A stability and solubility study of riboflavin and some derivatives

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

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Subjects / Keywords:
Riboflavin   ( mesh )
Pharmacy thesis Ph.D   ( mesh )
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bibliography   ( marcgt )
non-fiction   ( marcgt )

Notes

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

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University of Florida
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All applicable rights reserved by the source institution and holding location.
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oclc - 25743313
notis - AEK7303
sobekcm - AA00004956_00001
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Full Text












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