Day
Amber 48 0.096 64.4 1.288 70,5 1.410
Flint 0.0 0.000 2.2 0.044

Ten Do"e
Amber 3.0 0.060 56.1 1.122 70.1 1.402
Flint .. .... 1.0 0.020

Fifteen Days
Amber 1.6 0.032 46.8 0,936 69.5 1.390
Flint *.. ,.... 0.4 0.008

Twenty Days
Amber 0.5 0.010 32.1 0.642 69.0 1.380
Flint ,* *,... 0.0 0.000

Thirty Days
Amber 0.0 0.000 27.2 0.544 68.6 1.372
Flint ...* ..... 1,. ****

Sixty Days
Amber ... ..... 1.1 032 2 1


0. 4


.











TABLE 30
THE STABILITY OF RIBOFLAVIN-5'-?IOSPRATE SODIUM I 0.1 PER
TWEE 80 IN DISTILlED WATER STORED UNDER VARIOUS CONDITIONS
IE FLI!T AND MER BOTTIXS

Sundlta, Diffurred Lht DanaresB
I ,metron Iametron Inmetron
Reading Mg./Ml. Reading Mc,/g. Reading Mcg./M1

Limetron Reading of Freshly Prepared Sampler 75.0 or 1.500 cmg./l.

one Day
Amber 19.2 0,384 72.8 1.456 75.0 1.500
Flint 1.9 0.038 30.9 0.618

Three Days
Amber 3.9 0,078 71,3 1.426 75,0 1.500
Flint 0.6 0.012 12,2 0.244

Five Days
Amber 2.7 0.054 68.0 1.360 75.0 1.500
Flint 0,0 0.000 1.8 0.036

Seven Days
Amber 1.5 0.030 65.1 1.302 75.0 1.500
Flint ... *..., 1.5 0.030

Ten Day
Amber 0.9 0.018 60.5 1.210 74.8 1.496
Flint ,...*** 0.3 0.006

Fifteen Days
Amber 0.4 0.008 57,0 1.140 74.2 1.484
Flint *0 ,..... 0.0 0.000

Twenty Days
Amber 0.0 0.000 55.6 1.112 73.4 1.468
Flint 0.* .... .****

Thirty Days
Amber ... **... 52.4 1.048 72.5 1.450
Flint ..* .*... .** 0. 7*

Sixty Days
Amber ,*. *.... 41.5 0.830 70.4 1.408











TABLE 31

THE STABILITY OF RIBOFLAVIN-5 -PHOSPHATE SODIUM IM 0.5 PER CENT NIAOI
IN DISTILLED WATER STORD ITD-R VARIOUS CONDITIONS
IN FLINT AND AMR BOTTLES

e laglM T J eDifgus.d Li.ht DinMsE.
ueatran Imrmtron Lumetron
Reading Mcg,/M. Reading M LgKM. Reading HMgo l.


Lumetron Reading of Freshly Prepared Sample


70.6 or 1.412 mcg./al.


One Day
Aaber
Flint

Three Days

Flint

Five Days
Amber
Flint

Seven Day
Amber
Flint

Ten Days

Flint

Fifteen Dayo
Amber
Flint

Twenty Days
Aaber
Flint

Thirty Days
Amber
Flint

Sixty Days
Amber


15,0
2.0


4.9
1.1


2.8
0,7


1.9
0.0


0,8



0.1


0.0
.0*0.
0,0


0.300
0.040


0.098
0.022


0.056
0.014


0.038
0,000


0.016



0.002



0.000
S....


70.3
42.9


68.6
14.4


66,7
7.0


64.0
2.5


60.8
1,5


58.1
1.0


56.4
0.0


51,8


1.406
0.858


1.372
0.288


1.334
0.140


1.280
0.050


1.214
0.030


1.162
0.020


1.128
0.000


1.036


70.6



70.6


70.6



70.6



70.2



69.7



69.2



68.7


1.412



1.412


1.412


1.412



1.404



1.394



1.384


1.374


66.6 1.332


41,0 0.820


















TABIE 32
THE SOLUBILITY OF RIBOFLAVIN-5'-PHOSFPHTE SODIUM
IN SOME AQE S SOLUTIONS AMD OTHER SOLVENTS


Lunetron Mg.AI,
...... J "eading _,,, .
Distilled Water 4,9 35.71

0.9% Sodium Chloride 5.2 37,14

0.9% Potassium Chloride 5.2 37,14

1.0% Sodium Aoid Phosphate 3.5 25.00

1.0% Potassium Acid Phosphate 3.0 21.43

1*0% Niacinamide 6.0 42.85
1.0% Urea 5,8 41.42

Propylene Glycol 8.0 5,71
Glyoerin 12.5 8.93

Alcohol 4.0 0.08











Results with Flavazin Soluble

Winthrop-Stearnsa Flavaein Soluble or riboflavin sodium-sodium

tetraborate was selected for study because of its solubility and its

popularity on the market.

The assay of this preparation was evaluated fluorophotometri-

oally. It showed a 50.0 per cent riboflavin content. Accordingly,

0,5 (a. of Flavaxin Soluble was equivalent to 1 Ga. of riboflavin.

One milligram of Flavazin Soluble was added to each ml. of the

solvents used for stability study* Since the lumetron was set for

readings up to 2 mog. per ml., each solution had to be further diluted

by adding 3.4 al. to a sufficient quantity of distilled water to make

1000 ml. Twenty-five milliliters of this dilution were used for fluoro.

photometrio analysis.

The solubility of Winthrop-Stearns' product in some aqueous

solutions and other solvents was determined. Further dilution with

distilled water was found necessary with several solvents to be able

to successfully determine the results on the lumetron.












TABLE 33

THE STABILITY OF FLAVAXIN SOLUBLE: IN DISTILLED WATER STORED
UNDER VARIOUS CO;iDITIONS IV FLITT AiD AMB R BOTTLES


Lumetroun n umetron
Reading Mcg./M1. Reading Mcg./M1. Reading Mog./Ml.


Lumetron Reading of Freshly Prepared Sample. 82.0 or 1.640 meg./al.

One Day
Ambr 34.5 0.690 82,0 1.640 82.0 1.64
Flint 2.7 0.054 45.5 0.910

Three Days
Amber 14.0 0.280 79.8 1.596 82.0 1.64
Flint 1.1 0.022 28,2 0.564

Five Days
Amber 2.0 0.040 78.3 1.566 82.0 1*64
Flint 0.5 0.010 20.6 0.412

Seven DQBys
Amber 1,5 0.030 77,5 1.550 82.0 1.64
Flint 0.0 0.000 14.5 0.290

Ten Days
Amber 1.1 0.022 74,2 1.484 81.6 1.63
Flint .* ,..... 8.9 0.178

Fifteen Days
Amber 0.8 0.016 70.0 1.400 81.0 1.62
Flint ... ..... 5.2 0.104


0



0



0



0



2



0


Twenty Days
Amber
Flint

Thirty Days
Amber
Flint

Sixty Days
Amber


0.0
*.


0.000


.... *


65.3
2.41.


61.9
1.2


1.306
0.048


1.238
0.024


0.936


80.2



79.4


1.604



1.588


77.2 1.544












TABIE 34

THE STABILITY OF FLAVAXIN SOLUBLE IN DISTIL,:D WATER BUFFERED AT pH 6
STORED UNDER VARIOUS CONDITIONS IN FLINT AND AMBER BOTTLES

agnlight Dusred Lgnht Darknef
Lumetron Letron 1mtron
Reading Mag./MI. Reading ag./Ml. Reading Mog./ML.


Lanmtron Reading of Freshly Prepared Samples


83.9 or 1.678 og.s/al.


One Day
Amber
Flint

Three Day
Amber
Flint

Five Days

Flint

Seven Days
Amber
Flint

Ten Days
Auber
Flint

Fifteen Days
Amber
Flint

Twenty Days
Amber
Flint

Thirty Days
Amber
Flint

Sixty Days
Amber


33.8
2.6


15.2
1.3


2.2
0.6


1.6
0.0


1.0



0.6
*..


0.0
*to



***


0.676
0.052


0.302
0.026


83,9
47.6


81.5
29.0


0.044 80.0
0.012 21.2


0.032
0.000


0.020



0.012



0.000
*o...


*.. *

**.. *


78.3
15.3


75.5
9.4


72.3
5.8


68.5
2.8


63.8
1.5


48.9


1.678
0.952


1.630
0.580


1.600
0.424


1.566
0.306


1.510
0.188


1.446
0.116


1.370
0.056


1.276
0.030


0.978


83.9



83.9


83.9



83.9



83.9



82.8


82.1



81.5



79,0


1.678



1.678


1.678



1.678



1.678



1.656


1.642



1,630



1.580












TABLE 35

THE STABILITY OF FLAVAXIN SOLBLE IN DISTILLED WATER IBFFEfRED AT p1 5
STORED UND'-R VARIOUS CONDITIONS I! FLIJT AND AMBLR BOTTLES
il -- --- ..- .- .. .- --.- --- ]--- -;--- ... --- IN -- ....... .. I i
L ta hg Difrofsed imht armreal
LuDmtron Luastron Lumetron
Reading MIg./M1. Reading Mcg./M1. Reading Mcg./M1.


Lmetron Reading of Freshly Prepared Samples


One Day
Amber
Flint

Three Days
Amber
Flint

Five Days
Amber
Flint

Seven Days
Amber
Flint

Ten Dase
Amber
Flint

Fifteen Days
Aaber
Flint

Twenty Days
Amber
Flint

Thirty Days
Amber
Flint

Sixty Days
Amber


36.2
2.9


16.3
1.5


3.0
0.8


2.1
0.0


1.8



0.9



0.2
0*4


0.0
oe


0,732
0.058


0.326
0.030


0.060
0.016


0.042
0.000


0.036



0.018



0.004
**0**


0.000
.*..*


82.0
47,5

81.4
28.4


80.8
20.2


78,9
15.0


76.0
8.9


72.8
5.1


68.8
2.5


63.5
1.4


82.0 or 1,640 Mg./Mal


1.640
0.950


1.628
0.568


1.616
0.404


1.578
0.300


1.520
0.178


1.456
0.102


1.376
0.050


1.270
0.028


82.0



82.0


82.0



82.0
82,0



82,0



81.2



80,7



79.8


1,640

1,640


1.640



1.640



1.640



1.624



1.614



1.596


48.8 0.976


77,6 1.552











TABIE 36

TIE STABILITY OF FLAVAXIN SOLUBLE II DISTILLED WkATR BUFFERED AT pH 4
STORED UNDER VARIOUS CONDITIONS IN FLIIT AND AMWBR BOTTLES

a.nlight u ae. ...a. D. mange -
Lumnetron umeBtron IUmetron
Reading RMg./el. Reading Meg./Ml. Reading Mcg./XI1


Lumetron Reading of Freshly Prepared Samples


84.5 or 1.690 mcg./ml.


One Day
Amber
Flint

Three Dayu
Amber
Flint

Five Days
Amber
Flint

Seven Days
Amber
Flint

Ten Days
Amber
Flint

Fifteen Days
Amber
Flint

Twuety Days
Amber
Flint

Thirty Days
Amber
Flint

Sixty Da.-s
Amber


39.6
3,3


19.2
1.8


3.8
1.0


1.8
0.0


1.0
4..


0.8
0.*


0.0
,0#


0.792
0.066


0.384
0.036


0,076
0.020


0.036
0.000


0.020



0.016



0.000



0***0
*e* *e
...ee


84.5
52.5


83.4
33.2


82.3
24.4


81.7
17.2


80.2
8.8


79.1
5.3


75.3
2.6


70.1
1.5


1.690
1.050


1.668
0.664


1.646
0.488


1.634
0.344


1.604
0.176


1.582
0.106


1.506
0.052


1.402
0.030


84.5



84.5


84.5



84.5



84.0



83.6



82.7



82.1


1.690



1.690


1.690



1.690



1,680



1.672



1.654


1.642


*.... 70.1 1.402


82.1 1,642











TABLE 37

THE STABILITY OF FIAVAXIN SOLUBU3E I 25 PER CEIT GLYCERDI IN DISTILLED
WATER STORED UND.R VARIOUS CONDITIONS IN FLINT AND AWMB BOTTLES

audtff ht Diffut ed ;Lbht Brgtadiens
LumBtron Lumetron Lumetron
Reading Mag./1. Reading Mcg./M. Reading Meg./1.

Lumetron Reading of Freshly Prepared Sanple: 84.4 or 1.688 mcg./ml.


One Day
Amber
Flint


Three Days
Amber
Flint

Five Days
Amber
Flint

Seven Days
Amber
Flint

Ten Days
Amber
Flint

Fifteen Daye
Amber
Flint

Twenty Days
Amber
Flint

Thirty Days
Amber
Flint

Sixty Day
Amber


4.0
2.9


29.5
1.5


8.3
0.9


7.0
0.0


5.2



3.0
00*


1.2



0.7


0.0
0.0


0.880
0.056


84.4
66.5


0.590 82.5
0.030 41.5


0.166
0.018


0.140
0.000


81.5
30.2


78.5
18.4


0.104 75.8
*.... 11.8


0.060



0.024


0.014
O*0O0
****,


73.9
7.2


70.1
408

65,0
2.2


0.000 49.2


1.688
1.330


1.650
0.830


1.630
0.604


1.570
0.368


1.516
0.236


1.578
0.144


1.402
0.096


1.300
0.044


84.4


84.4



84.4



84,4



83.9


83.3


82.8



82.3


1.688


1.688



1.688



1,688


1,678


1.666



1.656



1.646


0.984 80.0 1.600











TABLE 38
THE STABILITY OF FIAVAXIN SOLUBLE IN 50 PER CENT GLYCERIN IN DISTILLED
AFTER STORED UNDER VARIOUS CONDITIONS I FLINT AND 4ER BOTTLES

gunligh Diffused Light a eana
IAmt .ron Luatr, Lu.metron
Reading Mb./Ml. Reading g,/1, Reading Mg,/M1.


Lanetron Reading of Freshly Prepared Samwple


83.9 or 1,678 mog.,/l,


One Day
Amber
Flint

Three Days
Amber
Flint

Five Days
Amber
Flint

Seven Days
Amber
Flint

Ten Days
Amber
Flint

Fifteen Days
Amber
Flint

Twenty Days
Amber
Flint

Thirty Days
Amber
Flint

Sixty Days
Amber


58.3
3.1


42.8
1,8


7.6
1.0


6*5
0.0


4,5


3.5


81,



0.8

00*

0.0


1.166
0.062


0.856
0.038


83.9
75.0


82,4
52.1


0.154 81,1
0.020 37.0


0.130
0.000


0.090


0.070


0.036


0.016
,..o.o


79.1
19.2


77,5
12.4


75.5
8,1


71.2
4,5

66.3
2.2


1,678
1.500


1,648
1.042


1.622
0.740


1.582
0.384


1.550
0,248


1.510
0.162


1.424
0.090


1.326
0.044


0.000 50.2 1.004


83.9



83.9


83.9



83.9



83.5


82.9


82.3


81.9


1,678


1.678


1,678



1.678



1,670



1.658


1.646


1,638


80.0 1.600












TABLE 3


THE STABILITY OF FILVAXID SOLUBLE IN 25 PER CENT PROPYLENE
DISTILLED WATER STORED UINDR VARIOUS CONDII
IN FLNT AND AMBER BOTTLES


OGLCOL IN


Sun0lish Diffused Liaht DarknesJ
Lumetron Lumetron Lmetron
_- Reading Mog./ Reading Mpe.Al. Reading Mcg./Ml.

lmnetron Reading of Freshly Prepared Seaples 83.9 or 1*678 mog./al.


One Dqa
Amber
Flint

Three De
Amber
Flint

Five Days
Amber
Flint

Seven Days
Amber
Flint

Ten Days
Amber
Flint

Fifteen Days
Amber
Flint

Twaty Days
Amber
Flint

Thirty Daw
Amber
Flint

Sixty Daya
Amber


37.5
2.5


21.5
1.4


4.5
0.8


3.7
0.0


3.0



1.5
***


0.3


0.0
0,0


0*
O.O


...


0.750
0.050


0.430
0.028


0.090
0.016


0.074
0.000


0.060



0.030
* ***.

0.006



0.000
0**ee


83.9
63.3


82.5
32.7


81,7
20.5


80.8
11.7


77.3
7,5


72.3
4.5

67.2
1.8


63.3
0,3


48.2


1.678
1.266


1.650
0.654


1.634
0.410


1.616
0.234


1,546
0.150


1.446
0.090


1.344
0.036


1.266
0.006


83.9


83.9



83.9



83.9



83.3


82.8


82.0



81.7


0.964 79.6


1.678



1.678



1.678



1.678



1.666



1.656


1.640


1.634


1.592











TABLE 40


THE STABILITY OF FLAXIN SOLUBLE IN 50 PER CEMT PROPFILNE
DISTILLED WATER STORED UNDER VARIOUS CONDITIONS
IN FTLT AND A R BOTTLES


GLYCOL IN


Saism'ht Diffused IMpar mesp
Lamtron IAntron Lamtron
Reading mcg./. Reading Mc./Mi. Reading Mcg./M1.

Lnuetron Reading of Freshly Prepared Samplsi 85.0 or 1.700 mcg./Als


One Day
Amber
Flint

Three Days
Amber
Flint

Five Days
Amber
Flint

Seven Days
Amber
Flint

Ten Days
Amber
Flint

Fifteen Days
Amber
Flint

Twenty Days
Amber
Flint

Thirty DaBe
Amber
Flint

Sixty Dayr
Amber


45.0
3.0


25.5
1.8


5.3
1.0


3.9
0.0


2,8



2.0
e,,

0.8
eto


0.0
#60


0.900 85.0
0.060 69.9


0.510
0.036


0.106
0.002


83.9
44.9


82.2
29.6


0.078 81,4
0.000 16.9


0.056



0.040


0.016
o*e*o


79,7
8.9


76.4
5.0


72.3
2,1


0.000 67.0
,..,, 0,8


81.1 1.622


85.0


1,700
1.398


1.678
0.898


1.644
0.592


1.628
0.338


1.594
0.178


1.524
0.100


1.446
0.042


1.340
0.016


85.0



85.0



85.0


85.0


84.6



83.8


83.0


1,700



1i700



1,700


1,700



1.692



1.676


1.660


50.2 1.004











TAB 3 41
THE STABILITY OF FLAVAXDI7 SOLUJDL IN A SATURATED SOLUTION OF ET1YL
AMINOBE~I0ATE I~ DISTILTED WATER STORED UND-R VARIOUS CONDITIONS
IN FLINT AND AMEFR BOTTLES

Smnl'fiht Diffsed Light ... Darkness
LuImn tn Lumetron uametron
Reading MogA/. Reading ../M. Reading Me ./Ml.

uImetron Reading of Freshly Prepared Samples 85.6 or 1.712 mcg,/ml,

One Day
Amber 40.2 0.804 85.6 1,712 85.6 1,712
Fint 3.2 0.064 65.0 1.300

Three Doys
Amber 32.2 0.644 84.2 1.684 85.6 1.712
Flint 19 0.038 52.2 1.044

Five Days
Amber 12.5 0.250 83.7 1.674 85.6 1.712
Flint 1.1 0.022 47.0 0.940

Seven Days
Amber 7.4 0.148 82.4 1.648 85.6 1.712
Flint 0.C 0.000 40.0 0.800

Ten Dys
Amber 42 0.084 80.0 1.600 85.6 1,712
Flint ** *.... 31.9 0.638

Fifteen Days
Amber 1,8 0.036 79.0 1.580 85.6 1,712
Flint .,,* .,,, 19.2 0.384

Twety Days
Amber 1.0 0.020 77,6 1.552 85.0 1.700
Flint ... *..*. 8.8 0.176

Thirty Days
Amber 0,6 0.012 74,3 1.486 84.8 1.696
Flint ... *.... 3.8 0.076

Sixty Days
Amber 0.0 0.000 65.8 1.376 84.4 1.688


-- --


-r











TABIE 42
THE STABILITY OF FLAVAXIN SOLUBL. IN 0,01 PER CENT QUININE BISULFATE
IN DISTILLED WATER STORED UNDER VARIOUS CONDITIONS
IN FLINT AND AMBFR BOTTLES

afiight Dfgaand t DarineawB
uImetron ietron Lunetran
Reading M g./M. Reading Mcg./M. Reading cg./M1.


Lumetron Reading of Freshly Prepared Samplet


83.7 or 1.674 mcge/nl.


One Day
Amber
Flint

Three Days
Amber
Flint

Five Days
Aaber
Flint

Seven Days
Amber
Flint

Ten Day.
Amber
Flint

Fifteen Days
Amber
Flint

Twenty Days
Amber
Flint

Thirty Days
Amber
Flint

Sixty Dap
Amber


51.7
3.0


38.0
1.8


14.1
0.9


4.3
0.0


2.4



1,1
0*S


0.5
0.*


0.0
00#


1.034
0.060

0,760
0.760
0.036


0,282
0.018


0.086
0.000


0.048



0.022



0.010
*e*.


83.7
57.3

83.1
48.8


82,2
37.3


81.5
2460


80.7
17.2


79.3
12.1


77.2
7.6


0.000 72.2
..... 2.7


59.3 1.186


1.674
1.146


1.662
0.976


1.644
0.746


1.630
0.480


1.614
0,344

1.586
0.242


1.544
0.152


1.444
0.054


83.7



83.7


83.7



83.7



83.5


83.1



82.8


82.4


1.674


1.674


1.674



1.674


1.670



1,662



1.656


1.648


81,2 1,624




Full Text

PAGE 1

:. A STABILITY AND SOLUBILITY STUDY OF RIBOFLAVIN AND SOME DERIVATIVES By JOEL JOHN _!IERTZ A DISSERTATION PRESENTED TO THE GRADUATE COUNOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITI OF FLORIDA June, 1954

PAGE 2

ACKJ.OWLEDGMENTS The author wishes to express his sincere appreciation to Dr. C harles H Becker, Chairman of, e Supervisory Committee, for his untiring patience a m e n couraging guidance i n t h e direction of this work. The valuable assistance o f D r Verner M Lauter is also gratefully acknowledged The author wuld also like to exp:ress his appreciation to Eli Lilly and CompOllY for their financial assistance to him and for purchasing equipment and supplies. 11

PAGE 3

TABI.E OF CONTENTS LIST OF TABIES INTRODUCTION REVlEW OF THE LITERATURE Historical Sketch Isolation Occurrence Physical and Chemical Pro~rties ssrq Determinations Increasing Solubility EXPERIMENTAL Materials Used Preparation of Riboflavin Derivatives PyruV'ic Acid, Lewli.nie Acid and Citl"S.coni c Anhydride Derivatives Scope of the Fluorop h otomater Standardization of t h e Lumetron Pr¶tion of lutions Adjusting the Lumetron for Analysis Stability Study Methods Solution& Used Procedure Solubil1 t y Study Method$ Solvents Used Procedure iii Page V 1 3 3 7 9 11 15 19 26 26 29 29 .31 34 34 .34 37 37 /I) 43 43 43

PAGE 4

thod of Riboflavin Derivatives Results wi tl Riboflavin Results with Ribofi vin-5'-Pho phate SodiUlll Results with Flavaxin Soluble sults vi.th a Pyruvic Acid Derivative of Riboflavin Results vith a I.evullnic Acid Deriva-tiv of Riboflavin Results with a Citraconio Anhydride Derivative of Ribofl vin DISCUSSIO OF aSULTS SUMMARY iD COl CLUSIO S BIBLIOGRAPHY B!OGRAPHICAL I cm n REPORT iv Page 45 47 63 79 95 111 127 129 1'51 139 144 145

PAGE 5

LIST OF TABLES Table 1 DesQription ot Drugs d Chemicals Used 2-. Lumtron Rea.dings of Various Dilution s of a Standard Riboflavin Solution Containing 2 Mg./Uter .'.l~ Tho Stability of Riboflavin in Oistilled Tater Stored Under Various Conditions in Flint and Amber Bottles 4 The Stability of Riboflavin in Distilled Water Buffered at pH 6 Stored Under Various Conditions in Flint and Page Amber Bottles 49 5 The Stability of boflavin in Distilled Water Buffered at pH 5 Stored Under Various Conditions 1n Flint and Amber Bottles 6 The Stability of Riboflavin in Distilled ater Buffered at pH 4 Stored Under Various Conditions in Flint and Ambor Bottles 51 7 The Stability of Riboflavin in 25 Per Cent Glycerin in Distilled }.ater Stored Under Various Conditions in Flint and Alli r Bottle-a 52 8 The Stability of Riboflavin in 50 Per Cent Glycerin in Distilled rater Stored Under Various Conditions in Flint and Amber Bottles 53 9 The Stability of Riboflavin in 25 Per Cent Propylene Glycol in Distilled Water Stored Under Various Condi tions in Flint and Amber Bottles 10 The Stability of bofla.vin in 50 Per Cent Propylene Glycol in Distilled Yater Stored Under Various Condi -tions in Flint and Amber Bottles 55 ll~ The Stability of Riboflavin in a Saturated Solution of E thyl Aminobenzoate in Distilled Water Stored Under Various Conditions in Fl~nt and Amber Bottles 56 12 The Stability of Riboflavin in 0 01 Per Cent nine Rt.sulfate in Distilled ter Stored Under Various Conditions in Flint and Amber .Bottles V

PAGE 6

Table 13 The Stability of Riboflavin in a Satur ted Solution of ta-tbyl Umbellif erone 1n Dis tilled Ii ter Stored Under Various Conditions in Flint and .Amber .ttles U.. The Stability of Riboflavin in l O Per Cent Urea in Distilled ator Stored Under Various Conditions in Flint and AI!lber Bottl"8s 15. The Stability of bofl vin in 0 1 Per Cent en 80 1n Distilled tr Stored Under Various Conditions in v1 Page 59 Flint and ber Bottles 6o 16. Tho St bility of bofl vin in 0 5 Per Cent Miacin in Distilled ater Stored Under Various Conditions 1n Flint and ber Bottles 61 17 The Solubility of flavin in Some Aqueous Solutions .. and other Solvents lS. The Stability of bofla.vin-51Phosphate Sodium in Distilled ter Stored U?Xler Various Conditions in Flint and be r ttles 19 The Stability of boflavin51Phoaphate Sodium in Distill Water Buffered at pH 6 Stored Under Various 62 64 Conditions in Flint and Amber Bottle s 65 20 The Stability of boflavin-51-Phoophe.te Sodium in Distilled ter Buffered t pH 5 Stored Umer Various Conditions in Flin t and Amber Bottles 66 21. The St bilit of Riboflavin 5'-Pliosphate S ium in Distilled ater fered at pH 4 Stored Under Various Conditions ill Flint and r Bottles 67 22. Tho Stability of boflavin51Pbosphate S um in 25 Per Cent Glycerin in Distilled Water Stored Under Various Conditions in Flint and Amber Bottles 68 23. The Stability of Riboflavin-5'-Phosphate Soditm 1n 50 Per Cent Glycerin in Distilled rater Stored U?Xler Var ious Conditions in Flint and Amb r Bottl s 24. The Stability of Riboflavin-5'-Phoaphate Sodium in 25 Per Cent Propylene Glycol in Distilled Pater Stored Undor Various Conditions 1n Flint and Amber Bottles 70

PAGE 7

v11 Thl p 25 The Ste.bill ty of bofls.vin-5 '-Phosphate Sodium in 50 Per Cent Propylone Glycol in Distilled tr Stored Under Various Condition in Flint and ber ttles 71 26. o Stabill ty of ; boflnvin-51Phosp t Sodium in a Saturated Solution of Ethyl Atninobenzoate in Distilled !e.t-r Stor d Under Various Conditions in Flint and Amber Bottle 72 27. e Stability of Riboflavin-51Phosphate Sodium in 0 0 1 Per Cent nine Bisulfate in Dis tilled later Stored Under Various Conditions in Flint and Amber Bottles 73 28. Th Stability of Riboflavin-5'-Phosph t e Sodium in a turated Solution of Beta 1ethyl U llifcrone in Dis till ed Tater Stored Under Various Condi tions in Flint and Amber ttl s 7 4 29, The Stability of Ribof'lavin-51-Phosphate Sodiuo in 1 0 Per Cent Ure in Diotilled at r Stored Under Various Conditions in Flint d Amber Bottles 75 ~ The S tability of boflavin-5'-Phosphate S ium in 0 1 Per Cent Tween 80 in Distilled ater Stored Under Vari o us Conditions in Flint and Amber Bottles 76 31. Tho Stability of Ribof'lavin51Phosphate Sodium in 0 5 Per Cent Niacin in Distill d tor Stored Under Various Conditions in Flint nd Amber Bottles 77 32. The Solubility of P.ibofie.vin 5 -Phosphate Sodiuo in Some Aqueous Solutions end Other Sol vents 7 8 3 3 The Stability of Flavaxin Soluble in Distilled -late r Stored Under Various Conditions in Flint and AI:lber Bottles &> 34. Th Stability of Flo.vaxin Soluble in Distilled :ator Buffer d at pH 6 Stor d Under Various Condition 1 n Flint d her Bottlos 8 1 35. The Stability of Flnvaxin Soluble in Distill d ater ferod t p 5 Stored Under Various Conditions in Flint ber Bottles 82 36. The Stability of Flavoxin Soluble 1n Di tilled ter Buffered at pH 4 Stored Under Various Conditiono in Flint and Anlber Bottles 8 3

PAGE 8

Table '3'1. The Stability of Flavaxin Soluble in 25 Per Cent Glycerin in Distilled rater Stored Under Various Condition in viii Page Flint and Amber Bottles 84 38. The Stability of Flavaxin Soluble 1n 50 Per Cent Glycerin ill Distilled ater Stored Under Various Conditions in Flint and her Bottles 85 39. Th Stability of Flavaxin Soluble in 25 Pr Cent Propylene Glycol in Di tilled Water Stored Under Various Conditions 1n Flint and Amber Bottles 86 40. The Stability of Flavaxin Soluble 1n 50 Per Cent Pro pylene Glycol in Distilled ter Stored Under Various Conditions in FliDt and Amber Bottles 41. The Stability of Flavaxin Soluble in a Satur ted Solution of Ethyl Amino nzoate Stored Under Various Conditions in Flint nd Amber Bottles 88 ~ The Stability of Flavaxin Soluble in 0 01 Per Cent Quinine Bisulfate in Distilled Water Stored Under Various Condition in Flir:t and Amber Bottle s 89 4.3. Th Stability of Flavaxin Soluble in a Saturated Solution of tathyl Umbelllferone Stored Un r Various Conditions in Flint and Ambe r Bottles 90 44. The Stability of Flavrodn Soluble in 1 0 Per Cent Urea in Distilled ater Stored Under Varioua Conditions 1n Flint and Amber Bottles 91 45. Tbe Stability of Fls.va.xin Soluble in O l Per Cent Tween 80 in Di tilled 'ater Stored Under Various Conditions in Flint and Amber Bottles 92 46. The Stability of Flavaxin Soluble in o 5 Per Cent ? ie.cin in Distilled ater Stored Under Various Conditions 1n Flint and Amber Bottles 93 47. Tb lubili ty of Flavaxin Soluble in So Aqueoua Solutions and Othe r Solvents 48. The Stability of a Pyruvic Acid Derivative of Riboflavin in Distilled ater Stored Under Various Con dit.ions in 94 Flint and b r Bottles 96

PAGE 9

Table 49., e Stability of a Pyruvic Acid Derivative of Ribofi v1n in Di tilled 'ater Buffered at pH 6 Stored Under Various ix Page Conditions in Flint and Amber Bottles 97 50 The Ste.bill -cy or a Pyruvic Acid Derivative of boflavin in Distilled ater Buffered at pH 5 Stored Under Various Conditions in Flint and Amber Bottles 98 51. The Stability of a Pyruvic Acid Derivative of boflavin in Distilled Water Buffered e.t pH 4 Stored Under Various Conditions in Flint and Amber Bottles 99 $2. The Stability of a Pyruvic Acid Derivative of Riboflavin in 25 Per Cent Glycerin in Distilled Water Stored Under Various Conditions in Flint and Amber Bottles 100 53. The Stability of a Pyruvic Acid Derivative of Riboflavin in 50 Pr Cent Glycerin in Distilled ter Stored Under Various Conditions in Flint and Amber Bottles 101 54-The Stability of a Pyruvic Aeid Derivat!v of Ribofiavin in 25 Per CeD t Propylene Glycol in Distilled later Stored Under Various Conditions in Flint and Amber Bottles 102 55. The Stability ot a Pyruvic Aeid Derivative of Riboflavin in 50 Per Cent Propylene Glycol 1n Distilled ater stored Under Various Conditions in Flint and Amber Bottles 103 56. The Stability of a Pyruvic Acid Derivative 0 Riboflavin in a Saturated Solution of Ethyl Aminobenzoate in Dis tilled Water Stored Under Various Conditions in Flint and Amber Bottles 104 '57. The Stability of a Pyruvic Acid Derivative of Riboflavin in 0 01 Per Cent Quinin Bi.sulfate in Distilled ater Stored Under Various Conditions in Flint and AI:lber Bottles 105 58. The Stability of a Pyruvic Acid rivative of Riboflavin in a turated Solution of Beta Methyl Umbelliferone in Distill ed Water Stored Under Various Conditions in Flint and Amber Bottles 106 59. Tho Stability of a Pyruvic Acid Derivative of Ribofiavin in 1 0 Per Cent Urea in Distilled Water Stored Under Var-ious Conditions in Flint and Amber Bottles 107

PAGE 10

X Table Page 60., The Stability or Pyruvic Acid Derivative of Riboflavin in 0.1 P e r Cent Tween 80 i n Distilled 1ater Stored Under Verious Conditions in Flint and Amber Bottles 108 61., 'l'he Stability of a Pyruvic Acid Derivative of Riboflavin in 0 5 P e r Cent Niacin in Distilled Water Stored Under Various Conditions in Flint and Amber Bottles 109 62., The Solubility of a Pyruvic Acid D erivative o Riboflavin in Some Aqueous Solutions and Other Solvents 110 6J. The Stability of a Levulinic Acid Derivative of Riboflavin in Dis-till d 1ater Stored Under Various Conditions in Flint and Amber Bottles 112 The Stability of a Levulinie. Acid Deriv tive of Ribofiavin in Distilled ater Buffered t pH 6 Stored Under Various Conditions in Flint and Amber ttlos 113 65 The Stability of a Levulinio A e1d Derivative of Ri.boflavin 1n Distilled Water fered at pH 5 stored Under Various Conditions in Flint and Amber Bottles 114 66. T.be Stability of a Levul1nic Acid Derivative ot Riboflavin in Distilled Water fered at pH 4 Stored Under Various Co ditions in Flint and Amber Bottles ll5 67 The Stability of e. Levulinie Acid Derivative of Riboflavin in 25 Per Cent Glycerin in Distilled Water Stored Und r Various Conditions in Flint and Amber Bottles 116 68. The Stabilit.y o f a Lewlinic Acid Derivative of Riboflavin in SO Per Cent Glycerin in D-istilled Water S tored Under Various Conditions in Flint and Amber ttles 117 (:JJ. The Stability of a Levullnie Acid Derivativ of bofiavin in 25 Per Cent Propylene Glycol in Distilled Water Stored Under Variouo Conditions in Flint and lunber Bottles ll8 70 The Stability of a Levulinic Acid Derivative of boflavin in 50 Per Cent Propylene Glycol 1n Distilled Water Stored Under Various Conditions in Fil.tit and Amber Bottles 119 71. The Stability or a Levulinio Acid Derivative of bofl nn in e. Saturated Solution of Ethyl Aminobenz.oate in Distilled later Stored Under Various Conditions in Flint and Amber Bottles 120

PAGE 11

Table P 72. The Stability of a Levulinie Acid Derivative of Ribofl :vin in 0.01 Per Cent Quinine Bisulfate in Distilled ater xi Stor d Under Various Conditions in Flint and Amber Bottles l2l 73 The Stability of a Levulinic Ao.id Derivative of Riboflavin in a Saturated Solution .of Beta {ethyl Umbellif rone in Distilled ater Stored Under Various Conditions in Flint and Amber Bottles 122 74. The Stability or a Levulinic Acid Derivative of Ribotla\l'in in 1 0 Per Cent Ure in Distilled Water Stored Under Var-ious Conditions in Flint a.nd Amber Bottles 12.3 15. Tho Stability of Levul.inic Acid Derivative ef Riboflavin in 0 1 Per Cent l een 80 in Distilled tar Stored Under Various Conditions in Flint and Amber Bottles 124 76. The Stability of a Levulinic Acid Deri tive of Ribonavin in 0 5 Per Cent Niacin in Distilled Water Stored Under Various Conditions in Flint and Amber Bottles 125 77. The Solubility o f Levullnie Acid Derivative of Riboflavin in Some Aqueous Solutions and Other Solvents 126 78. '!'he Solubility of a Citracon1c Anhydride D rivative 0 bofiavin in So!:!e Aqueous So.lutions and Other Solvents 128

PAGE 12

TRoDUCT!ON Almost since the discav ry and isolation of vitamin~ or riboflavin, tho probl m of solubility end stability in solution has been th caus of mu concern Numerous raetboda ha been sug gested for preparing solutions containing a r lative.cy high conoentr tion of riboflavin. Most or these sug ted thods, hovever, do not show any increase in stability' greater t t L\t of the pure Vitamin itsel.. Ono of the purposes of this investigation s to pr pare a more soluble form or derivative of this vitamin. Another aim was to prepare sol.utions of riboflavin, or a deri tive ereof, ich wuld bo ore stable to light and yet retain active phy iolog.teel activity. An evaluation of riboflavin and so derivativ s was made vi.th regard to solubility and stability in various solvents and under differant stor ~e conditions. It is often desirable to e.dmini .. ter riboflavin parentorally, and to do so, it is necessary that the vitamin be present in a therapeutically effective amount and in a reasonable quantity of hormless dilueDt, It is likewise desirable to administer such riboflavin solu tions by Qral route. Sueh a form of vitamin B2 could also be us d for the enrichment or foodstuffs, for infant preparations and in many other pharmaceuticals Riboflavin is only very sparingly soluble in both water and in aqueous acidic solution11 At 2()0 c (1) only 0 12 mg. per Dl.. of wter 1

PAGE 13

2 will dissolve. Al ough more riboflavin is soluble in al a.line aqueous solutions, suo solutions are extremely unst ble d the riboflavin soon loses its physiological activity. It is of advantage to h ave a salt producing an acid pH present in t he preparation of such solutions. This typ e of salt could serve to maintain the pH of t he solution near the isoelectric point of riboflavin, thor by 1ncreasinp, its stability. In human beings (2) natural as woll as artificially induced riboflavin deficiencies have bee n observed. Lesions on the lips, and fissures at t h e e.ngles of the mouth (cheilosis) are characteristic a tons which are promptly relieved by the administration of pure ribo flavin Human pellagra is often accompanied by definite symptoms of riboflavin deficiency, and in general, some riboflavin deficiency probably exists u hether t he outv d sympto are detectable or not. Thus, it is apparent that the value of a concentrated and stable solution of vit n B 2 is of prim e importance. An attempt has been made in this inveetigntion to prepare such types of solutions.

PAGE 14

A REVIEW OF T.lE LITERATURE Historical Sketch The chemioru. nature of the yellow en fluore cent pigmen t of whey, now referred to as riboflavin (synonymous wit lactoflavin, vita,.. min G and vitamin}) eoded the attention of chemists ( 3 ) as early as 1879. A co nsiderable concentration of this pi ent was effected and certain of its more obvious chemical properties were clearly set forth by Bleyer and Ka.llmann ( 4) in 192$ ?fo unusual significance vas ass~ oiated with this pigment by these. early workers, who apparently regarded it only as one of the minor co nstituents or milk. The chemical nature of the pigment was still quite obscure In the course of an investigation into the nature of pellagra, Goldberger and Lillie (5) produ ce d deficiency disease in rats, characterized by ophthalmic and bilaterally symmetrical denuded areas. The factor that prevented these lesions waa heat-stable, in contrast to vitamin B1 which Ya.a heat-labile. It was termed by Goldberger, the P P. (pellagra-preventing) factor but was later designated vitamin B2 in Great Britain and vitamin Gin the United States (6). It is now known that vitamin B2 or riboflavin is not the rat pellagra-pre'O'entat1ve factor but owing to the lack of knowledge at that ti oft e existence of ot.er members of tho B complex, this sconception was widely prevalent.

PAGE 15

In 19.32 farburg and Christian (7) described a new oxidation enzyme obtained fro:i aqueous extracts of yeast. The enzyme in rater solutions uas yellov and eyJrl.bited green fluorescence. It has now been established (32) tat t his 11ye llov e n z n is presen t in every livin g cell or at least 1n the cell o f all the higher forms of life. 4 The s~nto s ported by othe r workers s characteristic of Vitamin B2 deficiency varied co nsiderably, howe v er, and frequently dif-fered kedly from those observed by Ool d b erg~ r and Lillie ( 5) In particular, so?:18 work ers reported o n l y an abse n ce of growth vhilo others noted e o.ppearance of a dermatitis i n so. e of t l experimental animals In 1934 OyorfI1 (8) (9) showed t h e fallacy of ldberger and Lillie's experiments. This observer showed that rats ntai ed on a vitamin B free diet, with Bi concentrate and lactoflavin added, devel oped a number of pellagra-like changes which w re not only unrelieved, but even ad wors by t h e addition o f more vitamin B2. These lesions, so p roduced, whic h wer of a so m ewhat differen t character fron those produced by Goldberger and L.i.ll1e, were cured b y an unknown factor, tentative named by Cqorgy vitamin B(,. It was containe d in e "Peter's E luntofl from charcoal as prepared from yeast extract. This author admitted that certain skin l esion s can be p roduced by deprivation of vitamin~ but m ea sharp distinction b etween the manifestations ao pro duced and those due to deprivation of vitamin B6 us, i ti ly, the t rm vitamin B2 was intended to describe the faotor that caused pellagra, now known to be identical with nicotinic

PAGE 16

5 acid Subsequently, it came to be used to denote the rt growth factor, riboflavin. The first step to ards an underst 1ng of the nature of vita min was taken by Kuhn, Gyorgy and l a er-Jauregg (10),. who isolated from egg-white a compound 'W'i.t a strong y llowisb-een nuoreocence. They called ~s subst ce uovofle.vintt and showed that it stimulated the growth of rats. In the a journal containing t h e papor by Kuhn .!Y., there appeared a paper by :Ellinger and osohar (11). They reported t e presence of similar fluorescent substances in milk, liver, ddney, urine, muscle, y east and in certain plant terials. They described the isolation of a cry-stalline fluor scent substance from whey. This subst ce obtained fl-om whey they called "lactofl rin" and they prop ose d _o name "lyochromes" for the group to which all these substancos belonged This te was in contradistinction to a .group of naturally occurring fat-soluble pi@'.!lents called "lypoohromes Bo-th Kuhn !! and Koschnra (11) s gested th t the pi onts mi t be related to the "yellow enzyme disco red in yeast. In fact, Kuhn sho d that one and the s substance, lumiflavin, vas produced by irradiation of the yellow enzyme and of riboflavin. Shortly after the publication of ese pa.pors, Booher (12) re ported the prep ation of a concentrate fro :i 'Whey o'Wder th t shoved a strong yellow fluorescence and had grouth-pro oting properties for the rat. t first, these pi ents isolated from various substances (13)

PAGE 17

6 were given speeifie n es according to their origin, for exa.rirple: ovoflavin, lactofl vin, urofiavin and hep ato.t'lavin It slater realized that they-vere all probably i dentic with o ne another. This as con fir.nlod by direct c o .. ?parison of some of t..'1e cam.:, ounds, bu t sevoral were isolated in such small amounts that rigid proof of i dentity was not po.
PAGE 18

7 Isole.t1on boflavin has been isolated from a 'Wide variety of an1.mal and plruit products (15) including egg-Yhite, milk, liver, kidney, urine, barley malt, dandelion blossoms, grasses, egg yolk d retinas of fish eyes !t can be stated with absolute eerte.inty that the erysta.lline f'laV"in obtained from ee.ch of these various sources vas chemically iden tical with riboflavin. At lee.st such is the case for thos~ to ldrl.ch adequate determinative tests have bee n applied. The methods of isolation (16) veried .somewhat in different laboratories and wit the raw-materio.1 employed, but nearly all the workers used adsorption on fuller t s earth ( or in aom.a inata.'lces lead sulfide) from. a slightly acidaqueous or aqueous -alcoholic extract. The result ing adsorbate wo.c eluted with pyridine, or pyridine-methanol ... water mix-ture or diluto nia, and the eluate, .aftor being concentrated, was treated with a heavy tal., such as silver or thallium, to precipitate the flavin i n t e form of a salt. The free flavin we.s recov a red from the precipitate by suitable treatment and reerystallized froo vater, dilute alcohol or dilute acatic acid. I n their earlier work Kuhn and his coworkers (10) obtained from 100 Kg. of dried egg albumin, corresponding to about .33,. 000 egg 1.0 rag. of thrice roorysto.llized flavins .. According to subsequent measurements (17} of t h e quantities of flavin normally present in dried egg albumi n this yield would correspond to about 7 pe r cent of the total flavin present in the egg albumin Similorly the yield of crystalline

PAGE 19

8 fl vin from lk, r ortad in th earlier or (18), w not greater than 5 per cent of t~ e total qu tity present~ The use of heavy tal precipitation increased the yiold to about 18 to 20 per cent of the q tity re orted (17) to be normally preeent in milk 5'nthetic fle.vin first prepared in 1934 by Karrer and Kulm (19 ) and also by Reine d and iey d (20). This synthetic flavin was shown in both oases to be C! emieally identical w1 th t e flavin ioole.tod fr d to h ve the s biolo cal. value for r ta. Karrar (21) first used t he te riboflavin d its synthesis proved it to be an alloxaz1ne structure corab1Ded with ribo e

PAGE 20

9 Occurren ce bofla.vin (l) is "1id ly distributed in nature in both plants 8lld animals, being found as a free pi nt or c bined with a protein. It is an essential co nstituent of all living cells. In the pl t world analysis (22} has shown t t riboflavin occurs naturally in the een actively growin g leaves and that it per sists there in higher co ncentr tion tho.n 1n other parts of t he plant. Conseque ntly, green stems and leaves are a much richer source of the vitamin t han t he flower or root. H ovever, the vitamin is present in small amounts i n practically all root vegetables and tubers. There is re o n to believ that as the leaves mature and dry, t he ribofl vin contont may be correspo ndingly d.1.m1nished ( 23). This may have a bearing on the vitamin content ot milk since it h s been found that cows fed o n fresh youn g grass yield mi richer in ribofla vin t h animals receiving a ture and drier grass { 24). Ho .rever, milk, either fr-ash or processed, seems to be a relatively rich and con stant eource of riboflavin. The co ncentratio n of the Vitamin in seeds ( 23) is subjected to considerable vari tion and rea ches it ma:rl.mum in the germ portion., Legumes, peas and bean provide a erately rich source wile nuts and cereal gra,ins are somewhat oror .in their content. Fruita generally, and particularly citrus have been proved to provide only a trifling amount o f this substance The glandular organs of animal (24) co nstitute e richest ot all foodstuffs in their ribofl :Vin content. The lean muscle flesh

PAGE 21

10 contain v ey considerable quantitie. Many species of mieroorg i (25) are cap ble of synthesizing riboflavin, and because ot the exttlnsive bacterial grovth in the human intestinal tract, this supply, fom important and constant source or The retinas of the eyes of many species of sn1maJs have been reported (26) to contain relatively high concentrations of flavin. It was supposed that the flavins are involved in some light sensitized re actions concerned Yi.th dim vision. Riboflavin is synthesized co rciel.l.y (1) on a large scale for addition to bread, nour and other dietary and pharmaceutical preparations.

PAGE 22

11 Physical and Chemical Properties Riboflavin (27) is a. yellow to orange-yellow crystalline powder having a slight odor It melts at abou t 2800 c. end its saturated solution is neutral to litmus. Riboflavin is quite stable in strong mineral acids. .Jhen dry 1 t is not appreciably affected by diffused light, but in solution, especially in the prese nce of alkali, it deteriorates quite rapidly, the deterioration being accelerated by light. Riboflavin is so sensitive to light that on irradiation with ultraviolet rays or s ble light (l) it undergoes irreversible decomposition bofl :vin is 6,7 ethyl-911-ribitylisoalloxazine. It is us a nitrogenous p olyhydroxy alcohol (l). At le one of the methy l groups in position 6 or 7 is essen-tial in order that t h e flavin lecule shall possess vitamin activity. The absence of both t he 6 d 7 methyl groups actually appears to be acco mpanied wit toxicity (28). With regard to t he side-chain, o nly the D-ribose or D-arabinose residue attached to t he nitrogen atom in position 9 has t hu s far p roved to be compatible with vitamin activity of e flavins. ce edingly small variations in t he sidechain often cause co p l ete l a c k of vi t n activity. The flavins1 as a group, all share the tricycllc c h romophoric nucleus t ha t confers on them the yellow color to which they owe their group name. The vitamin activity (29) of the various members of this group of yellow p igments is profoundly influenced by the position and nature of t he substituent groups in the benzene nucleus and by the nature of the sidechain attached to the pyrazine ring.

PAGE 23

12 Riboflavin, thee irieal formula of 'Which is Ci,112fY1406, has a solubility in water of 12 .,:,. per 100 ml. at 27. 5 C Some vari tions i n solu lit ha been noted Blld this is due to differeneeo 1n the intern \l crystalline structure of t e vitam.in. The aqueous solution has a strong yellowish-green fluoresc ence which is discharged by ncid or alkali. is is used as a basis for identification by the u. S. P (27) borl vin (25) (27) is sparingly soluble in ethyl ale ho l (4. 5 mg. per 100 J!ll.. t 27 so C ) amyl alcohol, cyclohexanol, phenol or amyl acetate, but insoluble in acetone, ether, benzene or chloroform It is more soluble in isotonic sodium chloride solution and ve-ry soluble in dilute elkali., splittin orf the D-ribityl sid chain (25) tbe resultant lecule becomes soluble in chloroform To incro so the solubility of ribofie.vin in water (for injection use) the U. s ? e.llows sue. preparations to co tain nicotinamide, uro or other suitable h es solubilizing agents., In neutral or acid ueo,m solution, ribonavin (30) sho s no rotation, but in alkalin solution it is str y I -rotatory. Riboflavin is amp ot ric inn ture, with an isoelectrie point at pH 6 (31)., The dissociation eonst ts ere: On acetylation (10}, a tetraacetato with a o.eltin point of 21;!' C is formed On 1rradi tion in a.1.kaline solution (:32), riboflavin yields lur.ui'lavin, Ci.3H~4~, d this being sparingly soluble in ter,

PAGE 24

13 separates fro the i adi ted solution. Irradiation of neutral or acid solutions of riboflavin (33) is attended 'With the form tion of 6,7-dimethyl-alloxazine or lumichrome which exhibits intense blue fluorescence. Goldblith and Proctor ( 34) showed that eloct-ro s or X-rays (3 roogavolts from a Trump generator),\ i ere used to irradiate solutions of pure ribofl vin i n et lie petri dishes, caused destruction o f this fluoreseent substance a ccording to nccepted methods of assay. The higher t he concentration of irradiated vitamin, the less was the percenta of destruction. The products of irradiated riboflavin were lumichrome plus fragments. Ellinger d (11) f ound vitami n B2 t o be reversibly reduc ed by sodium dithio nite solution by zin c in acid solution, by hy dro n sulfide in alkaline solution, by hydro n in the presence of e. ea.talyst, or by tits.nous chloride to a leuco-com.pound wich was reox idized to riboflavin and t he color and fluorescen ce restored on exposure to air By aintaining riboflavin (35)., bot synthetic and natm-al, in a redu ee d state \lit sodium hydrosulfite, it can be protected from sun light destruction. The reduced state c an be reoxidized by vigorous shaking th excess air. c., nclusions -were that duced controls shololed a 90 pe r cent destruction in a thirty minute exposure boflavin was said t o be rendered more stable to light by the presen ce of sodium dithionite (36) or by hea ting with boric acid (37). Solutions containing boric acid are recommended for injection and are

PAGE 25

14 said to be self-sterilizing 11 as photo -stable. Ribof l avin {25) has a acteristie absorptions ctrum, the po s of t.e nbsorption bands being 221, 266, 359, and 445 mu. Crystalline riboflavin is stable in the dark at ordinary tem per tures but decomposes on exposure to light. Vitamin~ (38) is reltivel_y heat-stable in acid solution; and the ~ate of destruction is rapidly incx-3ased with increasing alkalinity. In alkaline solutions it is unstable, especially wen these solutions are exposed to light. Ellinger and Holden (39) showed that at hif')l concentrations. of riboflavin in solution, the effect of ttquenching" comes into play It was considerably affected by certain anions, sue as halides, cyanide, thiocyanide, sulfite nd nitrite. Ferrous and ferric salts (en oxidation-reduction process) has o1milar "quenching" effect. Epley and Hall (40} experimentod with several of the F. D and C. colors and showed that riboflavin was unstable to F. D and C. green number .3. Iowever F. D and C red number 3 and F D and C orange number 1 seemed to protect Vitamin B2 from photochemical dest ction. Com.on cork, unless previous soaked in a large volume of water (41), contains a subst ce which strongly inhibits tl fluorescence of riboflavin. o app ciable destruction occurred wen milk was incubated by SUre and Ford (42) for twenty-two hours at 31 to 31 C. or during the c ooking of foods (4.3) 'When, on the othe r hand, milk in bottles was exposed to sunlight by Peterson 21 :1. (44), more than half t he riboflaVin w destroyed wit n two hours.

PAGE 26

15 Assay Dote nations The U s P (27) givos both a crobiological assay p rocedure and a flu oro photo etric thod for determining riboflavin. The fluoro photometric method is b ed on t he measure of fluorescence in acid solu tion. By comparing the concentration of an unknown solution with t hat of a prepared standard using a rl.u.orophotom ter that can accurately measure riboflavin activity in approximately 0 1 to 0 2 mog. pe r ml., tho amount present can accordingly be calculated. The microbiological method is carried o u t in acid media. It is also baaed on a comparison of an unknown with a standard solution uein a pure culture of lac;wpacillua casei Visual methods for the determination of nuorescence have been employed, but photoelectric t e chniques (45) have been almost univ really adopt ed in more recen t years. Ioy (46) de a study of the fluorometrio method and the microbiological method of assay o f riboflavin. H e foun d that there were no ~tatistical differences between t he results oft two methods -When an amber transparen t she.de of t he t ype c only used in department store display windows for filtering out ultraviolet rays was placed ovor laboratory windows, it was found to minimiz e the destruction of riboflavin (47) by ligh t rays during the courso of ass~. The use of added ar~ificial light resulted in approciable destruction of riboflavin. So sensitive is riboflavin to t he action of ligh t t hat riboflavin asseys should be carried out i n dim light and preferably in red

PAGE 27

16 ligh t De rre d Brom (48) recommended a 150-watt lamp screened with a red cellophane filter. The light from the lamp normally employed in a Coleman spectrophotometer, however, does not cause appre ciable destruction. Various standards hav been used for comparison 'With t he intonsity of fluorescence of tho unknown, for xemple, pure riboflavin (49), po tassium di o te (50), fluorescein (51) and uranium glass {52) have been used as such standards, Cohen (53) used Kleinmann nephelomater with light from a ercury lamp filtered through screen of nickel oxide for the determination of vitamin~ by means of its fluorescence. To prepare a solution for fluorescent analysis, eisberg and Ievin (50) reco!llllended the use of a clear solution. Various dilutions of this solution wer e made, up to 50 ml. in square 60 ml. bottles and compared under ultraviolet light with standards of sodium fluorescein. The standards were ms.de up in terms of 0 1 1 0 me. of riboflavin per ml. Riboflavin oan be det rmined in concentrations of 0 .00;-2. o mg. per liter as show by Kavanagh (54). He used a two photocell balanced circuit with a galvanometer as a null poin t indicator to measure the ratio of the fluorescence of t he unknown to that of a standard glass or quinine solution. With the use of proper filters, small amounts of suspended material did not interfere with t he experiment For determining riboflavin content, Hodson and Uorris 45) based their methods on the utilization of certain properties of ribo-

PAGE 28

flavin. 7 ing their work on fluoro atric thods their ciaims veret l. bofl .vi n fluoresced green we rs.di tod 'Wi a blue li t 2. It as not destroyed by mild oxidation or reduction. ,3. It could be redue d to a non-fluoreseing form m.th sodium hydrosulfite and reoxidized re dily by ohnldng with air. 4. !twas not reduced by st nous chloride. 5 The intensity of t l o fluo acence could be asurod with a photoolectric cell. In furthe r exp e r ntal wor, Kuhn and Moruzzi (31) took measurements 0 t h fluorescence o f ribone.vin olution o with graded pH ve.lues and showed that at a pH of 1 7 on the acid side and pH 10 2 on the alkaline side, the fluorescence bri~tn ss seeme d to be proportional to the riboflavin concentration Jones an d Christiansen (55) reported t hat riboflavin gave a maximum. fluorescence at a pH of 6 to 7 whereas arrer and Fritsche (56) ointed out that maximum fluorescence was exhibited by a 0 003 per cent solution of riboflavin at pH 7 0 Conner and Straub (57) shoved that a linear relationship between fiuoreseence an d concentration of riboflavin exists betw en the limits of 0 013 to 0.13 cg. per ml. Hanson and r iss (58) recomt!ended that in determining riboflavin concentrations, a standard olution co ntaining 50 mcg. par ml. (50 parts p e r llion) shoul d be used. Alt u th foun that it was difficult to get that much ribofl vin into perfect solution, they

PAGE 29

18 obs rved th t fluoresc c e uas proportional up to about .30 parts per million. In soi:ie ca.Ge e straight-line relationship between concen -tration and fluoros ce n oa would ssibly co.tinue up to 50 p ts per million. It was said to be se.f'or to work at a con eentratio-n not greater than 30 p ts per million. At the University of Witwatersrand in Joh ne -sburg, Alper (59) cl ed that substances whic h fiuor see exhibit fatigue when exposed con tinuously to the radiation which causes fluorescence A dilute aqueous solution of riboflavin gave straight line when the logarithm of the intensity of t he fluorescent light wns plotted against timo very trial ended, not vi.th an equilibrium sto.te but with rate of fading \Jhich could no lon r be a.a d This photofatigue was impor t tin the fluoro. etric assay o f substances lik :ribofl:vin. Slater and .:brell (60), -who worked vitb the Klett photoelectric fluo:rometer-colorimeter and the Klett photoelectric fluorometer gave detailed procedures for a..ldng tbiochrome .d riboflavin solutions. It was shown that many precise asurem nts of tho fluor-esoont intensity of solutions ero possible 'With fluorometor.

PAGE 30

19 Incre ing Solubility In viev of the lo solubility of riboflavin, numerous methods havo been suggested and many derivatives made for the preparation of solutions containing relatively high co ncentration of the vitamin. One or the earliest patents obtained to increase t h e solubility of riboflavin vas received by uhagen (61) in 1941. He olaimed to have gotten up to 0 .2; Gm. of riboflaVin in 100 ml. of a 10 per cent nico tin de or salts of nicotinie acid in ter. Frost (62) showed that riboflavin-nicotinami de solutions may be physiologically tabilized by adjuatinff t he pH value of the solutions to 2 6-6 6 end prefer bly from 4 4 6 6 In a later work, Frost (63) proved that t he solubility of ribofla in nicotinanide solutions de creases progressively at pH values more acid than 5 0 st e nico tinamid e concentration was inerensed from 5 to 5 0 per cent, the solu bility of riboflavin increased t pH 5 from about 0 1 to about 2 5 per cent. The observed strong solven t ef'fect of niacin on riboflavin appeared to be r elated to its chemical eonstitution 'With both C~4 end CONH2 groups being i n volved. An acid which for:ned an addition salt reduced t he solvent action of nicotinamide but did no t ellmnate it. Various details v ere tp..ven for the production of double salts of riboflavin, suc h as sodium riboflavin and an alkali metal borate by Auerbach (64) He claim d that the use of borax with an alkali vas said to give e complex:

PAGE 31

20 In 1942, Frost (37) showed that a riboflavin-boron solution of good stability, eontD.ining up to 0 3 per cont riboflavin can be ob tained by heating an aqueous solution of riboflaVin and up to 5 per cent boric acid t pH 6 5 for ~hree hours at 950 C With taboric acid leso heating vas re uired. The specific rotation of ribofiavin belo pH 6 wos ent~ ced in a sitive direction by boron but the sol-vent effects of boron con pounds wero l balo pH 6 0 and were inere s d nbove pH 6 o It was found that tho ribityl group was i n volved in the solvent reaction dth boric acid and that the effect was independent of t.he v ory insolublo isoallo:xa.zine group Any attempt to bonzoyl te riboflavin in the esenoo of borates gave no reaction. boflavin tetrabenzoate and riboflavin rnonoborate vere prepared Isotonic proparations of t he riboflavinboron comple vere self-sterilizing townrd molds and bacteria and were suitable for injection. ran and Stein (65} experimented with the sodium salt of ribof'l vin and polybasic carboxylic acid such. o.s phthalic or succ1nic acid or t eir a.nhydridos refluxed in pyridine. After the reaction vas completed the pyridine was evaporated, the residue dissolve d in water and acidified. Crystals that separated were recrystallized from boiling lt!ater. Th~so derivatives, particularly t h e succinate, were ahown to have increased the solubility in water v ery favorably as compar~d to that of pure riboflavin. Hoffe r (66) showed that in 180 ml. of a 2 per cent solution of a lower alkylolami.de of gentisic acid in water he was e.ble to got up to 0 .33 Gm. of riboflavin soluble,

PAGE 32

21 Hoffer in collaboration with Furter (67) sho1.1ed that in aqueous solution containing 5 p a r cent gcntisic acid and 5 per ce~t sodium gantisate, e.t pH 5 .0, ribo.fla.vin, up to 16 0:1. as soluble in each 100 ml Jurist {68) showed tha.t concentrate d ribofl rln nolutions were obtained by dissolving the vitamin in an aqueous solution 0 a phamacodyn cally unobjectionable aliphatic amidine ncid dition product, such as acatomidine hydrochloride. 20 per cant aqueous solution of acetru.udine hydrochloride w11l carry 1900-2000 mcg. per ml. Solutions did not deposit riboflavin Yhen chilled to 80 c~ d they were heatsterilized and sto d vithout changes in color o:r clarity,. By using live r extra.ct as a. solvent, having 250 to 350 :mg. per ml. of live r solids, Shelton ( 69) showod tat it as possible to get up to 0 2 Gm. of :riboflavin soluble por 10 0 ml. Bird and Kuna (70) demonstt-ated that riboflavin was readily brought into solution with gal.lie acid or its ................. $. salts. Ten milli liters of a 10 per ce n t solution of gal.lie acid in 50 pe-r cent aqueoua ethyl alcohol dissolved 14 mg. or ribo:f'l. nn. Sodi gal.late in a 10 per cent aqueous aolution at pli 6 7 dissolved 58 of r boflaVin in 10 ml at 24 5 C., Dry mixtures of the vitamin and the salts of gallic acid dissol~ed readily in water Riboflavin ws co nverted into a eQlttblo complex by treatment \Tith gal.lie acid in the presence of water and an inorganic acid by Zentner (71). Pr iswerk (72) reported a solubility of 4 Qui. of ;riboflavin per 100 ml. of an aqueous solution containing 25 per cent or more of a

PAGE 33

22 water soluble salt of 2,4-dihdroxybenzoio acid or its lower monoalkyl ethers. The ortho, meta and para compounds of the latter vere specified. Water -soluble salts of benzoic aoid and its amino or hydroxy substituted d rivat1ves w re used as solubiliziug a.gents in aqueous solutions by 6.ller (73). He showed that alkali benzoates (including sodium p-hydrox:,benzoate and sodium aminobenzoate), magnesium or sodium salicylata and monoetha.nolamine salicylate all helped to increase the solubility of riboflavin. Haas (74) claimed to ha\te gotten up to 8.0 G:n. p r ml. soluble as t h citra.t of diothyl noacetyl riboflavin. Upham (75) prepared stable, sterile and elear solutions of ribo flavin citrate in propylene glycol. Ho reported up to 40 mg. pe r ml as the poss1bl solubility. ~olutions containing additional substa.~ces in the same sol vent wre also gi vcn ?: os and Upham (76) prepared citric, malic a.nd tartaric acid estars of riboflavin by heating the acid and vitamin in phenol at 120 to l.$ C The esters var epa.rated by pouring the cooled mixture into ether. The ester were water soluble nd stable at pH 5 5 7 5 which were suitable for solutions.for parenteral administr tion. Knauf and Kirchmeyer (77) prepared solutions of riboflavin con taining 0 1 to 0 3 per cent using water and 1 to 4 per cent veratyl alcohol as the solubilizer. Solutions suitable for oral or parenteral administration and containing 0 .15 to 0 3 per cent of riboflavin were reported by Charney (78)io These solutions of ribo:flavin, alone or with other substances,

PAGE 34

23 ~ere prepared by using 1 p e r c e n t vanillin as t h e solubilizing agent in water or propylene glycol. Charney (79) also reported t h e solubility of riboflavin in 4 p e r cent aqueous L,.tyrosin e amid e a.t p H 5.0 a.."ld in 4 per cent aqueous L,..tyrosine amid e plus 10 p e r ce n t nicotinamide at pH 5 o The effeet of sodium chloride, glycerin, urea, boric acid and sodium salicyl ta as solubilizers and st bilizors for co ncentrated solutions of riboflavin uere studied b y Gupta and Gupta (80) Boric acid v s found to be the mot efficient stabiliz r but was painful when administered intramu cularly. Sodium sali 1la.te (5 pr cent) :tn a eon centra.tion ot 2 5 l!lg. of riboflavin r llU. 'WO.S foun d effectiw. Gerlough rud Smith ( 8 1} found t hat acetyltryptop han had a olubilizing action on riboflavin an d coul d be used in the preparation of solutions for parenteral administration, The solubility of riboflavin in aqueous media s increased by the addition of 20 parts of p yridylcarbinol (82) to 80 parts of ter. Schoen an d Gordon (8.3) wor d with ,1ater soluble metbylol derivatios of riboflavin. By reacting fo alde hyde with vitamin B 2 the monomethylol and dimethylol derivatives Y~re obtained. Such compounds ue-re found to be stable to potassium permanganate at room temperature but not at 500 C. When riboflavin w s fused with an amide, such as urea, urethe.n acetrunide or niacin, a product was obtained which yielded water solutions containin g up to 6 per cent ribofiavin. Steeber (84) showed that on acid salt, suoh as Nal:J2P04-H20 may be i n corporated in the melt or

PAGE 35

added to the d material. The com sition of the fus d product was not determined but it was smned to contain equi lecular amounts of riboflavin and amide Stone (85) made a water solub1 derivative of riboflavin by dissolving it first in concentrated sulfuric acid, neutralizing 'With calcium hydroxide d freeze-drying. This calcium salt of a sulfate ester of riboflavin in aqueous solut~on w s found to be table in air and 1000 t ea as soluble as U.S. P Riboflavin It was soluble in methyl alcohol, glycerol and propylene glycol and slightly soluble in ethyl alcohol. Analysi indicated an empirical formula: portion oft e sulfur was present as the sulf t. Fluoro tric ass9 gave riboflavin content of 57. 2 per cant. boflavin-51phosphate ester monosodium salt (86) prepared byphosphoryl tion of ribofi vin with chlorophosphoric acid. The solu bility in water was claimed to be more than 100 tim->s that of riboflavin. The empirical formu.l was reported as: It was fully aetiv biologic ly, mierobiolo cally and enzymatically However, the greater sensitivity of the phosphate ester to destruction by ultraviolet light necessitated eful protection of dilute solutions from exposure The monodiethanolamine salt., vi.th a water solubility of mor th 200 times that of riboflavin, was also prepared. This salt ..

PAGE 36

25 w a s slightly acid in ueous solutions. A method of solubilizing ribofla v.1.th sodium ,3-hydroxy 2 naphthoate was developed by Arnol d and Auerback !. (87) In the procedure the riboflavin itself was not treated. An aqueous solutio n with the naphthoate salt was prepared with t h e co ncentration being about doublet.at of the desired concentr tion of riboflavin. The vitamin was then added and after a little stirring it went i nto solution. An exa c t mechanism of the solubilization effect was not proposed but it was be lieved that so a sort of complex wao formed

PAGE 37

t erials Used The drugs and c h emicals used in this i n v stigation together with the sourc, grade d lot number are show in Table l. The manufacturers of thes terials are indicated by letters as follows: L Eli Lilly an d Co. M rek an d Co., I nc. W S WinthropStearns I nc. H -LR Hoffmann-Le. Roche, Inc M theson Co., Inc. G General Chemical Division S Sargent Chemical Co. E Eastern Chemical Oo. S Swift and Co. D Dow Chemical Co. F Fisher S cientific Co. K Koppers Co. I nc. ML Mallinckrodt Chemical 'orks SNY Smith N e w York C Carbide and Carbon C h emicals B r Chemical Co. A Atlas Powder Co. 26

PAGE 38

TABIE l DESCRIPTION OF DRUGS AND CHEMICMS USED terial R1bofla.'111n Flavaxin Soluble {Riboflavin Sodium Sodium Tetraborate) Ribofle.vin-5 P .... osph te Sodium Pyruvic Acid Sodium Hydroxide Barium Hydroxide Potassi Acid Phth ate Fluorescein Sodium Ethyl Ether Glycerin Prop1lene Glycol Ethyl Antlnobenzoate Quinine Bisulfate fleta,-Metllyl Umbelliferone 1:ieotinic Acid {Niacin) Oitraeonic Anhydride Liquefied Phenol Levulinic Acid {Liquid) Ethyl Alcohol Manufacturer L M 'W'-S H -1..R MA G SA E M M s D F M K ML SNY ML MA 0 Grade USP USP C P Reagent Reagent Reagent USP USP USP USP USP USP USP USP CP USP Lot Number Semple 42989 510123 5300 J089 24392 92353 50223 0638 800 1855550 13847 43587 C5P--2132 2697 Investigational 0024 2/JJ4

PAGE 39

TABIE 1--Continued teria.1 ut'aeturer Gr de wt Number Sodiuza Chloride M agent 41439 Potassium Chloride M Reagent 42'378 Sodiuni Acid Phosphate ML USP 7868 Potas"'iUill Acid Phosphate B USP 2251 raeotinamide M USP 50180 Urea B USP 1490005 P olyoxyethylene Sorbitan Monooleate (Tween 80) A USP

PAGE 40

Preparation of Riboflavin Derivatives fn:v,ie Aoid 1 roilinic Acid and ca.tmconie Anhrid9 ~rivativea 29 To 5 Gm. of' ribofla111n, pl ced in a 250-ml red glass flask, wero added 5 ml. of pyruvic acid; levulin1o acid or citraconic anhy dride (whichever was indicated), and 50 ml. of llqu0fied phenol. The flask \mS ,set in an oil bath ond refluxed fr,cxn four to six hours at a temperature ran o ran 100-110 C The reaction mixture was al.10\led to cool to room temperature and then poured, with constant stirring, into 500 ml, of etho:r in which the riboflavin derivativ precipitated, The color.ad, crystalline precipitate was sepM'e.ted from the ther mixture by filtration on a Buchner funnel, and the product was washed with several portions of ether and dried betveen sheets of filter pape r 'lbe dried derivativ was further purified by placing it in a mortar and tr1turating with a 100 ml ~ portion ot ether and filtering. This procedure was repeated three times after wicb the preparation was again dried between sheets of filter paper. It was dried in an oven at 60 c for our hours and then laced in the dark in a desiccator over phosphor-0us pentoxide A working yield of derivativ0 vas obtained by this method Ano er method of syntbetis, which did not prove as successful as that just mentioned, involved the refluxing of 2 Gm. of riboflavin and 8 ml. ot the organic acid or anhydride in edia. made up of 2 al. of' -concentrated s lf"uric cid 1n 30 ml. of distilled water This mix ture wa..s also refluxed in an oil bath at a temperature range from

PAGE 41

100 1100 C for four to six hours After the re etion mixture was cooled to room temperature, the sulfuric acid was carefully neutralized with a slurry of calcium oxide in water The precipitated calcium sulfate was remov; d by filtration and t he aqueous solution containing the derivative was evaporated to dryness in an oven at 600 C. This ethod was not generally employed in the preparation of soluble derivative due to the apparently l o w riboflavin yields and the difficulty encoun tered with the isolation. other ethod tried for isolating the riboflavin derivative from the re ct1on mixture rere vacuum distillation and ste distilla~ tion. Althou the oo und was 1sol ted with vacuum distillation, this proeedur found too time consumi ng Ste distill tion de -stroyed most of the vitamin

PAGE 42

31 Scope of th Fluorop otometer The inst ent used to determin the stability and solubility of ribofl :vin and some of its deriv tives s the lumetron photo electric fluoresc nee ter odel 402 The oper tion of this type of fluorophotom ter (88) is based o n the light of rcury vapor lamp which is condensed by an optical sy tem to form a parallel beam. Thia be is p soed through a narrowband filter which isolates t exciting li t of t he proper vave length. The exciting be is split into two parts. On~ part enters the sample holder whic 1s provid d wi thin front window of low ul tr :violet absorption. The fluoresc n ee or t he liquid is registered by two larga barrier-I er photocells 'Whi are rr gad late ally on bot sides of the s :mple hold r 1n the fluor seance pie up unit. lt rs between sample holder d photocells serve to isol t e t h specific fluorescence band d to elicdne.te e influ n c of pri ligbt mich be scat-tered by particles susp nded in t he liquid. The other or the beam is deflected by a fron t surface mirror snd acts upon th bal ce which is ounted so that it c turned through an angle of 9()0. Tho two uring photocells and tho balance photocell er connect din a brid circuit with a slide wire d vith a galvano tar as th zero indicator. The purpose of t he bridge circuit ia to el nate the influenc of light intensity variations of e mer vapor lamp The ge.lvano ter was st to t he zero mar in th center of th scale by means o.1' e galvanometer zero adju nt knob This etting w o check d from time to ti.J:le and r adjust d when n cessary. Ho ev r

PAGE 43

32 it vas not found necessary to have the 1i t pot alw s exactly on the zero mark of tho scale. The high sensitivity of the multiple re flection gal ano eter made it necessary to troat t e balancing operation in a manner differing sli tly from th balancing of circuits e ployed in less ensitiv galvo.no tors. A very co n veni nt electrical thod to acco1:1plish this is used in the instrument. Th middle position of the alvanom ter key switch (off position) is de not to disconnect the galvano ter, but to connect th alvano ter and the other parts of the circuit into a substitute circuit in which all interferences but no photocurrents regist r on the galvanometer The lumetro n is furnished with 10 b8k lite plat numbored from 1 through 10 Plat 1 has no aperture were s plates 2 through 10 are provided with apertur s ranging in diameter from 1./16 of an inch to 3/4 of an inch. These plates fit into a slot on the right of th filter bolder compartment a.Dd serv t e purpose of r ducing or blocking out the light on the balance hotocell or on the casuring photocell. The most suitable reduction plate is selected by trying out the various ap ertures starting with the larger apertures and proce ding to t h e smaller ones Finally, av ry small aperture is found 'Which no longer permits balancing even though the bal ce control is turned all the way counter clockwise Tho reason fort sis that \Ii.th this reduction plate the balance beam has been reduced so much that t h e bal ce cell, ev n 1f turned to face the bale.nee be squo.rely, can no lon r be.lance the cur rent of the measuring cells. The smallest a.p rture with wicb it is still possible to balance the circuit or t h e next larger aperture should

PAGE 44

33 be u d The selection of t e aperture h no err ct upon the nuor s ee n co ading obtained but only upon t he conv nionce in balancing with tho balance cell controls. In ord e r to asure, by fluorescence, the cone ntration of an ingredient in a solution, it i s Decessary to haves e solvent alone as well as of t h e ingredient alon. A known amount of the in dient is dissolved in the aolv nt and serves as t he standard tor which the instrument is balanced with the slide wire on 100 The sol ve n t alone s erv as the standard blank. If this blank show a reading o n the instrum en t (oi er du to inherent fluorescen c of t h e solvent or due to scattered pri light), t s r ading is suppres ed by means of zero suppressor knob so s to e the blank rad z ro on t he slid& wire dial. of e slide wire scale from O to 100, then cov rs t he r8llge of co n e ntr tion fro: zero to tho known concen tration of th tandard

PAGE 45

34 Standardization of the Lulnetron Pt9paration of Solutions To preps.re the luinetron f o r ste.bili t., tudies of riboflavin and some of its derivatives, it vas necessary to adjust t h e instrument in such a manner so t hat t here would be a linear relationship between oonoentr ation and nuore seence. Such a r elationship exists in concen trations up to 2 mg. per liter (54). The plottin g of calibration_ curve is not necessary in this range, since the aJ110unt of fluorescence ls directly proportional tote concentration. The prepe.ration of a standard stock solution of riboflavin was made by taking exactly 50 mg. of.' riboflavin, previously dried at 105 c for two hours an dissolving it in enough distilled water (acidified with l ml. of glacial cet1c acid per liter) to make 1000 ml. To prepare a solution dilute enough for fluorophotometric analyais, this olution had to be further diluted by taking a /J) ml. aliquot and diluting with a sufo1ent amount of distilled water to 1000 ml. The re-muting concentration of this standard solution was 2 mg. per liter or 2 mcg. per cl. Adjusting t e L'umet>;gn fgr Anal.ysia After setting the galvanometer to the zero ark, the primary and secondary filters war inserted into the lwnetron The sample holder, Wit h 25 ml-. of the ribofla'Vin standard solution containing 2 meg. per ml. was placed into t h e flu oreacen-e.a pick-up unit. Wit h the slide wire dial set e.t 100 and after t h e insertion of the proper reduction

PAGE 46

35 plate, the lumetron balance cell was adjusted. This gav a reading of 100 with a solution containing 2 mcg. per m.1. The slide wire dial was t hen set at zero d a blank solution (solvent only) was placed 1n the p 1ok-up unit, Here, t he lumetron was adjusted to give a r ading or zero by turning t e su pp r ssor knob counter-clockwise. solution containin g 1 m cg per ml. of nuoresc 11i sodium vas similm-ly prepared using distilled water. The fluorescence re6ding vas taken after adjUBtmen t of the lUD1etron liith th riboflavin solution and the blank Thia fluorescence :reading was used as a standard so t h t the instrument could be properly checked and adjusted each day before use After the lumetron was set for the inaximum and minimwn values, various dilutions were made of the standard riboflavin solution and fluorop hotometric readings were taken as described in Table 2.

PAGE 47

TABIE 2 LUMETRON READINGS OF VARIO U S DlLUTI O N S OF A STANDARD RIBOF VIi SOLUTION CONTA!NING 2 m./LITER Ml. of S tandard ? (I. o f Distille d Lumetron Riboflavi n Solution Watwr Added Mcg, /lO.., Readin g 0 .50 24.50 0 .04 1 5 1 .2; 23.75 0 1 4 7 2 .50 22.50 0 2 9 8 3 ,75 2 1 25 0 3 15 0 5 00 20.00 0 4 20. 5 6 .25 1 8 .75 0 5 2 5 3 7 .50 17,50 o 6 30 5 8 .75 16 ,25 o 7 35,5 10 00 15 00 o s 40, 5 11.25 11.75 0.9 45 .3 12 ,50 12 .50 1 0 50. l 1 3 ,75 11 .25 1 1 5 5 3 15 00 10 00 1 2 6o. 5 16,25 8 .75 1 3 65 3 17 ,50 7 ,50 1 4 7 0 2 1 8 ,75 6 .25 1 5 75, 2 20 00 ; .oo 1 6 80. 4 21,25 ,,75 1 7 8 4 8 :?2.50 2 .50 1 8 90.0 2.3.7$ 1 .25 1 9 9 5 0 25.-00 o .oo 2 0 100 0

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St bility Study thods S v.u;!:ions Used The solutions used as solv nts for t h e 1nvesti tion of the stability of ribofiavill and some of i t a deriv tives were as follow: Buffer Solution at pH 6 o Duffer Solution at p H 5 0 Buffer Solution at pH 4 0 25 Per Cent Glycerin in Distilled Water 50 Per Cent Glye rin i n Di&tilled Water 25 Per Cent Propyle11e Glycol in Distilled Water 50 Per Cent Propylene Glycpl in Distilled ater Saturated Solution of Ethy l Aminobenzoate ill Distilled Water o .Ol Per Cent nine Bi.sulfate in Diatilled water Saturated Solut1Gn of Beta.-,.!-ietht l Umbelll.ferone in Distilled Water 1 0 Per Cent Urea in Distilled Water 0 1 Per Cent Tween SO in Distilled Water o,.; P e r Cen t Nicotinic Acid in Distilled Water Buffer solutions from pH 4 to 6 were used in view of the lit erature .reports that this range we.s moat favorable to t h e stability of riboflavin solutionB Glycerin and propylene glycol in distilled water vere selected because these are widely used vehicles in pharmacy Aqueous solutions of ethyl e.min obenzoate, q uinirie bisulfate and beta-methyl umbelliferone vere used as solventa since it was thought that they might delq destruction of the vitamin in e presence of 1 gh t due to their

PAGE 49

s-un screening properties. th ur a and nicotinic aeid are used by so phe.rmaceuticn.l houses as solubilieera for riboflavin, snd it was thought those ght be effectiv for stabilizing solutions of the riborlavin derivatives prepared in this investi tion. Tween 80 ws selected becciuse it is commonly used in oral vitamin drops The distilled water us to up all solutions throughout this inve:rti at:1on vas la.bore.to distill$d ter. The pH varied from 6 l to 6 4 .. The solutions used in the pr pa.ration or buffer mixtures were prepared aecording tote Oler and Luba procedure (27) as follow: Bri Hydroxid, Tost Solution A saturated solution of barium hydroxide was prepared by adding an excess-amount to recently boiled distilled water, s ng thoroughly and then filtering~ The test solution was freshly prepared e tL~e it ws needed 012 t Sodiun Rydroxide Sclut.i.ou This solution s prepared by diaaolving 9 .00 am. of sodium hydroxide in 950 ml. of distilled wate r A freshly propared saturated solution or reagent barium eydroxide we added dro_p by drop until no more precipitate was formed Th mixture vas tho roughly sh en d allowed to stand overnight in a stoppered bottle. The next~. t he precipitate was filtered of and the resulting product gave oa.rbonate fre e solution of sodium hydroxide To standardize the product, 10 ml. of normal sulfuric acid w s diluted with 50 ml. 0 carbon dioxide free distilled vater and two drops or phenolphthalein T S vas added This solution vas titrated with the sodium hydroxide solution until a permanent pink color was produced The normality of

PAGE 50

'.1J the sodium hydroxide solution was calculated and adjusted to exactly 0 2 M vith freshly boiled and coole d distilled w ter. 092 M Potassium Biphthalate Solution Exactly ,40.843 Gm. of potassium biphthalate was dissolved in 900 ml. of diotilled wter and t hen sufficient distilled water added to make 100 0 ml. The molarity was t hen determined Blld adjusted to exactly 0 2 ) by titration with the prepared 0 2 M sodium hydroxide solution using phenolphthalein as the indicator, 012 M Monob@.sic Potassium .Pho@phate Solution. This preparation w.s made by dissolving exactly 27 218 Gm. of oonobaaie potassium phos phate in distilled Yater and diluting with sufficient distilled vater to make 1000 ml. The molarity was determined and adjusted by titrating against 0 2 r sodium hydroxide solution. Th buffer solution t pH 6 0 was prepared by adding 50 ml or 0 2 M monobasio potassium phosphate to 5 ,64 ml. of 0 2 M sodium hydrox ide solution a.nd diluting to 200 ml. with distilled vater The buffer solution at pH 5 0 we.a prepared by taking 23 .65 ml. of 0 2 M sodium hydr-onde and adding it to 50 ml. of potassium biphthal ate. This was diluted to 200 ml. with distilled water. The buffer solution at pH 4 0 was prepared by a dding 0 40 ml. or 0 2 M sodium hydroxide to 50 ml. of potassium biphthaJ.ate and diluting to 200 ml. with distilled water, Solutions of glycerin, propylene e l.ycol and polyoocyethylene aorbite.n monooleate (Twe en 80) in distilled water were prepared on a volume to Volume basis whereas solutio e of quinine bisulfate, urea Blld

PAGE 51

nieotinic acid in distill d ter wero prepar don a weight to vol basis Tb satur ted solution of ethyl a.minobonzoate in distill d wa~ ter was prepared by taking an amount ot thyl aminobenzoate that would nomall.y dissolv in the desired quantity of water and dissolvin it first in the smallest amount of ethyl alcohol. Thia alcoholic solution waa then added to the distilled water little at t e with thorough agitation. The preparation was allowed to tand overnight 1n a wll stoppered bottle and then filtered the next day. Th saturated solution of beta-methyl umbellife rone in diotilled wat r w.s prepared by adding 1 Gm. of beta-methyl U?llbelliferone to a liter of boiling stilled ter wit eonstant agitation, allowing it to cool to room temper tu.re o.nd s ttin g it aside overnight in a 11 stoppered bottle. Tho next day the needle-like cryetals ich precip., itated out of solution re removed by filtr tion. rocedre Stability studies w ere valuated for t h e following preparationss bofiavin Pyruvic Acid Derivative of Riboflavin L0vulin1e cid riv tiv of Riboflavin Flavaxin Soluble Riboflavin-51Phosph t Sodium Solutions of t h e abov were prepared in different concentrations in 250-ml. red volumetric flasks. Ea solution was kept in the dark

PAGE 52

41 ove ight in red colored and well stoppered bottles. An initial reading was taken just before the start of stor ge under various conditions of light. Flint and amber bottles, commonly employed in the stor g e of pharmaceutical products, were used as the containers for the solutions. Thre e sets of each of' the vitamin solutions were prepared for each sol vent. One set of solutions was kep t in direct sunlight by placing the co ntainere o n the flat roof of t he building. Another set was kept in the diffused light of the laboratory by placing the containers on a table, The thil7d s e t ws k~pt in total darkness by s tting t he bottles in clos$ d cardboard boxes in a labor a tory desk locker. Twenty-five milliliters were stored in each type of container, .Approxi tely 1 ml. of toluene, enough to produce a layeX' on t he top of t he solution, w s added to each container to prevent mold owth The lumetron va.s set to give a reading of 100 with a standard riboflavin solution containing 2 mcg. per ml. and a res.ding or zero 'With the part1culo.r solvent used A certain quantity oft e more con centrated solutions had to be diluted with enough distilled water to tall into the range of t he lum.etron. Alon g with the store. of t he solu tions co ntaining riboflavin or its derivatives, a set of blanks (the solve n t o ) was also stored under the s e conditions. Tho blanks also were stored in amber and flint bottles with a layer of toluene on t he top-The use of blanks was deemed nec essary especially vi.th so1vants of 0 01 por ce n t quinine bisulfate in distilled water and with a saturated solution of beta..-metbyl

PAGE 53

42 umbollifcrone in distilled wtor which nom ly h ve their ow inher nt fl~oroacence Accordingly, before a fluorophotometric reading was de termined, the lumetron 'WaS adjusted tor o.d zero with the insertion ot t e blank. The vitamin content of all solutions in bo flint and her bottles ws evaluated fluorophotom trie ly at t h e end of the following time interval: one day, three daya, fiv days, s ven days, ten days, fifteen days twanty days, thirty day and sixty days boflavin s used as a control in the stability study. Both Flavexin Soluble and riboflavin-5' hosph te sodium were selected be-cause they reco z d as fairly soluble ribofiavin deriv tives and are available on e market These wero compared for stability with the riboflavin derivatives prepared in this investi tion.

PAGE 54

43 Solubility Study thods Solvents Us~d The solvents and solutions used tor t h e investigation of the solubility of riboflaVin ands e of its derivatives were follows& Distilled ter Glycerin P ropylene Glycol 0 9 Per Cent Sodium Chloride in Distilled Water 0 9 Per Cent Potassilllll Chloride in Distilled Water 1 0 Per Cent Sodium Acid Phosphat& in Distilled Water 1 0 Per Cent Potassium Acid Phospbo.te. in Distilled ater Ethyl Alcohol l O Per Cent Niacinamide in Distilled Water 1 0 Per Cent Urea in Distilled Water The abov e p rcentage solutions w ere prep~ed on a weight to volume be.sis. A sufficient quantity of salt was vei ed out., dissolved in a pc>rtion of distilled w.ter and then made up to volume with more distilled water ptocedure The method used tor deter1nin1n g the solubility of riboflavin and some of its derivatives consisted o-f preparing saturated solutions 1n the solvents and solutions listed, Saturation was attain ed by placing an excess amount or riboflavin or a derivative t hereof i n the solvent,

PAGE 55

44 heating to 600 c for soverlll minutes with constant agitation and then coolin g to room temperature. Care s en to avoid any unneces.aaey e~sure to light in this proeedure. After cooling, the preparations t-r.3N well stopnered and stored overni t in total darkness. 'nle next daw, the ex cess unt of crystals was removed by centrifuging. One llilitor or the solution 'W'S$ oaretully pipetted into 950 ml. of distilled water and then further diluted to 1000 ml. with more distilled wter. A 2$-ml. aliquot of this solution ws used to obtain a fluorophot1':netric r,aading ith the ore soluble deriv tives, it lJtlS found necessary to further dilute 1 ml. of the above solutions to 250 ml. in a red volumetric flask. Thia s sometimes found nece ao.ry becauso the lumetron was adjust d to giv a r ading of 100 tdth the standard riboflavin solution end e. reeding of zero with a distilled mter blank. The J>t:'SPBration of the standard riboflavin solution ws the same as that described under assay procedure Solubilitieo wre determined for en.eh of the followinga Riboflavin Pyruvic Acid Derivative of Riboflavin I.ewlinic Acid Derivative or Ribofiavin Citraeonic Anhydride Derivative 0 Riboflavin Flavaxin Soluble Ribofla'rln 5'Phosphate Sodium

PAGE 56

45 Assq thod of Riboflavin Derivatives The det rmi.nation of t h a.mount of riboflavin quivalent to a specific quantity or deriv. tive was determined fluorophoto trically. A tands.rd riboflavin stock solution ra.s first prepared Riboflavin, U s P was dried at 1050 C for two ho s o.nd stored in the dark in a desiccator ov r phosphorous pentoxide. Exactly 50 mg. vere oo.rotully weighed and diosolved in distilled wnter to e one liter. This solution was stored in a red glass bottle under toluene and placed in a refrigerator which waa set t Soc. Each ml. represented 50 m cg of U S P Riboflavin A standard riboflavin solution was prepared from t he above stock solution by placing 10 ml. of t he above preparation in a red glass vol umetric flask and diluting to 250 ml. with distilled water. E aoh ml represented 2 mog. of riboflavin. A 25-ml aliquot of this solution 1 was placed in a asmpl co ntain r and t he l umetron adjusted to read 100 vith this concentr tion. The s amount of distilled water was used as the blank and e zero suppressor knob was adjusted to read zero ccordingly, t e eparation of solutions of riboflavin derivatives w re made in a similar manner as t hat descri d for riboflavin solutions. The resulting concentrations were also 2 mcg. per ml. After adjusting the lu tron with the standard riboflavin solution and the blank a 25-ml aliquot of the derivative solution was placed in sam ple container. The percentage of riboflavin was determined directly by the amount of fluorescence registered on the lumotron

PAGE 57

manner: The following derivative s of riboflavin were assayed in this Pyruvic Acid Derivative Citraeonic Anhydride Derivative Lovulinic Acid Derivative Flavaxin Soluble Ribotlavin-5'-Phosphate Sodium 46

PAGE 58

47 Results, w1 th Ribollavin Stability studies were evaluated for riboflavin in various sol~ents and the results were used as a control for the derivatives syn tbee1aed in this investigation. Fifty milligrams o riboflavin were added to a liter of each of the solvents listed under solubility studies~ Since the llll!letron was set for determinations up to 2 mcg. per ml_., the ribofiavin solu tions were diluted by adding 1 ml. of the vitami. n solutions to 24 ml. 0 distilled water. An ini ti.al lumetron reading was determined before the onset of storage Ribofiavin was used as the basis for' assay of the other deriv atives studied in this investigation. The solubility of riboflavin in various solvents vas determined and the mn.ount. of riboflavin present in ea.ch ml. of saturated solution was evaluated fluorophotometrieally.

PAGE 59

TABIE .3 THE STABlLITY OF RIBOFLAVIN IN DISTI i D TER STORED UND VARIOUS C011)ITIO SIN FL T Al D AMBER BOTTLES Slmlight Diffut!ed IJ.abt Dergess Lumetron Lumetron Lumetron atling J:tcg,/Ml. ReJeding. 11cg,/m., Reeding Mcg./Ml, Lu.matron Re ding of Freshly Prepared Sample: 95. 4 o~ 1 9()8 mog./ml. One Day Am: r 67. 9 1.358 92. 0 1 .840 95. 4 1 .908 Flint 2 5 o .o;o 46. 5 0 ,930 Three Days Amber 43. 0 0 .86o 90. 5 1 810 95. 4 1 .908 Flint 2 0 0 040 IJ) O o .soo Five Days Amber 37. l 0.742 89, 8 1 .79 6 95. 4 1 908 Flint 1 8 0 036 23 0 0 .460 Seven Deys Amber 19.-2 0 .384 as. 4 1 .768 95 4 1 .908 Flint 0 .. 7 0 .014 10 5 0.210 Ten Days Amber 14 8 0 .296 87. 8 1 .756 95. 4 1.908 Flint 0.1 0 002 4 5 0 090 Fifteen Days Amber 9 8 0 .196 86. 5 1 .7'30 95. 0 1 .900 Flint o o 0 000 2 2 0 044 Twenty Dey-s A:m:ber 3 5 0 .090 76 2 1~524 95 0 1.900 Flint ., ... 1 1 0 022 Thirty Days Amber 2 0 0 040 71. 5 1 .430 94. 5 1 890 Flint o s 0 016 Sixty Days Amber o o 0 000 54 .3 l.086 93. 4 1 868

PAGE 60

49 TAB 4 THE STABILITY OF OOFLAVI. II DISTIL V'i'.D WA R D AT pH 6 STO :n UN VARIOUS CO IDITI011S IN FLINT AND A1=..:u, OOTTIES DiffusJjld Light Imnetron Sunll$\ Lumetron Rending g ./1-11. Reading . g ./JA. Darkness Lumetron Rea.ding Meg,/ ll, Lumetron eading of Freshly Prepared SSlll:Ple: 91. 5 or 1 830 mcg./ml. On DB Amber Flint Three Days Amber Flint Five Days Amber Flint Seven Days Amber Flint Ten Dqs Amber Flint Fifteen Days Amber FllDt Twenty Days Amber Flint Thirty Days Amber Flint Sixty Days .Amber 66. 1 2 JS. o o a 19 1 0 2 12 9 o o 0 5 .. o o 1 .064 0 028 o ,760 0,.016 0 382 0 .004 0 .258 0 000 0 152 0 028 0 010 0,,000 87 7 8 9 85 0 2 1 8 4 2 o 6 75. 4 0 3 68 2 o o 54. 5 1 820 0 .848 1.802 o ... 778 1 .784 0 .442 1.754 o .178 1 .700 0 ,01.2 1 .684 0 012 1 .508 0 006 1 ,364 0 .000 91. 5 1 .830 91 5 1 830 91 5 1 830 91 5 1 .830 91 3 1 826 91. 0 1 820 90 8 1 816 l 808 89. 2 1 .784

PAGE 61

50 TABIE 5 THE STABILITY OF RIBOFLAVIN IN DISTILLED WATER BUFFERF.D AT pH 5 STORED UNDER VARIOUS CO ITION S IN FL AND R BOTTI.ES SUnllght Diff'useg ~eh! Darkp~S Lumetron Lumetron Lumetron I I B&adil)g l gs:.Lm.. HQag~s li2i 1LW.11 Rea
PAGE 62

51 TABIE 6 -THE STABILITY OF RIBOFLAV l n DISTILLED WATER BUFFERBD AT pH 4 STO D UNDER VARIOUS CO DITIO:S IN FLINT AND AMBER ~TTLES Splip.bt Dit~a~;Ufh i Darlgl~ss tumetron hmietron Lumetron Reading Mcg./MJ.. Readine; Ncg,/Mt, Reading .cg,hg, Lumetron Reading of Fr~shly Prepared Sample: 93+ 3 or 1 .866 ocg. /ml One Dau Ambei-68. 2 l .364 93. 3 1 866 9.3. 3 1 .866 Flint 2 0 0 040 43. 1 0 .862 Three ~s Amber 55. 4 1 .108 92. 6 l 852 93. 3 1 .866 fl.int 1 5 0 .030 36. o 0 120 Five na,s Amber 40,.1 0 002 91.8. l.836 93. 3 1 .866 Flint l O 0 020 24.2 0 .484 Seven ~s Amber 20 0 0 .400 90. 9 1.818 9 3 J 1"4866 Flint 0 2 0 .004 9. 2 o .1s2 Ten Days Amber 15 2 0 .)30.4 90,l 1 .$02 93. 3 1 .866 Flin t o o 0 000 2 0 044 Fifteen Days Amber 10 9 0 21s 89. 4 1 .788 92. 9 1.858 Flint .... .. -. 1.4 0,-028 Twenty D~s Amber 2.. 5 0 050 ?9 8 J...596 9~ .. 5 1 ~850 Flint .. .. .. - o.s 0 010 Thirty Days Amber 1.1 0 .022 75. 0 1 .500 92 0 1 .840 Flint 0 0 .000 Sixty Daus Amber o o 0 000 60. 1 1 .202 91:-.3 1 826

PAGE 63

TABIE 7 THE STABILITY OF OOFLAVIN IN 25 PE CENT GI3:CERI Ill DISTILLED t -TBR STORED UND'1R VARIOUS CONDITIO s Ill FL!NT A ID ., BOTTLES Sunlight Dif' ~od tiat Lumotron t;umetron ad!Dfl kcs, h ed C. cg,/.fl.., Lumetron a.ding of shly Prepa.rad Samples 98 4 0 or 1 .96o meg. /ml Ona~ Junbor 70. 5 1 4].0 98. 0 l 960 90 0 l .96o Flint 3 0 0.-06o 46. 5 0 .930 Three :O~s Amber 50. 4 1 .008 9 s 1.890 98. o 1 ,960 Flint 1.-0 0 4 020 40. 1 0 802 Five l)qs 1 ,860 98, 0 l .960 :ber 41. 2 0 .824 93. 0 Flint o s 0 016 29, 0 o .560 Seven Dey-s her 2s. o 0 .560 00. 9 l.778 98. 0 1 .960 Flint 0 3 0 006 18. 2 0 ~ 364 Ten btws Amber 12. s 0 256 88.:, 1 .766 9e. o 1 .960 Flint o o 0 000 9 ,2: 0 184 Fifteen Days Amber 7 4 0 ,148 87. 3 l 7/1) 98 0 1 .960 Flint 5 0 0 100 Twenty Dey-s Amber 4 6 0 092 77 J 1 .542 97. 7 1 9;4 Flint o o .-060 Thirty D~s r ::;. o o .060 71. 5 l .430 97 2 1 944 Flint . . ...... 1 4 0 028 Sixty Days Amber o o 0 000 60. 2 1.204 96, 0 1.920

PAGE 64

53 TABIE 8 THE STABILITY OF RIBJFLAVI IN 50 R CENT GIXCE, DISTIUE D WATER STORED tn D ,R VARIOUS CO ITION S I FLIN T MID AMBF.R OOTTIES Sunlight ~,[fued L!P-ht Darkness Lumetron Lumetron Inmetron Reading g,L n Reading fcg,LM!s ~adins; iSct:.Lm .. Lumetron Reading of Freshly Prepared Sample: 92. 0 or l .840 m c g ./ml. One Dey Ambor 80. 5 1 .610 91. 0 1 .820 92. 0 1 .8,40 Flint 2 5 0 .050 53. 5 1 070 Three Days Amber 59. 0 1 180 90 2 1 804 92. 0 1 .84() Flin t 1 0 0 .020 46. 0 0 920 Five Days Am r 49. 6 0 992 89 1 1 .782 92.0 1 s40 Flint 0 7 0 140 35. 2 0 704 Seven Dt:cy"s Amber 38. 4 0 768 88. 3 1.766 92. 0 1 .840 Flint 0 1 0 002 24 2 0 484 Ten Deys Amber 34. 6 o .692 86.1 1 .722 92. 0 1 .840 Flint o o 0 000 13. 2 0 .264 Fifteen Days Amber 29. 8 0 .596 84. 5 1 690 91 8 1 .8.36 Flint ...... 9 4 0 188 Twenty Deys Amber 21. 5 0 .430 76. 1 1 .522 91. 6 1 .832 Flint 7 4 0 148 Thirty Days Amber 15. 2 0 .304 72. 3 1 .446 91 3 1 826 Flint 5 0 0 100 Sixty Deys Amber o o 0 .000 59. 8 1 196 91 0 1 .820

PAGE 65

54 T DIE 9 THE STABILITY OF RIBOFLAVIN IN 25 PER N T PROPYIE m GU'COL IN DISTILIBD WATER STORED DER VARIOUS c m ITIOUS I NF T AND AMBER BOTTLES Sunlight Diftl!aed Liftht Darkness Lumetron Lumetron Lumetron ~a.ding cg1LM1, Reading Mcg1.lm. Reading Mcg.LM1.1 Lumetron Reading of Freshly Prepared Sample, 94 8 or 1 .896 mcg. /ml One Day Amber 63. 5 1 .270 94. 0 1 .tso 94 8 1 .896 Flint 1 5 o .o;o 42. 1 0 .842 three Days Amber 42. 8 o .856 92 0 1 840 94 8 1 896 Flint 0118 0 .016 33. 5 o .. 670 Five Days Amber r,.-6 0 752 90. 6 1 .812 94. 8 l .896 Flint 0 1 0 002 24. 5 0 .490 Seven Days 1 .896 Amber 23. 1 o .462 89. 2 1 784 94 8 Flint o o 0 000 16 l 0 322 Ten Deys Amber 9 4 o 188 ss. 1 1.762 94. 8 1 896 Flint 1.2. 0 0 2/IJ Fifteen Days Amber 5 8 0 116 s s o 1.760 94. 4 l .888 Flint ... 6,.,3 0 126 Twenty Days Amber 4 1 0 082 so.a 1 ,604 94. 0 1 8/J) Flint 5 2 0 104 Thirty Daye Amber 2 0 0 040 77. 4 1,548 93. 7 l 874 Flint .. .... 3 8 0.076 Sixty Days Amber o o 0 .. 000 56 8 1 136 93,.0 1 .860

PAGE 66

55 TAB?E 10 Tm!! STABILITY OF RIBOFLAVDl I 50 PER CE T PROPYLENE GLYCOL D DISTIL! D WA R STORED UND"' VAHIOUS COIIDITIOl S J 1$ F T A AMBE R BOTTtES D;ftused Wzgh~ barkness Lumetron IAk"n8tron Sunligbt Lumetron Ree.ding Mcg,./Ml.. Rea.ding Mcg. /m Rea.din 4cg,/Ml.. I.umetron Reading of Freshly Prepared Sample: 89. 5 or 1 .790 mcg. /ml One D~ Amber Flixrt Three 0& ber Flint Five Days Amber Flint Seven D~s Amber F lint Ten Daya Amber Flint Fifteen Days Amber Flint Twe:nty Deys Amber Flint Thirty Days Amber Flint ... Sixty Days Amber 19 0 12 1 o o 1 .246 0 022 0 .978 o .006 0 .516 O j.00 0 .000 88 5 49-.0 86.,1 41. 2 1 .. ??o 0 980 l .722 o .s24 1 .680 0 .598 1 .576 0 080 1 .536 0 060 1 .790 1.790 l ?90 l.'790 89. 1 1 .782 1 .780 88.7 1.774 1 .7&J

PAGE 67

TABLE 11 TUE STABILITY OF RIBO uvn I N A SATURAT D SOLUTION OF ~THYL AMINOB iZOATE n DISTILLED WATER STO D mm R VARIOUS CONDITIO?t S I FL AND A R BOTTLES ~ll~ D1!:f,eS,l ~SJ~ Darlales Lumetron Lumetrcm Lumetron Reading t0A, Jmading Mcg,/Ml, n&a41ng. Mcg./Ml.1 Lumet.ron Reading of Freshly Prepare d Sample: 90 0 or 1 .800 mcg./ml. Ono Dey Amber 75.5 1 .510 90.0 1 .800 90. 0 1.800 Flint 4 0 0.080 67.o 1 .340 Tbre& Dqa Amber 45 .. 0 O t 900 8 8 .. l 1.762 90. 0 1.800 Flint 2 s 0 .056 61. 5 1 230 Five Days Atiiber 34. 1 0.,682 87. ; l.-750 90. 0 1,800 runt 0.7 0 014 ;4 8 1 096 Seven Dl;ifs Amber 28 2 0 .564 87. 2 1 .744 90 0 1.800 Flint 0 2 0.004 40. 6 0 812 Ten !)eye Amber 23. 7 0 .474 87 .0: 1 7/IJ 90 0 1.800 Flint o o 0 000 40, l 0 802 Fifteen Days Amber 12 1 0 .242 84 6 1 .692 90. 0 l .800 Flint .... 20 3 o .4()6 Twenty Beys Amber 6 4 0 128 80 5 1 .610 90. 0 1,800 Flint 7 3 o ~l.46 Thirty Deys Amber o.s 0 010 77. 2 1 ,544 89, 5 1 .790 Flint ....... 2 5 o .o;o Sixty Days Amber o .. o 0 000 69. 2 1.384 88. 5 1 .770

PAGE 68

57 TABIE 12 THE STABILITY O F RIBOFLAVU IN 0 .01 PER CENT QU INE BISULFATE IN DISTILLED WATER STORED UND~"lt VARIOUS CONDITIONS n FL.11-iT AND AMBER BOTTLES Sunlight .. Dif:O:!sed I,4gbt De;:knesf Lumetron brinetron Lumetron Reading Mcs:1LMJ... Rsadins Mes-Lm Reading Mcg1LID.1 Lumetron !leadin g of Freshly Prepare d Semple: 90.0 or 1 .soo mcg, /ml One Dey Amber 68 o 1 .360 90 0 1 ,800 90, 0 1 800 Flint 17.1 0.:342 47,0 0,9/J) Three D~s Amber 6;. o 1.300 85,4 1 708 90,0 1 .800 Flint 10,1 0 .202 44 2 o .ss4 Five Days Amber 53,,6 1,.072 84,2 1*684 90 0 1 800 Flint l.~ 0 030 36, 3 0 .726 Seven Deys Amber 37.8 o .756 84 0 1 .680 90. 0 1;800 Flint 1.0 0 020 26. 8 0 ,536 Ten Days Ambe;r 33. 4 o .668 8J. O I .66o 90 0 1 800 Flint 0 3 o .006 10. 8 0 .216 Fifiieen Days Amber 30 2 0 .604 82 1 1,.642. 90. 0 1 800 Flint o o 0 000 3 1 0 062 Twenty Days Amber 21 0 0 420 81 4 l .628 89, 4 1 788 Flint ' ...... 1 5 0 030 Thirty D&Q'S Amber 10 .. 6 0 212 76 2 1 .524 89, 2 1 .784 Flint ~- 0 5 0 010 Sixty Days Amber o o 0 000 63. 4 1 .268 88 0 1 .. 760

PAGE 69

TAB1.E 13 Tim STABILITY OF RIBOFLAVIl I N A SATURATED SOLUTION OF BETAl TBYL UMBELL!FEROl D D I STILLED WA R STORED UNDER VARIOUS COUDITIO S D i FLINT AND AMBER BOTTI.ES Sunlight D-ii'f\Wed k!flht Darkness Lumotron Lumetron Lumetron flading k,c1 L n., Beading Mcg,bn.. ReS!D~ . Mcg./Ml, Lumetron Reading of Freshly Prepared Sample: 90 5 or 1 8 1 0 m c g /ml One Day ~r 69. o 1 ,380 90 5 L 810 90. ; 1 .810 Flint 2 2 0 044 67. 5 1 .350 Three Dey'S Amber 4.3.. 2 0 864 90 0 l .800 90. 5 1 810 Flint 1.2 0 024 63. 7 1 214 Five Days Amber 32. 1 o .642 87.1 1 .742 90. 5 1.8 1 0 Flint 08 0 ,016 54, 0 1 .080 Seven Ds.,ra Ainber 28. 5 0 .570 s; s 1 .716 90. 5 1 .s,.o Flint 0 1 0.002 43. 2 0 .864 Ten Do, Amber 14.1 0 282 84. 5 1 690 90. 5 1 810 Flint o o 0 000 22. 9 0 .458 F1riteen D~s Amber 6 1 0 122 82 i 0 1 .640 90. ; 1 .810 Flint 1 5 4 o .,oa Twenty Daye Amber 42 0.084 75 .. 0 1.;00 90 0 1.aoo Flint ,, . .... 4 2 0 004 Thirty D~s .Amber 0 2 0 040 71 0 1 .420 89, 8 1 ,796 F lint 0 1 0 002 Sixty Deys Amber o.o 0 000 63 1 1 .262 88 6 1 .772

PAGE 70

59 TABIE 14 THE STABILITY OF RIBOFLAVIU IN 1.0 PER CENT UREA I N DISTILLED WATER STOR D mm VARIOUS cm ITIO N S III FLI N T ID R BOTT~ Enlllrllt Diffl!f!ed List!t Darkness Lumetron Lumetron hlmetron Read;tng Mog,/W,, Reing -&,h I.KL, Readipg j fog. /!Q. tumotron Reading of Freshly Prepared Sample: 92 0 or 1 .840 mcg. /ml One Day Amber 64 5 1 .290 92 0 1 .840 92 0 1.840 Flin t 1.5 0.030 40. 1 0 .802 Three Dqs Amber 44. s o.896 90.,0 1 .800 92. 0 1 .840 Flint 0 8 0.016 32 l 0.642 Five Days Amber 33 0 o .660 s; 2 1 704 92 0 1 .840 Flint o o 0 .000 14~8 0.296 Seven Deye Amber 17 0 0 ,34D 79. 4 1 .588 92. 0 1.840 Flint o.o o~ooo 6.; 0,.130 Ten ~s Amber 4-5 0 090 75, 2 1.504 91 5 1 .830 Flint 0\ 2.1 0 042 Fif'teen Days Amber 2 4 0 048 69. s 1 .396 91 J 1 826 Flint .. .. 1 2 O 024 Twenty Days Amber o s 0 .016 62. 5 1 .250 91 0 1 820 Flint Ot8 0.016 Thirty Days Ambol0 1 0.002 53. 1 1 .062 91 0 1 .820 Flint II o.o 0.-000 Sixty Days Amber o o 0 000 42. s o .856 9 0 4 1 .808

PAGE 71

, 60 TAB!E 15 THE STABILITY OF RIBOFUV!N IN 0.1 PER OENT TWEEN 80 IN DISTILIED WATER STORED U N R VARIOUS comITIO s I H FLINT MID AMBER BOTTIES Soolight Dj;f!'.J!ge~ !ti{{ht P!,rkpel@ Lunletron tumetron :u.imetron Rafi41ng Mcg,/Ml-ReqdiQS Mpg,/MJ., Rea.ding fog./Ml, Lumetron Reading of Freshly Prepared Sample: 91. 0 or 1 .820 g ./ml. One Day Amber 62 8 1 .256 ss. o 1 .760 91. 0 1 .820 Flint 4.0 0 080 11). 5 o .s10 Three ~s Amber 41, 9 o .838 86. 1 l.722 91 0 1 820 Flint 2 8 0 .056 3 5 0 0 100 Five Day3 Amber 32. 0 o .640 85. 2 1 704 91 0 1 820 Flint 1 4 0 028 20. 1 0 402 Seven Dqs .Amber 1 5 8 0 .316 83 .. S 1 .676 91 0 1 .820 Flint o e 0 .016 8 5 0 170 Ten Dqs Amber 2 7 0 .054 82 5 1 .650 91,0 1 .820 Flint 0 4 0 008 4 1 0 ,082 Fifteen Dqs Amber 1 8 o .o.36 so. 2 1 .604 90.7 1 814 Flint o o 0 000 o s 0 .016 Twenty ~s Amber 0 4 0,.008 71. l 1 .422 90. 4 1 .808 Flint .. ... 0 3 0,.006 Thirty ~e Amber 0 1 0 002 65. 1 1 .302 89. 8 1 .796 Flint o o 0 000 Sixty Days o o 0 000 51. 7 1,.034 88. 9 1 .ns

PAGE 72

61 TABIE 16 THE STABILITY OF RIBOFLAVIN IN 0 5 PER CENT N !ACU IN DISTILIED WATER STORED UNDER VARIOUS COlIDITIONS IN FLINT D AMBER BOTTLES Sunlight Dif~eg Lieht Darkpess Lumetron Lumetron Lumet.r-on Regding, g,/Ml. Jmading Mcg,/Ml, Reagiyg !et;,/Ml, tumetron Reading of Freshly Prepared Samples 94. 2 or 1 .884 cg./ml. One Day Amber 68. 8 l .376 94. 0 1 .880 94. 2 1 884 Flint 1.0 0 .020 40. 0 o .soo Three ~s Amber 53.8 L.076 93. 1 1 .862 94,.2 1 884 Flint o 6 0 012 34 2 0.684 F ive Days Amber 39. 2 o .784 92. 4 1 .848 94. 2 1.884 Flin t 0 2 0 .004 22 0 o .1+1JJ Seven Days Amber 17 9 0 .3,8 91.l 1 .822 94.-2 1 .884 Flint o o 0.000 7,.2 0 .44 Ten Days Amber 11. 1 0 .262 89. 4 1 .788 94.2 1 .884 Flint 1 0 0 020 Fifteen Days Amber 8 9 0 .178 se. 2 1.764 93 9 1 878 Flint .. ..... 0 4 o~oos Twenty Days Amber 2 l 0 042 79 0 1.500 93. 4 1 .868 Flint o o 0 000 Thirty Days Amber o.s 0 016 74. 2 1 .484 9.3. 0 1 .860 Flint .. , Sixty Dqs Aln.ber o o 0 000 59. 2 1,184 92. l 1 842

PAGE 73

TABLE 17 SOWBILITY OF RIBOFLAVIN IN S01 A QUEOUS SOLUTION S Arm O m sot !, TS A v e rage ~ ./Ml. wmetron Rja.ding Distilled Water 7 2 0 14 0 .9% Sodium Ohloride 10 0 0 .20 0 .9% Potassium Chloride 9 , $ O .l9 1 o i Sodium Acid Phos phate 5 5 o u 1 0 % Potassium Acid Phosphate ; 5 o u 1 .0% Niaeinamide 20 0 0 .40 1 0 Urea 17. 0 0 .34 Pr opyl ene Glycol 26. 4 0 52 Glycerin ,42~0 o .84 Alcohol 2 2 0,.04 62

PAGE 74

63 esults with Riboflavin-5'-Pho ate Sodium Hof ... wc...u.&.1-La Roche's riboflavin-; '-phosphate sodium salt ws selected for study because of its unusually high solubility and its availability on the mark t. The 858 of this salt by the fluorophoto tric procedure showed a 70 0 per cent riboflavin content. Thu l or ribofiavin-51pbosphate sodium was quivalent to 0 7 of riboflavin. Five m1111 of ribofle.vin-51-phospb.ate sodium vere added to each ml. of e solve ts used for stability study. Since the lumetron s et for determinations up to 2 g por ml., each solution had to be furthe r diluted by adding 0 4 ml. to a sufficient quantity of distilled ter to 1000 ml. Twenty -five milliliters of this dilution wre used for f uorophoto tric analysis. The solubility of Hoffman-La Roche's product in so e solutions 8.Ild other solv nts we determined Due to its unusunlly high solubilit, furthe r dilutions with distilled water ere nee ssary for evaluation Yi.th the lumetron.

PAGE 75

64 TAB 1-8 THE STABILITY OF RIBOFIAVIN-51--PHOSPHA'l'E SODIUM IN DISTllJ'.ED WATER STORED mm VARIOUS OOlIDITIONS IN FLTh"T Alm AMBBR BO'l'TIES Di{fused LiBh:fs Dar tumetron Lumetron .cg, @ ,Readi:n ff Meg.ftp., Reading .feg, /;D, Lumetron ading of Freshly Prepared Sample: 70 0 or l .400 cg./J:l.1.. One Dey Amber 15 2 0 .304 66. 2 l .. 324 70 0 1 .400 Flint 1 0 0 020 33.,0 o .66o Three Days Arri r 3 5 C,.070 64 .. 1 1.282 70.0 1 400 Flint 0 1 0.002 16. l 0 .322 Five Days Amber 2 0 0 040 ~,.4 1 .208 70 0 1 .400 Flint o o 0 000 1..-. 8 0 .036 seven ays Amber 1,.5 0 030 59. 8 1 .196 70.,0 l .4DC) Flint ... 1 0 0 020 Ten Days Ambe:1 0 0 020 57_.8 1 1;6 70 0 l .lJ)O Flint ... ...... o 6 0 012 Fifteen Days Amber 0 4 o .oos 54. 5 1 090 6S. 7 1 .394 Flint 0 1 0 002 Twenty Daye Amber o o 0 000 52. s 1 .056 68. 5 1 .,370 Flint .... -o o 0.-000 Thirty Days Amber 49. 4 0 988 67. 7 1 .354 Flint .. .. ..... SixtyDqe Amber .... 38. 6 0 .772 65 .. 5 1 .310

PAGE 76

65 TABIE 19 S TABILITY OF RIBOFLAVIN 5'-PHOSPHA SOD I N DISTILL WA R UF'FE D AT pH 6 STORED UNDER V ARIOUS CO IDITION S IN FLINT AND R BOTTLES Sunlight Ditfustd I4all1( Darlgaess Lumetron Lumetron Lumetron Reading Mog~Lme Re ing Mqg./Ml. Ree.ding Meg.bg. Lumetron a.ding of Freshly Prepared Sampl.&: 72,5 or 1 .450 mcg./inl.. One Dey Amber Flin t Three D~ Amber Flin t Five Days Amber runt S even Dqe Amber Flint Ten Days Amber Flint Fifteen Dqs Amber Flint Twenty Daye Am'b&r Flint Thirty Days Amber Flint Su.-ty Days Amber 2 5 o o .. . 1 1 o 6 o o ". .. 0 3;0 0 036 0 096 0 012 0 050 0 000 0 .036 0 022 ..... 0 012 t 0 000 ...... 62, 1 2 8 5 4 0 o o 51. 6 1 .342 0 .682 1 196 0 030 l .l.48 0 020 1 120 o .oos 1 ,088 0 000 Ill o .806 12. ; 72. 5 1 .440 71. 6 70. 9 70 0 1 .400

PAGE 77

66 TAB 20 STABILITY OF RIBOFLAVIN51.,.PHOSPHATE SODIUM IM DISTILTfi'D WATER BUFFE DAT pH 5 STORED UNDER V OUS CO!IDITIONS IN FLINT AUD AMBER BOTTI.ES Sunlight ~y:rueed Yi~h~ Dm:isQe~H! Lur.ietron Lunetron wmetron 1'9ading Mcs,,Lm Rerul;inEI H2a./Ml &adinS MeiLm.. ~tron Reading of Freshly Prepared Samples 7 4 2 or 1.,484 mcg./ml One Day Amber 18. 7 0 374 73. 0 1 .460 74 2 1 ,482 Flint 2 0 0 .040 .35. 0 0 700 Three !>$ye Amber 5 0 0 100 71. 4 1 .. 428 74. 2 1 .482 Flint o s 0 .016 1s 5 O T/0 Five Days Amber 3 .,1 0 ,062 69. 5 1 .J,o 74 2 1 .. 482 Flint 0 2 0 004 3 3 o ,066 Seven Days Amb&r 1 5 0 .030 67. 6 1 .352 7 2 1 .482 Flint o o 0 000 2 3 0 .. 046 Ten Days Amber 1 0 0 020 63 .. 2 1 .264 7J. 8 1 .476 Flint + 1 8 0 036 Fifteen Days Amber 0 7 0 014 58. 5 1 .170 73. 3 l .466 F'llnt ;; ...... o 6 0 0 1 2 Twenty Days Ambe~ o o 0 000 55~ 5 1 .116 72~ 6 1 .452 Flint 0 2 0 .004 Thirty De3"s Amber .... ..... 53, 4 1 .068 72 0 1 .440 Flint . . ., ... o o 0 000 Sixty Di\YB Amber "-.... 42~ 5 0 850 70 ~ 1 1 ~402

PAGE 78

6 7 TABIE 2l THE STABILITY OF RIOOFLAVIN-5t ... pnQSPHA'l'E S ODIU!: I N DIS TILIED WATE BUFFERED AT pH 4 ST "'"'D UlIDER VARIOUS COIIDITIO! S n t FLI N T D AMB E R BOT'l'LES Snp]i!P~ 12!~~2A Id.t3bt Pe,rkpeas Lum.etron Lumtron .tumetron Reading Mcg.fM!.. ead1ns Mcg,/llle Readins MCif(Ml. Lumetron Reading of Freshly Prepared .Sampla1 7'J, 6 or l 472 mcg.-/ml One~ Aniber 18 0 0.360 72,.0 1 .440 73. 6 1-.472 Flint 1.8 0.,,036 42,5 0,850 Threo Days Amber 47 o_.094 70.4 1,408 73 6 1 /{12 Flint 1.0 0.-020 17 l 0 3.42 Fi"'8 Days ofo;6 Amber 2 8 69l5 l.-390 73 6 1 472 Flint 0 4 o .oos 5 6 0 112 Se'V'an Da;y's .Amber 1 3 0 026 68. 0 1 ~360 73~ 6 l 472 Flint o 0 000 3 .. s 0 .076 Ten Da;ts Amber 08 0 016 64 2 l 284 73 0 1 .46() Flint .. 1.:$ 0 030 Fifteen Days Amber 0 2 o .oo,~ 62.-2 1.244 72 4 1 .448 Flint 4 1,0 .Q 020 Twenty Days Amber 00 0 000 60 ; 1.210 71 9 l,438 Flint .... ,~ .. o .o 0 000 Thirty Daye Amber "" .. ;;.o l 100 71 5 1 4:30 Flint .. ,. -. Sixty Days 69 8 1,39g Amber ,. 440 o.aso

PAGE 79

6$ TAB!E 22 STABILITY OF RIBOFLAVIN .. ;1-PHOSPHA S ODIUM IN 25 PER CE GUCERIN Ill DISTILLED WATER STORED UNDER VARIOUS CONDITIONS IN FLIN T AMD AMBER BOTTLES SunlJ.P.!1t Diffused Lif?ht Darlmess LU!l'I:) tron Lumetron Lumetron Reading 1-k:g./Ml. Readin g Mcg. /Ml Reading Mcg. /Ml wmetron Reading of mshly .Prepared ~les 73.1 or 1__. 478 m.cg./ml. One Day Amber 2.3. 1 0 462 73 0 1 460 73. 9 1 .478 Flint 2 1 0 .042 57. 5 1 150 Three Days r 1 0 1 0 .202 7:?. 8 1 .456 73.'1 l .478 Flint 1 4 0 .028 40~7 0 814 Five Days Amber 2 1 0 042 12. 1 1 .442 73. 9 1 .478 Flint 0 1 0 014 26. 6 o .;32 Seven ~s Amber 1 s o .o.36 71 0 1 .420 73. 1 1 ~ 478 Flint 0 1 0 .002 18 .8 0 .376 Ten Dav& Amber 1 ; 0 .0.30 67. 0 1 .340 73 3 1 .476 Flint c o o .coc s o o .16o Fifteen Days Amb&r 0 9 0 .01g 6.3, $ 1 .270 72 8 1 .456 Flint ... 2 3 0 .026 Twenty Days AI!lber 0 3 o .006 60. 8 1 .216 72. 6 1 .452 Flint 0 9 0 .018 Thirty Days Amber c c c .ccc 54. 1 1 082 72, 4 1 .,448 Flint .,. o o c .occ Sixty Das Amber ii ... 44. 8 o .896 70 l 1 .402

PAGE 80

69 TABLE 23 THE STABILITY OF RIBOFLAVIN5'-PHOSPlIATE SODIUM IN 50 PER CENT GLYCERIN I DISTILLED WATER STORBD UNDER VARIOUS cru DITIONS IN FI;.INT AUD AMBER BOTT SJmUffht ~t~1aL1~ pe.r1cness Lumetron twnetrotl wmetron Reading hcg. fai~ Mcg./t-0.. Refl:ding Mcg. /l,n Lumetron Reeding of n-~ably PrepB.l'ed Sample: 69+ 3 or 1 .,386 mcg./ml. One Ds1 Amber 23. s 0 476 69. 0 1 ~380 69 3 1 )86 Flint 2 2 O .OM, 46. 1 0 922 Three Days Amb&r 16. 1 0 322 68.,.; 1 .366 69-.3 1.386 Flint 1 3 0 016 Z9, l 0 .. 582 F1.ve ~s Amber 8 9 0 11s 67 8 1 .356 69. 3 1.386 Flint o 6 0 012 20 o .u.2 Seven "1.s Amber 4 6 0 .-092 66. o 1 ,320 69. 3 l .386 Flint 0 2 0 .004 18 2 o .364, Ten Days Amber 3 0 o .060 62. l 1 ,2,42 69. 3 1 .386 Flint o o 0 000 12. 5 0 250 Fifteen Days Amber 21tl 0.042 61, l 1 ~22 68. 9 1 .378 Flint . .. 9 1 0.182 Twenty Days Amber 1,,1 0 022 59. 6 1 .192 68. S 1 .370 Flint ... 6 6 0 .132 Thirty Days Amber o o 0 000 56".3 1 126 67. 8 1 .356 Flint :3. 2 0 .064 Sixty na,s ber .. 48,.5 0 .970 66.1+ l .328

PAGE 81

70 TA.BIB 24 S TABILITY OF RIBOFLAVIN51-PHOSPHATE S O D ~ IN 25 PER T PROPY ,.. m GLYCOL IN D I STILLED WATE R STORED UNDER VARIOUS CO -IDITION S Ill F LI T AIID Al R BOTT SU!}light Dif"!)med ~gb~ ~ss Lumetron Lumetron Lumetron Reading Mei./Ml. I Reading McfitLMl Readins Mcg./m. mmetron Readin g of Freshzy Prepared Sample: 72. 0 or 1 .44{) mog. /ml One Day Amber 19 0 o .3ao 67. 7 1 .354 72 0 1 .440 Flint 1 9 0 .. 038 52. 3 1 .046 Three Dqs Amber 9 2 o 184 65. 7 1 .314 72 0 1 .440 Flint 1 0 0 020 28..,6 0.,572 Five Days Amber 2 0 0 040 64. 2 1 .284 ?2 0 1 .440 Flint o 6 0 012 14. 7 0.294 Seven Days Amber 1.,6 0 032 62 2 1 .244 72. 0 1 .440 Flint 0 2 0 004 10 s 0 .216 Te-n Amber 1 0 0 020 57. 9 1 .158 71. 6 1 .432 Flint o o 0 .000 4 3 0.,086 Fifteen Days Amber 0 .3 o .006 $6. 0 1 120 71. 2 1 .432 Flint 3 4 o .068 Twenty~ Amber o o 0 000 55. 3 1 .. 106 70 9 1 .4).8 lint 1 8 o .o,36 Thirty Days Amber 53. 8 1 076 70. 7 l11W.. Flint .... o o 0 000 Sixty Daus Amber ...... 44. 2 0 884 69. 8 1 .396

PAGE 82

7l TABLE 2; THE STABILITY OF RIBOFLAVIN51-PHOSPllATE SODIUM IN 50 PER CENT PROPYLENE GUCOL I t DISTILIED WATER T O D UNDER VARIOUS co. !TIO S FLINT AND .AMBER BOt'l'IES S3mUgnt Lnmetron m,weg. Wawt Lumetron J)&.rknesp Lumetron Reading {5& @ ~adins Meg @ Reading Meg./Ml, wmet~n Reading of Freshly Prepared Sample: 72. 4 or l .448 ncg ./ml. One Dt\y Amber 20 2 0 1./)4 72, 0 1 .440 72. 4 l .MS Flint 2 0 0 040 55. 0 1 100 Three Days Amber s o o .16o 71 S 1 .430 72. 4 1 .448 Flint Oi9 o .008 29 3 o .586 Five Daye Amber 1 6 0 032 69 0 1 .380 72. 4 1 .448 Flint 0 .3 o .006 16. 1 0 .322 Seven~ Amber 1 0 0 .020 67 .. 7 l .354 12. 4 1 .448 Flint o o 0 000 12. 1 0 242 'fen ~s Amber 0 9 0 ,018 65. 2 1 .304 n .o 1 ,440 Flint ,. Iii s o 0 100 Fifteen De.yo Amber 0 1 0 002 62. 4 1 .248 71. S 1 .436 Flint 4 4 o oss Twenty Dey Amber o o o~ooo 58, 5 1 170 11 6 1 .4:32 Flint .,._ ... .,. 2 5 0 .050 Thirty Dqs Amber 56,3 1 .126 71.,0 1 .420 Flint ll o.o 0.000 Sixty Days Amber 47 3 0 946 69.,4 1 .388

PAGE 83

72 'l'ADIE 26 STABILITY OF RI ~IAVIN-5 -PHOSPHATE SOD! IN A SATUR.i\ SOLUTION OF ETHYL OBE ZOATE N DISTI 'D WATER STORED UNDER VARIOUS CONDITIO rs I l FUNT AND AMBER OOTTI.ES i t Lumatron Darkness Lumetron Reading Mcg./Ml.. Meg~ / ~a.ding tumetron a.tling or Freshly Prepar d Sam:plos 7i. 2 or 1 .424 m cg./ml~ One D~ Am r Flint Three Deys her Flint Five Dn.ys Amber lint Seven Ds,s Amber Flint Ten D93a .Amber Flint Fifteen Deya Amber Flint Twenty Deys Amber Flint Thirty Deys Amber Flint Sixty Days Amber 27. 2 3 2 ll. l 1 8 3,.9 0 6 1 2 0 2 0 5 o o o o 0 .. 802 0 082 0 .544 0 .064 0 222 0 036 0 .. 128 0 020 0 078 0 .012 0 .. 0 1 0 0 000 0 000 .... 68 0 31.0 67,0 21.0 6;3. 5 5,.8 6().5 2 9 1 418 1 .. 024 1,.408 o 866 1 .310 0.246 1 270 0 116 1 .210 o.o;s 71. 2 70.8 70.0 7 1 .424 1 .. 424 1 416 1 .. 408 l 400 1 .390 1 374

PAGE 84

73 TABLE 27 THE STABILITY OF RIBOFLAVIN-5 ... pnosP HATE SODIID n 0 .01 PER CENT UI?t!N E BISULFATE I N DISTILIE D WATER STORED UND R VARIOUS CONDITIOU S I N FLINT AND AMBE R BOTTLES Jm1ight m:r~!~ ~ah~ Darkness Ull!letron tumetron Lume~n Reading Mcg,/Ml.. Reading Mcg./Ml. Reading Mcg.fl,n.. ; L -umetron Reading of Freshly Prepar d Sample 1 76, 2 or 1 .524 cg./ml,, One Day Amber 23, 4 o .468 71, l 1 .42:a 76. 2 1 .524 Flint 3 8 o .rn6 48. 8 o .976 Three Daya Alnber 19 2 0 .:394 70 l 1 .402 76 2 1 .524 'Flint 2 2 0.044 2;. 2 0 .504 Five Daye Amber 12 8 0 .256 69. 6 1 ,392 76. 2 1 .524 Flint 1 0 0 .020 16 2 0 .324 Se"ren. ~s Amber 10 6 0 212 68. 9 1 .378 76. o 1 .520 Flint o s 0 .010 14. 4 0.288 Ten Dtqs Amber s o 0 100 64 1 1 .282 75. 9 1 .518 Flint. o o 0 000 10 1 0 202 Fifteen ~a Amber 3 4 o.068 62. ; 1 .250 75 7 1 .514 Flint 8 0 o .16o Twenty ~s Amber 1 9 o .o.3s 61.,8 1 ,2% 75. 5 1 .510 Flint .... 6 8 0 .1.36 Thirty Days Amber 0 2 0 .004 58. 7 1 114 75. 2 1 .504 Flint 3 2 0 .064 Sixty Dqs Ar.tber o o 0 000 51. 5 1 .0.30 74. l l.482

PAGE 85

74 TABI.E 28 STABILITY OF RIBOFU 51-PHOSPHA SODIUM n A SATURA D SOLUTIO? OF T -fETI YL UMBELLIFERO I N DISTILI.ED WATER STORE D UNDER VARIOUS CO.LID!TIOJ IN FLIN T ADD AMBE R BOTTLES Sunlight Diffused MM Uarknees Lullletron Lumetron Lu!netron Reading Mog./Kl. Rea.din Mcg .. /Ml. Readin~ Mcfi./Ml Lumetron Reading of Freshly Prepared Sample; 69 7 or 1 .394 mcg. /ml One De.y Amber Flint Three Days Amber Flint Five Days Amber Flint Se'V'en Days Amber Flint Ten Days Amber Flint Fifteen De.ya Amber Flint Twenty Days Amber Flint Thil'ty Dqs Amber Flint Sixty Deys Alnber 1.5 o .. o o s 0 2 o o o .;s2 0 .064 0.-220 0 040 0.130 0 022 o ~ o .oos 0.0.30 0 000 0 .016 .. .. o .oo,~ 0 .. 000 .... 64. s 43. 8 63 0 20 0 47. 2 1 ,290 0 ~690 1 ,280 0 .504 1 260 O .IJ)O 1 J-40 0 040 1 .. 082 0 028 0 944 1.394 69. 7 1 .394 1 .394 69. 7 69. 5 68, 9 1 .378 68, 9 1,378 68. 5 67. 2

PAGE 86

?S TABLE 29 STABILITY OF RIBOFLA VD -5 -HOSP sonrmi m 1 0 R CENT UREA n DISTIIJ.ED i TER STORED U!~PER VARIOUS CONDITIO IN FLI A ID AMBER BOTTLES ll D .od mess Lumetr011 hunetron Lumetron adin J-Icg./!:llr &ding Met,t./m I I Reading Mcg. /Ml Lumetron a.ding of Freshly Prepared Sample: 70. S or l .416 m cg /ml One Day Amber 6 l 0 .122 68 8 l .376 70 8 1 416 Flint 1 1 0 022 Jl. 1 c .622 Three Days Amber 5 a 0 116 67 .e. l .356 70 8 1 416 lint o s 0 .016 11.0 0 220 ive D8.JS Amber 5 2 0 104 65. 6 1 .312 70. 8 1 .416 Flint 0 5 0 010 5 2 0 104 Seven Days Amber 4 8 0 096 64. 4 1 2se 70. 5 1 410 Flint o o 0 000 2 2 0 .044 Ten Days Amber ,;!lo o .o60 56. 1 1 122 70.,,l 1 402 Flint .... ...... 1 0 0.020 Fifteen Days her 1 6 0 .032 46, 8 o .936 69. 5 l .390 lint 0. 4 o .oos Twenty Days Amber o ; 0 010 .32. 1 011642 69. o 1 .380 Flint .,. o o 0 000 Thirty Days Amber o o 0 000 27 2 0 544 68. 6 1 .372 Flint -~, . .. Sixty Days Amber .... 15 1 o ~ 66. 2 1 324

PAGE 87

76 TABIE 30 THE STABILITY OF RIOOFLA vn 51-PJ OSPHATE SODIUM I' O l PER CE 'l'tlEEK 80 I.1 DISTILLED fl STORED UNDER VARIOUS COJDITIOl S FLi r rm AMBER TTLES i~ Diffused !J.~ Lumetron tron ad.i n/;! i!cg./Ml. Ree.dinI Mc!l./Ml. Mcg./MJ.. Imnetron a.ding of Freshly Prepared Sample: 75,0 or 1.500 cg./ml. One Dar Amber 19 2 0 .384 72. 8 1 .456 75. 0 1.500 Flint 1 .. 9 0 033 30. 9 0 .618 Three Days Amb z, .3. 9 0 078 71. 3 1 .:426 75. 0 1 .500 Flint o 6 0 .012 12 2 0 .244 Five Da;ys ber 2 7 0.054 68~0 1.36() 75~0 1.500 Flint o.o 0 000 l 8 0 .. 036 ven Days Amber 1 .. 5 0 .. 030 65.l 1 302 75. 0 1 .500 lint ... ~--5 0 .. 030 Ten Days Amber Oo9 0 .. 018 60.5 1,,210 74 8 1 .496 Flint . 01"3 o.006 Fifteen Days Amber 0 4 o .oos 57. 0 1 .140 74.2 1 .. 484 Flint ,.. ... ., o .. o 0,000 Twenty Days 1.468 Amber o.o 0 000 55,6 1 .. 112 73. 4 Flint .. .. ,, -... "' .. Thirty Days Amber ...... 52. 4 1.048 72. 5 L .450 Flint w ... ... ii." Sttty Daya Amber ... 41,5 o ,830 70 4 l,408

PAGE 88

TABIE .31 THE STABILITY OF RIBOFLAVIll51-PlIOSPHATE ,SODIUM I N 0 5 PER CEl~ T NIACIN IN DISTILLED WA R STORED UND.,....R VARIOUS CONDITIO S FUNT AND .AMB'3R BOTTI.ES Sunligb~ Di!:~S!d .Y.sbt Darkness Lumetron Lumetron Lumetron Reading Mcs;./Ml. Reading ,Mcg./Ml. Reading Mg;./Ml. tumetl"on Reading of Freshly Prepared Sample1 70. 6 or 1 .412 mcg./ml+ One Day Amber 15 0 0 ..300 70. 3 1 .~6 70 6 1 .412 Flint 2 0 0 040 42. 9 o .sss Three Days Amber 4 9 0 ()98 68. 6 1 .372 70. 6 1 .412 Flint 1 1 0 .022 1 4_.4 0 288 Five nays Amber 2 a 0 .056 66 7 1 .334 70. 6 1 .412 Flint 0 7 O Ollt7 o 0 .140 Seven Days Amber 1 9 0 .038 64.0 1 280 70 6 1 .412 Flint o o 0 000 2 ; 0 .050 Ten Days Alnber 0 8 0 .016 60. s 1 214 70 2 1 .404 Flint 1 5 0 030 Fifteen Days Amber 0-.l 0 002 58A l 1 ,162 69. 7 1 .394 Flint ,. .. 1 0 0 .020 Twenty ~s Amber o o 0 000 56. 4 1 128 69. 2 1 .384 Flint .. o o 0 000 Thirty Days Amber ,., . .,., 51. 8 1 .0.36 68. 7 l 374 Flint 4 ..... Sixty Days Amber .... 41. 0 0 820 66. 6 1 .332

PAGE 89

TABLE 32 T SOLUBILifi OF RIBOFLAV1N ... 5 t.,.PHOSPUATE SODIUM ItJ SO A OEOUS SOLUTIO l AND OTHER SOL TS Distilled Water 0 .9j Sodium Chloride 0 .9:i; Potassium Chloride 1 .0% Sodium Acid Phosphate 1 .0% Potassium Acid Phosphate 1 .0% iaeinamide 1 .0% Urea Propylene Glycol Glycerin Alcohol A~era Lumetron ~ ~eadint3 4 9 5 2 5 2 3 5 .3. 0 6 o ; 8 s o 12. 5 4 0 .. 35.71 37 .14 37.14 25 00 21.4.3 42.85 41.42 5 .71 a .93 o .08 78

PAGE 90

79 Results wit Fl :vaxin Soluble Winthrop teams avaxin Soluble or riboflavin sodium--sodium tetra.borate selected for study because of its solubility d its popularity on th ket. The ass of this preparation wa valu ted nuorophotometri cal.ly. It showd a 50. 0 per cent ribofl :vin content. ccordingly 0 5 Gm. of Flavexin Soluble as quivalont to l Gm. of riboflavin. One milligram of Flavaxin Soluble ,as added to each ml. of the solvents used for stability study~ Since tho lumetron was et for readings up to 2 cg per ml. each solution had to be further diluted by adding 3 4 to a sufficient quantity of distilled water to make 1000 ml. Twenty-five lliliter or thio dilution vere used for fluoro photom&tric analysis. The solub111 ty of nthro teams' roduot in so aqu ous solution and o r solvents was determined Further dilution 'With distilled water ws found nocessacy-with everal solvents to be able to s ccesstully dete n tho results on the l etron.

PAGE 91

80 TAB 33 STO BOTTI.ES t Darlmess Lumetron Lumetron Readi n g ./Ml. Lumetron Readi of Freshly Prep ed Sampler 82,.0 or 1 .6.40 eg /ml One Day Amber 34 5 o .690 82 0 1 .640 82 0 1 .640 Flint 2 7 0 054 45. 5 0 910 Thre Days Amber 14.0 o .280 ?9 8 1 .596 82. 0 1 .640 Flint 1111 0 .022 28 2 o .564 Five :ys Amber 2 0 0 .040 78. 3 1 .566 82 0 1 .640 Flint o ; 0 010 20. 6 0.1~ Seve n 8 her 1 5 0 030 77 5 1 .550 8 2 0 1 .640 Flin t o o 0 000 14 5 0 290 Ten Days :r 1 1 0 022 74.2 1 .,484 81.6 1 .632 Flint 8 9 0 178 Fifte ys bar o s 0 016 70 0 1 .400 81 0 1 620 Ji'llnt I 5 2 0 104 Twenty Days ber o o 0 .000 65. 3 1 .306 80 2 1 .604 Flint 2 ~ 0 048 Thirty Days Amber 61 9 1 .238 79. 4 1 .588 Flint . 1 2 0 024 Sixty D~s Amber 46. ~ 0 936 77. 2 1 .544

PAGE 92

81 TABIE 34 STABILI'fi OF FIAVAXIN SOLUBLE IN DISTILLED YA R BUFFERED AT pH 6 STO D U!DER VARIOUS CONDITIO S F rf AND AMBER BOTTLES SuaJ+mt D~~ed.W~ PJtkness Lumetron Lumetron wmetron Reading Mcg,/m. Reading Mc./Ml. Reading Meg./Ml. Lutnetr.on Reading of Freshly Prepared Samples 83. 9 or 1 .678 r1cg./ml. One Day' Amber 33. 8 o .676 SJ 9 1 .678 83. 9 1 .678 Flint 2 6 0 052 47. 6 0 .952 Three Deya Amber 15 2 0 302 81. 5 1 ,630 s3,.9 1 .678 Flint 1 3 0 .026 29. 0 o ;so Five Days Amber 2 2 0 044 80. 0 1 .600 83. 9 1 .678 Flint o 6 o.o 2 21. 2 0 .424 Seven Days Amber 1 6 0 032 78 .3 1 .566 83. 9 1 678 Flint o o 0 000 15 .3 o .306 Ton De.ya Amber 1 0 0 020 75 .. 5 1 .510 8.3. 9 1 .678 Flint 9 .. 4 0.188 Fifteen Days Aniber o 6 0 -012 72, 3 1 .446 82. 8 1 .656 Flint 5 8 o u6 Twenty Daws Amber o o 0 000 68. 5 l .370 82. 1 1 .642 Flint 2 8 0 056 Thirty Days Amber 6;3. 8 1 .276 81. 5 1 630 Flint II 1 5 0 .030 Sixty Dey-s Amber 489 0 .978 79. 0 1 .580

PAGE 93

82 TAB!E 35 R STABILiff OF FLI\VAXIN SOLUBIE IN D!STILIED WATER BUFFERED AT pH 5 STORED UNn VARIOUS CONDITiors IN FtIN'.t A 'D AJ,ffiir' BOTTLES unllgh Dit1)a~ed Y.mll Degae;; Lmnetron Lu.matron Lumetron ~a.dins cs-LMl Read1ns Mes. @ ~ Re~ns Meg., n lmtletron Rea.ding of Freshly Prepared Sample: 82 0 or 1 .640 c g /ml One Day Amber 36. 2 0 .732 a2'io l\!61+0 82. 0 1 .640 Flint 2 9 0 .058 47. 5 0 950 Three Daye Amber 16 3 0 .326 81. 4 1./ 628 s2. o 1 .640 Fl.int 1 5 0 030 28" 4 o .;68 Five Daye Amber 3 0 o .060 so. s l .616 82 0 1 ,640 Flint o s 0 .016 20 2 0 404 Seven Days 1 .640 Amber 2.,1 0 042 78. 9 1 .578 82 0 Flint o o 0 000 1; o 0 .300 Ten Days Amber 1 8 0 .0.36 76. o 1 ;20 82. 0 1 .640 Flint a 9 0 17s Fiftean Days Amber 0 9 0 .018 72 8 1 ,456 111. 2 1 .624 Flint 5 1 0 102 Twenty De.y,a Amber 0 2 0 .004 68. S 1 .376 80 7 1 .614 Flint 2 5 0 050 Thirty Days Atnbe:r o o 0 000 63,.; 1 ,270 79, 8 l 596 Flint 1 4 0 028 $1.ny Days Amber ~ 48. S o .976 77. 6 1 .552

PAGE 94

83 TABIE 36 M STABILI'IY OF FLAV s o wn IN DIS 'l'ILIED STORED UNDER VARIOUS CO!IDITIOl S IN FLU T D!,fued L!Sbt P!rkms-1 !; tron Lumetron 1.iur.!etron Readmg iii p Mcg. /Ml ; Rea.ding eg./11..-ad1ng Mc ./Ml. J Lumetron Readi ng of F reshly Pr e d Sam.pl r 84 5 or 1 .690 m e ./ml. One Day A.tlber 39. 6 0 792 84 5 1 .690 84 5 1 .690 Flint 3 .:; o .066 52. ; l .050 Three Days Amber 19 2 0 .384 8.3~ 4 1 .668 84 5 1 .690 Flin t 1 8 0 036 33-. 2 0 664 Five Days Amlr 3.,8 0 076 82 3 1 .646 84 5 1 .690 Flint 1 0 0 020 24114 0 .488 Seven Days Allber 1,8 0 036 8 1,.7 1 .634 8 4 5 1 .690 Flint o o 0 .000 17112 0 .344 Ten Dayo 1 ,68o Amber 1.0 0 020 so. 2 1 ~604 s 4 o Flint .. "~. 8:. 8 0 .176 Fifteen Days Amber o s 0 016 79. l 1 .582 8,3. 6 lij672 Flin t l> 5 3 0 .106 Twenty Dey-a ber o o 0 000 75. 3 1 .506 82 7 1 .654 Flin t ,. 2.. 6 0 052 Thirty ~s Amber .... 70. l 1 .402 8 2 l 1 .642 Flin t ... . 1 5 Oit0.30 S i xty Amber .. 70. l 1 402 8 2 1 1 .642

PAGE 95

84 TABI.E 'Y/ St BIL?TY OF FLAVAXlll SOLUBLE IN 25 R CENT GUCE 1 I?i DISTI WATER STORED mm VA..'fUOUS CONDI'i'IO S ni FL:ll AfID BOTTtES s 1 t !Jifty,Qg,g Liight Ekness L tron turl1etron Ltunetron Reading -~og./Ml. Reading Meg./)n. Reading fcg.,/ a Luwetron Reading of Freshly Prepared Sample: 84 4 or 1 .688 g ./m.1. One Day Amber 44 0 o .sso 84, 4 1 .688 84-4 1 .688 Flint 2 S 0 056 66. ; li,.330 Thre ~a Amber 29. $ 0 590 82. 5 1 .. 650 84. 4 .688 Flint 1 5 0 030 41. 5 o .830 ve Days .Amber 8 .,3 0 166 Sl. 5 l .63() 84. 4 1 688 Flint 0 9 0 .018 .30. 2 0 .604 Sov n Deys Ambel" 7 .0 0.140 78. 5 1,.570 84. 4 1 688 Pl.int o o 0 000 10. 4 o .. 368 Te Days Am ... 5 2 0 104 75. S 1,.516 83 9 l .678 .. Flint ... ., u s 0 .236 Fifteen De;ys lunbor 3 0 o .060 73 9 1 .578 83 .3 1 .666 Flint 7 2 0 144 T-t.renty Daye Amber 1 2 0 024 70 l 1 .402 82. 8 1 ,656 Flint .. 4 8 o 096 'l'hirty Dey Ariber 0 ~ 7 o .ou 6$ 0 1 .;00 82,.3 1 .6.46 Flint 2 2 0 044 Sixty Deya Amber o o 0 000 49. 2 0 984 80 0 1 ,600

PAGE 96

85 TADL'E 38 THE STABILITY OF FLAVAXIN SOLUBIE IN 50 CE?IT GLYCERIN IN DISTILLED WA R STORED UNl)R VARIOUS CONDITIONS '.IN FLIHT AND Al' R BOTTIBS SunlluJlt )21f'3!sed.Isttmi Dglmes a Lumetron Lumetron Lumetron Reading M c ,./Ml.. Reading cg./Ml. Read.in Mc;./M.1.. Lumetron Roe.ding of Freshly Prepared Samples 83 9 or 1 .678 mcg./ml. One Day Amber 58.,3 1 166 83 9 1 ,678 83. 9 1 .678 Flint 3 1 0 .062 75. 0 1 ,500 Three Days Amber 42. 8 0 856 82 4 1 .648 83. 9 1,678 Flint 1 8 0 ,0.38 52. 1 1 .042 Five Days Amber 7 6 0 .154 81 1 1 622 83. 9 l .678 Flint 1 0 0 020 Yl, O o .7t..O Seven Deya Amber 6 .5 0 130 79. 1 1 .582 83. 9 1 6 7 8 Flint o o 0 000 1 9 2 0,384 Ten Days 1 670 Amber 4 5 0 090 77 5 1 .550 83. 5 Flint 12 .. 4 o .248 Fifteen Days :Amber 3 5 0 010 75. 5 1 .510 82. 9 1.,658 Flint 8 1 0 .162 '!'wenty Daya Amber lit8 o .o.36 71.2 1 .424 82. 3 1 ,646 Flint 4 5 0 090 Thirty Days Amber o s 0 016 66. 3 1 :,26 81. 9 1 .638 Flin t ... 2 2 0 .044 Sixty Days Amber o o 0 .000 50. 2 1 ,004 80 0 1 .600

PAGE 97

86 TAB:tK 'J:} THE STABILITY OF FLAVAXIlf SOLUB t u 25 PER CEl-RO !ENE GLYCOL D I STILIED WATER STO D UNDER VARIOUS C O xIDITIONS IN FLINT AND AMBER BOTT s 1101\ftll t DU:fl!ied lt!tm1 Darkness Lumetron Lumetron Lumetron Ree.ding eg./Ml. a.ding !:U/1- aping Mcg./Ml. wmetron a.ding of esbly Prepared Sample ~ 83, 9 or 1 .678 mog./ml. One Day Amber Y/. 5 0 750 83. 9 1 .678 8.3. 9 1 .678 Fllnt 2 5 o .oso 6) .3 1 .266 Tbree ~s Amber 21. 5 0 430 82 5 1 650 83. 9 l .678 Flin t 1 4 0 02s 32.7 o ,654 Five ~s Amber 4.5 0 090 81, 7 1 .634 8'.3. 9 1 6 7 8 Flint o s 0 016 20 ; o .a.o Seven Days Amber 3 7 0 074 ao. s l .616 83. 9 1 6 7 8 Flint o o 0 .000 11. 7 0 .. 234 Ten Daye Amber 3 0 o .o6o 77, 3 1 .;.t.6 83. 3 1 .666 Flint ,. .. ... ,.. 1 0 .150 Fifteen Days Amber 1 5 0 .0.30 72 J 1 .446 82. 8 1 ,656 Flitlt - 4 5 0 090 'lventy Deys Amber 0 ,.3 o .006 6'7. 2 1 344 82 0 1 .6.IJ) Flint 1 8 0 036 Thirty Days 1 .266 Amber o o 0 000 6.3, 3 81. 7 1 .6.34 runt 0 3 o .006 Sixty Daye -Amber Ill , ... 48. 2 0 964 79. 6 1 .592

PAGE 98

TABIE 40 STABILITY OF FLAV. n~ SOLUBlE I 50 P.,.,, a T PROPY "" GLYCOL IN DISTILLED WATER STO D UMDER VARIOUS co1mITIONS lli' FLni T AND MBER BO'l.'TIES sun;u,mt, Lumetron e De.rknese ~tron Lu:mtron Reading Mcg. /Ml Readinf I Mc./M'.l. Reading icg. /Ml. Lumetron Reading of Freshly Prepar d Samples 85 0 or 1 700 mcg./rel. One~ ber Flint Three ~s iber Flint Five ys Amber Flint Seven Days .Amber Flint ten Days Amber Flint Fifteen Days Amber Flint Twenty Dqs Amber Flint Thirty Days Amber Flint Sixty DE11S Amber 45. 0 3 0 25. 5 1 8 2 8 2 0 o 8 o o 0 900 o .060 0 .510 0 .036 o .lo6 0.002 o .078 0 000 0 056 .... 0 040 0 ,016 0 000 . .. 85 0 if:J, 9 79.,7 8 9 67 .. 0 o s 1 700 1 .398 1 .594 0 178 1 .524 0 100 1 .446 0 042 1 .. 340 0 .016 85 0 85. 0 85 0 s3. a 81. l l .700 1 700 1 .700 l .692 1 .676 1 .660

PAGE 99

88 D SOLUTIO OF L ous cm DITIO s t fcg. /Ml cg./Ml. fumetron a.di n of r ably Prep ed l : 85. 6 or 1 ,712 cg ./lll.. One Day r 40. 2 o .804 85 6 1 ,712 85. 6 1 .712 Flint .3. 2 o .064 65. 0 1 .300 Three Days r 32. 2 0 644 84. 2 1 684 85. 6 1 .712 Flint 1 9 0 ,0.38 52. 2 1 044 Five Days Aaber 12. 5 0 250 S3. 7 1 .674 85. 6 1 .712 lint 1 1 0 022 47, 0 0 940 Seven Days Amber 7 4 0 .11..s 82. 4 1 .61+8 85. 6 1 .712 Flint o o 0 000 J/) 0 o .soo T n Daye .Amber 4 2 0 084 80 0 1 .600 85. 6 1 .712 Flint .31. 9 o .638 Fi.fte n Days Amber 1 0 036 79. 0 1 .580 85. 6 1 .712 Flint 1 9 2 0 384 Twenty ye Am r 1 0 0 020 77. 6 1 .552 85. 0 1 .700 Flint s 0 176 Thirty Deya Amber o 6 0 012 74. 3 1 .486 84 8 1 .696 Flint .3. 8 o .<116 Sixty Days Amber o o 0 000 65. 8 l .Y/6 84. 4 1 .688

PAGE 100

89 TABIE 42 THE STABILITY OF FLAV I 1 SOLUBIE I 0 .01 PER CEl T !UINI BISUIFATE IN DISTILIED W TER STO D UND,.. VARIOUS CONDITIONS IN FtnT D A.MIF.R BOTT s Sup]J.Rht Diffused Litm~ Darkness Lumetron LumetroD Lumetron Reading Mcg. /Ml Reading Mcg./Ml. Reading cg. /Ml Lumetron Reading of Fr sh Prepared Samples 83. 7 or 1 674 cg ./ml. One Day Amber 51. 7 1 034 83. 7 1 674 83. 7 1 .674 Flint .3. 0 o .060 57. 3 1 .146 Three D91s Amber 38. 0 0 .760 83. 1 1 ,662 8.3. 7 1 674 Flint 1 8 0 .036 48. 8 0 .976 Five Days Amber 14 1 0 282 82 2 1 .644 8.3. 7 1 674 Flint 0 9 0 .018 37. 3 o .746 Seven Deys Amber 4 3 o .086 81. 5 1 .630 83. 7 1 674 Flint o o 0 .000 24.0 0 .400 Ten Days Amber 2 4 0 .048 80. 7 1 614 83. 5 1 .670 Flint 17. 2 0 .344 Fifteen Days Amber 1 1 0 022 79. 3 1 .586 83. l 1 .662 Flint 12 1 0 ,242 Twenty Days Amber 0 5 0 010 77 2 1 .544 82 8 1 .656 Flint 7 6 0 152 Thirty De.ya Amber o o 0 000 72 2 1 .444 82. 4 1 .648 Fli:ct 2 7 0 .0;4 Sixty Ds3S Amber 59f.3 1 186 81 2 1 ,624

PAGE 101

90 TABLE 43 THE STABILITY OF FLAVAXIN SOLUBLE A SATURATED SOUJ'!'IO OF BETAMETHYL UMBELLIFERONE I N DISTILI.ED WATER STORE D UNDER VARIOUS CO!IDITIOU S N FLiti':F A N D AMBER BOTTLES ynllghi L:1m1ic Darlglees wmetron Lumetron Lumetron BL'iing Mcg./Mt, Reading Meg./Ml. Reading Mog./Ml. u.mietron Reading of F.res ly Prepared Samples 85.5 or 1.71 0 mes /ml. One Day Amber 45.3 o .906 a;.s lQ710 85. 5 1 ,710 runt 2 8 0.0;6 54. 5 l .090 Three D~s .Am.be~ 29.3 0 .586 84 8 1.696 85.5 l .710 Flint 1 2 0 024 39. l 0.,782 Five Days: Amber 12 l 0 .242 8.3. 6 1 6 7 2 85. 5 l.'710 Flint 0 1 0,014 28. 9 0 .578 Seven Dqs Amber 5 4 0.108 81 8 l 636 85, 5 1.710 Flint o o 0.000 17.8 0.356 Ten D aye Amber .3. 1 0.062 80. 1 l.,602 8 5. 2 1.704 Flint . s.2 0 ,164 Fifteen Days Amber 1 5 0 030 79 2 1 .584 84 8 1.-696 Flint 4 9 0 ,198 Twent Daya Amber 0.-8 0 016 78 .3 l.566 84. 5 i.690 Flin t 2 .. 1 0 .,042 Thirty Days Arrtber o o 0 000 73. 2 1.464 84,2 1,684 Flint . ., .. 0 8 0 .016 Sixty Daya Amber et 6o. 4 1 208 83 0 l .660

PAGE 102

91 TAB.IE 44 THE STABIIJTY OF F!AVAX! S OLUBIE Il\ 0 1 PER CENT T\ 80 DI S TIL IE D WATER STO D UNDER V IOUS CO!IDI'l'IONS I ft 1 FLINT AUD Al OOT'l'LES 2W)light Lumet on Rea.din L Lumetron Reading 0 Fre hly Pr p e Saq,le; 84 5 or 1 .690 mcg./ml, One~ .Amber Flint Three Days Amber Flint Fi\Te Days Amber Flint Seven~ Am r Flint Ten Days Amber Flint ifteen Days J\tnber Flin t Twenty Days Amber Flint Thirty Das Amber Flint 1 .. 8 0 9 1 0 o o o J o o .. 0 724 o .060 0 .324 o ,o.36 o .o:36 0 018 0 020 0 000 0 018 o .006 ii 0 000 .... .. ,. .... ... .. 80. 5 23. 0 79. 5 12 9 76 .:, 9 60. o 1 1 47. 0 1 69 0 0 960 1.610 0 .460 l .590 0 .2,s 1 30 0.,098 1 .200 0 022 o 940 84, 5 84.5 8 0 8 2 81.0 79 7 l .690 l 690 1 .680 1 674 1 .644 1 .594

PAGE 103

92 T BLE 4, S'l' ILITY OFF VAXIl SOttJB I l_...O PER CE T WA R STORED UNDE R V RIOUS CO! DITIO Il FLINT i,t Dif~od Litmt Darkne s L'Wlletron Lumetron Lumetron Reading Meg./Ml.. Re s I ing Mcg./Ml. Reading :fcg./Ml. Lumetron Reading of Freshly Pl'epared Sample~ 84. ; or 1 .690 mcg. /ml One Day Amber 36,3 0 .726 84 5 1 690 s4. 5 l .690 Flint 2 5 0 .050 69. 5 1,,.390 Three Days Amber 10 0 0 .200 81 2 1 .624 84. 5 1_.690 Flint 1 8 0 036 37,,5 o .750 Five Daya bar 7 5 0 150 79 5 1 ,590 84. 5 1 .690 lint 0 9 0 .018 15 8 0 .316 Seven Days oor 4.6 0 092 75. 8 1 .516 84. ; 1 .690 Flint c o 0 000 9 6 0 .192 Ten Daya Amber ) 8 0 .(f/6 64. 6 l 292 84. 0 1 .680 Flint . - 5 5 o no .Fifteen Days r 1 0 .. 0.30 ss. 2 1 .164 83. 7 1 .674 Flint .. ., ... 4 ; 0 090 Twenty Daya Amber 0.9 0 018 ;o. o 1 .000 82. 9 1.658 Flint 1' II ..... 2 8 04056 Thirty D9s r o o 0 .000 38. 2 0 .764 82 2 1 .644 Flint . ,,., 1 2 0 024 Sixty D9s Amber 'Z"/, 8 0 ,556 so. o 1 600

PAGE 104

93 TABIE /.6 STABILITY OF FI.AV: ll{ SOLUBLE IN o 5 PER CE rr NIACIN m DISTILI.ED WA R STORED mm-:-VARIOUS CONDITIONS IN FLTI AND AMBER BOTTIES ~iffuaed iJ~! emai,.ght De.rlpless Lumetron llll?letron tumetron Reading Mog./Ml. Readinfi Nog./Ml. Ree.ding Mcg./Ml. Imnetron Reading of Freshly Prepared S8111ples 84 6 or 1 .692 .ncg,/ml. One Day' Amber 39. 5 0 790 84. 6 1 .692 84.6 1 .692 Flint :,. 2 0 064 55. 2 1 104 Three Days Amber 19 0 o .380 83. 3 1 .666 s4 6 l .692 Flint 1 7 0 .034 30 1 o .6o2 Five Days Amber 3'. 5 o .o7o 82. 1 1 .642 84. 6 1 .692 Flint 1 0 0 020 25 0 o .;oo Seven Days Amber 1.6 0 .0.32 81 3 1 .626 84 6 1 .692 Flint o o 0 .000 16 1 0 .322 'ron Days Amber 0 9 0 01s 80. 6 1 .612 84.2 1 .684 Flint ...... 7 8 0 15 6 'Fifteen Days Amber 0 7 0 014 79, 2 l .584 83 8 1 .676 Flint 4 9 0 098 Twenty Days Amber o o 0 000 75. 4 1 .508 83. 2 1 .664 Flin t 3 0 o .060 !rty Days Amber 70 2 1 .404 82, J 1 .646 Flint ..... 1 8 0 036 Sixty DEey"s Amber .. ... . 54. 6 1 .092 so. 2 1 .604

PAGE 105

TABI.E 47 SOLUBILIT'.l OF FIA V IN SOLUBLE n SO}, AQUEOUS SOLUTIONS MID OTH"'~ SOL TS J Diatillod Water 0.9 Sodium Chlorid e o .9j Potassium Chlorid 1.0$ Sodium Acid Phosphate 1 .0% Potassium Acid Phosphate 1 0 % 1 ie.cinrunide 1 .0% Urea Propylene Glycol Glycerin Alcohol Avera tumetron Readin -99.0 99. 0 54,2 48. 0 10 5 1;. o 21, 6 Mg. / Ml 10 ,50 4 .20 13 00 0 .45 94

PAGE 106

95 Results wit a Pyruvic Aeid Derivative of Ribofiavin The pyruvic acid derivative of riboflavin was prepared accord~ ing to the procedure listed under the prep t1on of riboflavin deriV atives. This derivative was a yellowish-orange c sta.lline p owder, differing in color from pure riboflavin by being so'!newhat lighter. It was hygro~copic When dry', it was not preciably affected by diffused ligh t The melting point ms 'between 168-172 C Assay of the pyruvic acid derivative by the fluorophotom.etric procedure shoved a 70. 0 per cent riboflavin content Accordingly, 0 7 Gm, of ribof"lavin was equivalent to 1 Gn4 oft pyruvic acid deriva tive. One u1 em of t h e derivative was added to each ml. of the solvent~ used for stability studl. Since t l lwnetron wa sat fr de termiriations up to 2 mog. per ml. each solution bad to be ~urther diluted. by adding 2, 5 ml. to a sufficient quantity of distilled ua.ter to make 1000 ml. Twenty .. five 11111.ters of this ilut.ion were used for nuorophoto:metric snru.ysis The solubility ill the various aolvents entioned previously was evaluated tor the pyruvic acid derivative,.

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96 TABIE 48 THE STABILITY OF A PYRUVIC ACID D ERIVA'l'I 1 oF RIBOFLA vn, DISTILLED WATER STORED mm' VARIOUS CONDITIONS I N FLIN T AMD Ar BOTTLES Diffused Light Lumotron Darkness Lumetron g ./},O.. Rea.ding Mcg./tn. Ras.ding Mcg.,hn. lametron ading of shly Prepared Sample: 86,.2 or 1 724 m c g./ml,. One Day ber 50. l 1 .002 85. 0 1 700 86 2 1 .724 Flint 1 ~ 0 024 50 8 1 016 Three Days Amber' 20 .. 4 0.408 82 3 1 .646 86. 2 1,.724 P'llnt o s 0.016 34. 0 0,.680 Five ~s Amber 7.2 0 144 81 l l .622 86 2 1 .724 Flint 0 3 Oooo6 1s.5 o .370 Sewn Days Amber 3 8 0.076 79.4 l .588 86.2 1 724 Flint o o o ~ooo s o 0.160 Ten D~ Amber 1 5 0.030 79.5 1.570 86.o 1 720 Flint ... . $ 5 0110 Fifteen Day Amber 1 .3 0 026 76 8 1.536 85 .. 8 1 716 Fllnt .. ~ II. 3 9 0 ... 078 Twenty Days ber l O 0 020 70 1 1 .402 85. 4 l .708 Flint ... 2 0 0 040 Thirty Days Amber 0 4 o .oos 64. 0 1 280 85 0 l._700 Flint o .. o 0 .000 Sixty Days Amber o o 0 000 46,. 2 0 924 9 3 4 1 .668

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TAB 49 ~TABILITY OF A PYRUVlC ACID D ERIVATIVE RlBOFIAVIN U DISTILLED ATER BtJFFE&---i) T pH 6 STO D UNDER VARIOUS CO!WITI0 1 S IN FLINT AND Al,ID.;, BOTTLES Sunlight Diffused Li&?ht Darkfl,ess Lumetro:o Lumetron Lumetr011 Readint, Mcg./,n. Reading fcg ./Ml Reading Mcr;./Ml. Lumetron Reading of Freshly Prepared SBtllple: 86 2 or 1 .724 mog./ml. One An r 52. 1 1 .042 8 4 8 1,696 86 2 1 724 Flint 1 5 0 030 47 .'J 0 950 Three Days A.moor 23. 4 0 .. 468 a2.o 1 .640 S-6.2 1 724 Flint 0 9 0 .018 28. 4 o .568 Five D ys r 9 1 o .1s2 s 1 .3 1.626 86. 2 1 724 Flin t 0 4 o .oos 14. 5 0 290 Se-VI n Deya r 4 2 o o 4 79 8 1 .. 596 86 2 1 724 Flint o o 0 000 6 ; 0.1.30 Ten Days Anibor 2 8 0 .056 78 .. 5 1.,570 86. o 1 .720 Flint 4 3 0 086 Fifteen Days Amber 1 5 0 030 74,9 1 .498 8 5. 7 1 714 Flint ... 3 5 o .07o 'twenty Days Amber 1 1 0 022 68, 2 1 .364 85 2 1 704 Flint 1 9 0 038 Thirty Days Amber 0 7 o .OlA 6.;. s l 276 84 g l i 696 Flint o o 0 000 Sixty Deys Amber o o 0 .000 45~8 o 896 83. 2 1 .664

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9 TABIE 50 STABILITY OF A PYRUVIC A,CID DERIVA T OF RIBOFLAVIN DISTILLED WA:J: mFFERED AT pH 5 STORED VARIOUS CONDITIONS I N FLINT AMD AMBE R BOTTI.ES Sunligbt Diffued Li,mt Darkness L'Ullletron Lumatron Lumtron Reading Mcs./Ml Readini Mc;./Ml. Readini M ci ./MJ. Lumetron Reading of Freshly Prepared Samples 82. 3 or 1 .646 meg. /ml One ber 55. 4 111108 80 8 1 .616 82. J 1 .646 Flint 1 5 0 030 ;o 2 1 004 Three Days Amber 25 2 0 .504 79 0 1 .980 82. 3 1 .646 Flint 0 8 0 016 30. 4 o.600 Five Days Atlber u 3 0 226 78~ 2 1 .564 82. 3 1 646 Flint 0 3 o .006 16 0 0 320 Sewn Days ber 5 8 o n6 75.,7 1 .514 82. 3 1 .646 Flint o o 0.000 4 2 0 084 Ten Days ber 3 C o .c6C 74 2 1.,484 82 1 l .642 Flint . ,3. 1 0 .062 Firtoen Days Amber 1 9 o .o:,e 72. 4 1 .448 81 S 1 636 Flint ...... 1 2 0 024 Twenty Days Amber 1 1 0 022 70 2 1 .404 81 0 1 ,620 Flint 1 0 0 020 Thirty Days Amber 0 5 0 .010 65. 4 1 ,308 80 7 1 .614 Flint 0 3 o .006 Sixty Days Amber o o 0 .000 45. 8 0 916 78. 8 1 .576

PAGE 110

99 TABIE 51 THE STABILITY OF A PYRUVIC ACID D ERIVATI OF RIBOFLA.VIN IN DISTILLED WATER B D AT pH 4 STORED tTh"DER VARIOUS l DITIOM S Ill FLINT AND BOT1' s Sunllp.ht Dif!:ged lr!eh~ Derknees Lumetron Wl!Jetron Iiumetron Reading Mcg./Ml.. Rea.din~ Meg./Ml. Reading l cg./m L: t -Imn.etron Reading of Freshly Prepared Selnpl&s 80, l or 1 .602 One Dey Amber 56, 2 1.124 78, 9 1 .578 80 l 1 ~602 Flint 1 4 0 028 49. s o .996 Three Days Amber 25~ 3 o .506 77. 4 1~548 8 0 l 1 .6o2 Flint 0,7 0.014 29. 5 0 $90 Five Daya Amber 10 4 0 .208 76. 8 1 .536 80,1 l .602 Flint 0 2 0 004 15. 4 0 308 Seven Days Atnber 6 o 0 120 76 0 1 .520 80. l 1 .602 Flint o o 0 000 5 0 0 ,100 TM Days Amber 2 9 0 .0;8 75.4 1 .508 SO. l 1 ,602 Flint J 8 0 076 Fifteen Days Amber 1 s 0 036 74,.6 1,492 79. 7 1,594 F1lint 1 9 o .o:;s Twenty Deya Amber 1 0 0 020 12. 0 l .458 79,l l .582 Flint 1 5 0 030 Thirty Days Amber o 4 o .oos 66. l 1 ,322 78. 3 1 .566 Flint ... .. 0 2 0 .004 Sixty Dfqs Amber o o 0 .000 46. 6 0 .932 77 2 l .544

PAGE 111

100 TABLE 52 THE STABILITY OF A PYRUVIC ACID DERIVATIVE OF RIBOFLAVIN IN 25 PER CEMT GLYCERIN I DISTILLED :ATER STORED UNDER VARIOUS CONDITIONS lN FLINT AND AMBER BOTTIES Sunlight ~UJi\1ea Y.mat Darknes@ Lumetron Lumetron Lumetron Readins Mc1./Mt. Readina Me,/Ml. ReadiDl Mc1./M1. Lumetron Re ding of shly Prepared Sample: 86, 2 or 1 .724 mcg./ml. One Day Amber 61. 3 1 .226 86 2 1 .724 86 2 1 724 Flint 1 5 0 0.30 82 l 1 .642 Three Deya Amber 21. 1 0 .422 s; s 1 716 86. 2 1 724 Flint o s 0 016 62 2 1 .244 Five Days Amber 10 3 o .2o6 8 4 2 l .684 86 2 1 .724 Flin t 0 1 0 002 47. ; 0 950 Sevon ntws Amber 7 0 0 140 8). 6 1 672 86 2 l ,724 Flint o o 0 000 32. 8 o .656 Ten Day s Amber 3 3 0 066 81. 5 1 .630 86. 2 1 .724 Flint 22. s 0 .456 Fifteen Daye .Amber 2 9 0 058 7 8 5 1 .. 570 85 8 1 .716 Flint ...... 14-. 3 0 286 Twenty De.ye Amber 2 2 0 044 76. l 1 522 85, 4 1 708 Flint 8 5 0 170 Thirty Days Amber 0 9 0 018 66. 5 1 .:no 84.0 1 .68o Flint . 2 4 0 048 Sixty Days Amber o o 0 000 49,/) 0 990 82 4 1 .648

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101 TABIE 53 STABILITY OF A FIRUVIC CID DHRIV TIVE OF RIBOFLAVIN IN 50 PER CfilI T GLY DISTILLED W A R STORED tm R VARIOUS CO ITIO S FLI. AND Al R BOTTIBS D~ft!:!sed Ligbt Darkness Lumetron wmetron cg. /1.fi. Ming Meg /Ml. Reading Mog. /Ml. wmetron ading or Freshly Prepared Ssmplei 89. 5 or L 790 cg. /ml Ono Day Amber 68.9 1.378 89.5 1 790 89. 5 1 .790 Flint 1 9 o .o~ 83. 3 1 .666 Three Days Amber 33. 5 o.670 88 8 l .776 89. 5 1 .790 Fl.int 0 9 0.018 65. l 1 ,.302 Five Days Amber 15.5 0.310 ss. o l .. 7(:/) 89. 5 1 .790 Flint 0 2 0 .004 50 7 1 .014 Seven Deys Amber 9 2 0.184 87.1 1 742 89. 5 1 790 Flint o o 0 000 34 2 0 684 Ten De.ye ber 3 5 0 070 85. 2 1 704 89. 5 1 .790 Flint 20 ,.3 0 .406 Fifteen Days Amber 3 0 o .060 82 5 1 .650 89. 0 1 780 Flint 10 2 0 204 Twenty Deys ber 2 4 0 .048 79. 6 1 592 88. 6 1 772 Flint ...... 4 6 0 092 Thirty Days Amber 1 4 0 028 71 5 1 .4.30 87 8 l .756 Flint 2 8 0 0 5 6 Sixty Days Amber o o 0 000 65. 0 1 .300 85. 9 1 .718

PAGE 113

102 'l'ABJ.E 54 1 S T ABILITY O F A PYRUVIC ACID DERIVATIVE OF RIBOFLAVIN Dl 25 P E R C E PROPY J .!. GLYCOL D DISTILI.ED ATE STORED UNDER VARIOUS Cffi."DITION S I FLINT Am) AMBER BOTTLES Sunligp t Dif!3~ed .Y:rJ!t Darkne.ss Lumetron Ltu00tron Lumetron Rea.ding fcg. / n Readintg Mcg./1-0.. Reading 1cg. / 0. Lumetron adin g of Freshly .Prepared Samplo: as. 4 or l 768 cg ./ml. One Day Amber ;6. o 1 .. 120 88. 4 l 768 8 8 4 1 .768 Flint 1 7 0 .. 034 ?8 2 1 .564 Three DSS Amber 20. 6 0 .412 87. J 1 .746 88.,4 l .768 F lint o 6 0 012 54. 6 1 092 Five Days Amber 10. 0 0 200 86, l 1 .722 88. 4 1 768 Flint O l 0 002 40. 3 o .806 Seven DayQ Amber 6 9 0 138 85. 2 1 704 88. 4 1.768 Flint o o 0 000 Z'l. 4 0 .548 Ten Days. Ambei-2 9 0 058 8;. 7 1 674 8 8 0 1 .760 Flint 1s. o 0 .360 Fifteen D~s Amber 2 5 0 .050 so 5 1 .610 87. 6 1 .752 Flint 10 6 0 .212 Twenty Day s Amber 1 9 0 038 76. 6 1 .532 86. 9 1 .738 Flint 6 4 0 128 Thirty Daya Amber o 0 010 67 8 1 .356 86 2 1 724 Flint 3 .. 2 0 064 Sixty Deys ber o o 0 000 62. 3 l 246 84 0 1 .680

PAGE 114

103 TABLE 55 THE STABILITY OF A PYRUVIC ACID DERIVATIVE OF RIBOFLAVIN IN SO PER CE! T PROPYID1E G!XCOL I N DISTILIED WATER STO D UNDER VARIOUS co~.DITIOJS I N FLlNT AND AMBER BO'l'TLES Sunlight wrused Lig!!~ Darkness Lumetron uunetron Lumetron Reading ~g./Ml. Readin Mcg./Ml. Reading Mcg./m. huuetron Reading of Freshly Prepared Sample: 89. 8 or 1 .796 mcg./ml One Dey Amber 62-.3 1.246 89 0 1 780 89. S 1 796 Flint 1 6 0 032 82. 6 1 .652 Throe Deus Amber 29. 0 0 .580 sa. e 1 .776 89. 8 1 .796 Flint 0 8 0 016 64 .,, l .298 Five D~s Amber 12 4 0 .248 87 8 1 .756 89. 8 l .796 Flint 0 2 0 004 53.a 1 076 Seven Days Amber 7.0 0 .140 86. o 1 .720 89. S 1 .796 Flint o o 0.000 38. 6 0 712 Ten Deys Amber 4.2 0 084 84. 7 1 .694 89. 2 1.784 Flint ... 25. 8 0 .516 Fifteen Days Amber 1 8 0 .036 81.-0 1 620 88. 7 1 .774 Flint . 16 2 0 .324 'l'wonty Days Amber 1 5 0 030 78 6 1 .572 88 l 1 762 flint ... 9 7 0 194 Thirty Daya Amber 0 9 0 .018 71 0 1 .420 87.,2 1 .744 Flint 4.,3 0 .086 Sixty Deys Amber o o 0.000 64 e 1 .296 86. e 1 736

PAGE 115

04 TABIE 56 THE STABILITY OF A PY C ACID D':RIVATIVE OF RIBOFLAVIl IN S TURATED SOWTION OF ETHYL fINOBENZOA IN DISTIL ?D WATER STORED UNDER VARIOUS CO IDITIO rs I FLI AND AMBER BOTTI.ES Sunlight 1>1:rused Ugn.~ Darkness Lumetron Lumetron Lumetron Rea.ding og./Ml. Reading Meg. / n. Reading Mcg./Ml. Luootron Reading of Fr shly Prapar d Semple: 83 a 5 or l .670 meg. /ml .. One D~ Amber 69 7 1..394 8245 1.650 83 .. 5 l.670 Flint 2.5 0 030 79.8 l.596 Three D83'S Am r 11).9 O 818 81 .. 8 1 .. 636 83~5 L670 Flint 1 .3 0 026 581J4 l 168 Five Day ber 29 8 0 596 81 0 1 .. 620 8.3 5 1 .. 670 lint 09 0.018 45 3 O 906 Seven Days r 22.2 o.M.4 80 2 1.604 83,.; 1.670 Flint 0 5 0.010 36.4 0 728 Ten Days Amber 17.3 0,346 79 .5 1.$90 83.5 1.670 Flint o o 0 .. 000 2249 0.458 Fifteen Da;rs Amber 7.4 0.1.48 77.,7 1.554 83._l 1.662 Flint ~ ........ 18,6 0 ';72 Twenty ~s Amber 2 8 0.056 75,6 1.516 82. 8 1 656 Flint o.. 11~0 0 220 'Thirty Days Amber 1.7 0 .034 72. 8 1 .456 82. 5 1 ,650 Flint 6.1 0.122 Sixty Das Amber o o 0,000 f:R. 5 1.390 81. 7 1 .634

PAGE 116

105 T A B 57 S T BIU'l'Y OF A PYRUVIC ACID DERIVATIVE OF R tBOFLAVIN IN 0 01 PER CEl1 T unINE B ISULFATE IN D I S TILLED WATER STORED UND R VARI OUS CO?lDITIONS I N LINT AND AMBE R BOTTLES Sunligh~ D,Ufused _Li~ Darkness Lum.etron Lumetron Lumetron R ee.ding Mcg./Ml. adine Meg./1'fJ... Reading Mcft/Ml. Lumetron Reading of Freshly Pre p n r ed Se.mplet 8 3 8 or 1 .676 mcg, /ml One Day Amber 55. 3 1 ,106 81. 3 1 .626 83.,8 1 .676 Flint 9 0,,.038 ?6 3 1 ,526 Three Days Amber 35. 0 o .680 80 0 111600 8,3. 8 1 76 Flint 1 0 0 .020 5?, 3 1 .146 Fiire Days Amber 20 0 0 .400 79. 2 l.584 83. 8 1 .676 Flint o s 0 016 l,J). 5 0 810 S even Days Amber 12 4 0 .248 78, 0 1 ,560 8 3 8 l .676 Flint 0 2 0 004 3.3. 4 0 ,668 Ten Days Ambe r 7 6 0 152 7"!, 5 1 ,5SO 83, 8 1 .676 Flint o o 0 000 19, 5 0 .390 Fifteen rs Amber 5 1 0 102 74 l 1 482 63. 5 1 .670 '* Flint ... 15. 3 o .306 Twenty D~ Amber 3 8 0 016 71. 8 1 .436 8.3, 1 1 .662 Flint ~ii. i, 9 5 0 0 190 Thirty: ~s 1 .656 Amber 1 0 0 020 68, 9 l .378 s2. s Flint .... 42 0 .084 Sixty Days 141634 Amber o o 0 000 62. 3 ., 246 81 7 .....

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TAB 58 S'l' BlLITY OF PYRUVIC CID D F V A T OF RIDOFIA Ill A S TED SOLUTIOl OF TA ... J 'THYL IDmELLIFE ONE IN D ISTILLED HATESTORED U?ID V IOUS C ITIO IN FLINT Al D AMB'"'R BOT'l'!.BS 1 tumetron ading Mcg./ln. D iffu sed Light Iumotron ading Mcg;,/m., 106 ss Meg./Ml.. tumetron ading of shly Prepared S let 81~3 or 1 626 meg./1111~ One Day Amber Flint Tbroe Days Atlber Flint Five Days Amber Flint Seven 3.18 A:niber Flint Ten Deys Amber Flint Fifteen Days Amber Flint Twenty Dayi:, Amber Flint 'lblrty Days Am: r Flint Sixty Days Amber 63. 0 1 8 22. 2 0 7 14. 3 0 1 10 5 o o J 5 1 1 o o 1 .260 0 .036 o 724 0 020 0 ./+44 0 .014 0,.286 0 002 0 .. 210 0 000 0 000 80 .. 5 75 5 77.0 19 8 70.2 1.2 J..610 1.510 1 .. 592 1.086 L564 o .788 1.552 o ,.;66 1 .. ;40 0.,396 1 .. 504 o .o66 l.l..04 0 024 81 .. 3 80, 6 79. 0 78. S l 626 1.626 1 620 1 .580 1 .576

PAGE 118

lf/1 TABIE 59 THE STABILITY OF A PYRUVIC ACID DERIVATIVE OF RIBOFLAVIN D l 1.0 PER C T UREA IN DISTILLEP WA R STORED UNDER VARIOUS CONDITIO N S IN FLIUT MID AMBER BOTTIES Sunlight !?1ff'lwed Umt Darkne&S Lumetron Iannetl"Qn :tu?netron Reading M c g./Ml, Reading M~g @ Reading Mcs./Ml. uim.etro-n Reading of Freshly Prepared Semple : 85. 6 or 1 .712 mog./ml. one Dey Amber 36. 2 o .714 84 5 1 .690 85. 6 1 .712 Flint 1 0 0 020 71. 9 1 .438 Three Days 1 .654 Amber 12 3 0 246 82. 7 85 6 1 ,712 Flint 0 7 0 .014 ~ o o .640 Five Days Aillber s 2 0 164 81, 9 1 .638 85, 6 1 ,712 Flint 0 3 o .006 24. 4 O J.88 Seven na,s Amber 4 6 0 .092 76 3 1 .526 85, 6 1 .712 Flint o o 0 000 10 1 0 202 Ten Days Amber 4 0 0 080 67. 3 1 .346 85 6 1 .712 Flint . 4 2 0 084 Fifteen Days Amber 2 3 0 .046 54 0 1 .oso 85 0 1 .700 Flint 1 9 o.o.38 Twenty Days Amber 2 1 0 042 41. 8 o .e.36 84,5 1 690 Flint o 6 0 .. 012 Thirty Days Amber 0 9 0.018 27 .. 2 0 .544 83. 5 1 .670 Flint o o 0 000 Sixty Days Amber o o 0 000 19. 4 o .388 81. 2 1 624

PAGE 119

108 TABm 60 STABILITY OF A PYRUVIC A CI D DERIVATI :. RIBOFLAVIN IN 0 1 P R CE T 80 I DISTILLED WA R STORED UNDER VARIOUS CO DITIONS IN Ftn T AlID AMBER :OOTTLES Sypl;\ght ~~~-fltmt Dar lgle s s Lumetron Lumetron umietron Reading Mog./Ml. Reading Meg./Ml. Reading cg./m. Lutnetron Ree.ding of Freshly Prepared Samplet 85, 8 or 1 .716 mcg./ml. One Day Amber 49. 1 0 982 85 0 1 700 85 8 1.,716 Flint l O 0 020 48. 2 0 ,964 Three Days Amber 19. 7 0 ,394 81 2 1 .624 85. 8 L 1 716 Flint 0 7 0 .014 33, 3 o .666 Five Days Amber 6 o 0 .120 80 l 1 602 85. 8 1 .716 Flint 0 2 0 .004 16 8 0 .336 Seven D~s Amber 2 8 0 0;6 78 .3 1 .566 85, 8 1 716 Flint o o 0.,000 9 2 0 .182 Ten Days Amber 1 4 0 028 76.,8 1 ,536 8 5 8 1 ,716 Flint . ... 5 5 O.,llO Fiftee n Days Amber 1,.1 0 022 75, l 1 .502 85 2 1 704 lint .3. 4 o .o68 Twenty Days Amber 0 7 0 014 71, 0 1 .420 84. 8 1 ,696 Flint .. 1 8 0 ,0.36 Thirty Days. Am r 0 2 0 004 6; ; 1 .:no s4.2 1 684 Flint .. 0 ., 0 0 000 Sixty Days Amber o o 0 ,000 45 ,.3 0.,906 82 7 1 .654

PAGE 120

TABIE 1 ST BIU'fi OF A PYRUVIC CID D IVATI t:; OF RIBO LAVIN IN 9 5 pt> CEtiT 1 IAC I DISTI IJED WATER STORtD UN VARIOUS C DITIOrS IM FLI?IT Am AMB'R BOTTLES wmetron a.ding 11,.t l09 Lumetron Read One Day of Freshly Prepar e d Sample: 82. 5 or 1.650 c g ,/ml. Amber Flint Three Days Amber Flint Fi Daya Ati r Flint Seve n Dqs Amber Flint Ten Dqs Amber Flint Fifteen Days Amber Flint Twenty Days Amber Flint Thirty Days Amber Flint $5,xty Days Amber 26. 3 o s 11 0 0 1 5 2 o o 3,0 1 9 1.1 o ; o o 1 .168 0 028 0 .526 O .Ql6 0 220 0 002 o ,060 o .o;s ., . .. ; .. 0 '!01 0 0 .000 '17, 9 7 6 74,l 2 0 48. 0 1.622 1.,036 1 .584 0 268 1 .572 o .366 1,558 0 ,152 1 .536 0 ,080 1 ,26 0 070 1 380 0.000 0 ,960 82.,5 1 ,650 82 5 1 ,650 82 0 81 6 8 1 1

PAGE 121

110 T B 62 THE SOLUB!LI'lY OF PYRUVIC ACID D FJiIVATffi OF RTOOFLA.VIll I S AQUEOUS S-OLUTIONS AND OfflE R SOLVENTS Average Mg./Ml, Lumetron ~a(iing Distilled Water 70.0 2 .00 0 ,91' Sodium Chloride 74., 0 2,ll 0 .9% Potassium Chloride ?9 5 2 .27 1 0% Sodium Acid Phosph te 75. 0 2 .14 1~0% Potassium Acid Phosphate 75 0 2.14 1 .0% N iacinamide 95. 0 2 .71 1.0% Ur a 76.l 2 17 Propylene Glycol 78, 3 2 .2, Glycerin 63. 3 1 .80 Alcohol 26. 5 0 74

PAGE 122

lll Results with a Levulinic Acid Derivative of Ribofl :vin The 1 vulinio acid dorivative was prepared according to the general procedure loyed in this investigation for making riboflavin derivatives. This derivativ was a y llov cryatallino powder It was by oscopic and elted bet en 224-228 c Assay of th lewlln1c acid derivativ employing flu.orophotometric procedure yielded an 80 2 per cent riboflavin content. Thus, 0 802 Gm. of riboflavin was equival nt to 1 of the derivative. One m.1111 of e derivative 'tmB ded to each ml. of the sol~ nts previously ntioned for stability study. Since the lumetron was set for determinations up to 2 cg. por ml., aoh ~~lution had to be further diluted by adding 2 2 ml. o f th vitamin solution to eui'f1c1ent distilled tar to make 1000 m.l. Twenty-five milliliters of this dilution were used tor fluorophoto trio analysis. Solubility studies re evaluated for the lewlinic acid deriv tive.

PAGE 123

ll2 Table 63 ST.ABILITY OF A LEVUL IC AOID DERIVATIVE OF RIBOFLAVIN IN DISTILLED WATER STOR.:'., D UNDE R V OUS CONDITIONS IN FLI AND ffl.3R BOTTLES Sunlight l!!!:fus~ W:imt Daress Lumetron Lune tron Lumetron Reading cg./Ml. R-eadin Mc. /Ml. Reading cg /Ml Lumetron ading of Freshly Prepared Sample, 88. 6 or1 .772 mcg. /ml One Day Amber 53. 4 1 .068 86. ; l .7'JQ 88. 6 1 772 Flint 2 s 0 .056 59. 2 1 184 Three D~s Amber 26. o 0 .520 84. 8 1 696 88, 6 1 .772 Flint 1 4 0 .028 35. 5 0 110 Five Daye Amber 12 8 0 .256 83. l 1 .662 88. 6 1 m Flint 0 4 o .oos 16. 6 0.332 Sovon Days Am.ber 4.5 0 090 82. o 1 .640 88 6 1 .772 Flint o o 0 000 9 0 0 180 Ten D8fS Amber 2 9 o .oss 78, 0 1 .560 ss. 2 1 764 Flint 4 s 0 .090 Fifteen Days Amber 1 5 0 030 77 0 1 .540 87 8 1 .756 lint 3 3 o .066 Twenty Days Amber 1 1 0 022 71, 6 1 .432 87. 6 1 .752 Flint 1 7 0 034 Thirty Days Amber 0 3 0 006 68. 4 1 ,368 87 2 1 744 Flint o s 0 016 Sixty Days Amber o o 0 000 49. 5 0 990 85. 7 1 714

PAGE 124

113 'l'ABIE 64 S'l'ABILITY OF A r.vutnnc ACID DERIVA'l' RIBOFIAVI I N DISTILLED A R '!?D AT pH 6 STORED mm: VARIOUS CO ITIONS IN FLINT AND AMB"'1'.R BOTTLES Diffused Light Dp:kpess Lumetron Lumetrori Lumetron Reading Meg. / Reading Mcg./Ml. Reading Mcg./Ml. Lumetron Reading of shly Prepared Sample; 88. 4 or 1 ,768 cg./inJ.. One~ Amber Flint Three Days Amber Flint Five Days Amber flint Seven Days Amber Flint Ten Deys .Amber Flint Fifteen Days Amber Fl.int Twenty Days Amber Flint Thirty Daye Amber Flint Sixty Days Amber 54. 6 2 7 13 0 0 5 4 8 o o 3 2 1 5 1 .3 o o l 092 0 ,054 0 .$00 0 030 0 260 0 .01.0 0 ,096 0 000 o,064 0 030 0 026 o .oos 86. o 48. 2 s; o 15. 0 s1. a 6 8 70. $ 2 0 67. 9 o o 1 720 0 .964 1 .688 o .,so 1 .. 660 0 .300 1 .564 0 096 ss. 4 88, 4 8 7 5 86. a 85. 3 1 .768 l .768 1 .768 1 .768 1 768 1 .758 1 .750 1 7:,6 1 .706

PAGE 125

114 TABIE 65 THE STABILITY OF A LEVULIUIC AO DF.RIVATIVE OF RIBOFIAVIN ll DISTILLED TP....R BUFF D AT pH 5 S ED ID DER VARIOUS CONDITIO S TI FL.INT A? D AMB BOTTLES Snllght 01:gys~d JA t: Darlglens Lumetron Lumetron Lumetron Reading Mcg. /Ml Reading Mog .. /1.n. Reading Mcg, /Ml. Lumetron Reading or Freshly Prepared Sampl~, 86 4 or 1 .728 cg /ml One Day Amber 54 .3 1 .086 84 2 1 .684 86. 4 1 728 Flint 2 6 0 052 47.,2 0 .944 'lbr e O~s Amber 24 8 0 .496 82.,'.3 1 .646 86. 4 1 .728 Flint 1 5 0 030 28. 5 o .570 Five Days Amber 12 6 0 252 81 l 1 622 86. 4 1 .728 Flint 0 4 0 008 15. 4 0 308 Seven Days Amber 4.5 0 090 79. 8 l .596 86. 4 1 728 Flint o o 0 .000 '7, 0 0 140 Ten Days Amber 3 0 o .060 76,,2 1 .524 86, o 1 720 Fllnt 4 5 0 .090 Fifteen Days Amber 1 6 0 032 74. 9 1 .498 85 7 1 .w. Flint .... 3 6 O e 072 Twenty Days Amber 1 0 0 020 7'J. 4 1 .468 s;. 4 1 708 Flint 2 0 0 040 Thirty Days Amber 0 3 o .006 69 o 1 .380 s; o l .700 Flint .... 0 3 0 .006 Sixty Days Amber o o 0 000 51. 8 1 .036 83. 8 1.,676

PAGE 126

11$ TABIE 66 STABILITY OF A IE LINIC ACID D ERIVATIVE OF RIBOFLAVD IN DISTILLED WATER BUFFE D A T pH 4 S'l'ORED UND ffi VARIOUS CONDITIONS IN FLIT AND AMBER BOTTLES Sunlight mt~s29.1.1i~i Darlgless Lume~n Lumetl"On Lumetron Reading cg./n Reading Mcg./Ml.. Reading g ./Ml. L'wnetron ading of Freshly Prepared Sample: 86 6 or 1 .732 DlCg. /ml One Day Amber 55. 8 1 .116 84. 8 1 ,696 86. 6 1 .732 Flint 2 s 0 .056 49. 1 0 .982 Three Days Am r 27. J 0,.546 82 4 1 .648 86. 6 1 .7.32 Flint 1 4 0 028 29. 8 0 ,596 Five Days Alnber 1;. 4 o .308 s1 3 1 ,626 86. 6 1 .732 Flint o 6 0 0 1 2 16 2 0 .324 Seven D~s Amber s o 0 100 79. 6 1 .592 86. 6 l .732 Flint o o 0 000 8 1 0 162 Ten l)eys Amber 3 8 0 076 7?. 8 l .556 86. 2 1 .724 Flint 4 .s 0-.096 Fifteen Days Amber 2;1 0 .042 76 8 1-.5.36 86. o 1 720 Flint 3 5 0 010 Twenty Dqs Amber 1 6 0.032 74.5 1 490 85 9 1 .718 Flint . 2 2 0 024 Thirty Days Amber o s 0 .016 71 8 1 .436 85 7 1 714 Flint .... ii ., ... o s 0 010 Sixty Days Amber o o 0 000 54.4 1 .088 84 5 1 .690

PAGE 127

116 TABLE 67 STABILITY OF A EVULD I C A CI D D RIV TI O F RIBOFLA VD IN 25 PER CE! T GLYCERIN DISTI r.n W A STO.RED UNDER V OUS CO,DITIONS F TAD BO'l'TLES Sunlight P&Jcness Lumetron Lumetron Reading Mcg./Ml. Reading Mcg./Ml. Lmletron ading or Freshly Prepared &mpl z 88 8 or 1.7'6 cg ./ml. One~ Amber 65. 5 1 .310 ss. o 1 .760 88. 8 1 .776 Flint .3. 8 o .076 80. 1 1 .602 Three ~s Amber 35. 5 0 .710 87. 1 1 .742 88. 8 l .776 Flint 3 1 0 062 61. 2 1 .224 Five Da,ys Am r 20 .3 o .JJJ6 85. 5 1 .710 88. 8 1 .776 Flint 2 2 0 044 51. 6 1 .032 Seven Days Amber 10 4 0 .208 83. 4 1 .668 88. 8 1 ,776 n1nt 0 9 0 018 40. 6 o .s12 Ten Dqs At:lber 4 0 o .oso 80. 4 1 .608 88. 2 1 764 Flint o o 0 000 30. 0 o .600 Fifteen ~s Amber 2 4 0 .048 77. 5 l .550 87, 7 1 .754 Flint 14.7 0 .294 Twenty ~s Amber 1 5 0 030 73 2 1 /1,4 87 1 1 .742 Flint 7 5 0 150 Thirty Day Am r o 6 0.012 68. 6 l J72 86 8 1 .7'36 Flint 2 7 0 054 Sixty Days Amber o o 0 000 53.6 1 .072 s;. o 1 .700

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117 T BIE 68 STABIUTY' OF LEVU I C ACI D D . :RIV TIVE OF RIBOFLI VIN 50 PER CE T GLYCERI IN DISTILLED UA R STOHED UNDER V. ous ca DITIO s I N FLINT A?JD mER BOTTUS Sunl!ff,ht .ffysed Li.pht Lumetron Lumetron Reading ~cg. /Ml. Reading Mcg ~/Ml. Darkness IAJJnetron e.ding Mcg./1'0.. tumetron Reading of Freshly Prepared Sample, 90. 0 or 1 .. 800 mcg. /ml One Day Amber Flint Tbreo Days Amber Flint Five~ Amber Flint Setren Days Amber Flint Ten D~s Amber Flint Fifteen DEcy"s Amber Flint Twenty Daye Amber Flint Thirty Da.ys Amber Flint Sixty D~s Amber 67. 0 4 0 16,.7 1 0 7 0 o o .. o.o 1 ~340 o .080 o .754 0 .070 0 .504 0 .052 0 .334 o._020 0 .190 0 004 0 140 0 000 0 0 88 89. l 81. 0 1 .782 1 .620 1 .. 760 1 266 1 .708 1 .044 1 6 48 0 .t59 8 l.,508 0~1s6 1.314 90 0 90. 0 90 0 90. 0 89. 7 89. 1 86. 8 1 .800 1 .800 1 .800 1 .soo 1 .794 1 .'782 1 776 l_.766

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us TABIE f8 STA.Bl TY OF A LEVULIUIC ACI D n ~RIVAT OF R BOFI n .. 25 C E PROPY JIB GIXCOL IN DISTILLED ATE. STO D mm VARIOUS C01 ITIOlS DI FL T AND AMBER BOTT!ES cg. /.n. Mcg,/i D lmess J;umetron Readin Mcg./Ml.., Lumetron ding of Freshly-Prepared Semple . 90 4 or 1.808 eg./ml .. One Day Amber Flint Three ys 'ber Flint Five Days Amber Flint Sev Days Amber Flint Ten Dys ~r Flint Fifteen Day Amber Flint Twenty D Amber Flint Thirty Deys Amber Flint Sixty Day: Amber 57.9 1 7 16 4 0 7 11 0 0 2 49 o o ... 0 8 1 158 0 .. 034 O 490 0.030 0.,.328 0 ,014 0 220 0 004 0 098 0 000 0 050 .. if 0 .016 ... ~ 0 .000 88 3 55.8 79 8 12 0 78 0 7,.0 1766 1 116 1 .. 368 0 .. 022 90 4 90 4 86 8 l 808 1 08 1 808 1 ,794 1.780 1 76 9 1~7J,6

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119 TABLE 70 STABILITY OF A LEVULINIC ACID D""RIVATIVE O F RIDOFUVD IN 50 PER CEN T PROPY"' G!XCOL I N DISTILIED IA R S TORED mIDER VARIOUS ca ITIONS I N FLIN T D BOTTIES Sunlight Diffused !4g~ nar1mess Lumetron Lumetron Lumetron Reading g /M.l, Reading Mcg./Ml. Reading 1e;./Ml. tum.etron Reading of Freshly Prepared Sample: 89. 5 or 1 .790 mcg. /ml One Day Amber 63 .. 8 1 .276 88 6 1 .772 89. 5 1 790 Flint 2 0 0 040 74. 4 1 488 Three ~s Amber 27 0 0 .540 87. 5 1 750 89. 5 1 .790 Flint 1 6 0 .0.32 58. 3 1 .166 Five Days Amber 14. 8 0 .296 86. 4 1 .722 89.5 1 .790 Flint o 7 0 014 37 2 0 744 Seven navs Amber 9 8 0 .196 85 l 1 .702 89. 5 1 .790 Flint 0 .3 o .006 26. 3 0 .526 Ten Days Amber 4.5 0 .090 83 2 1 .664 89. 5 1 790 Flint o o 0 000 15. 4 0 .30s Fifteen Day-s Amber 2 6 0 052 82 3 1 .646 89. 0 l 780 Flint 9 2 o.1s4 Twenty Daya Amber 1 6 0 .032 78.3 1 .566 88 7 l .774 Flint 4 5 0,090 'fllirlq Deys Amber 1 0 0 020 69 5 1 .390 88. 2 1 764 Flint .,, 2 4 0 048 Sixty Days Amber o o 0 000 61 4 1 .228 86. 7 1 .734

PAGE 131

120 TAB 71 THE STABILITY IEVULINIC ACID D F..RIVATIVE OF RIBOFIA IN A SATURATED SO 0 OF ETHYL AMUOBENZO TE DISTILLED WATER STORED UNDER VARIOUS CONDITIONS IN FLINT AlID AMBER BOTTLES SunJJ;ght J21ffus1g Y:mt J?&kness Lumetron tmnetron Lumetron Reading Mcg. /Ml. Rea.ding Mcg. ,/Ml. Rea.ding .r. cg./MJ.. -Lumetron ReQding of Freshly Prepared Sample: $7. S or 1 .756 mcg./ml. One D~ Amber 55. 1 1 102 87 0 l 7JJ) 87. 8 1 .756 Flint l 9 0 038 so. o l .600 'l'hree Deys Amber .32 0 o .640 86. 1 1 .722 87 8 1 /756 Flint 1 4 0 .028 6,3. 5 l 270 Fiw Days Amber 24. 4 o .488 s;. 4 1 .708 87. 8 1 756 Flint o s 0 016 55. 4 1 100 Seven Days Amber 17 0 0 .340 84 6 1 .692 87. 8 1 .756 Flint 0 1 0 002 42. 4 o .848 Ten Da;ys Amber 6 8 0 136 83, 2 1 664 87. 2 1 .744 Flint o o 0 000 31. 3 0 626 Fifteen Daye Amber 4 5 o .090 s2 4 1 .648 87. 2 1 .744 Flint 20 .. 4 0 .408 Twenty ~s Amber 2 5 0 050 Sl. 2 1 .62 4 86 9 1 .738 Flint ...... 13. s 0 .276 Thirty Daya Amber 1 0 0 020 79,.3 lt586 86. 5 1 .7~ Flint ; o uo Sixt y D ays Amber o ... o 0 000 74. 7 1 .494 85. 9 1 .718

PAGE 132

121 TABIE 72 THE STABILITY OF A LZVULI lIC ACID D.filIVATI OF RIBOFLAVII n 0 01 PER C ,rrr UI nm BlSULFA l!, u DISTILIED WATER STO UND VARIOUS CONDITIONS IN FLIU r AND A 'R BOTTLES Dil'i'us d Ligbt tumetron cg./Ml. Reading Mcg./Ml. Mog./m. Lumetron Rea.ding of Freshly Prepared Sampl e : 89. 2 or 1 .784 mcg. /ml One Day Amber 62.3 1.-246 88. 8 1 1 776 89. 2 1 .784 Flln. t 2 1 .. 042 81, 0 l .620 Tht-ee s r 3J. s o,.696 87,.1 1.742 89. 2 1 .784 Flint 1 .. 7 0 .. 034 6.3 .3 1 266 Five Days AmbE!r 26. 8 0~536 86 .. o 1 720 89.-2 1 .784 Flint 0 9 0 .01 8 44. 3 0 886 Sewn Days Amber 15. 2 0 304 84~2 1 684 89. 2 1 .784 Flint 0,.2 0 004 39. 8 0 796 Ten Days Amber 3.9 0 .,07 8 82 7 1 .6$4 89. 2 l .784 n1nt o o 0 .000 26 l 0 .522 Fifteen Days Amber 2 8 0 .056 80 l l.6o2 89. 2 1 784 Flint ... ~ 17. 5 0 .350 1.7 0.-0.34 77 8 1 ;56 88 8 1 .776 ,.. ... 6 5 0,.1,30 Thirty Dys Amber 09 0 018 73. 7 1.474 88. 3 1 766 Flint .,. f ') 3415 0.070 Sixty Days Amber 0,0 0.000 67 1 1~342 87. l 1 742

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122 TABm 73 TllE S T BILITY O F A IEVULINIC ACID DI'RIVAT OF IBOFVI Ill A SATURA! :1) SOWTIO? OF nET -METHYL UMBELLIFERONE I D I S TILIED W. TER STORED UNDSR VARIOUS CONDI T IOI.S n r FLn T D JM"l.DJ;:)/;\ BOTTLES unllght Diffued I4ght Lumetron Lumetron a
PAGE 134

123 TAB 74 STAB! TY OF A LEVULI T I C ACID D.2:RIVATI .FRI FLAVDI IN 1.0 R CE.ri' U .... I DISTI T.El) WA ~:It S T RED mm VARIOUS COxDITIONS I FLINT Ar D AMBT.' BOTT s Sunlit.mt Darkness Lumetron L'umetron adint.J Mcg. /Ml cg .. /Ml. Reading Mcg./Ml. -LumetroD atli ng of Freshly-Pr pared S l f 86. 9 or 1 .738 mcg./ml. One Day Amber 67. l 1 .342 85 3 l .7o6 86 9 1 .738 Flint 2 0 0 040 ;8. 1 1.,162 l'b.r,ee Days Amber 28. 7 0 .574 83. 5 1 670 86 9 l .738 Flint 1 3 0 026 17 5 0 .350 Five Day Amber 10 0 0 200 78 0 1 .560 86. 9 1 .738 Flint 0 8 0 016 13 0 0 260 Seven Deyoe Amber 7 1 0. 142 76 1 1 .522 86 ; 9 l 738 Flin t o o 0 .000 8 8 0 176 Ten Dey~ Amber 5 0 0 100 61 0 1 220 86.5 l .. 730 Flint .. .... 3 9 0 01 s Fifteen ye Amber 3 3 o .066 42. 3 0 .846 86. o 1 .720 Flint .... 1 6 0 .0.32 Twenty D ays Amber 2 6 0 052 /.1). 7 o .s14 85. 3 l .7o6 Flint 0 4 0 008 rty Days Amber 1 0 0 020 34. 2 o .684 84. 2 I .684 Flint o o 0 .000 Sixty Days Amber o o 0 000 .23. 3 0 ./.1:>6 82. l 1 .642

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124 TAB 75 THE STABILITY OF A VULIN IC ACID DERIVATI OF RIBOFIAVIN IN 0 1 Cfil' T TWEE 80 I DISTILLED WA'l'ER STORED mm One Day Amber Flint Three Days Amber Flint Five Days Amber Flint Seven Days Amber Flint Ten Days Amber Flint Fifteen Days Atlber Flint Twenty Days Amber Flint Thirty Days Amber Flint Sixty D~ys Amber VARIOUS CONDITIO S I N FLINT AND AimER BOTTIES Sunlight Lumetron Reading -fcg./Ml. 66. 3 1 7 .)6. 1 1 0 10 3 o o .... o o 1 .326 0 .034 0 .122 0 020 0 .41.s 0 012 0 206 0 000 o.oso 0 048 0 034 0 022 0 000 Diffused LigM Lumetron a.ding Mcg,/?-O 76. 3 1 5 71 2 0 5 67. 9 o o 48. 8 1 .690 0 .360 1 .630 o._110 1 ;60 0 .014 l .526 0 ,030 1 ,358 0 .000 0 .976 Pff:knees Lumetron ruling tcg j1.n. 1 .762 88. 1 1 .762 88,.l 1 ,762 88. l 1 .762 87 8 l .756 87. 5 1 .750 87. 2 1 .730 85. 0

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125 TABIE 76 THE STABILITY OF A VULnuc CID D OF RIBOFLAVlM OF o 5 PER CENT tUAC IN DISTILLED \TA -STORED UMDER VARIOUS CONDITiors n FLINT AND t.'R .BOTTIES Sun ight Lumetron Readitlg Diffused Lieb\ Darlmeps umetron Lu.matron Mcg./MJ.. Reading M cg /Ml Reading lcg. /Ml Lumetron Reading of Freshly Prepared Sample: 84. 5 or 1 .690 cg. /ml One De.y .Amber Flint Three Days Amber Flint Five Days Amber Flint Seven Deya Amber Flint Ten Days Amber Flint Fifteen Days Amber Flint Twenty Days Amber Flint Thirty Dqs Amber Flint Sixty Days Amber 1.3 .3 0 5 3 5 o o 1 8 1 5 ... o 6 .... o o 1 044 0 050 0 .528 0 026 o .266 0 010 0 010 0 000 0 020 ., .... 0 .012 0 000 76,6 :;. i 51. 3 1 .652 1 048 1 .630 0 622 1 584 0 .406 1 .548 0 100 1 .5.32 0 .064 l.506 0 040 1 ,428 0 020 1,,326 0 000 1 026 1 690 l .690 1 .690 1 690 1 ,684 1 .680 83. 6 1 .672 82 7 1 654 80, 5 1 .610

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126 TABLE Tl SOLUBILITY OF A LEVULIUIC CID DFJUVATIVE OF RIBOF:U VIN I N SO! A UEOUS SOLUTIOMS AND 0TH SOLVENTS Average Mg. / Lumetro!l Reading Distilled a.tel" 75. 0 l .87 0 .9% Sodilllll C oride 77. 5 l .93 0 9 % Potassium Chlorid e 77. 5 1 .9.3 1 .0% SodiUll1 Acid Phosp ate 75, 0 l.87 1 0 Potassium A cid Phosphate 75. 0 1 .87 l o i N iacinamide 87.2 2 17 1 0 % Urea.. so. o 2 00 P ropylene Gl.ycol 96 .,3 2 .39 Glycerin 63. 0 1 .57 Alcoho l 15. 5 0 .39

PAGE 138

Results wi tb Citro.conic Anhydr1de Deri vative of Riboflavin The oitr conic hydride deriv tive of riboflavin was prepared accordi ng to ntioned under p p tion or deriv tives. This derivative dark oran in color a nd crystalline. It was v ry hygroscopic The elting point rang was between 224-22So c When dry, it ws found not to be appreci bly affected by diffused light. ss of eitraconic anhydrid derivativ of r1bofl v1n showed a 76 5 p r cent riboflavin content. ccording l.y 1 of ribo-fl :vin wo.s equivalent to o ?65 of the deriv tiv This was deter mined fluorophoto trically. Solubility studie wer valuated for e eitraconic anhydride derivati of riboflavin in the same manner oth r derivatives. that determined for the

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TABLE 78 SOU: ILITY O RIBOFIA CITRACO?, IC ilil'DRIDE DERIVATIVE OF I N SO, AQ OUS SOLUTIONS OTHER SOL 8 Average Mg./ Luinetron Reading Distilled Water 76 0 1 99 o.9% Sodium Chloride 78 0 2 04 o .9j Potassium Cblori 78. o 2 .04 1 0 % Sodium Acid Phosphate 80. 5 2.u 1.0% otassium Acid Phosphate 80.5 2 11 1 .0% N i cinamide 100.0 2 .62 1 .0% Urea "/9,;2 2 07 Pro pylen e Glycol 63. 5 1 .66 Glycerin 52.0 1.36 Alcohol 25. 2 o .66 128 .,

PAGE 140

DISCUSSIO OF ~ULTS Although number of deriv tive of ribofl v1n were ma.de 1n this investigation, only th p vie acid, 1 vulinic acid and citra conic anhy id
PAGE 141

130 All solutions of riboflavin derivatives stored in flint bottles and placed in direct sunlight precipitated eventually out of solutio11 In ell instances there was a color cha:nge from brilliant orange or yellov-orange tot at of dark.brow or ollve!l Solutions of riboflavin derivatives stored in flint bottle-a and placed in diffused light did not precipitate out of solution. With regard to color changes in this instance, hovever, all solutions of the derivatives lost their brilliancy and some developed a green or brown tint. In no case did a precipitate occur in solutions stored in amber bottles in either diffused light or in direct sunlight. Solution~ stored in the dark in either flint or ember bottles remained clear and retained t hoir original. color after the sixty-day storage test. Stability studies with riboflavin and derivatives in distilled water and in aqueous buffered solutions, stored in amber bottles and in diffused light, shoved a gradual deterioration over a sixty-day exposure period. Those solutions stored in flint bottles in dif'used light .showed over a ;o per cent destruction of tbe riboflavin p6-tency at the end of three days of storage. It was also noted that stability was favored at lower pH values. This is in agreement v.1.th the investigations of Conner and Straub (57). Riboflavin was observed to be somevbat more stable in distilled water and in buffered solutions than any of the derivatives studied. It was evident that as the riboflavin molecule was alter d the tabllity w.s affected, especially in aqueous or buffered solutions. In direct sunlight, all solutions in distilled water as well as

PAGE 142

131 those buffered at various pH valuee and stored in flint bottles e~ibited almost complete destruction at the end of az:i exp osure period of one day. With riboflavin solutions in amber bottles placed in direct sunlight, a rapid deterioration vas observed with ore than 50 per cent destruction of the vitamin content at the end of five days. Riboflavin seemed to stand u p lo e r to the direct rays of the &un in amber bottles, w hen dissolved in distilled vater and in buffered solutions a.tan acid pH, than any or the othe r derivatives studied. Popular pharmaceutical solvents such as, g l ycerin and propylene glyeol in distilled water were also chosen ,as solvents tor stability st'UQY. The pyruvic acid derivative and the lewllnie aeid derivative ,as well as riboflavin-5f.-phosphate sodium seemed to exhibit a greater stability with solution s in amber bottles containing 50 per cent glycerin and propylene glycol than in th e with 25 per cent of the same solution. In fact, t he stability was greater here t hall that of solutions in distilled water and at various btlffered pH values. The fact that part of tho vater was replaced with an organi c solvent which, accordingly, decreased the amount or the aqueous phase coul d have affected the degree of hydrolysis of the riboflavi~ derivatives. With solutions of t... e levulinie acid and pyruvic acid deriva tives of' riboflavin in flint bottles stored in diffused light, replaeing part of the aqueous phase with glycerin or p ropylene g'.cycol seemed to delay the dostruction of riboflavin initially.-How3ver, with longer exposure to diffus d light, there was alJnost complete destruction of the vitamin content in these solutions.

PAGE 143

132 Solutions of the lewlinic acid and pyru~ic acid derivatives stor-ed in flint bottles Md in which part of t he aqueous phase had been replaced with either glycerin or propylene .glycol exhibited almost complete destruction \then stored in direct sunlight. H o ever, the same solutions stored 1n amber containers were o~erved to possess a so what greater initial stability and t hen rapid deterioration. In view of this, it appears that th~e should be no odvantage in the use of aqueous solutions of glycerin or propylene glyco l as solvents for riboflavin or any of its derivatives Vb.en stored in flint bottles and in direct sunlight. Stability tests with riboflavin in an aqueous olution of glycerin ~ d propylene g].ycol in flint bottles showed only a slight edvan tege over the use of distilled water as t he solvent. Ethyl m:dnobenzoate, quinine bisulfate and be thyl umbell-iferone p o s sess sun screening properties. Aqueous solutions of these were prepe.red and studied as solvents for :riboflavin and its deriva tives. It was felt t ha t sueh substances might screen out certain light rays and t he reby ad d to the stability of ~iboflavin preparations in solution. The selection of a suitable soo screening agent presents considerable difficulty. Here, such an ideal substance should have a desirable solubility in water and yet remai n physiolQgieally inactive. With solutions of riboflavin or derivatives thereof stored in direct sunlight din flint and ember bottles, the use of these sun screening agents proved to be of no advantage. f1 th solutions stored in amber bottles and in the presence of diffused light, t he presence of

PAGE 144

13.3 these agento influenced considerably the stability of all solutions of riboflavin and its derivatives. In all cases, satur ted sol.ution ot ethyl aminobenzoate se8111ed to contribute most to the stability of th fluorescent portion of tho mo1ecul. A saturated solution of ethyl sminobenzoate we.a mol'o r avorable to the stabill ty of the pyruvic. acid and levulinic ao.id deriv tives than solutions or propylene glycol and gly:cerin in distilled ter. Thus, tbe significance of ligllt causing th destruction of r:i.bofla.Vin mu.at again bo brought out as perhaps the most inq)ortant single factor contributing to the deterioration of the Vitamin in solution. Urea and nicotinic acid are frequently used as solubillzers fo r riboflavin,. Aqueou"' solutions of those wei-e prepared and studied as solvents for riboflavin d its derivatives. These solutions, 'When stored in diffused light exhibited a marked instability in the presence of 1.U"ea. This was probably due to the alkaline degradation products of urea plus the effect of light on these solutions .. Aqueous solutions o~ urea exposed t o sunlight 1n both ber and flint bottles exhibited rapid deteTioration, It w.s intGresting to ote, ho-rev r, that only negligible amount of deterioration was: observed with aolutions of urea stored in total darkness, boflavin and riboflavin derivatives in an aqueous solution of nicotinic oid showed stability similar to that of solutions buffered at an acid pH. The stability of riboflavin d its derivatives in an aqueous solution of polyo2;1etbylene sorbi.tan monooleate or Tween 80 was similar to that exhibited by solutions of the vitamin in distilled water. It

PAGE 145

1'4 w.s felt that :i; reen 80 might exili ce stability of the solutions because it is frequently used in oral vitamin preparations.-The phen non of fluor scenoe is itlherent to riboflavin solutions and, a..eeordingly, addi t.ional invostigations should be made vi th to bope of finding a ... tabl so vent and st billzing ag nt. The answer to the stability of riboflavin and any o its derivat ves might very well rest th e elimination of all harmful light rays which contribute to its deterioration. Thi could be accomplished with the selection of a proper container tor solutions and th us of a sun screening agent. The solubility of riboflavin in various ueous solutions sbo~d th tin the presence of sodium chloride aoo potas iUJ:1 chloride th_ solubility w.s actually increased somewhat whereas in the pr enc of odium acid phosphate and potassium cid phosphate the observed solubility seemed to deereaso slightly as com.pared to distilled water., Riboflavin $bowed the eatest solubility in aqueoua oolut1ons of niacinamid and urea as comps.red to the other aqueous solutions. Of all e solvents evaluat din the solubility study, riboflavin was the moat oluble in glycerin. It uas soluble to the extent of 0 84 g er ml. This is a solubility six times greater than t in distilled vater. However, this is not su.ffic1 nt to of practical value ill ph ma.ceutical preparations. Propylene glycol was the next best solvent studied, \lhereas riboflavin was praetl~ insoluble in aloobol. Riboflavin was solubl in water to the extent of 0 .14 per ml.

PAGE 146

135 Due to the unusually high solubility of ribof'lavin-51phosphe.te sodium., e solubility had to be deter.;dnod by additional dilution in orde r to fall in t he range oft e lu:metron Even when placing 1 ml. of the saturated solution in anou distilled water to e 1000 ml., the observed t'lu.oreseenco was greater than 2 mog. per ml. Ace rdingly, further dilution was necessary and this w.s accomplished by placing in e. red volUill.'~tric flask 10 ml. of the liter dilution made vith each of the s turated solutions of elycorin and propylene glycol and diluting to 250 ml. 'Wit distilled w ter. The saturated solution of distilled water and other aqueous solutions used as solvents also had to be further diluted by placi 1g 1 ml o.f tl e original liter dilution 1n a rod vol tric flask e.nd diluting to 250 ml. vi distilled water. The saturated solution in cohol required no dditional dilution. Results sJlo d tl t in distilled w tor riboflavin-51 hosp ate sodium had solubility or 3$ 71 mg. per ml. It found to oo slightly more solublo in aqueou solutions of sodiUil'l chloride and potassium chloride and so wha.t les soluble in aqueous solutions of sodiuo acid phosphate and potassium acid phosphate than in distilled water. Glycerin propylene glycol nd alcohol were not as satisfactory solvents for riboflavin5'-phospba.te sodium s distilled water or t he various aqueous solutions studie. Tho unusually high solvent effect with 1 0 per cent niaeinamida and urea in distilled. 1,10.ter was p robably due to the increased solubility of the un esterified portion of the salt .. lloff'mann-le. Roche' s salt has one cf e highest solubilities of any riboflavin deriv tive on tho o.arket but in dilute solution it appears to be more sensitive to

PAGE 147

136 light than pure ribofla'V':1.n. Solubility studies with Flavaxin Soluble or riboflavin sodi odium tetra.ho te also had to be further dilut d to fall in the ran e of t h e lum tron. A ueous olutions 0 urea and niaeinamide were diluted by placing 10 ml. of the saturated solution,, ich vas previously diluted to o n e liter, in a 250-: red vol tric flask d en ad ne sufficient quantity of sti ad water Solu ilities in prop lene glyco l and glycerin were tr -ated in th& s e manner Results sho ,ed tl t the solubil1 ty of Fl :vaxin Soluble in pro pylen glycol d glycerin were unusually high. However the solubility in alcohol w s very s1ig..ltt It is pos iblo however, to attain gher concentrations of ribotle.Vin sodium-sodium tetraborate by prolonged heating and at higher perotures. In gener borates ar only s l ovly soluble and possess a greater solubility at elevated temperatures The solubiliti s of t h e pyruvic acid levulinio acid and citraconi c anhydride derivatives of riboflavin were also studied. These d rivatives were found to be more soluble -than riboflavin in t,,ator They were all about fifte t es ore soluble than .ibofl a.vin Propylen ~eol was also found to be better than glycerin as a solvent. It uas observed that urea and niacinm:dd increased the co ncentration s of these derivatives in aqueous solut ons Sodium chloride and potassium chloride seemed to a a s1mj]ar effect.

PAGE 148

SUMMARY D CONCLUSIONS 1 A search of the literature revealed that numerous thods have been suggested for preparing solutions containing a relatively high coneentration of riboflavin. 1Dst of these suggested methods do not show any increase in stability greater than that of the pure vita.min. One of the purposes of' this inve ti tion was to pr pare more soluble derivatives and to evaluate the stability and solubility of these in various types of solutions. Solubility and stability studies vere also evaluated for riboflavin, riboflavin-51pbospbate sodium and Flavaxin Soluble 2 The levulinic acid, pyruvic acid and citraconic anhydride derivatives of riboflavin wre pr pared Solutiono of these derivatives were placed in flint and bottles and stored 1n sunlight, dif'fused light and darkness Solutions of riboflavill, riboflavin-51phosphate sodium and Flave.xin S oluble were also evaluated for stability in the same manner. 3 Amber bottles were much better than flint for intaining the vitami n potency of solutions o f riboflavin or any of its derivatives. Complete darkness caused only a negligible destruction of the vitw.n with all types of solutions. rapid deterioration oecured with all solutions placed in direct sunlight 1r specti ve of the type of container used 137

PAGE 149

138 4 Riboflavin and its derivatives were more stable at lower pH values. By replacing part oft aqueous phase with glycerin or propylene lycol, testability of solutions of the derivatives prepared in this investigation and that of riboflavin-stphosphate sodium was better than solutions \lit. distilled water. The use of some sun screening agonts, especially saturated ueous solution of ethyl amino benzoate, delayed destruction of the vitamin 5 Solubility studies with riboflavin and some of its derivatives showed that riboflaVin51phospha.te sodium had the highest solubil-ity in water e pyruvic acid, lewlinio acid and citraconie anhydride der-ivatives sh
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BIBLIOGRAPHY 1 Cook, E F and Martin, E 'W., "Remingto n's Practice of Pharmacy The Ma.ck Publishing Co. Easton, Pa., i?lth Edition, 191.S, p 908. 2 llliams, R. J end Beerst&cher, E "An Introduction to Bio-chemistry," D VM rostrand Co. Inc Ne, York, Second Edition, 191.S, p '270. 3 Blyth, A. Y., JI Chem, Soq , .22, 530 (1$79) 4 Bleyer, B and KeJ.lmann, o Btoehem z,, 112, 54 (1925) 5 Goldberger, J., and Lillie, R D TJ,s blie Health Rep !.!, 1025 (1926) 6 Sure, B J J Med, Assoc. 22,, 26 (1932), 7 Warburg, o., and Christian, w B1ochem1 z ~, 438 (1932) 8 Gyorgv, P liayure. lll, 498 {19.34). 9 Birch, T w Oyorg:,, P and Harris, L. J Biochem, J., ~ 28.30 (1935). 10 ll. Kuhn, R Gyor~, P ; and W agner..Jauregg, T Ber. Deut, Chem, Ges., ftl., l.4.60 {1934). Ellinger, P an d K osehara, s Ber, Deut. Chem, Ges, gJ_; 1460 (1934) 12. Booher, L. E J Biol. Chem, J.27., 591 (1934). 1.3. Sherman, H c and Smith, s L "The Vitamins," Little and Ives Co. ew York, Second Edition, 1931, p 111 14 Council o n Pharmacy and Chemistry, J Am, Med. Assoc., .uu!, 1340 (193?). 15 Booher, L E., J1 B iol Chem, 119; 223, (1937). 16 Greene, R D end Black A J. Am~ Che_ Soc , 22, 1 820 (1937) 17 Kuhn, R Wagner..Jauregg, T,, and Kaltschmitt H Ber, Deut, Chem, .9wb., f!l, 1452 (1934) 1.39

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18 Kuhn, QyorftY, P., 10.34 (19.33) d agner-Jauregg, T., Ber Deut, Chem, Ges., 19. Karrer, P., and Kuhn, R., Helv, Ch1Jn1 eta, ll, 1010 (1934). 20. Kuhn, R., Reinemund, K and eygand, F., Ber, Deut, Chem, Ges1 gJ_, 11/40 (1931.) 21. Von er, H Ilelv, Ghi I eta-,~, 522 (1935). 22 ronsell, E J&, .311 (1940). 2.3. She H c (1938). d Lanford, c. s J:, 1278 25. Robinson, F. A., "'l'b e Vitamin B Complex," John 1111' and Sons1 Inc e Y ork,. First Edition, 1951, p 132. 26 or, and Adler, .ir.&...-=~~.,;:.;=,=--' 22 105 (1934). 2'7. "The Unit States P copeie.," Easton, Pa. Fourteenth erlsion, 1950, p 512. 28 Kuhn, R d Boulanger, P Z1 physik. Chea ,,~ 233 (19.36}. 29. Boohor, L E J Biol, Chem., ~. 39 (19.33) 3() Kuhn, R and Rudy H -.i.s--;=-::::-.a......_;;;;;a._...~ ~, 169 (1935) 31. Kuhn, R ~..a..::~u....~;a.~1.1 fiL, 888 (19.34). 32. Warburg, o d Christian, w W!.2W5a._., ~, m (19.33). 3.3. Karrer, P et al., Helv, Chim. eta. l2, 1010 (1934). 34. Goldblith, s d Proctor, B Jucleopics 5, &, 50 (1949) 35. 011-Blly, c and Seivert, C. ., _,Ja.,.,=:.=-=~::;::.=;,;;a., m, 621 (1939) ,36. 0'1-nlly, c and Seivert, C W .,_....,...,;;,i;~-~ JI., lll7 (1942) 37 Frost, D v., J ,Biol, Che:m, 693 (1942) ;38. Roscoe, M Biochem, J., il, 1540 (1933) :39. Ellinger, P and Holden M Biocheme J,-~ 147 (1944)

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u Goui-ev1teh, A auu, soc. ohm. 1o1,, :.m,, 111 {1948). 43 Williams, R R and Cheldslln, v Seienge, 22, 22 (191,2) 44. Peterson V. J Haig, F M.,. and Shaw,. A o J Am, Chem, Soc., 662 (19 1 ;4) 45 Hodson, A z and Norri~,. c ,;;,,J~.::::.:i:.i~.:.iC....:;;:m~ JJ!, 621, {19.39). 46. wy, H \I, ~Ja...A~!i.a..~.:liii.l~~~lal!&, ~' 461 (1949) 47 -otles, R T and Roberts, F J .Ag;roc, Qf fic. AQ:, Chem.,, B, 797 (1949) 48. De Merre, L. J and Brown, W s , Arcp ; Biochem., i, 181 ( 1944) 49., Cohen, F H Reg, Tre.v, Chim0 j4, 133 (193S). 50. eisberg, s. M., and Levin, I., Ind, Eng. Chem .. Anal. Ed., 2 523 {1937). 51. Hand, D1o B I nd , 'Ir Chem,. Apal, Ed., JJ., 306 (19.39). 54 Kavanagh, F., ;tnd Epg Chem, ,Anal Ed;, 2, 108 (1941) 55. Jone$, W s 270 { 1941). d Chri t1ana.en, W G JI Arq, Phtp"Dl, ssoc,. JQ, -. 56. Karrer, P and Fritsche, H Helv, Qhim, Acta,!!!, 911 (1934). 57. Conner, R T and Straub, G J J;nd E ng Chem,, &ml, Eg. .. 1l, 385 (1941} 58 .. Ha:l.son, s. and iss, A F Analy;s;y, ZQ., 48 ( 1945 ) 59"' Alper, T nture. 451 (1946) .. 6o. SJ.a ter, E C t and Morell, D. B ustraJ.ia.n Chem, Inst, J Proc .. .li, 221 (194SJ.

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61. Auhagen, E (to Winthrop Ch meal Co. Inc .), u s pat. 2 256,604, Sept 23, 1941. 62. Frost, D,. v (to Abbott Laboratories), u S. pat. 2,407,412, Sept. 10, 1946. 63. Frost, D V J, Am, Chem. oc , 1064 (1947). 64. Auerbach, (to Winthrop Chemical Co. Inc .), u. s pat. 2,332,548, Oct. 26, 1944 65. Stein; G ruid Moran, J., (to rok and Co. In c .), u s pat. 2,358,356, Sept. 19, 1944 66. Hoffer, (to Hotxmann-La Roche, Inc .), U.S. t. 2,463,461, fa.rah 1, 1944. 67. Furter, and Iloffer , (to Hof n-La Roche, Inc .), u s pat. 2,438,880, ch 30, 1948 68. Jurist, A. E (to E R Squibb and Sons), u. s. pat. 2,358 ,.331, Sept 19, 1944. 69. Shelton, R s (to vim.. s. rrell Co.), u s. pat. 2,379,644, July 3, 1945. 70 Bird, J c and Kuna, A .. ( to S rrell Co.), u s. pat. 2 ,407,624, Sept 17, 1946. 71. Zentner, R (to Hof11naI1D-Le. Roche, Inc .), u. s at. 2,423,074, Jun 24, 1947. 72. Preisverk, E (to Hofi"mann-La Roche, Inc .), u s pat. 2,349 ,986, 30, 1944 73. Miller, H (to Eli Lilly and Co.), U s pat. 2,.395,378, Feb 19, 19/+6. 74. Haas, G. J , (to Hoffmann-La Roche, Inc .), u s pat. 2,;398,706, April 16, 1946. 75 Upham, s. (to Sept 7, 1948. rican Cyanamid Co.), u s pat. 2,~9,041, 76. Moos, and Upham, s (to American Cyanamid Co.), u s pat. 2,449,003, Sept 7, 1948. Tl. auf, A and Kircbmeyer, F J (to Abbott Laboratories), U._ s pat. 2 440,050, April 20, 1948.

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143 78* Charney, J., (to Wyeth Inc.), u .. s p~t 2,449,611), Sept. 10, 19.48. 79" Charney, J,, (to Wyeth Inc.), u s pe.t. 2,445,208, July 13, 19~. 80. Gupta, s B and Gupta, H J, Proc. Ipstt Chemists, ( India), l (1949) . 81. Gerlough, J M., and Smith E L., (to E R Squibb and Sons), U s pat 2,459,518, Jan 18, 1949. 82,. Schlapfer, R., (to Hof Jan. 4, 1949, 83., Sch oe n K., end Gorden s., Arcll1 Biochem., 22, 149 (1949 ),. 84Stecher P., (to Merck and Co., lnc,), u s pat, 2,480,517, Aug, 30, 1949. 85, Stone, G., Science .l, 283 (1950), 86 Flener, F., Ab;t,ractg of l)pers, XIIth Internatione.1 Oongreoa of Pur and Applied Chend:stry P 1111 ~, Murphy, .aa.~....:=.:w&~~ 88 WJl)8tro n Pho;to: Eleetric Fluot9ssence 1 ate,: Instructions Prepared by the Photovol t C o rp of ew Y ork.

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IOGRAPUICAL ITEMS Joel John Hertz was born in Po.lo Alto, California, on ?love ber 22, 1926. After graduation from high school, he attended St ord University for two years beore enlistiDg in the Unit d State$ Arrrq While in Service, he was th the i dical Department for tvo years 1n Occupied Japan He entered the University of Florida in September, 1948, and received t e degree of Bachelor of S cience 1n Pharmacy, "mth high honors," in February, 1951. In April,. 1951, he ua.s avtarded the Lehn and Fink Gold ,iedal Avard tor excellence in pharmacy, pharmacognosy and pharmacology .. lle is a licensed phaxmncist in the St te of lorida and was engaged in active practice for nine nths following his graduation He entered Graduate S chool at the University of Florida :in September, 1951, and completed his ~ducat.ion on an Eli Lilly Fellowship and an Amorican Foundation Grant He is a member of the .American Pharmaceutical Association, Rho Chi, & national honorary pharmaceutical society, Kappa Psi, a na tional pharmaceutical fraternity, and Phi Kappa Phi, a national honorary scholastic ~ociety.

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This dissertation was prepared under the direction or the chai of the candidate s sup ervisory committee and bas been approved by all hers of ttee. It ws subnitted to e Dean or the College of Pharmacy and to the Graduate Council and was approved as partial fulfillment of th req uirements for the degree of Doctor ot Philo ophy. June 7, 1954 Et~~ Dean, College of Pharmacy Dean, Graduate School SUPERVISORY COMrIITTEE:

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UNIVERSITY OF FLORIDA 111111111/I Ill I l l lllll lllll I I IIIIII I III III I I I IIII I IIIII Ill llll I I 3 1262 08554 3709


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