Group Title: Characteristics and application of a laser ionization/evaporation source for tandem mass spectrometry /
Title: Characteristics and application of a laser ionization/evaporation source for tandem mass spectrometry
Full Citation
Permanent Link:
 Material Information
Title: Characteristics and application of a laser ionization/evaporation source for tandem mass spectrometry
Physical Description: xiii, 195 leaves : ill. ; 28 cm.
Language: English
Creator: Perchalski, Robert John, 1948-
Publication Date: 1985
Copyright Date: 1985
Subject: Mass spectrometry   ( lcsh )
Lasers in chemistry   ( lcsh )
Lasers in biochemistry   ( lcsh )
Chemistry thesis Ph. D
Dissertations, Academic -- Chemistry -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
Abstract: An instrument system is proposed that is based on tandem mass spectrometry (MS/MS) and a focused laser for vaporizing selected areas of samples within the ion source so that the molecular components of the sample can be identified and quantitated. This instrument is similar in principle to a commercially available laser mass spectrometer (LAMMA), but additional selectivity is possible with the proposed system because it has two stages of mass analysis, making molecular analysis feasible. The instrument used for this study consists of a triple quadrupole mass spectrometer and a single-shot, coaxial flash lamp-pumped dye laser, interfaced through a simple system of mirrors and a lens,mounted near the ion source, which serves as a vacuum seal and focuses the laser radiation on the sample. The system is evaluated by observation of short-lived (less than 1 ms) and long-lived (greater than 1 ms) events after the laser is fired. Ion signals lasting several seconds are observed when samples are irradiated in a heated chemical ionization (CI) source. These signals are a combination of a desorption event and a vaporization event, and desorption CI is used as a model to characterize the laser-initiated processes. The latter are called Laser-Induced or Laser-Enhanced Desorption CI. The current system proved unsatisfactory for quantitative analysis because the time limitations of the instrument combined with a concentration-dependent ion signal lifetime produced calibration curves with low correlation coefficients. Qualitative confirmation of the presence of a drug in rat liver was obtained, however, after laser vaporization of a small sample of intact tissue within the ion source. Recommendations are made for overcoming the time limitations of the present system so that a microprobe capable of molecular analysis of heterogeneous and compartmentalized biological samples might be developed.
Thesis: Thesis (Ph. D.)--University of Florida, 1985.
Bibliography: Bibliography: leaves 185-193.
General Note: Typescript.
General Note: Vita.
Statement of Responsibility: by Robert John Perchalski.
 Record Information
Bibliographic ID: UF00099340
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000898073
notis - AEK6764
oclc - 015477434


This item has the following downloads:

PDF ( 6 MBs ) ( PDF )

Full Text







To my parents with love.


I wish to express my sincere appreciation to Dr. Richard A. Yost

for his direction and faith in this project. I am also grateful to the

members of my committee: Dr. James D. Winefordner whose initial advice

should have been given more serious consideration and whose instrumenta-

tion helped to make this project more successful; Dr. B.J. Wilder whose

continuing personal and professional support over the last 13 years has

been invaluable in the attainment of this goal; Dr. John G. Dorsey who

offered advice and encouragement; Dr. L. James Willmore who offered

counsel and expertise; and Dr. John Eyler who took over where

Dr. Willmore left off. I also appreciate the generosity of

Dr. Martin Vala who loaned us the laser.

Thanks are also due to Drs. Neal Brotherton and Dean Fetterolf who

got me started, and to the members of the research group who freely gave

advice and assistance. Special thanks are due to Dr. Jodie V. Johnson

who kept the instrument running, and to Dr. Edward Voigtman for help

with the laser and the storage oscilloscope experiments.

The LAMMA experiments were completed through the kindness of

Dr. David M. Hercules of the Department of Chemistry, University of

Pittsburgh. The assistance of Dr. Edward J. Hammond in the animal

experiments and Dr. William Ballinger in the preparation of samples for

LAMMA analysis is appreciated.

This project could not have been completed without the help and

cooperation of my children, Matthew, Abby, Ben, and David.

Finally, for her support and patience, I thank my wife, Lynn.



ACKNOWLEDGEMENTS................................................ iii

LIST OF TABLES............................................. vii

LIST OF FIGURES............................................ viii

ABSTRACT.................................................... xii

I INTRODUCTION .................................................. 1

Overview of Current Technology............................. 1
Overview of Present work................................... 13

IN MASS SPECTROMETRY ......... ............................ 15

Effects of Absorption of Laser Radiation
at Solid Surfaces......................................... 16
Laser-Induced Vaporization and Particle Emission........... 18
Analytical Usefulness of Laser Desorption
Mass Spectrometry........................................ 25

III EXPERIMENTAL ................................................. 27

Laser Microprobe Study..................................... 28
Preliminary Triple Quadrupole Mass Spectrometry
Study: Regional Distribution of Phenobarbital
and Phenytoin in Rat Brain................................ 30
Laser-Triple Stage Quadrupole.............................. 35


LAMMA Experiments.......................................... 47
Regional Distributions of Phenobarbital
and Phenytoin in Rat Brain................................ 55
Laser-Triple Stage Quadrupole Characteristics.............. 62


V SHORT-LIVED PROCESSES......... ................... .... ..... 68

Triple Stage Quadrupole Control............................ 69
Comparison of Storage Oscilloscope and Data System.......... 75

VI LONG-LIVED PROCESSES.................... ..... ........... ... 99

Long-Lived Signal Profiles.................................. 100
Quantitative Analysis and Application of
Laser Induced Desorption Chemical Ionization............. 147

VII CONCLUSIONS AND FUTURE WORK ............................... 170

Conclusions Based on the Present Work...................... 170
Future Work.... ............................................ 178


TEST COMPOUNDS............................................ 183

LITERATURE CITED ............................................ 185

BIOGRAPHICAL SKETCH .................... .................... 194



I Common Operational Modes of the Triple-Stage
Quadrupole Mass Spectrometer............................... 33

II Masses and Flight Times for Nickel, Molybdenum
and Lead Isotopes in LAMMA Calibrant........................ 49

III Effects of Programmed RF Function and Rod Polarity
Switch Settings During Various TSQ Operational Modes......... 73

IV Weight of Phenytoin Removed from Copper Grid
by a Single Laser Shot...................................... 126

V (M+H)+ Ion Intensity Ratios for LIDCI of PHT and
and PHT* Deposited with Parlodion on Copper Grids.
Comparison of 2 Shots at Different Sites on
Each of 5 Grids ............................................. 130

VI Graded Melting Point Standard Solution Containing
Compounds Having Melting Points from 76'C to
Above 300C ................................................. 131

VII Comparison of Maximum and Summed Ion Currents
for DCI of Graded Melting Point Stock Dilution
from Copper, Nickel, and Silver EM Grids.................... 135

VIII Calibrant Solutions Made from Graded Melting Point
Standard for DCI/CAD from Nickel and Teflon
Substrates ........................... ............ .......... 152

IX Phenytoin Calibrant Solutions for LIDCI
from Nickel Grids ........................................... 158

X Coefficients of Determination for DCI calibration
with Nickel Grids and Teflon Paddle, and for LIDCI
Calibration with Nickel Grids................................ 161

XI Phenytoin Concentrations in Rat Brain and
Liver Samples and in Fixative Solutions..................... 163



3.1 Analyzer section of Finnigan TSQ mass spectrometer.......... 32

3.2 Diagrams for machining blank side flange of TSQ
for fused silica lens, and spacers for adjusting
focal point of lens within ion source........................ 37

3.3 Diagrams for machining 900 and 45 stainless steel
solids probe extensions..................................... .40

3.4 Layout of lasers and beam directing optics in
relation to TSQ ion source.................................. 42

4.1 Plot of flight time vs. the square root of ion
mass of LAMMA calibrant .. .................. ................ 52

4.2 LAMMA spectra for nickel-molybdenum-lead
calibrant, section of rat brain from cobalt lesion
site, and section of rat brain from in control
animal...................................................... 54

4.3 Ion current profiles for direct solids probe
vaporization and and selected reaction monitoring of
phenobarbital and phenytoin in rat brain extract............. 58

4.4 Concentrations of phenytoin and phenobarbital in
various areas of rat brain (ug/g) and in plasma
(pg/mL)................................................... 60

4.5 Variation of focal spot diameter with distance in
front of and in back of focal point......................... 64

4.6 Laser pulse shapes from photodiode signal as
monitored by storage oscilloscope at various
driver voltages and laser cavity diameters.................. 67

5.1 Stability diagram for quadrupole mass filter................ 72
5.2 Plots of total ion current vs. time for Ni
(m/z 58) showing storage scope data and profile
data for nickel EM grid target............................... 77


5.3 Plots of total ion current vs. time for cationized
phenytoin (m/z 275) showing storage scope data
and profile data for phenytoin over Na2CO3 on
copper EM grid.............................................. 83

5.4 Plots of total ion current vs. time showing the
efficiency of Q1 and Q3 mass filtering (profile
data) for phenytoin over Na2CO3 on copper EM grid ........... 85

5.5 Plots of total ion current vs. time showing
efficiency of Q1 mass filtering and effects of
programmed RF function in Q1................................ 89

5.6 Plot of total ion current vs. time for 4-vinyl-4-
aminobutyric acid (mol wt 129) and reserpine (mol
wt 608) over Na2CO3, showing time resolution of
respective cationized molecules with m/z 152 and
m/z 631 ..................................................... 92

5.7 Plots of total ion current vs. time for phenytoin
over Na2CO3 showing effects of reagent gas on
signal for cationized molecule.............................. 95

5.8 Plot of total ion current vs. time for phenytoin
in parlodion film (no Na2CO3) with auxiliary
CI (m/z 253) ................................................ 97

6.1 Plot of reconstructed ion current vs. time
for laser evaporation of carbamazepine epoxide
(mol wt 252) in CI source.................................... 102

6.2 Plots of ion current vs. time for laser
vaporization of tetrabutylammonium
perchlorate, menthol glucuronic acid,
adenosine, and acetazolamide................................ 105

6.3 Plots of RIC vs. time and solids probe temperature
vs. time for desorption chemical ionization of
phenytoin from copper grid floating in CI source
at the electron entrance aperture........................... 109

6.4 Plots of RIC vs. time and solids probe temperature vs.
time for direct probe evaporation from a glass solids
probe vial, and desorption chemical ionization from a
400 mesh nickel EM grid of hydergine........................ 112


6.5 Plot of RIC with respect to position of sample in CI
ion source for phenytoin on copper grid...................... 115

6.6 Plots of RIC vs. time for DCI of sodium phenytoin
with filament turned on at various times.................... 118

6.7 Plots of RIC vs. time and solids probe temperature
vs. time showing DCI and LEDCI of phenobarbital............. 122

6.8 Plots of RIC vs. time for a mixture of phenytoin
and 1,3-15N-2- 13C-phenytoin with parlodion
evaporated on copper grids.................................. 128

6.9 Plots of ion current vs. time for DCI of graded
melting point stock dilution from copper, nickel
and silver grids............................................ 134

6.10 Plots of ion current vs. time for DCI of graded
melting point standard (60-76 ng of each drug)
from nickel EM grid, and Teflon probe extension............. 138

6.11 Plot of negative ion RIC vs. time for DCI of
sodium phenytoin at various source temperatures.............. 142

6.12 Plot of positive ion RIC vs. time for DCI of
sodium phenytoin at various source pressures................. 144

6.13 Plot of ion current vs. time for LIDCI of
components of graded melting point standard................. 146

6.14 Plots of RIC vs. time for DCI/CAD of 0.558 ng and
5.42 ng of hydergine on nickel grid showing
dependence of signal lifetime on sample size................ 150

6.15 Plot of response ratio (analyte/internal standard)
vs. amount of analyte on grid for DCI/CAD of 1 pL
of graded melting point calibrant solutions on
nickel grids................................................ 154

6.16 Plot of response ratio (analyte/internal standard)
amount of analyte on grid for DCI/CAD of 1 ul of
graded melting point calibrant solutions on Teflon
paddle..................................................... 156


6.17 Plot of response ratio (PHT/PHT*) vs. amount of PHT
on nickel grid for LIDCI/CAD of PHT calibrant
solutions................................................... 160

6.18 Plots of ion current vs. time for phenytoin parent
ion, major daughter ions and ion recorded at laser
firing for LIDCI/CAD of rat liver tissue section............ 166

6.19 Daughter spectra of phenytoin (m/z 253) from laser
shot for rat liver tissue section, and sample of
pure PHT on nickel grid..................................... 169

Abstract of Dissertation Presented to the Graduate School of the
University of Florida in Partial Fulfillment of the Requirements for
the Degree of Doctor of Philosophy



Robert John Perchalski

May, 1985

Chairman: Richard A. Yost
Major Department: Chemistry

An instrument system is proposed that is based on tandem mass

spectrometry (MS/MS) and a focused laser for vaporizing selected

areas of samples within the ion source so that the molecular components

of the sample can be identified and quantitated. This instrument is

similar in principle to a commercially available laser mass spectrometer

(LAMMA), but additional selectivity is possible with the proposed system

because it has two stages of mass analysis, making molecular analysis

feasible. The instrument used for this study consists of a triple

quadrupole mass spectrometer and a single-shot, coaxial flashlamp-pumped

dye laser, interfaced through a simple system of mirrors and a lens,

mounted near the ion source, which serves as a vacuum seal and focuses

the laser radiation on the sample.


The system is evaluated by observation of short-lived (< 1 ms) and

long-lived (> 1 ms) events after the laser is fired. Ion signals

lasting several seconds are observed when samples are irradiated in a

heated chemical ionization (CI) source. These signals are a combination

of a desorption event and a vaporization event, and desorption CI is

used as a model to characterize the laser-initiated processes. The

latter are called Laser-Induced or Laser-Enhanced Desorption CI. The

current system proved unsatisfactory for quantitative analysis because

the time limitations of the instrument combined with a concentration-

dependent ion signal lifetime produced calibration curves with low

correlation coefficients. Qualitative confirmation of the presence of

a drug in rat liver was obtained, however, after laser vaporization of a

small sample of intact tissue within the ion source.

Recommendations are made for overcoming the time limitations of the

present system so that a microprobe capable of molecular analysis of

heterogeneous and compartmentalized biological samples might be





The purpose of this work was twofold: first, to design, construct,

and evaluate a laser evaporation/ionization source for a triple

quadrupole mass spectrometer, and second, to determine the feasibility

of developing a milli- or microprobe, based on a microscopically-

directed sampling laser interfaced to a tandem mass spectrometer that is

capable of molecular analysis of heterogeneous samples. Such an

instrument could be used for chemo-morphological mapping of geological,

metallurgical, environmental and astronomical samples, and of intact

plant and animal tissues by direct, in situ elemental and molecular

analysis. The major use of this instrument, however, is anticipated in

the biomedical sciences, in which samples and their chemical

constituents are highly compartmentalized from the organism to the

ultrastructural level.

Overview of Current Technology

Much of the work of medical, agricultural, biochemical, and

pharmacological research involves experiments designed to provide

insight into the chemical and physical processes occurring at the

cellular and subcellular levels. Elucidation of these processes is

sought to define, in molecular terms, the normal chemical condition of

organisms of interest from microscopic flora and fauna to man,

abnormalities in this chemical condition that are evident as disease or

deficiency, and effects and mechanisms of action of the various curative

measures employed to produce and maintain an optimal state of being.

The experiments are conducted in five general areas: morphology,

physiology, pharmacology, histochemistry, and chemistry. Morphological

studies relate shape and structure with physical function.

Physiological and pharmacological studies define organism and

substructure reaction to endogenous and exogenous (both therapeutic and

anti-therapeutic) chemical species, respectively. Histochemical studies

relate, usually indirectly and qualitatively, chemical species, and

anatomical structure. Chemical analysis also relates chemical species

with structure but has greater potential for quantitation through direct

determination of the compounds of interest. Each of these

methodologies, when used in this context, is site-specific at various

levels of spatial resolution, and depends wholly or in part upon

microscopy (1, 2).

Methodologies for Physical and Chemical Analysis

Morphology is generally the initial subject of interest and is

pursued at three levels, delineated by the instruments used.

Macrostructural surveys include all work done with the unaided eye and

therefore are limited to gross anatomical correlations in large

organisms and organ systems with dimensions measured in meters to

millimeters. Microstructural studies are conducted with the light

microscope in its various forms and can delve deeply into organ systems

in large species or give information of more general nature for one- or

few-celled organisms. Dimensions are measured in millimeters to

micrometers. Ultrastructural investigations are conducted with the

electron microscope and give information at the cellular and subcellular

levels with dimensions normally measured in micrometers to nanometers.

Physiology and pharmacology are usually restricted to micro- to

macrostructural studies, since the probes used to measure or observe the

chemical or physiological response are incapable of spatial resolution

below the cellular level (5 to 10 pm) in a living organism, or in a

normally functioning tissue or cellular preparation. These studies

include clinical evaluations of drug effects on animals and humans

(pharmacological), and nutritional studies designed to evaluate effects

of and requirements for biomolecules such as vitamins, amino acids,

hormones, and transmitter substances necessary for proper nervous system

function (physiological). In many cases, subject response coupled with

chemical analysis of easily obtained body fluids (blood or urine) can

give the information sought, since all foreign substances and most

biomolecules present in an organism are in dynamic equilibrium with the

circulatory system. At the lower limits of spatial resolution for these

studies, test chemicals are usually applied by microiontophoresis (3)

and responses are monitored by potentiometric (4) or voltammetric (5)


The gross chemical analyses which necessarily precede physiological

studies and coincide with pharmacological studies can be carried out by

usual methods (chromatography, ultraviolet, visible and infrared

spectrophotometry, nuclear magnetic resonance and mass spectrometry,

atomic absorption, emission and fluorescence spectroscopy) applied to

physiological fluids and tissue homogenates. As the work progresses,

however, to the stage at which a more exact localization of active

substances is desired, more spatially selective techniques must be used

which usually place more stringent requirements on the analytical

techniques, particularly with regard to sensitivity. The techniques

that are most used for localization studies are based on three general

methodologies, histochemistry, autoradiography, and direct chemical

analysis of microdissected tissue samples or subcellular fractions

obtained by density gradient centrifugation.

The histochemical methods rely upon chemical, enzymatic or

immunological reactions which involve the molecule of interest, an

enzyme specific for the molecule of interest, an inhibitor of that

enzyme, or an antibody created to bind specifically to the molecule of

interest, as reagents to give, either directly or indirectly, a product

visible with a light or electron microscope (6). The method is devised

so that all reactions occur rapidly in succession, and the final visible

product is synthesized at and does not diffuse from the location of the

reactant of interest. Potential for qualitative analysis is predicated

on the specificity of the chosen reagent, enzyme or antibody for the

molecule of interest. Insuring this specificity presents the most

challenging problem during method development. Quantitative analysis is

possible by densitometry if normal stains are used, or by fluorometry if

the final product is naturally or can be made fluorescent with a

fluorogenic reagent or fluorescent-labeled enzyme or antibody (7-9).

The introduction of raster beam microscopes, coupled with computerized

data acquisition and imaging techniques, have contributed greatly to

improving the accuracy of these methods (10).

Autoradiography is a direct method of localizing radioactively-

labeled molecules of interest at the light and electron microscope

levels (11). The system under investigation is sacrificed and sectioned

at an appropriate time after the labeled compound is introduced. The

sections can be treated as usual for the microscopic method of choice

(staining) and are then coated with a thin layer of silver halide

emulsion (gelatin). Resolution is determined by the homogeneity of the

emulsion, the size of the particles, and the thicknesses of tissue and

emulsion layers. After exposure of the emulsion for a suitable period

of time, which depends upon the half-life of the nuclide (normally

several weeks to a few months), the emulsion is developed and the sample

is subjected to microscopy.

Direct chemical analysis of anatomical structures isolated by

microdissection or density gradient centrifugation can sometimes be

performed using scaled-down versions of normal analytical methods.

Typical methods for microdissection of brain, however, allow

differentiation of more than 220 cell types and areas of a rat brain

(whole brain wet weight: 1.5 g) with samples measuring 200 500 um

square x 100 pm thick, a volume of 4 25 nL (12), and methods for

isolation and analysis of single brain cells (20 50 pL) have been

reported (13). As sample size is decreased to effect a more exact

localization, enzymatic cycling methods must be used (14) which take

advantage of the chemical amplification (104-108) afforded by the

continuous recycling of enzymes in a reaction. This catalytic effect

increases the concentration of the substance chosen as an indicator to

the point where it can be accurately measured. These methods have

generally been applied to determination of endogenous substances,

biomolecules and enzymes, rather than exogenous substances.

Microbeam and Probe-Type Instruments

Another methodology that has been developed is the use of

microbeams of electrons, ions or light, not only to visualize

microstructures, but also to obtain a simultaneous chemical analysis of

the surface by analysis of emissions of electromagnetic radiation,

electrons and ions resulting from the interaction of the incident beam

with the sample. The first such instrument, patented in 1947 and

commercially available in the late 1950s, was the electron microprobe

(15) which uses a focused (0.1 to 3 pm) beam of electrons to produce X-

rays characteristic of the elements in the sample. Other probe-type

instruments that followed include those based on Auger electron

spectroscopy, ion backscattering spectrometry, electron spectroscopy for

chemical analysis (ESCA) and secondary ion mass spectrometry (SIMS) (16,

17). The SIMS instruments gave rise to the ion microprobe and ion

microscope, which differ in the method of obtaining an image of the

sample. The microprobe uses a focused beam of ions to scan in raster

fashion over the sample surface. The microscope uses a flood beam about

250 to 300 im in diameter to image an area of the surface. Final images

of the secondary ions emitted maintain a direct relationship to the

point of origin at the sample surface (18).

Laser-based instruments. Shortly after the introduction of the

laser in 1960 (19), it was incorporated into a microprobe instrument

based on emission spectrography (20), and emission of electrons, ions

and neutral atoms was observed from conductors, semiconductors and

insulators irradiated in a mass spectrometer (21). Several reviews

discuss the early work in both of these fields, concentrating primarily

on emission spectroscopic instruments, laser characteristics, and

interactions of laser radiation with solid surfaces (22-24). The most

recent spectroscopy-based instrument to be introduced commercially was

the laser Raman molecular microprobe (MOLE) (25). Point or global

illumination was possible to allow recording of a complete Raman

spectrum of a particular portion of a sample or mapping of a larger area

by selecting a Raman line characteristic of an analyte of interest.

Although this instrument was capable of identifying some molecular

species, the complexity of the identifiable molecules and of their

environment was limited. Most applications to date have shown

identifications of inorganics, or simple organic deposited in

biological systems in concretions of essentially pure crystals. This

system is no longer commercially available, but work is still continuing

in this area (26). Another microprobe instrument based on nitrogen-

pumped dye laser-excited atomic fluorescence of analytes after site-

specific sample evaporation by a second microscopically-directed laser

(ruby) has also been reported (27).

Laser-based mass spectrometers. Lasers have been applied

extensively in mass spectrometry (MS) over the past 20 years as a means

of rapid vaporization and ionization of samples in the ion source.

Conzemius and Capellan (28) have reviewed much of the work conducted

prior to 1980, including instrument system characteristics (lasers and

mass spectrometers) and applications. This review has been updated to

include all work published through 1982 (29). In 1975, Hillenkamp et

al. (30) reported the use of a LAser Microprobe Mass Analyzer (LAMMA)

which was designed primarily for in situ determination of

physiologically significant elements in intact biological samples. The

instrument, introduced commercially in 1977 (LAMMA 500), is based on a

time-of-flight mass spectrometer with a Q-switched, frequency-multiplied

Nd-YAG sampling laser and a He-Ne spotting laser, both directed and

focused through a high quality microscope. The system has been

described (31), and two reviews have covered sample requirements,

performance characteristics (32), and applications to structural

analysis (33). Proceedings of a symposium on analysis with LAMMA have

been published (34) and include articles on characteristics of laser

spectra of various molecular classes (35, 36), preparation of biological

specimens (37-39), and techniques for quantitative standardization (40,

41). A second generation instrument (LAMMA 1000) has been introduced

(42-44) which allows for analysis of bulk samples rather than restrict-

ing the analyst to the use of thin sections, as did the LAMMA 500.

Molecular analysis with current probes. The characteristic that

links all of the above microprobe instruments is their almost exclusive

application to elemental analysis. This is not to say that they have

not been used to obtain spectra of molecules; however, there is an

almost complete lack of applications for which the microprobe capability

has been used in real, heterogeneous samples for qualitative or

quantitative determination of molecules. In fact, in a search of the

literature, only four such applications were found (two for the MOLE and

two for the LAMMA). In three of these (25, 39, 45), the analysis

succeeded because the spatial resolution of the laser beam allowed

sampling of microcrystals or pure concretions of analyte, and not

because of the resolution or chemical specificity of the spectrometric

method. The only application that succeeded in identifying a molecular

species in an intact, heterogeneous sample involved detection of a

fungal metabolite, glyceollin, in plant tissue with LAMMA (46). In this

study, two ion masses indicative of the metabolite were observed as the

infection boundary was crossed. Part of the problem is that these

instruments were never really designed to do molecular analysis. It is

not, however, unrealistic to expect that the considerable selectivity

necessary for molecular analysis is obtainable with appropriate

combinations of currently available methodologies. In fact, several

review articles on mass spectrometry and tandem mass spectrometry that

have come out of the laboratory of R.G. Cooks in the past four years

have included some reference to a "molecular microscope" (47-51),


although no specific instrument has been described. The current project

grew out of a desire to begin working toward this ideal.

Projections for a molecular probe. Of the three instruments

described above that are capable of localized molecular analysis, the

laser mass spectrometer was thought to be the most promising. Due to

the non-exclusive nature of the electromagnetic spectra of molecules

(i.e., all organic molecules contain essentially the same types of bonds

which give rise to signals contained within a limited portion of the

electromagnetic spectrum for any given method), the selectivity and

specificity of a microprobe instrument based upon optical (Raman)

spectroscopy would be seriously limited. The molecular reordering that

occurs during surface bombardment with energetic ion beams (50) would

ultimately limit the resolution obtainable with an instrument based upon

SIMS. The primary deficiency that precludes molecular analysis of

heterogeneous samples with the LAMMA is the lack of confirmation of the

identity of an ion corresponding to a specific mass peak. Elemental

analysis is possible because molecular interference can be eliminated

by using a high power density (greater than 109 W cm-2) and confirmation

can be based on relative isotopic abundances for the analyte. This

deficiency might be corrected by substituting a tandem mass spectrometer

for the current single stage mass spectrometer to allow online verifi-

cation of the identity of selected ions by sequential fragmentation and

mass analysis steps.

Tandem mass spectrometry. Modern tandem mass spectrometry (MS/MS)

was developed in the laboratories of Cooks (52) and of McLafferty (53)

using reversed-geometry, double-focusing instruments for Mass-analyzed

Ion Kinetic Energy Spectrometry (MIKES). The introduction of the triple

quadrupole mass spectrometer by Yost and Enke (54) simplified the

automation of tandem mass analysis, increased the allowable speed of

data acquisition, and brought the technique within reach of laboratories

other than those specializing in design and construction of unique MS

systems (similar instruments are now available from several

manufacturers). A recently published volume (55) summarizes the

developments and applications of this relatively new field. A tandem

mass spectrometer consists of an ionization-fragmentation region

followed in space (tandem sector and quadrupole instruments) or time

(Fourier transform ion cyclotron resonance instruments (56)) by a mass

selector-analyzer region. Therefore, a molecular ion or fragment

produced in the ion source can be selected by the first mass analyzer,

fragmented, usually by collisionally activated dissociation (CAD), and a

complete spectrum of the fragments can be obtained by scanning the

second analyzer. Carrying this process to its limit, a single pure

compound can be totally characterized by obtaining CAD spectra for the

molecular ion and each fragment produced in the ion source; or a mixture

may be characterized by using a soft ionization technique producing

primarily molecular ions with little fragmentation, and obtaining a CAD

spectrum for each component. Used in this way, the instrument is

analogous to a chromatograph/mass spectrometer system; however, the

relatively lengthy chromatographic separation is replaced by a nearly

instantaneous mass separation. This decreases analysis and method

development time, but more importantly, allows additional modes of

operation that require simultaneous presence of signals from all

analytes of interest. An example of full use and an explanation of

these special MS/MS operating modes are given in a report describing a

rapid, general method for detection and structural elucidation of drug

metabolites (57).

The recording of mass spectra is a statistical process in that the

reproducibility of relative ion intensities is dependent upon the length

of time the ion signals are present, and the length of time available

for monitoring each mass of interest. Therefore, the primary problem

associated with interfacing a laser with a mass spectrometer is a

temporal one, since the ion signal resulting from interaction of a

pulsed laser (pulsewidth = 10-8 to 10-6 s) normally has a lifetime of

less than a millisecond. Zackett et al. (58) used a laser pulsed at

10 Hz to obtain MS and MS/MS spectra of sucrose deposited on a silver

foil. They reported that a 10 ns pulse gave ions for 300 us in a

chemical ionization (CI) source maintained at 0.1 torr with isobutane

and for 1.5 ms at a source pressure of 0.5 torr. The same group

identified a naturally occurring quaternary amine in cacti using laser

desorption MS/MS after extracting the plant material and evaporating the

extract on a silver or platinum foil (59). They used a CI source

maintained at 0.5 torr with isobutane, and concluded that the CI

conditions contributed only to increasing the ion signal lifetime by

causing more efficient extraction of ions from the source, and not to

the actual ion formation process. Cotter (60), however, has used an

auxiliary chemical ionization source to take advantage of the abundant

and relatively long-lived neutral species desorbed by the laser as

discussed by Ready (61). A molecular ion for guanosine was recorded

with a lifetime of 4 ms after a 40 ns laser pulse.

Overview of Present Work

The scope of the present work will include all aspects of

interfacing a single shot-laser with a tandem quadrupole mass

spectrometer, and prolonging the signal obtained upon interaction of the

laser radiation with a solid sample. Chapter II will cover the work

that has been done to develop an understanding of the interaction of

laser radiation with matter and the processes leading to ion formation

in laser desorption mass spectrometry. Chapter III will describe the

instrumental systems and procedures used in this study. The

capabilities of a commercially available laser microprobe (LAMMA

500) will be evaluated along with those of the triple quadrupole mass

spectrometer for localization of species of interest (Chapter IV). The

characteristics of the new system will be studied with respect to short-

lived (Chapter V) and long-lived (Chapter VI) processes, so that

recommendations can be made for future research efforts (Chapter VII) in

the development of a microprobe capable of site-specific molecular

analysis of heterogeneous and compartmentalized samples. Finally, an

attempt will be made to answer a question which may make the development


of a molecular microprobe-microscope for analysis of biological samples

somewhat academic; that is,

Although a molecule may be present in tissue, is it in a

form that is recoverable by direct vaporization or is it

incorporated into membranes, proteins and anatomical

structures in such a way as to make it unrecognizable?

This question may not have a single answer covering all species of

interest; however, it is important to show that discrete molecular

structures can be identified by vaporization from intact tissue.



This chapter is primarily a literature review covering the most

notable work directed toward developing an understanding of the laser-

induced processes leading to ion formation. The initial discussion of

absorption of laser radiation at solid surfaces covers the physical

basis for prediction of the extent of temperature increase at the target

surface upon laser impact. This treatment assumes that no vaporization

or melting of the target material occurs, and is applicable to

experiments conducted on bulk samples or substrates at power densities

at which little visible surface evidence exists of laser impact. The

second section deals with the investigation of the laser vaporization-

ionization processes. The primary questions that have been asked

include (1) what is the energy spread of particles of mass spectral

significance emitted (ions, atoms, neutral and ionized molecules), and

does the system exist in a state of thermal equilibrium? (2) how does

the nature of the laser-induced surface emission change with time? and

(3) what are the mechanisms of laser-induced ionization? The chapter

ends with a discussion of the techniques used to enhance the analytical

usefulness of laser-initiated processes.

Effects of Absorption of Laser Radiation at Solid Surfaces

The unique properties of laser light include a high degree of

brightness, directionality and monochromaticity, these features

resulting from the coherence of the emitted radiation. The laser is,

therefore, a versatile source of spatially and temporally controllable

spectral and thermal power. In laser desorption mass spectrometry

(LDMS), it is the thermal power that is of primary interest because of

the need for sample evaporation and ionization. The advantages of

laser-induced vaporization over more common methods involving resistive

heating lie in the high rate of heating which can approach 1010 C/s and

the extreme localization of the effects to areas as small as 10-8 cm2


Experimental systems used for LDMS are of two basic types: (1)

those in which samples are mounted on thin substrates such as electron

microscope grids and sample and substrate are completely vaporized by

the laser (MS instruments based primarily on time-of-flight); and (2)

those in which samples are mounted on relatively thick substrates (thin

foils to bulk metal stages) and are partially or totally vaporized with

no apparent effect on the substrate (studies employing time-of-flight,

quadrupole and sector instruments). Because the latter process is

viewed as an effect on the substrate which is transferred to the sample,

much effort has been expended in determining the temperature rise

induced in the sample substrate by the laser radiation, and the time-

course of laser-induced thermal effects.

Ready (63) has developed equations for predicting temperature

variations in metallic substrates for lasers having pulse widths greater

than 10-8 s. An assumption of local equilibrium at the site of impact

is based on the premise that the energy is absorbed by electrons whose

collision frequency is 1013 s-1 (the Debye frequency) so that

distribution of the energy to other electrons and the lattice is

instantaneous with respect to the duration of the laser pulse. A

generalized dimensionlesss) equation was presented by Ready to allow

calculation of curves applicable to any material. This treatment was

used by van der Peyl et al. (64) to calculate the time-dependent

temperature variation for a stainless steel surface irradiated by a

pulsed (200 ns) C02 laser, whose pulse profile was experimentally

determined. A comparison of stainless steel (m.p. 1808C) and quartz

(m.p. 1883C) indicated that a lower energy was required to bring quartz

to its melting point (2.4 mJ) than was required for stainless steel

(5.6 mJ). Lincoln and Covington (65) used a specially designed time-of-

flight mass spectrometer (66) which allowed variation of specimen

distance from the ion source (30 or 100 cm) and monitoring of ions or

neutrals. They calculated surface temperatures for two different types

of graphite having different thermal conductivities by measuring the

expansion velocity of the vapor plume and calculating the vaporization

temperature from gas dynamic models for the vapor expansion process.

Both of these studies indicated a more rapid increase in surface

temperature for materials with lower thermal conductivities, the

rationale being that materials of low thermal conductivity do not allow

for rapid redistribution of the absorbed energy to the bulk substrate.

Knox (67) has reviewed some of the earlier work in which surface

temperatures were determined experimentally. Results for normal pulsed

lasers with power densities of 106 to 108 W cm-2 were 2200'C to 9700C

for metals, and were relatively reproducible. Q-switched lasers with

power densities of 108 to 1011 W cm-2, however, gave widely variant

results for similar materials.

Laser-Induced Vaporization and Particle Emission

This discussion will focus on the vaporization processes that occur

when laser radiation strikes a target. The initial section will cover

vaporization from the point of view of the substrate, primarily with

respect to spatial and temporal characteristics. The second section

will cover vaporization from the point of view of the vapor plume, and

will summarize most of the work that has been done to elucidate the

mechanisms of laser-induced ion formation.

Laser-Induced Vaporization

Ready (68) again appears to be the jumping-off place for discussion

of the vaporizing actions of normal-pulsed and Q-switched lasers. Data

have been given for depths of holes produced in a number of thick metal

targets. For a normal-pulsed laser (700 ps pulsewidth), the depth was

dependent on thermal conductivity of the substrate at low laser power

density, but became independent of this property at higher power density

and dependent on latent heat of vaporization of the metal. Therefore,

at low power, the material having the greater thermal conductivity

redistributes the energy to the bulk phase rapidly, experiencing

relatively little vaporization, whereas at higher power, energy is

absorbed too fast for redistribution, regardless of the thermal

conductivity, and deeper holes are produced in the material having the

lower heat of vaporization. The power density, F (W cm-2), at the point

where heat of vaporization becomes dominant is given approximately by

Eq. 2.1:

F > 2LpK t- (2.1)

where L is the latent heat of vaporization (J g-1), p is the density

(g cm-3), Kis the thermal diffusivity (cm2 s-1), and t is the laser

pulse length (s). This view is consistent with the types of laser

interactions outlined by Kovalev et al. (69) and Furstenau (70), who

described interactions for metals, semiconductors and dielectrics based

on a model in which the extent of the interaction was determined by the

concentration of free charge carriers (electrons) in the substrate and

the difference between the valence band-conduction band energy gap and

the photon energy of the laser radiation.

For Q-switched lasers (F > 109 W cm-2), vaporization is not a

simple matter of ablation of material at its boiling point at a front

that advances into the substrate. Other processes occur, including

exertion of pressure on the target by the vapor plume, allowing

superheating of the surface and leading to explosive ejection of

material, and absorption of incoming laser radiation by the plume. In

general, much less material is removed by short (ns), very high power

density pulses than by longer pulses of lower power density.

Scott and Strasheim (71) have studied the spatial and temporal

characteristics of vapor plume generation by normal-pulsed (850 us),

semi Q-switched (30 us) and Q-switched (50 ns) lasers. Metal substrates

were used and impressive photographic evidence was presented in each

case. In the normal mode, vapor emission began at about 30 us after

initiation of laser action and ended at about 400 ps, even though lasing

continued. This was thought to be due to scattering of incoming light

by particles emitted from the surface. The vapor expanded at

8.3 x 103 cm s-1 with the solid angle of ejection decreasing as the

crater became deeper. The semi Q-switched laser provided radiation at a

higher power density and emission began after about 20 us. No particle

ejection was observed and the plume expanded at a rate of

3.2 x 105 cm s-1. For the Q-switched pulse, a small spherical plasma

was visible for about 20 us. The expansion rate was greater than

106 cm s-1 and no particle ejection was seen. In all cases, the vapor

plume expanded in a direction normal to the target surface regardless of

the angle of incidence of the laser beam (33, 72).

Laser-Induced Particle Emission

The primary mathematical relationship found in articles dealing

with LDMS is the Langmuir-Saha equation (Eq. 2.2):

+/i0 = (g/g0)exp[(O I)/kT]


where i+ and i0 are the positive ion and neutral molecule fluxes

leaving the surface at temperature T. The quantities g+ and go are the

statistical weights of the ionized and neutral states, k is the

Boltzmann constant, is the work function of the surface (eV), and I

is the ionization potential (eV). The equation gives the temperature

dependence of the degree of ionization of surface emissions (73). For

substances for which I is greater than D, evaporation occurs at a

temperature less than that required for efficient ionization (74). Datz

and Taylor (75) reported that the alkali metals, the species most

commonly implicated in laser-induced ionization processes, follow this

equation when adsorbed on platinum and tungsten. From a review of the

literature, Conzemius and Capellan (28) divided ionization efficiency

into two general regions based on power densities of lasers employed.

Below 108 W cm-2, ionization efficiency was low, about 10-5. Above

5 x 109 W cm-2, emissions were almost completely ionized.

Energy- and Time-Resolved Studies

Measurement of the energies and time-course of emission of desorbed

ions and neutrals is useful for elucidation of the mechanisms of laser

desorption-ionization, and also for providing a firm basis for increasing

the analytical potential of the technique. Cotter (76, 77) has recently

reviewed the area of energy- and time-resolved studies, some of the most

notable work having been done in his own laboratory. Numerous reports

of energy spreads of elemental and molecular ions have offered

conflicting evidence. The review by Ready (78) cited ion energies up to

5 keV and typical values of up to 400 to 500 eV. Bernal et al. (79),

using a Q-switched CO2 laser (775 ns pulsewidth; 7 x 106 W cm-2),

reported time-resolved energy spreads during the 1.3 us ion pulse as 0

to 60 eV initially and rising to about 200 eV. Ion energies of up to

20 eV have been reported for the LAMMA 500 for thin non-metallic

samples, and up to several hundred eV for bulk metallic samples (31).

Hardin and Vestal (80), using a quadrupole instrument with samples being

introduced continuously on a moving belt, reported energies of 6 to

25 eV for potassium iodide clusters and protonated and cationized guanosine

ions and fragments. They reported that the observed time-of-flight

distributions were dependent on laser power density, and the relative

concentrations of sample and alkali salt on the belt. All work was

performed at a power density of about 107 W cm-2. Energies as low as

0.26 eV were reported by van der Peyl et al. (81) for Na+ in a double-

focusing instrument.

Cotter and coworkers have published a series of papers in which

they report results obtained on a versatile time-of-flight instrument

designed to allow observation of ions produced directly by the laser, or

laser-produced neutrals ionized by a pulsed electron beam (82-85). An

ion-drawout pulse was provided to sample the contents of the ion source

at any time up to 500 us after the laser was fired. They have used this

system to resolve the convoluted profiles of laser-desorbed ions into

separate contributions from prolonged vaporization envelopes and finite

energy spreads. The energy spread for K+ was 15.3 eV at 10 ps after the

laser pulse, and decreased to 0.31 eV at about 44 ps. This is probably


the most accurate work that has been done to date, and while it does not

negate the value of the previous work, it certainly offers a much

improved method for solving the problems.

Mechanistic Studies

Early work in laser-assisted mass spectrometry was concerned mostly

with elemental analysis or pattern recognition after pyrolysis of large

molecules or molecular aggregates. Vastola et al. (86, 87) introduced

the concept of using laser radiation for volatilization and ionization

of molecules that could not be vaporized intact by the usual methods of

ohmic heating. Until 1978, laser desorption continued to take a back

seat to field desorption (FD), secondary ion mass spectrometry (SIMS)

and the newly introduced (88) plasma desorption (PD) in the field of

molecular analysis. The commercial introduction of the LAMMA 500 and

the report by Posthumus et al. (89) in particular provided impetus to

define the optimum conditions for laser desorption and the mechanisms of

ion formation. In this work, laser desorption spectra were presented

for important biomolecules which were generally considered intractable,

including oligosaccharides, cardiac glycosides, steroid conjugates,

antibiotics, nucleotides, nucleosides, amino acids, peptides, and

chlorophyll. All gave abundant molecular ions as alkali metal ion

adducts (M+Na+, M+K+), indicative of a process called "cationization".

This study, along with others (88, 90, 91), indicated that a rapid

heating rate was important for molecular ion production in preference to

fragmentation and decomposition. Although most researchers have used

laser power densities of 105 to 108 W cm-2, Stoll and Rollgen (92)

observed the cationization process and desorption of quaternary ammonium

ions (preformed ions) using a CW CO2 laser at a power density of about

20 W cm-2; however, after scrupulously cleaning the copper substrate and

purifying a sucrose sample to eliminate traces of alkali metals, no

molecular ions or protonated or cationized molecules were detected, even

though the sucrose did evaporate.

In an effort to determine the mechanism of the cationization

reaction which is predominant in laser desorption, van der Peyl et al.

(93-95) performed experiments using several different instrumental

configurations in which thermionically produced alkali metal ions

originated in a different area of the ion source than the test compound

(sucrose). Temperatures of both thermionic emitter and sample holder

were monitored during resistive heating and laser heating cycles and

plotted along with ion intensity curves. They proposed a model in which

the laser serves to produce a steep temperature gradient at the surface

with alkali metal ions thermionically emitted from the hot central zone

of the laser spot (greater than 427C) and neutral sample molecules

vaporized from the relatively cooler periphery. Ion formation occurs in

the gas phase as an ion-molecule reaction producing a stable, even-

electron species with a positive charge on the alkali metal atom. Stoll

and Rollgen (96, 97) arrived at a similar conclusion using a system in

which the thermionic emitter was not completely isolated from the sample.

Heinen (98) and Hercules et al. (33) have reviewed the types of

ions formed from molecules containing various functional groups.

Hillenkamp (99, 100) has summarized the work reported by the most

prominent LDMS research groups, and listed some features of LD mass

spectra. These features include (1) in contrast to the normal

situation with resistive heating, polarity and ionic character promote

desorption and ionization; (2) ions are generally even-electron species;

(3) cationization by alkali and other singly-charged metal ions is

common; (4) anionization has been observed but not frequently reported;

(5) positive and negative ions are produced in approximately equal

amounts, with less fragmentation of negative ions; (6) cluster ions are

frequently seen; (7) matrix effects are observed; and (8) extent of

fragmentation is dependent on laser power density. Cooks and Busch

(101) have compared the various desorption ionization techniques (SIMS,

FD, PD, LD) and have developed a general model for the ionization-

fragmentation events that occur. The model is based on the fact that

these techniques, which differ in the types of energy put into the sample

result in the production of similar ion types.

Analytical Usefulness of Laser Desorption Mass Spectrometry

The utility of a laser desorption/evaporation source in mass

spectrometry derives from the characteristics of the laser beam, i.e.,

it can be focused to sub-micrometer diameter spot sizes, contains a high

thermal power, can desorb or vaporize large, polar molecules intact,

and can ionize them directly or present them to the instrumental system

in a state suitable for ionization by other means. The primary tasks

associated with coupling a laser with a mass spectrometer are (1)

prolonging the laser-induced events so that they persist for the time

required for the mass spectrometer to acquire statistically relevant

data; or (2) designing the mass spectrometer so that it can acquire such

data during the lifetime of laser-produced ions. Ion lifetimes have

been extended by operating the ion source in the chemical ionization

mode (102). This not only takes advantage of the neutral species that

are desorbed, but also increases the lifetime of ions produced directly

by the laser. Using a renewable surface with a pulsed or CW laser

provides a continuous signal, but this is not ideal for microprobe work,

because the intrinsic nature of the investigation precludes the

assumption of a renewable surface (i.e., why use a microprobe if the

sample is homogeneous enough for the surface to be considered

renewable). A variation of this approach involves pulsing the laser in

synchronization with mass scanning so that only one mass is monitored

for each laser pulse (repetitive laser desorption (103)). McCrery et

al. (104) used ion storage in a Fourier Transform mass spectrometer and

were able to digitize and average twelve decay transients for each laser

pulse. Adapting the mass spectrometer to the laser duty cycle has been

done in the LAMMA instruments with time-of-flight mass analysis. Other

workers have used sector instruments with photoplate or array detectors

to simultaneously detect ions over a certain mass range. Since the

objective of this work is to evaluate a tandem mass spectrometer system

for application to laser microprobe mass spectrometry, emphasis was be

placed on obtaining as much information as possible from a single laser




This chapter describes the instrumentation and procedures used in

this three-part study. The first experiments, performed in the

laboratories of D. M. Hercules at the University of Pittsburgh, were

done to gain familiarity with a commercially available laser microprobe

and to illustrate the problems inherent in using a single stage mass

analyzer for analysis of heterogeneous samples. The second study was

done to evaluate the capabilities of the Triple-Stage Quadrupole Mass

Spectrometer (TSQ) for quantitative analysis of drugs in small (2-10 mg)

samples of rat brain in preparation for anticipated laser/MS/MS analysis

at some later date. The objectives of the final series of experiments

were (1) to design and construct a laser interface for the triple

quadrupole mass spectrometer; (2) to evaluate the instrument system with

respect to short-lived and long-lived events produced directly or

indirectly by the laser; and (3) to determine the feasibility of and

make recommendations for construction of a laser/tandem mass

spectrometer-based milli- or micro- probe capable of molecular analysis

of heterogeneous samples.

Laser Microprobe Study


Laser microprobe mass analyzer (LAMMA 500). The LAMMA 500

(Leybold-Heraeus GmbH, Federal Republic of Germany) has been described

in detail (31). The instrument consists of a time-of-flight mass

spectrometer with an open ion source, coupled with a Q-switched,

frequency-multiplied Nd-YAG laser through a high quality microscope. A

He-Ne laser aligned coaxially with the Nd-YAG laser is used for site

selection and aiming. The laser light is incident on the sample at 90

to the sample rear surface and ions are extracted into the mass

spectrometer at 90* to the sample front surface (the instrument operates

with a 90/-90 configuration, according to the convention established

by Conzemius and Capellan (28)). For this reason, samples are

restricted to thin films, or if granular forms are used, the laser must

be aimed so as to strike a grain edge. An ion reflector is employed to

minimize the effect of the initial ion energy spread on mass resolution

and to increase resolution by allowing a longer flight path without

greatly increasing instrument size.


Preparation of animals. An epileptic focus was created in an adult

male Sprague-Dawley rat by sub-dural deposition of powdered cobalt

(105). After 2 weeks, the lesioned animal along with a control (non-

lesioned) animal were sacrificed. The brains were removed (106) and

fixed in phosphate-buffered glutaraldehyde for 48 hours. The lesioned

area of the test animal and a corresponding area from the brain of the

control animal were dissected out and embedded for sectioning. Thin

sections (1 pm) from each sample were mounted on electron microscope

grids for use in the LAMMA, and serial sections were mounted on glass

microscope slides for pre-test selection of analysis sites by optical


Analysis. A calibration mixture of Ni, Mo, and Pb was made by

grinding together NiS04, MoS2, and PbSO4, and dusting the fine particles

onto an EM grid. Spectra were acquired with a 0.020 ps sampling

interval and a 1100 channel delay time. Delay time is the time in

sampling intervals that the transient recorder waits after the laser is

fired before beginning to accumulate data and store it in the 2048

channels available. Sampling interval is the time between each sample,

or channel width in ps, and determines the upper limit of the mass

range. The delay time multiplied by the sampling interval determines

the low mass recorded. The delay time plus 2048 multiplied by the

sampling interval is the total flight time that is covered in a run.

Laser energy before filtering (energy incident upon the sample is

selected very roughly by neutral density filters) is read in mJ after

each shot from a digital panel meter which receives a signal from a

photodiode. Energy from shot to shot is variable and uncontrolled.

Energies varied from 9.7 mJ to 32.5 mJ, but were usually between 10 and

20 mJ. Mass calibration was obtained from the Ni-Mo-Pb calibrant

spectrum by identification of mass peaks from the isotopic patterns and

correlation of flight times with chart distances. Numerous spectra were

recorded from the lesion sample and the control sample with delay times

of 1000 channels and a sampling interval of 0.020 js.Specimens were

observed in the LAMMA with the 32X objective (total magnification 320X).

The mass range covered was m/z 20-219.

Preliminary Triple Quadrupole Mass Spectrometry Study:
Regional Distribution of Phenobarbital and
Phenytoin in Rat Brain


Finnigan triple stage quadrupole (TSQ) mass spectrometer. The TSQ

(Finnigan MAT, San Jose, Calif.) (Fig. 3.1), consisting of an electron

impact (EI)/chemical ionization (CI) ion source followed in series by a

mass selector-analyzer quadrupole (Ql), an ion-focusing fragmentation

quadrupole (Q2), a second mass selector-analyzer quadrupole (Q3), and a

continuous dynode electron multiplier, has been described (107, 108).

Finnigan INCOS software (TSQ revision C) was used for instrument

control, and data acquisition, storage, and manipulation. The center

quadrupole (Q2) functions as a second fragmentation region when

pressurized with an inert gas (N2, Ar), operating by collisionally

activated dissociation (CAD) as described by Yost et al. (119). Table I

shows the primary operating modes of the instrument along with the

corresponding configurations of the mass analyzer and collision


Figure 3.1

Analyzer section of Finnigan TSQ mass spectrometer.



INLET i, nc -:i Hi $--

Table I. Common Operational Modes of the Triple-Stage Quadrupole Mass Spectrometer


(1) single stage mass

(2) parent ion experiment

(3) daughter ion

(4) neutral loss experiment
(linked scan)

quad 1

a) pass all m/z
b) scan m/z

scan m/z

select m/z

scan m/z

quad 2

no collision gas;
pass all m/z

collision gas.a
pass all m/z

collision gas,a
pass all m/z

collision gas,a
pass all m/z

quad 3

a) scan m/z
b) pass all m/z

select m/z

(a) scan m/z or
(b) select daughter
ions for each ion
passed through
quad Ib

scan m/z at same
rate as quad 1 but
with a constant
mass difference


normal mass spectrum
(CI, El, positive
and/or negative ion)

spectrum of all ions
that fragment to give
selected m/z

spectrum of all ions that
arise from fragmentation
of selected m/z

spectrum of all daughter
ions that result from
loss of selected
mass difference

a) If no collision gas is present, the unimolecular dissociation products (metastable ions) will be observed.
b) Selected reaction monitoring. Only daughter ions or ions characteristic of parent ion fragmentation are monitored.


Preparation of animals. Rats were given phenobarbital or phenytoin

(20 mg kg-l twice daily) for 5 days. Animals were sacrificed, the

brains were removed, and various structures cerebellarr gray and white

matter, sensory motor cortex, basal ganglia, hippocampus, corpus

callosum, occipital cortex, pontine white matter) were separated by

vacuuming into weighed glass pipets plugged with glass wool.

Analysis. The pipets containing the brain samples were reweighed

to obtain sample weight, and the section of pipet containing the sample

was crushed in a screw cap culture tube (Teflon-lined screw cap) for

analysis. Whole blood was also collected and plasma was analyzed.

Methylated analogues of the drugs [5-(p-methylphenyl)-5-ethyl barbituric

acid, 5-(p-methylphenyl)-5-phenylhydantoin] were used as internal

standards. Samples were homogenized by sonication in 300 uL of 1 M

phosphoric acid. After addition of 300 )jL of 1 M phosphoric acid

containing the internal standard, samples were extracted with 3 mL of

chloroform. The chloroform residue was dissolved in 100 uL chloroform

and 1 to 2 uL was placed in a probe vial for MS. The mass spectrometer

was operated in the selected reaction monitoring (SRM) mode by

sequentially passing the drug and internal standard molecular ions

resulting from methane CI through Q1, fragmenting those by CAD in Q2

(pressurized with nitrogen at 2.3 mtorr; collision energy of 18 eV) and

selecting the major daughter ions for each in Q3. Thus, phenobarbital

(mol. wt. 232) was monitored by selecting the m/z 162 daughter ion in Q3

while passing m/z 233 through Ql. Quantitation was based on the ion

intensity ratio of m/z 162 to m/z 191, the daughter ion of the

phenobarbital internal standard parent ion m/z 247. Phenytoin (mol. wt.

252) concentration was calculated from the ratio of the m/z 182 daughter

ion of the phenytoin molecular ion, m/z 253, to the m/z 196 daughter ion

of the internal standard (parent ion, m/z 267).

Laser-Triple Stage Quadrupole Studies


Laser. The laser was a coaxial flashlamp-pumped dye laser (Model

SLL-625, Candela Corporation, Natick, Mass.) operated primarily with

Rhodamine 6G (4 x 10-5 M in absolute ethanol) with driver voltages

between 16 kV and 20 kV. The output coupler was a sapphire window

without reflective coatings. Pulsewidth was 1.5 to 2 ps. The laser

could be operated only in the single shot mode with a maximum firing

rate of 3 shots min-1. An iris diaphragm, calibrated in 2 mm increments

between 2 mm and 18 mm, was inserted into the laser cavity between the

dye cell and the output coupler to reduce the diameter of the laser beam

and focal spot. Pulse energy, measured with a volume absorbing energy

meter (Gen-tec, Ste-Foy, Quebec, Canada), was 0.65 J, 1.49 J, and 1.64 J

for driver voltages of 16 kV, 18 kV and 20 kV, respectively.

Laser-mass spectrometer interface. The side flange of the TSQ was

machined according to the specifications of Fig. 3.2.a to accommodate a

1.5" diameter fused silica lens (focal length: 12.5 cm). Three spacers

(Fig. 3.2.b) were also machined to allow adjustment of the point of

Figure 3.2

Diagrams for machining (a) blank side flange of TSQ to hold 1.5"
diameter fused silica lens, and (b)spacers for adjusting focal
point of lens within ion source. Measurements marked are:
(A) 4.5 in.; (B) 1/4 in.; (C) 7/8 in.; (D) 5/8 in.; (E) 1 in.;
(F) 1/4 in. (G) 1/8 in., 3/16 in., and 1/4 in. (3 spacers).

focus over a 9/16" range in 1/16" steps. As it happened, the focal

point was about 1/16" from the reagent gas inlet side of the ion volume

with no spacers. Vacuum was maintained with O-ring seals. To allow

entrance of laser light into the ion source, a 1/8" hole was drilled

into the ion volume (El and CI) directly opposite the reagent gas entry

hole. Extensions to the solids probe were machined out of stainless

steel according to the specifications of Fig. 3.3 to allow insertion of

samples on electron microscope (EM) grids and angles of laser incidence

of 90 and 45. The 45 tip was later modified to allow grids to float

in the ion volume with no backing. This tip was used for the desorption

CI experiments. The dye laser was aligned (Fig. 3.4) by directing a He-

Ne laser (LI) through the dye cell (L2) with the retroreflector (MO)

removed, through the output coupler (WI) to a plane mirror (Ml) which

reflected the beam to another plane mirror (M2) mounted on the TSQ, then

to plane mirror (M3) mounted on a gimbal mount on a line axial with the

focusing lens (W2). Final adjustments in laser aiming could be made by

slight adjustments of M3 while determining the point of impact on an

electron microscope grid inserted into the ion source.

Detection systems. Short-lived events were monitored with one of

two MS detection systems. In the first, a storage oscilloscope (Model

549, Tektronix, Inc., Beaverton, Ore.) was connected directly to the

electron multiplier of the TSQ through a Type D high gain differential

amplifier plug-in, and was triggered by a TTL pulse from a photodiode-

to-TTL interface connected to the output of a fast photodiode (FND100,

EG&G Electro-Optics, Salem, Mass.). The laser light (about 8% of total

Figure 3.3

Diagrams for machining (a) 900 and (b) 450 stainless steel solids
probe extensions. The 450 extension was later modified for
desorption chemical ionizaton (DCI) by grinding the tip until it
was flat (dotted line). Measurements shown are: (A) 9/32 in.;
(B) 1/4 in.; (C) 0.0765 0.0005 in.; (D) 1/16 in.;
(E) 0.215 0.001 in.; (F) 1/64 in.; (G) 0.133 0.001 in.; (H)
13/32 in.. Depression is 7/64 in. in diameter and between 1/64
in. to 1/32 in. deep. A small leaf spring tightened by a screw
is used to hold the tabbed EM grid on the 900 tip, and a cylinder
made to just fit over the end of the 45 tip is used to secure
the grid on that probe extension.

a) 900 SOLIDS




i ;






' D T
*- H -t,


Figure 3.4

Layout of lasers and beam directing optics in relation to TSQ ion
source. Components are: (LI) He-Ne alignment laser; (L2) dye
cell of dye laser; (MO) 100% reflecting plane mirror for laser
back reflector; (Ml, M2, M3) plane mirrors for beam steering with
M3 on gimbal mount; (Sl) iris diaphragm calibrated in 2 mm
increments from 2 mm to 18 mm; (WI) sapphire window for laser
output coupler; and (W2) fused silica lens with 12.5 cm focal
length for focusing laser light inside ion source.

D; ----a--

N N~
N~ C~


t^ M3


I \

I M2





output) was reflected to the photodiode from a microscope slide placed

in the light path. Comparison of the TSQ instrument oscilloscope with

the storage scope indicated a sensitivity of 1.6 x 10-7 A V-1 for the

latter. The storage scope could not be run from the amplified

instrument signal, because the output of the amplifier contained the

instrument scope trigger signal and mass marker signals which disrupted

the storage scope signal and triggering. In the second detection

system, the TSQ data system was used for monitoring fast signals by

acquiring data in profile mode. In this mode, time resolution of 25 us

was obtained because the instrument acquires data at the maximum analog-

to-digital conversion (ADC) rate of 40 kHz. A single long scan (20 s)

of profile data was acquired during which time the laser was fired. By

setting the mass of interest with the unscanned quadrupole and turning

off the DC voltage to the scanned quadrupole, a signal of ion current

vs. time was obtained. This operation will be described in more detail

in Chapter V. The beginning of the scan was monitored by connecting an

oscilloscope (Model 2213, Tektronix, Inc.) to the positive Y-axis input

(Y+) at the rear of the TSQ scope and observing the scan signal (TSQ

scope set for multiple mass marker display) with a fast time base. Long

lived events were monitored with the TSQ data system operated normally,

but with the centroid sampling interval set at less than the normal 200

us. The centroid sampling interval is the number of ADC readings (1 to

8) summed to produce a single datum point. Therefore, with an ADC rate

of 40 kHz, the centroid sampling interval can be varied, in 25 ps

increments, from 25 ps to 200 ps with the latter being the default


High performance liquid chromatograph. A high performance liquid

chromatograph was used for quantitation of test drugs in animal samples

and to determine the quantities of sample vaporized in the ion source by

the laser. A high pressure syringe pump (Model 4100, Varian Associates,

Palo Alto, Calif.), 6-port rotary injection valve (Valco Instrument Co.,

Houston, Tex.), and a fixed-wavelength (214 nm) ultraviolet absorbance

detector (Model 1203, Laboratory Data Control, Riviera Beach, Fla.) were

used with a 4.6 mm i.d. x 250 mm stainless steel column, packed with a

5-pm particle diameter octadecyl silica gel stationary phase (Biosphere-

ODS, Bioanalytical Systems, Inc., West Lafayette, Ind.) and maintained

at 52C with a circulating water bath (Neslab Instruments, Inc.,

Portsmouth, N.H.). The mobile phase was pumped at 80 mL/h, and

consisted of acetonitrile:tetrahydrofuran:water (28.5:0.5:71) with

potassium dihydrogen phosphate added at a concentration of 2.7 g L-1


Laser system evaluation. The laser system was evaluated with

Coumarin 2 and Rhodamine 6G laser dyes (Eastman Kodak, Rochester, N.Y.).

The coumarin lased at about 450 nm, however, the intensity was low and

the useful life of the dye was only a few shots. Rhodamine 6G lased at

about 590 nm with high pulse energy and a single dye solution allowed

operation for several hundred shots. Pulse shapes were measured for

various laser cavity diameters at 16 kV, 18 kV and 20 kV by monitoring

the signal directly from the fast photodiode.

Laser-TSQ evaluation. The laser-mass spectrometer system was

evaluated with respect to short-lived and long-lived laser-initiated

processes. The short-lived events (ps to ms) were observed with an open

electron impact ion source at high vacuum, while long-lived processes

(ms to s) occurred with a closed chemical ionization source with reagent

gas present at greater than 0.35 torr. Phenytoin, a relatively

nonvolatile (m.p. 295 298C) drug, which was also used in the animal

experiments, was used in most of the system evaluations, since it was

readily available as the free acid and the sodium salt, and proved to be

a suitable test substance. A series of compounds (Appendix) having

various functional groups and molecular weights was also tested under

desorption chemical ionization conditions (110) in which only the source

block was heated in most cases, under normal direct probe conditions,

and under laser vaporization conditions to determine the versatility and

utility of the present system. In all cases, samples were inserted into

the source as solids packed into a depression in the stainless steel

probe tip or as residues from solutions evaporated on copper, nickel or

silver electron microscope grids (Ted Pella, Inc., Tustin, Calif.).

Preparation, collection and analysis of animal samples. To provide

animal tissue samples suitable for laser analysis, two adult male rats

were given a loading dose (150 mg/kg) of phenytoin intraperitoneally

75 min before decapitation. At the time of sacrifice, the animals were

ataxic and lethargic, indicating substantial drug absorption. Brains

and livers were removed and washed in isotonic phosphate buffer. One

set was frozen at -20C. The other set was fixed for 72 h in phosphate-

buffered glutaraldehyde (KC1, 0.0834 M; KH2PO4, 0.0127 M; K2HP04,

0.0846 M; 25% aqueous glutaraldehyde; 925 mL water). Samples from fixed

and frozen brains and livers were analyzed by high performance liquid

chromatography (111). Aliquots of homogenized weighed tissue samples

were deproteinized with acetonitrile. The aqueous acetonitrile phase

was washed with iso-octane and then poured into a tube containing

granular KC1. Vortexing caused the acetonitrile to be salted out of the

water, at the same time extracting the phenytoin and internal standard.

The acetonitrile layer was transferred to a clean tube, evaporated to

dryness, and the residue was dissolved in mobile phase for injection

into the HPLC system. Sections of the fixed tissues were frozen in

isotonic phosphate for later MS analysis.



This chapter includes discussions of (1) the LAMMA experiments with

thin slices of rat brain tissue taken from the cobalt lesion site in one

animal and a corresponding site from a non-lesioned animal, (2) the

determination of the regional distributions of phenytoin and

phenobarbital in rat brains by triple quadrupole MS/MS, and (3) the

basic characteristics of the laser-TSQ system, including laser pulse

shape and variation of diameter of laser spot size with target position

and cavity diameter.

LAMMA Experiments

The LAMMA instruments, initially the Model 500 and, more recently,

the Model 1000, were designed ideally to monitor atomic mass spectra

during the brief lifetime of laser-induced emissions from thin film and

bulk samples. The time-of-flight (TOF) mass spectrometer operates

within the time restrictions of Q-switched, single shot lasers and the

lifetimes of the ions which they produce, so that a complete mass

spectrum covering a significant mass range can be acquired in less than

100 us. After the laser fires, kinetic energy is imparted to the ions

in the source area by a high voltage accelerating pulse. This is


translated into an ion motion according to eqn. 4.1,

(1/2)mv2 = zeV


where m and v are the ion mass and velocity, respectively, z is the ion

charge, e is the electron charge, and V is the accelerating voltage.

The time of ion flight is determined by the length of the flight tube

(L) and the ion velocity (eqn. 4.2).

t = Lml/2/(2zeV)1/2


Since m is equal to the molecular weight divided by Avogadro's number

(6.023 x 1023) and the voltage in electron volts (eV) is the

accelerating voltage times the charge per molecule (ze), for singly

charged molecules with time measured in seconds, eqn. 4.2 becomes,

t = 7.199 x 10-7(L/V1/2)m1/2

t = cmI/2



where c is the instrumental constant, incorporating unit conversion

factors, length of the flight path and instrument accelerating voltage.

Table II gives the masses, their square roots and abundances for the

Table II. Masses and Flight Times for Nickel, Molybdenum and Lead
Isotopes in LAMMA Calibrant

Isotope Ion Mass (u) Ion Massl/2 % Abundancea dpb






tc dtd


a) % Abundance of naturally occurring isotopes.
b) dp = Chart distance from recorder start to peak in mm.
c) t = Total flight time in us.
d) dt = Total chart distance (dp + delay distance) in mm.


calibrant isotopes (Ni, Mo, Pb), and the distance and time data derived

from the calibration spectrum. The flight times (t) were calculated as

the sum of chart time (tc) plus the delay time (td) with

tc = nts(dp/D) (4.5)


td = ndts (4.6)

where n and nd are the total number of chart channels (2048) and the

number of delay channels, respectively, ts is the sampling interval, and

dp and D are the chart distance to the peak and total chart distance

covered by 2048 channels (393.0 mm at 0.020 ps sampling interval),

respectively. Regression analysis of flight time (ps) vs. the square

root of the ion mass indicated a 2.563 us lag (Y-intercept) between the

start of recorder timing and the ion acceleration pulse. The slope of

this plot (Fig. 4.1) gave the instrument constant, 3.944 ps u-1/2

Using this value with eqns. 4.3 and 4.4 gave a flight path length of

300.2 cm.

Figure 4.2 shows the calibration spectrum (A) with spectra acquired

from the lesion site (B) and the control site (C). Although the peak at

m/z 59 (*) is higher in the lesion site than in the control sample, it

is impossible to say if this signal is actually due to cobalt. The

presence of large peaks at almost all masses throughout this range

Figure 4.1

Plot of flight time vs. the square root of ion mass for LAMMA
calibrant (data from Table II). Data points cover all isotopes
for each metal (Ni: 58, 60; Mo: 92, 94, 95, 96, 97, 98, 100;
Pb: 206, 207, 208).

6.0 9.0

TIME (vs)








Figure 4.2

LAMMA spectra for (A) nickel-molybdenum-lead calibrant, (B)
section of rat brain from cobalt lesion site, and (C) section of
rat brain from corresponding site in control (unlesioned) animal
(m/z 59 designated by *).

precludes any accurate assessment of elemental or molecular composition.

These results point out the advantage of having two stages of mass

analysis (filtering and selection) for microprobe analysis. By

increasing the laser power density, the chemical noise from molecular

fragments could be decreased, but it would still be impossible to

definitely identify the ion at m/z 59 as cobalt since there is only one

naturally occurring isotope. Uncertainty of ion identification becomes

greater as mass increases because the number of possible elemental

compositions also increases. This deficiency is made obvious in a recent

report on LAMMA analysis of coal and shale samples (112). The laser was

fired at grazing incidence at 1/8" diameter chips, and ions produced

directly by the laser were recorded. Although the authors gave

explanations of the spectra and made mass assignments, primarily on the

basis of natural isotopic abundances and past literature reports of

laser pyrolysis gas chromatography and mass spectrometric studies of

homopolymers, a second stage of mass analysis would have added greatly

to the certainty of some of the assignments.

Regional Distributions of Phenobarbital and
Phenytoin in Rat Brain

At the time this project was started, very little quantitative work

on real biological samples had been done by MS/MS. A quantitative study

of typical samples amenable to analysis by other methods (i.e., gas-

liquid or high performance liquid chromatography) was considered to be a

necessary starting point, to define instrumental conditions,

particularly with respect to the CAD behavior of analytes, which would

give accurate, reproducible results. Since one of the primary

advantages of MS/MS is elimination of the need for pre-MS

(chromatographic) separation of mixture components, demonstration of

reliable analysis by direct vaporization of samples (extracts) from the

solids probe was also considered important.

Rapid vaporization of the entire sample from the solids probe vial

with as little temporal separation of analyte and internal standard

evaporation profiles as possible was found to be of primary importance.

Separation of the analyte and internal standard ion signals (because of

differences in their vaporization temperatures) caused the response

ratio to be dependent on the number of scans over which the signal was

summed. To minimize this effect, a heating rate of about 100C s-1 was

employed. This generally resulted in complete sample vaporization

within a 30 s period with a near-Gaussian ion current profile. Analyte-

internal standard pairs vaporized simultaneously within about 20 s (Fig.


Response ratios for drug and internal standard ion currents were

also dependent on instrument tuning, so that long-term (day to day)

reproducibility of calibration curve slopes was poor; however, linearity

was good, with correlation coefficients generally between 0.996 and

0.999. Figure 4.4 shows a histogram of the results for phenobarbital

and phenytoin in plasma and various brain regions. The numbers in

parentheses beside each bar give the number of samples (before slash)

Figure 4.3

Plots of ion current vs. time for direct solids probe
vaporization and selected reaction monitoring of rat brain
extract from an animal that received phenobarbital (m/z 233 to
m/z 162) and phenytoin (m/z 253 to m/z 182). Other signals are
from internal standards, p-methyl phenobarbital (m/z 247 to m/z
191) and p-methyl phenytoin (m/z 267 to m/z 196). RIC is
recombined ion current.

607 1 233' 162+

23201 253+ -182+

241281 247+ 191

30400 267+ 196

27601 233+ 233

249281 247+ 247+

845] 253+ 253+

51681 267+ 267

490241 RIC -

10.0 20.0 30.0 40.0
TIME (s)

Figure 4.4

Histogram showing concentrations of phenytoin and phenobarbital
in various areas of rat brain (ug/g) and in plasma (ug/mL).
Number before slash indicates the number of samples taken and the
number after slash shows the number of animals from which those
samples were obtained. See caption Fig 4.3 for reactions
monitored during analysis.




CORTEX (4/2)



CORPUS (6/3)


CORTEX (2/1)




10 20 30 40 50


60 70

taken from the number of animals (after slash) sacrificed. Regional

distribution of phenobarbital is relatively even. Although phenytoin

appears to be slightly more concentrated in white matter and less

concentrated in basal ganglia and hippocampus, it also is fairly evenly

distributed. Concentrations in structures that are possibly linked with

epileptic activity or drug effects (hippocampus, cerebellum,

sensorimotor cortex) are not greatly different from those in areas that

are not linked with seizures (pontine white matter, occipital cortex).

The averages of the histogram values for all structures are

38 + 2 ig g-1 and 11 + 3 jg g-l for phenobarbital and phenytoin,

respectively; brain to plasma ratios for these averages are 0.568 and

1.73, respectively.

The low relative standard deviations for these samples, 5.6% for 50

phenobarbital determinations, and 20% for 30 phenytoin samples, indicate

that monitoring analyte daughter ion intensities for selected reactions

with respect to those of suitable internal standards is a valid

technique for direct analysis of plasma and brain extracts by triple

quadrupole mass spectrometry. The response ratios are made more

reproducible by rapid, complete vaporization. This was assured by rapid

heating of the solids probe in this experiment. In the instrument

under consideration, rapid heating and sample vaporization would be

affected by using a pulsed laser as a sampling device.

Laser-Triple Stage Quadrupole Characteristics

Variation of Focal Spot Size with Distance from Focal Point and

Laser Cavity Diameter.

The laser beam, which is about 20 mm in diameter when emanating

from a totally open cavity, was reflected from a flat mirror 24 inches

in front of the output coupler to the 1.5 inch diameter fused silica

plano-convex lens used as the laser window in the TSQ. The target was a

black piece of cardboard that was placed from 12 mm behind the focal

point to 18 mm in front of the focal point. Spot size varied from

3.2 mm at the former to 5.0 mm at the latter and was 1.3 mm at the focal

point (Fig. 4.5). Spot size initially increased more slowly with

distance in front of the focal point. The cavity diameter was varied by

placing an iris diaphragm between the dye cell and the output coupler.

The target was located 4 mm in front of the focal point of the lens.

Spot size was 1.7 mm, 1.0 mm and 0.5 mm with iris openings of 18 mm,

12 mm and 6 mm, respectively. Below 6 mm the spot size was too small to

be measured accurately, but a flash could still be seen at the target

with the cavity diameter set to 2 mm. Normal cavity diameters used in

this study were 6 mm to 10 mm, depending on the age of the flashlamp and

laser dye solution. Focal spot sizes for these settings were 0.5 mm to

1 mm.

Figure 4.5

Variation of focal spot diameter with distance in front of (+,
toward) and in back of (-, away from) focal point for 1.5 in.
diameter, 12.5 cm focal length fused silica lens used for
focusing laser in ion source of TSQ.

Laser Pulse Shape and Duration.

Laser pulse shape and duration were measured using the output from

the fast photodiode to drive the storage oscilloscope directly. Figure

4.6 shows the tracings produced by varying the cavity diameter (2 to

12 mm) at 16 kV, 18 kV and 20 kV. In all cases, the light pulse was

reflected from two microscope slides to the photodiode so that all

pulses remained on the oscilloscope scale (5 V cm-1). Therefore, only

about 0.6% of the laser light was incident on the photodiode (8%

reflected from each slide). The time base of the scope was set at

2 ps cm-1, indicating a pulse width (full width at half maximum) varying

between 1 ujs and 3 us and depending on cavity diameter and driver

voltage. At 18 kV and cavity diameters of 6 mm to 10 mm (normal

settings), pulse width was 1.5 ps to 2 ps.

Figure 4.6

Laser pulse shapes from photodiode signal as monitored by storage
oscilloscope at driver voltage of (A) 16 kV, (B) 18 kV, and (C)
20 kV. Cavity diameter was varied from 2 mm to 12 mm in 2 mm
increments giving curves running from low intensity to high
intensity in each figure (drawn from photographs of storage
oscilloscope screen).






2 4 6 8
TIME (us)





2 4 6 8




2 4 6 8
TIME (ps)



Although the quadrupole mass filter scans the fastest of all the

scanning mass analyzers, normally covering 1000 to 2000 mass units each

second, it is still relatively slow when compared to the ion lifetime of

pulsed laser-generated ions. In spite of this incompatibility, an

attempt to observe the events immediately following the laser pulse was

thought to be desirable for this study for two reasons. First, the

laser used in this study was a single shot device (1.5 to 2 ius

pulsewidth) and prolonged signals were not expected, and second,

observation and verification of normal laser-induced processes such as

cationization required mass discrimination and time resolution in the us

range. This chapter will present results of experiments conducted using

an electron impact ion volume, modified to transmit the laser light, and

operated without reagent gas. Under these conditions, ion signals

lasted less than 1 ms and were monitored either with a storage

oscilloscope or with the TSQ data system. Limited mass discrimination

was maintained by selecting the mass of interest with Q1 and, with no

collision gas in Q2, selecting a range of masses which may or may not

include the mass of interest with Q3 operated in RF-only mode.

Triple Stage Quadrupole Control

The goal in these experiments was to have the TSQ operate as a

time-of-flight mass spectrometer for the selected ion, allowing data to

be obtained at a single m/z during the course of an experiment. This

had been accomplished in a single stage quadrupole instrument by Hardin

and Vestal (80), who used a pulsed laser with single ion counting.

Setting the analyzer to pass the selected ion mass, and increasing the

instrument resolution until only one event was recorded for each laser

pulse, they collected data until a statistically significant

distribution of ion flight times was acquired for each mass of interest.

The TSQ is electronically configured to operate with at least one

analyzer quadrupole scanning a range of masses (although that range may

be 1 u wide) in all modes. A system having both quadrupoles set to pass

a single mass had not been defined in programmable read only memory

(PROM). Since the laser used in this experiment was a single shot

device, the instrument had to be set up to provide a continuous signal

for the ion selected, with time resolution of a few microseconds. This

could have been done either by determining and adding the appropriate

instruction or instructions to the PROM to allow each analyzer to be set

to pass a single mass without scanning, or by causing the scanned

quadrupole to operate in the RF-only mode to allow a range of masses to

pass simultaneously rather than sequentially. Since modifying the PROM

could have had far-reaching and unforeseen effects on other parts of the

TSQ system, elimination of the DC voltage from the scanned quadrupole

was selected as the best way to solve the problem. This was

accomplished by changing the rod polarity switch located at the rear of

the quadrupole electronics module (QEM) from "+" to "off". This is a

three position switch ("+", "off", "-") that allows the operator to

change the DC polarity on the quadrupole rods. Under normal conditions,

there is no difference in response if the switch is set to "+" or "-"

The "off" setting removes the DC component from the rods. The range of

masses passed under these conditions can be determined from the

stability diagram for the quadrupole. The stability diagram (Figure

5.1) is a plot of the ratio of DC to RF voltages imposed on the

quadrupole rods vs. a function including the RF frequency, a dimensional

constant for the quadrupole (one-half the diagonal distance between the

rods in cm), the RF voltage, and the inverse of the mass-to-charge ratio

of the ion. Under normal conditions, the voltage ratio is set so that

only the mass (+0.5 u) under the apex has a stable trajectory through

the quadrupole. All other ions have unstable trajectories and are lost

by collisions with the rods. If the DC voltage component is removed,

all ions that fall under the curve have stable trajectories and will

pass. The ratio of the ordinate value under the apex (1/ml) to that at

the low-mass abscissa zero (1/m2) is about 7/9. Therefore, if an RF-

only quadrupole is set to pass ml, it will actually pass all masses

greater than m2 which equals 7/9 of mi.

Table III gives the effects on response for the most pertinent

combinations of operation mode (parent and daughter ion experiment),

programmed RF function (on/off) and rod polarity switch (+/off). Other

Figure 5.1

Stability diagram for quadrupole mass filter. Plot of DC to RF
voltage ratio vs. a function proportional to (m/z)-l. Shaded
region shows range of ion masses passed through quadrupole set to
pass mi with both RF and DC voltages imposed (normally, mi 0.5
u). The RF-only quadrupole will pass all masses higher than m2
where m2 = (7/9)m1. Parameters in the ordinate function are: RF
voltage, Vrf; quadrupole rod spacing (diagonal), ro; electron
charge, e; angular frequency of Vrf, w ; mass-to-charge ratio,

Table III. Effects of Programmed RF
During Various TSQ Operational Modes


Function and Rod Polarity Switch Settings

Operation Programmed Rod Polarity
Mode RF Switch
Q1 Q3


1 Daughter

2 Daughter

3 Daughter



on off

6 Parent

+ Daughter spectrum. Increasing Ql first
mass removes low m/z (all above 7/9 Q3
first mass passed).

off High broad signal. Low m/z and signal
intensity controlled by Q1 first mass.

off Broad peak displayed only when Q1 first
mass set on peak.

+ Parent spectrum. Increasing Q3 first mass
removes low m/z (all above 7/9 Ql first
mass passed).

+ Broad signal. Signal intensity 75% of
that with Ql rod polarity +.

+ Broad peak displayed only when Q3 first
mass set on peak. Signal intensity less
than that in daughter mode.

combinations were not listed because they were unremarkable or had no

bearing on the present discussion. The programmed RF function removes

the DC component from the unscanned quadrupole. The effects were

observed with the instrument under manual control with perfluoro-

tributylamine (FC43) being introduced continuously into the El source.

Effects that include a measurement of relative signal intensity for the

rod polarity switch on "+" or "off" were made by observing m/z 69 on the

instrument oscilloscope. Loss of mass resolution is expected when the

scanned quadrupole is operated in RF-only mode. Condition 3 was selected

for most of the work on short-lived processes because Q1 provides mass

discrimination and Q3 can be set up to pass or not pass the ion selected

by Q1. The advantage to this condition is that a long scan (20 s) can

be acquired to eliminate the delay time of a few milliseconds normally

required between scans. Further, since Q3 is not resolving masses, this

mode will provide an ion intensity vs. time response for the selected

ion rather than an ion intensity vs. mass response. In addition, the

beginning of the scan can be detected by an oscilloscope attached to the

"Y+" input of the TSQ oscilloscope. This is important for monitoring

fast processes with the data system because data acquisition begins at

the first regular scan after the experiment is initiated at the computer

terminal. Data acquisition does not begin immediately because the scan

sequence is not interrupted and restarted. Condition 6 is analogous to

condition 3 but Q1 is the scanned quadrupole (parent mode). Signal

intensities were generally lower for this condition, however, the major

drawback was that the scan signal was not visible at the "Y+" input as

it was for the daughter mode.

Comparison of Storage Oscilloscope and Data System

Signal for Ni+ from Nickel EM Grid

With the TSQ operating in daughter mode with Q3 rod polarity switch

off (condition 3), Q1 passing m/z 58 (Ni+) and Q3 passing all ions above

m/z 56 (scanning from m/z 71 to 72 in 20 s with rod polarity switch

off), the laser was fired at a clean nickel electron microscope grid

inserted into the ion source on the 90' probe tip. The storage scope

was attached directly to the electron multiplier through a high-gain

differential amplifier module (Type D, Tektronix, Inc.) operated at

1 my cm-1 with a time base of 50 jis cm-1. In Figure 5.2.A, traces 1 and

2 show response with the laser light blocked from the ion source and the

light admitted to the source, respectively. Trace 3 was obtained by

switching Ql to pass m/z 90 and Q3 to pass all masses above m/z 140

(scanning m/z 180 to 181 in 20 s), so that Ql and Q3 would be mutually

exclusive and neither quadrupole should pass any ion produced by

vaporization of the nickel target. In figure 5.2.B, traces 2 and 3 are

analogous to traces 2 and 3, respectively, in part A, except that the

response was monitored with the TSQ data system by acquiring in profile

mode. Also, Q3 was scanned from m/z 60 to 70 (passing all m/z above 47

to 54) for trace 2 and from m/z 170 to 180 (passing all m/z above 132 to

140) for trace 3. These data show that this method of recording

Figure 5.2

Plots of total ion current vs. time showing (A) storage scope
data and (B) profile data for nickel EM grid target. In A, trace
1 is the response with laser light blocked from ion source;
trace 2 in A and B is the response with Ql passing m/z 58 and Q3
passing all m/z above 56 (A) and all m/z above 47 to 54 (B).
Trace 3 in A and B is the response with Q1 passing m/z 90 and Q3
passing all m/z above 140 (A) and all m/z above 132 to 140 (B).
See text for explanation of differing Q3 scans for storage scope
and data system operation. Ion currents are given as counts


0.1 0.2
TIME (ms)

0.3 0.4




TIME (ms)


intensity vs. time data for fast events may be valid, that quadrupole 1

provides some mass discrimination, even for low mass elemental ions, and

that the storage scope and the data system give equivalent data. There

is always a minimum signal (rapidly decaying spike) observed by the data

system even when no sample is in the ion source, the laser light is

blocked before it reaches the source, and the electron multiplier is

off. This response is probably due to electrical interference from

discharge of the laser storage capacitor when the flashlamp is fired.

Instrumental Acquisition and Display Characteristics

It might be helpful at this point to describe the TSQ scan

parameter (SYSTem, System Status, Multiple Ion Detection), calibration

(CALIrate), acquisition (ACQUire), and display (PROFile, mass

CHROmatogram, and SPECtrum) programs. The above programs will,

henceforth, be referred to by the abbreviations used to access them

(underlined). Data can be acquired in two modes, centroid and profile.

Both are calibrated using the CALI program which establishes a linear

relationship between mass and time elapsed since start of a scan.

Therefore, even under normal circumstances, the mass of an ion is

determined by the appearance time after the scan starts, and the

spectrum is displayed as ion intensity vs. mass. Acquisition in profile

mode causes storage of all points detected at the maximum sampling rate

(25 js). Storage requirements are maximized, but a relatively

continuous plot of ion intensity vs. time is produced, allowing

observation of peak shapes. It is this mode that has been used in these

experiments on short-lived processes (Chapter V) to obtain a plot of


signal intensity vs. time. A threshold intensity can be chosen (in ADC

units), which allows immediate rejection of points having intensities

less than the selected threshold. Thresholding is the only decision

made by the data system at the full 40 kHz ADC rate. A setting of 1

means all data is recorded (no thresholding). For these experiments,

the threshold was routinely set at 2 to eliminate spurious electronic

noise. Therefore, a scan 20 s long, during which a signal is produced

for a brief instant by a laser shot, would result in a profile lasting

only from the time the signal exceeds threshold until it goes below

threshold. PROF is the program used to display profile data. Although

the program converts time to mass units, it plots essentially a time-

dependent, ion intensity curve, and calculates the time the signal is

above threshold. The plots of profile data shown in the figures were

calculated from these curves. Since time scales were usually different

for each profile curve, signal intensities were replotted on a common

time scale for comparison. The maximum intensity in a profile plot is

4096 counts, as determined by the 12-bit ADC. Q3 scan parameters were

most easily set using the MID program. Q1 mass was set using the SS

program. In this way, experiments could be run rapidly by writing the

required MID descriptors, and calling them as needed from SS. Q1 mass

was changed as required, also from SS.

Acquisition in centroid mode (used for experiments on long-lived

processes in Chapter VI) causes calculation and storage of the mass

centroid of a peak, minimizing storage requirements and making data

manipulation faster. In this mode, the centroid sampling interval is

chosen (using MID) in 25 ps increments between 25 js and 200 ps to give

a reasonable number of data points per mass peak. Therefore, data is

acquired at 40 kHz, and 1 to 8 data points are summed to give a single

datum point to be used in calculating the peak centroid. If a scan is

set up in SS, the highest possible centroid sampling interval (200 ps)

will be used. In SYST, a selection is available for centroid samples

per peak, however, the number actually used will be determined by the

mass range covered in the scan time. The only way to insure use of a

particular centroid sampling interval is to set that interval in MID.

The CHRO program is used to view the recombined ion current (RIC), or

the intensity profiles for each ion with respect to scan number (time).

This is analogous to the use of the PROF program for viewing short-lived

processes; however, an additional dimension is available (in the SPEC

program), which allows display of the mass centroids making up the RIC

(mass spectrum). Scan parameters for acquisition in centroid mode

(Chapter VI) were again set up using the MID program, both for normal

mass spectra and, as required, for parent and daughter ion spectra.

The centroid sampling interval was chosen so that scan time could be

minimized, and there would be at least 5 points/mass peak for

calculation of the peak centroid.

Filtering Characteristics of Quadrupoles 1 and 3

Although the quadrupole is supposed to effectively filter out all

ions of unselected mass regardless of ion energy, it is possible for

ions of inappropriate mass to pass through the quadrupole field without

deflection if their kinetic energy is high enough (109, 113, 114).

Therefore, a series of experiments was performed to determine the

filtering capabilities of the system under the conditions used for time

profile monitoring.

Figure 5.3.A and B show a comparison of storage scope and profile

data, respectively, for phenytoin. Ql was set to pass m/z 275, (M+Na)+

for phenytoin, and Q3 was scanned from m/z 343 to 353 in 18 s with the

rod polarity switch off (passing all m/z above 266 to 274). The

phenytoin (4 pL of a solution of 4.35 mg/mL) was layered over a Na2C03

residue (2 uL of a solution of 25 mg/mL) on a 400-mesh copper electron

microscope (EM) grid which was inserted into the ion source on the 90

probe tip. Trace 1 shows the first shot. Trace 2 is a second shot at

the same site. Although this figure shows an intense signal for

cationized phenytoin and significant reduction for a repeat shot (the

grid was totally disintegrated at the point of impact of the first shot

creating a hole about 0.5 mm in diameter), the signal is not necessarily

due to cationized phenytoin alone. Figure 5.4 shows responses for a

similar sample for which Ql was set to pass m/z 150 and Q3 was set to

pass all m/z above 202 to 210 (scanning m/z 260 to 270 in 18 s with rod

polarity off). In this case Q1 and Q3 are mutually exclusive and Q1 is

not passing an ion that should be produced by the sample; however, Q3 is

covering a mass range that includes ions that should be present. Trace

1 shows a shot at the center of the grid where the phenytoin

concentration, due to uneven evaporation of the methanol, was lowest.

Solids were usually concentrated in a ring describing the edge of the

sample droplet. Traces 2 through 4 show successive shots at the same

Figure 5.3

Plots of total ion current vs. time showing (A) storage scope
data and (B) profile data for phenytoin over Na2CO3 on copper EM
grid. In A and B Q1 is passing m/z 275 and Q3 is passing all m/z
above 267 to 275 (scanning m/z 343 to 353 in 18s with rod
polarity switch off). In both A and B, trace 2 is the response
from a second laser shot at the same site that gave trace 1.

0.2 0.4 0.6 0.8
TIME (ms)



2000 -

0.4 0.8
TIME (ms)

Figure 5.4

Plots of total ion current vs. time showing the efficiency of Ql
and Q3 mass filtering (profile data) for phenytoin over Na2CO3 on
copper EM grid. In traces 1-4, Q1 is set to filter out all m/z
produced by the sample (passing m/z 150) and Q3 is set to pass
ions that should be present (passing all m/z above 202 to 210.
In trace 5, neither quadrupole is passing ions produced by the
sample and they are set to be mutually exclusive (QL passing m/z
700 and Q3 scanning normally with rod polarity "+" from m/z 300
to 310. Trace 1 is at grid center where phenytoin concentration
is low. Trace 2 is at edge of droplet where concentration is
highest. Traces 3 and 4 are successive shots at site 2. Trace 5
is from an identically prepared grid at the edge of sample





TIME (ms)

site on the edge of this ring where concentration was highest. These

traces indicate that Q1 is not totally efficient at filtering ionic

species. Trace 5 was recorded with Ql set to pass m/z 700 and Q3

scanning from m/z 300 to 310 with rod polarity "+" (DC on) so that both

quadrupoles were mutually exclusive and neither was passing ions that

were produced by the sample. A minimum signal, generally present under

any conditions, was produced. This indicated that Ql and Q3 effectively

filtered out sample ions when set to pass masses that differed greatly

from the sample ion masses. Another experiment was conducted to

determine whether the filtering capabilities of Ql were lessened when

the selected mass was close to the mass of an ion produced by the sample

(i.e., is unit mass resolution maintained under the conditions produced

by laser desorption in the present ion source). Ql was set to pass m/z

275 and then, with a new grid, m/z 270 (test point arbitrarily chosen

5 u away from the ion mass), with Q3 passing all m/z above 202 to 210.

The signal intensity ratio for the former with respect to the latter was

about 5, indicating that unit mass resolution in Ql is not maintained

under these conditions. The particles that are causing these unwanted

responses may be fast ions or neutrals which do not "see" the low

voltage fields of the lenses and quadrupoles. Discrimination of a

selected ion mass from its nearest neighbors is less efficient than from

ions that have masses much greater or much less than the selected mass.

This problem was approached from another angle to show the total

ion signal with all quadrupoles operated in RF-only mode, and to

contrast that with the signal obtained when QI was operated as a mass

filter. Q1 was set to pass m/z 275 and Q3 to pass all m/z above 266 to

274 with the sample again phenytoin over Na2CO3. Successive shots were

fired at the same site (Figure 5.5.A, traces 1-3). Ql was then set to

pass m/z 350 and the programmed RF was turned on so that Q1 would

actually pass all m/z above 272. Two shots were fired at the same site

(new grid; Figure 5.5.B, traces 1 and 2). Programmed RF was then turned

off so that Ql would then only pass m/z 350 (an ion not produced by the

sample) and a single shot was fired at a new grid (Figure 5.5.B, trace

3). These figures show that Q1 does actually filter out a large portion

of the total signal that is possible under these conditions. Therefore,

the combination of quadrupoles 1 and 3 can be used to obtain limited

mass resolution in this rapid acquisition mode to allow monitoring of

the fast signal profiles.

Time Resolution of Low-Mass and High-Mass Ions

With these limitations in mind, an attempt was made to observe the

resolution of two compounds by time alone by operating the instrument in

a time-of-flight mode without mass discrimination in either analyzer

quadrupole. Q1 was set to pass m/z 160 with programmed RF on (actually

passing all m/z above 124), Q3 was set to pass all m/z above 117 to 124

(scanning m/z 150 to 160 in 18 s with rod polarity off). Data were

acquired with the TSQ data system in profile mode. The sample was a

mixture of 4-vinyl-4-aminobutyric acid (vinyl gaba, mol. wt. 129) and

reserpine (mol. wt. 608) over Na2CO3. Expected cationized species were

m/z 152 and m/z 631, respectively. All quadrupole offset voltages and

lens voltages were set at -20 V. The flight path length from the ion

University of Florida Home Page
© 2004 - 2010 University of Florida George A. Smathers Libraries.
All rights reserved.

Acceptable Use, Copyright, and Disclaimer Statement
Last updated October 10, 2010 - - mvs