A quadrupole ion trap laser microprobe for the mapping of pharmaceutical compounds in intact tissue

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A quadrupole ion trap laser microprobe for the mapping of pharmaceutical compounds in intact tissue
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A QUADRUPOLE ION TRAP LASER MICROPROBE FOR THE MAPPING
OF PHARMACEUTICAL COMPOUNDS IN INTACT TISSUE














By

FREDERICK J. TROENDLE


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


UNIVERSITY OF FLORIDA


2000














ACKNOWLEDGMENTS


More than anyone else, I wish to express my thanks to my wife,

Lorraine, without whom none of my accomplishments would have been

possible or had any real meaning. More than anyone else, she has supported

me and stuck by me as I pursued my wild dream of attaining a Ph.D. in

chemistry. I have no idea what she sees in me, but I hope that in some small

way I can come close to being who she believes I am.

I would also like to thank my parents, Gloria and Frank Troendle.

They had seven children, yet I never felt that I was just one of the crowd.

Despite the fact that I was the second born and the last in my family to achieve

a college degree, they never treated me like the black sheep that I truly was.

Their dedication to science, and more importantly to learning and

understanding anything which was interesting, has always inspired me to seek

out answers to things I did not know.

At the University of Florida, there are many to thank. First, I would

like to thank Rick Yost, my research adviser, for taking a chance on a student

who was nearly his own age with no real mass spectrometry experience. Rick

gives as much guidance as requested and as much leeway as needed by










each student. His encouragement and guidance will be sought long after I

have left the University of Florida.

Within the group of people I worked with at U.F., first, I wish to thank

Christopher D. Reddick. Another Silver Spring, Maryland lad (like myself),

Chris helped me get up to speed on Tubby so that I could do what was

necessary to graduate. Of course, Chris also put together Tubby in its original

incarnation, so everything I have done is simply a continuation of Chris's

original work. I would also like to thank Joe Mulholland, who came into the

group with me in 1996 and was always willing to debate (others call it argue)

with me about any trivial point of science without taking it personally (which

reminded me of my family). I also need to thank Brody Guckenberger for

answering every question I had about mass spectrometry and for just being

Brody. Joe McClellan deserves my thanks for his help with mass

spectrometry and anything related to U.F. and graduate school. Former group

members, Scott Quarmby, Jon P. DeGnore, and Steve Boue, all helped me

during the first two years at U.F. and deserve my thanks. Current members (in

addition to those mentioned above) who I wish to thank include James Murphy

(for thinking that I knew enough about mass spectrometry to ask me

questions) and Kevin McHale (for asking and answering questions and always

being there and willing to help out).

I would like to thank Todd Prox of the machine shop for his endless

patience and help in building a new, improved Tubby. I would never have










been able to do what I did without Todd's help. Also, I thank Joe Shalosky,

the machine shop supervisor, who bailed me out of several situations which

could have been disasters.

Finally, I wish to thank Bristol-Myers Squibb and the Florida Space

Grant Consortium for their financial support. Of course, no dissertation would

be complete without a thanks to Randy and the crew at Burrito Brothers for

making lunch what it was.














TABLE OF CONTENTS
page
ACKNOW LEDG M ENTS ................................................................................... ii

ABSTRACT....................................................................................................... vii

CHAPTERS

1 INTRO DUCTIO N .......................................................................................... 1

Mass Spectrometry Microprobe Instruments.......................................... 2
Laser M icroprobe M ass Spectrom etry............................... .......................... 10
Q uadrupole Ion Traps........................................ .................................... 12
History.................................................................................................... 12
Ion Trap Theory...................................................................................... 23
Overview of Dissertation.............................................................................. 32

2 INITIAL STUDIES .................................................................. ....................... 35

Overview of Instrum ent ................................................................................. 35
MALDI ...................................................... ................................................... 39
History.................................................................................................... 39
Theory of M ALDI.................................................................................... 41
Desorption ............................................................. ..................... 41
Ionization .................................................... ...................................... 56
Prim ary ion form ation ............................................................. ..... 57
Secondary ion form ation ................................................................... 63
Practical Aspects of MALDI on Tissue ................................................... 64
MALDI of Paclitaxel in Tissue............................................. ............ ............. 65

3 LASER DESORPTION/CHEMICAL IONIZATION .................................. 73

LD/CI.................................................................................................. 74
Laser Desorption ............................................................... ..................... 74
Chem ical Ionization.............................................................................. 79
LD/CI of Spiperone in Intact Tissue............................................... ........ 84
The X,Y-Stage.............................................. ............................................... 94
LD/CI w ith Spatial Resolution................................................................... ... 113





V









4 ELECTROSPRAYING OF THE MALDI MATRIX.......................................... 120

M A LD I D rop M ethod.................................................................................... 120
Electrospray A pparatus ............................................................................... 124
Electrospraying of the MALDI Matrix........................................................... 131
Infrared M A LD I ............................................................................................ 135

5 APPLICATION OF ELECTROSPRAYED MALDI MATRIX SOLUTION
AND LD/CI TO THE MAPPING OF PHARMACEUTICAL COMPOUNDS
IN INTACT TISSUE SAMPLES..................................................................... 153

S p ipe ro ne .................................................................................................... 15 3
Experimental Pharmaceutical Compound ................................................... 164
P a clitax e l ..................................................................................................... 17 8

6 CONCLUSIONS AND FUTURE WORK ....................................................... 195

LIST OF REFERENCES................................................................................... 207

BIOGRAPHICAL SKETCH ............................................................................... 215



























vi













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

A QUADRUPOLE ION TRAP LASER MICROPROBE FOR THE MAPPING
OF PHARMACEUTICAL COMPOUNDS IN INTACT TISSUE

By
Frederick J. Troendle

August, 2000

Chairman: Richard A. Yost
Major Department: Chemistry

Research was conducted on a custom built quadrupole ion trap laser

microprobe instrument (constructed at the University of Florida). Initial studies

with the instrument showed that it was capable of detection of pharmaceutical

compounds in intact tissue masses at trace levels by use of matrix-assisted

laser desorption ionization (MALDI); however, determining the exact spatial

location of those compounds within the tissue mass was not possible by

simple application of the MALDI matrix solution onto the surface of the tissue

(the MALDI-drop method).

An X,Y-micro-manipulation stage was constructed so that spatial

information about the location of drug compounds in intact tissue masses

could be obtained. The studies showed that, within half the diameter of the

probe tip (2 mm), all spatial information about the location of a drug compound

was lost during the MALDI-drop method. In order to preserve the spatial









distribution of drug compounds in intact tissue samples, two alternative

methods were investigated.

The first alternative method investigated to preserve the spatial

distribution of the drug compounds within intact tissue samples was laser

desorption coupled to chemical ionization (LD/CI). Studies demonstrated that

the spatial distribution of drug compounds is maintained during LD/CI and that

drug compounds could be detected at trace levels by LD/CI in intact tissue

masses.

The second method investigated was the electrospraying of the

MALDI matrix solution onto the surface of the tissue. An apparatus was

constructed to perform the electrospraying of the MALDI matrix solution.

Experiments showed that by electrospraying the matrix solution, the MALDI

matrix solvent evaporated quickly enough to prevent significant migration of

pharmaceutical compounds (within the resolution of the instrument-

approximately 150 pm).

Samples of tissue with a distribution of a pharmaceutical compound

were prepared and the ability of the instrument to map the location of a

compound within intact tissue masses was demonstrated. The drug

spiperone, at a level of 25 ng/mg of tissue, was mapped with a lateral

resolution of 150 pm in a tissue sample by both LD/CI and the electrospray

method. An experimental drug in animal tissue, at a level of 59 ng/mg, was

mapped with a resolution of 150 gm in an intact tissue sample by both

methods.













CHAPTER 1
INTRODUCTION


There are several chemical attributes that are important in

pharmaceutical research and development when selecting a lead compound

for development, including receptor/enzyme specificity, adequate potency, and

lack of toxicity in the therapeutic range.1 A significant, if not the most

significant, factor however, is the availability of the agent at the site of action.

Traditionally, in vitro cell-based assays have been used to determine the

binding efficiency of a drug with a receptor site. These types of screens can

give a good indication of a drug's activity, but they do not indicate whether or

not the drug will be transported to the disease target. Conversely, metabolic

profiling of bodily fluids using standard analytical techniques

(chromatography,2 nuclear magnetic resonance,3 and mass spectrometry

coupled with liquid chromatography4) can determine the systemic

bioavailability of a specific drug compound, but provides limited information

regarding the penetration of the drug into the tissues in either the intracellular

and/or extracellular spaces. To determine whether a drug reaches its site of

action and the chemical structure of the drug at that location requires

microprobe techniques with high sensitivity and good spatial resolution. The

combination of a microprobe instrument with high spatial resolution sampling









coupled with mass spectrometry for high sensitivity and selectivity makes a

powerful investigative tool for pharmaceutical research and development.


Mass Spectrometrv Microprobe Instruments


Mass spectrometry was a logical selection for application to

microprobe or micro-sampling analysis. Mass spectrometry has the detection

limits (typically in the low picogram range) necessary to keep the sample size,

and therefore the spatial resolution, small. Mass spectrometry is also easily

adaptable to micro-sampling techniques (as long as ions can be formed during

or after sampling). Additionally, and perhaps most importantly, mass

spectrometry yields a great deal of information about the chemical composition

of the substance being studied in the form of elemental or molecular

information.

One of the earliest examples of microprobe mass spectrometry was in

1963 when R. E. Honig and J. R. Woolston used a ruby rod laser to ablate

approximately 150 Ip.m by 125 p.m craters in a variety of metals and

semiconductor material.5 At around the same time that the first laser

microprobe instrument was being developed, the first ion beam microscope

mass spectrometer was being created.6 This new ionization technique

blossomed in the 1980's with the advent of fast atom bombardment (FAB) and

secondary ion mass spectrometry (SIMS). SIMS was able to bring the spatial

resolution of microprobe instruments down below the 0.5 pgm diameter range.









Since that time, microprobe mass spectrometers have undergone numerous

and drastic changes.

As microprobe mass spectrometry has evolved, three different

applications of the technique have emerged. One application is the field of

chemical imaging. This field has grown significantly over the last several

years. A quick search of papers published over the last five years (1994-

1999) indicated over 260 papers were published using both mass

spectrometry and imaging as keywords. For the purpose of this dissertation,

imaging mass spectrometry is defined as the systematic movement of a

sampling beam across a surface for the purpose of producing an image of the

surface which is based on one or more chemical signatures. This definition is

similar to the definition, given later, for mapping; however, the distinction

between the two is drawn from the intended purpose of the investigation.

Generally, one would image an object, while one would map the location of a

compound within a larger matrix. There are times when one would map the

location of a specific object (i.e. the location of a biologically important object

which is mapped by looking at a particular chemical signature from the object),

but generally mapping is done to plot the location of some chemical in a larger

matrix and imaging is done to produce an image of an object.

One of the reasons for the explosion of imaging mass spectrometry

has been the increase in computer technology and computing power. Bigger,

faster, and more powerful computers have allowed for computer control of the









sampling beam, which has created more accurate and more reproducible

scanning of the surface. The new, more powerful, computers have also

provided the storage space necessary to handle the enormous amount of data

that results from the imaging of a surface. Examples of imaging of surfaces

include producing an image of a cell based on its chemical composition,7 the

imaging of the surface between two polymer layers,8 the imaging of a metal

screw head to characterize the ability of the instrument,9 and many more.

The second field of microprobe mass spectrometry is mapping.

Mapping can be defined as the systematic sampling of a surface for the

purpose of plotting the location of a specific compound (or compounds) within

a larger sample matrix. The mapping of surfaces by microprobe mass

spectrometry include plotting the location of compounds separated by thin

layer chromatography,10 the location of phosphocholine in animal tissue,11 and

the mapping of proteins separated by gel electrophoresis.12 The main

purpose of this dissertation research was to develop a instrument which was

capable of mapping the location of pharmaceutical compounds in intact tissue

masses.

The third application of microprobe mass spectrometry is micro-

sampling. While spatial resolution would not seem to be an issue with micro-

sampling (simply stated as sampling a micro-volume of a substance), that is

not always the case. When examining particles in an aerosol, the ability to

ionize and analyze a single micrometer-sized particle can be of great









importance. A surface might be analyzed to determine its chemical

heterogeneity without the need to map or image the surface. There are times

when a thin layer of a substance needs to be analyzed without destroying the

entire sample. In biological systems there are micrometer or smaller sized

structures which may need to be interrogated to determine the presence of

particular compound or element. The simple sampling of a substance (without

mapping or imaging) at the micro-volume level is still an important application

of microprobe instruments. Some examples of micro-sampling mass

spectrometry include the sampling of micrometer-size glycerol droplets

containing dissolved inorganic salts,13 the sampling of rat hippocampal

neurons to determine the concentration of aluminum,14 and the

characterization of pigments in the fruiting bodies of microlichen.15

Another distinguishing difference between applications of microprobe

mass spectrometry today is whether the technique is applied to trace or major

components within a sample. A certain trade-off is necessary when

investigating trace components. If we assume that 10 picograms of analyte

must be ablated from the matrix for adequate detection of the compound, and

the compound is only present in the matrix at a concentration of 20 ppm, then

500 ng of matrix must be ablated for analytical detection. If on the other hand,

the substance under investigation is a major component of the matrix (assume

that it is 10%) then only 100 picograms of matrix must be ablated for analytical

detection. Additionally, matrix interference are more of a concern when









performing trace analysis. When looking for a major constituent in a matrix,

the ion of interest will typically be discernable above the background noise

caused by other matrix components and can be easily identified. However,

when the compound under investigation is only a trace component in the

matrix, the signal of the ion under investigation is typically buried in the

background noise. One way around this problem would be to select a matrix

which does not produce ions in the same m/z range as the ion of interest, but

that is not very realistic except when dealing with specially designed

experiments to test the sensitivity of the instrument. Of course, even when

investigating major components of a matrix, interference ions from the matrix

must be considered when performing quantitative analysis. This trade-off

between trace or major components in a sample can impact the size of the

spot used in sampling for microprobe analysis as well. If a greater volume of

matrix must be ablated for analytical detection, then either the spot size used

for sampling must be increased or the depth of penetration into the sample

must be increased (possibly by multiple sampling of the same location).

This issue of spot size has an impact on the maximum resolution

attainable with a microprobe instrument. Resolution is defined as the

minimum distance between two objects at which the two objects can still be

identified as separate. If the distance between each location sampled on a

surface is greater than the diameter of the spot used during sampling, then the

resolution of the instrument is defined as the distance between each sampling









location. This would be the minimum distance at which two objects (one

having the analyte under investigation present and the other not having the

analyte under investigation present) could be determined. On the other hand,

if the distance between each sampling location is equal to or less than the

diameter of the spot used for sampling, then the maximum attainable

resolution of the instrument would be defined as the diameter of the spot used

at each sampling location. For this reason, the distance between each

sampling location on a surface is typically greater than or equal to the

diameter of the spot used during sampling of the surface. It is obvious then

that the maximum resolution possible by a microprobe instrument is

determined by the diameter of the spot used during sampling.

For microprobe instruments using SIMS as a sampling and ionization

source, the spot size used to image organic samples is generally in the 1 to 10

|.m diameter range. In a typical mapping or imaging experiment, the primary

ion beam is rastered across the sample surface in a line by line fashion (each

line composed of several hundred individual spots, side by side).16 At each

spot a mass spectrum is collected. The intensity of the ion (or ions)

corresponding to the analyte of interest is determined for each spot and then

used to map or image the location of the analyte in the sample (using the

intensity of ion signal versus position of the sampling beam). Recently, a

SIMS microprobe instrument was used to produce an image of liposomes

based on the intensity of the ion at m/z 166, which corresponds to the









phosphocholine headgroup minus H20.17 The diameter of the spot used

during sampling and the distance between each sample spot was 200 nm

(well below the typical spot size used). To preserve the location of the

phospholipids in the membrane of the liposomes, a preparation of hydrated

liposomes (from lipids dissolved in an organic solvent, evaporated to dryness,

and then re-hydrated) was placed between two silicon wafers and quick

frozen by plunging the wafers into liquid propane. The frozen sample was

then fractured while under vacuum to prevent the deposition of atmospheric

water onto the exposed surface of the sample (SIMS penetrates only the

uppermost monolayer of the sample). The surface exposed by the fracturing

of the silicon wafers was then analyzed by a SIMS time-of-flight instrument

(TOF).

With a laser microprobe instrument, the spot used during sampling is

generally in the 25 to 50 plm diameter range. The larger diameter spot used

for sampling in laser microprobe instruments is the result of the physical

limitations of the light optics used to focus the laser beam. As is done with

SIMS mapping or imaging, the laser beam is rastered across the surface of

the sample and a mass spectrum is collected at each location analyzed (each

spot sampled across the surface). The intensity of the ion (or ions) which

correspond to the analyte of interest, along with the location on the surface

where that mass spectrum was collected, is used to map or image the location

of the analyte in the sample. Recently, a MALDI-TOF instrument was used to









image a copyright symbol () composed of coomassie blue dye which had

been stamped onto a cationic cellulose membrane target.18 The copyright

symbol was 1000 j.m in diameter. After stamping the symbol, the membrane

surface was electrosprayed with a MALDI matrix solution containing a-cyano-

4-hydroxycinnamic acid. The surface of the membrane was sampled by a

nitrogen laser (337 nm) with a beam diameter of approximately 25 [im. The

sample (which was attached to a moveable stage) was moved in the X and Y

direction in increments of 25 p.m. The final image produced was composed of

approximately 1600 pixels (sampling spots), which corresponded roughly to a

40 x 40 array. The image was visualized by two different methods. The first

method was simply the plotting of spots at 25 p.m intervals in the X and Y

directions. A spot was colored solid black if the signal/noise (S/N ratio) for the

[M+H]* ion of coomassie blue (mw=831) was greater than 2 at the sampled

location. If the SIN at the sampled location was less than 2, the spot plotted

was an open circle. The second method of visualization used was similar to

that used in the SIMS example given above, but when the S/N for the ion at

m/z 832 (the [M+H]* ion of coomassie blue) was greater than 2, the intensity of

the ion was plotted in the Z direction (with the location of that spot plotted in

the X and Y direction). When the S/N of the ion at m/z 832 was less than 2,

nothing was plotted. The image produced by MALDI-TOF was comparable to

a photomicrograph of the stamped image.









While the above examples are only a small fraction of the current

microprobe mass spectrometry work being preformed, they can be taken as

typical examples of the current state of the discipline.


Laser Microprobe Mass Spectrometry


The use of a laser beam to desorb or ablate target compounds in a

microprobe instrument was a logical selection. Lasers allow for a wide variety

of spot sizes to be used (from greater than 1 cm to smaller than 1 lIm in

diameter depending on the laser) and a wide variety of power densities

(usually in the 106 to 1011 W/cm2 range) capable of producing a large number

of gas-phase neutrals and ions from many thermally labile and nonvolatile

compounds. The first laser microprobe mass spectrometry instruments were

used primarily for mapping surfaces to determine elemental composition.19-21

Since then, the laser microprobe mass spectrometer has been utilized on

more diverse samples, ranging from geological specimens to human teeth.2226

The early laser microprobe devices almost exclusively employed time-

of-flight mass analyzers (TOF). TOF mass analyzers have experienced a re-

emergence in the last several years with the growing popularity of both laser

desorption and ionization (LDI) mass spectrometry and matrix-assisted laser

desorption and ionization (MALDI) techniques.27 The resurgence of TOF was

also carried along by advances in micro-computing which allowed for faster

electronics and more powerful storage of the large information gathered during









a TOF event. The time-of-flight mass analyzer determines the m/z of an ion,

accelerated in the ion source, by the time required to drift through the field-free

drift region and strike the detector. One of the first commercial laser

microprobe instruments was the LAser Microprobe Mass Analyzer (LAMMA).28

The LAMMA-500 (Leybold-Hereaus, K61n, Germany) instrument was based on

a TOF mass analyzer using a Q-switched Nd:YAG laser which was capable of

being focused to a 0.5 imrn diameter spot size (the actual ablated area was

usually around 1.0 jim depending on the selected power).29 A He:Ne laser

was used to mark the position of the ablation laser on the sample while viewed

though a standard quartz microscope slide which served as the vacuum seal

for the instrument. The sample was usually deposited on a copper grid

mounted on the vacuum side of the microscope cover slide. The entire stage

(cover slide and grid) was positioned for analysis by a x-y moveable stage.

The sample could be viewed by a standard optical microscope with high

resolving power and magnification up to 1200.29 The LAMMA-500 was

originally designed for the investigation of thin biological sections but was

quickly found to be useful for a wide range of organic and inorganic

applications.30 Recently the LAMMA-500 has been used to determine

chromium oxides clusters at a steelworks factory,31 the metal binding

properties of the algae Stichococcus bacillaris,32 and for investigations into

nucleosides and nucleotides.33









Since the laser in the LAMMA-500 impinged on the sample from the

backside (the laser beam struck the side of the sample facing away from the

extraction lens of the mass analyzer), it was limited to thin samples which

could be easily penetrated by the laser beam. Because of this limitation, a

second LAMMA instrument was introduced. The LAMMA-1000 (Leybold-

Hereaus, K6ln, Germany) brought the laser beam to the surface of the sample

facing the extraction lenses at approximately a 300 angle.34 The new design of

the instrument allowed the sampling of thicker and more diverse samples such

as thin layer chromatography plates,3537 organic compounds deposited on

nitrocellulose,38 and polymers.394

Since the introduction of the technique of using a laser beam for

micro-sampling, the laser microprobe has been coupled to every type of mass

analyzer including sector instruments,41 quadrupole mass filter instruments,42

Fourier transform ion cyclotron resonance ion traps,43 and quadrupole ion

traps.44


Quadrupole Ion Traps


History

Since the use and abilities of the quadrupole ion trap were critical to

the research described in this dissertation, a brief history and a quick review of

the theory behind the operation of the ion trap is in order.









The quadrupole ion trap was first introduced by physicists Wolfgang Paul and

Helmut Steinwedel in 1953 as one type of electrode arrangement out of many

possible (Figure1-1).45 The quadrupole mass filter arrangement of four rods

was another arrangement that Paul and Steinwedel also proposed. The

quadrupole ion trap consists of a hyperboloid ring electrode with two

hyperboloid end-cap electrodes (bottom two drawings in Figure 1.1). A

radiofrequency (RF) voltage is applied to the ring electrode along with a direct

current (DC) voltage applied between the end-caps and the ring electrode.

The quadrupolar electric field produced by the applied voltages traps the ions.

The trapped ions assume stable trajectories within the trap.

One of the earliest uses of the ion trap was to visualize the effects of

the electric fields on charged micro-particles to compare the theoretically

predicted trajectories to observed trajectories. Workers at the Ramo-

Wooldridge Research Laboratory in Los Angeles constructed an ion trap for

the purpose of viewing an ionized aluminum particle trapped in an ion trap to

experimentally measure the frequency of motion.46 The predicted trajectory

for the particle was that of a 2:1 Lissajous figure where the major motion of the

ion (in the z and r directions) would have a frequency of less than one half the

frequency of the applied RF voltage (Figure 1.2). In addition to the major

motion of the ion, a second "micromotion" was impressed on the ion which

was taken to be the result of the applied RF (the micromotion is actually the

applied RF frequency the secular frequency of the ion).












June 7, 1960

r De4 oc. 21. ;9


W PAUL METAL
APP AIUS 70BR SEPAR'."k; mAc.O ATIZCLS
of oinMec.1? s?::fxC CHTSAK


S54


4 'S


Fig. O


Flg )1


fNvEN,"Oes



i '-.-vVf~


Figure 1.1. Sketches of electrode arrangements by physicists Wolfgang Paul
and Helmut Steinwedel in original patent.45


2,939.952

4et*i-S.hOt 4































Figure 1.2. Retouched photograph of a singly charged aluminum dust particle (around 20 gm diameter) in an ion
trap. The operating conditions were: RF = 500 Vrms at 150 Hz, DC = 0 V. The motion is that of a 2:1 Lissajous
figure.46









Early use of the ion trap as a mass spectrometer determined the

mass-to-charge ratio (m/z) of an ion by mass-selective detection which

measured the resonant absorption of a 150 kilohertz (kHz) voltage applied

across the end-caps of the ion trap.47 Ions were continuously formed within

the ion trap by the introduction of electrons through a hole in one of the end-

cap electrodes. Ions which came into resonance with the voltage applied

across the end-caps absorbed a portion of the power applied and caused a

deflection on an oscilloscope. This method could be used to differentiate the

m/z of an ion because in the presence of a uniform electric field an ion

experiences a force which is converted into translational frequency of motion

which is determined by the ion's m/z. Thus by slowly increasing the amplitude

of the DC voltage, ions of increasing m/z came into resonance with the 150

kHz applied voltage.

Some years later, Rettinghaus developed an alternate means of

mass-selective detection.48 The detection method employed by Rettinghaus

was similar to that used today for the detection of ions in an ion cyclotron

resonance (ICR) mass analyzer. The RF voltage on the ring electrode was

slowly increased (with no applied DC voltage) while a special circuit detected

the induced current on the end-caps at a frequency of 410 kHz. As ions of

increasing m/z came into resonance with the detection circuit, a current was

induced on the end-caps due to the motion of the charged particles (ions) in

the ion trap and were detected by the special circuit. Both of the detection









methods involved complex detection circuits and had very limited mass ranges

over which detection could occur.

In 1967 P. H. Dawson and N. R. Whetten of the General Electric

Research and Development Center demonstrated the use of the ion trap as a

mass-selective storage device.49 The trap design included holes drilled in one

of the end-caps which allowed the stored ions to be drawn out of the device by

application of a negative (for positive ions) DC voltage pulse (1-10is in

duration) to the end-cap (Figure 1.3). The ions were then detected by striking

a Ag-Mg electron multiplier located just outside of the exit end-cap. This

detection method was both simpler and cheaper than previous detection

methods (electron multipliers were already being used for ion detection on

quadrupole mass filter devices). In addition, a grid placed in front of a hole

drilled into the ring electrode (for the introduction of electrons to produce ions)

could be pulsed negative just prior to the detection pulse so that ions would

not be continuously produced and only those ions in the trap with stable

trajectories would be stored. By holding the ratio of the applied RF and DC

potentials constant, only ions of a particular m/z would have stable trajectories

within the trap and so be detected during the end-cap DC pulse-out event.

This mass-selective storage operation of the ion trap is similar to the mass-

selective stability operation of the quadrupole mass filter. The advantage

demonstrated by the mass-selective storage of ions was a reduction of the

space-charge effect since only ions of a given m/z were stored in the device















Z AXIS


ELECTRON MULTIPLIER


CAP ELECTRODE


ELECTRON
GUN


ELECTRODE
TRAP VOLUME


Figure 1.3. Ion trap used to test mass-selective storage of ions. Ions were
formed in the trap by the introduction of electrons through the ring electrode.
Ions of specified m/z were ejected through drawout end-cap electrode by
application of DC voltage.49










prior to detection. Additionally, ion-molecule reaction products would have

unstable trajectories under mass-selective storage and so not complicate the

mass spectrum produced. The operation of the ion trap in mass-selective

storage mode was able to store ions in the trap for a period of days, although

there was demonstrated a loss of ion intensity due to ion scattering after a

period of about 16 hours. The major drawback to this detection method was

that only a single m/z could be detected for a given set of applied voltages.

That meant that a different set of voltages had to be applied to the ion trap

(and held stable long enough to pulse out the packet of ions) for each m/z

detected.

While mass-selective storage represented a step forward in the

operation of the ion trap, the ion trap remained mostly a device used for the

investigation of gas-phase properties of molecules and was not widely viewed

as a 'mainstream' analytical mass spectrometry device.

That changed with the introduction of a new operational mode for the

ion trap called the mass-selective instability mode.50 In mass-selective

instability mode, the RF voltage applied to the ring electrode is increased from

low amplitude to high amplitude. Ions stored within the ion trap assume

frequencies of motion which are related to their m/z and the amplitude of the

applied RF voltage. As the RF voltage increases, ions of increasing m/z

become unstable in the z-direction (the direction of the end-cap electrodes)

and are ejected from the trap. With holes drilled in one of the end-caps and an









electron multiplier placed outside the end-cap (similar to the design of Dawson

and Whetten), ions which exit the ion trap through the holes drilled in the end-

cap would strike the electron multiplier and be detected. It was also found that

a slight background pressure of helium gas (approximately lx10 03 torr)

increased the resolution of the resulting mass spectrum by collisionally

damping the ions in the trap, thus cooling the ions to the center of the trap so

that they were ejected in a tighter packet. This mode of operation of the ion

trap offered the increased benefits of a simpler operation (no need to apply a

DC voltage for pulsing out the ions), a greater scan speed (the entire m/z

range from 10 to 650 Daltons could be gathered in approximately 10 ms), an

increased m/z range (with no DC voltage applied to the end caps, the greatest

range of m/z's could be stored in the trap), and a reduction in the space-

charge effect (by operating the ion trap with no DC voltage applied during

scan-out).

Since the introduction of a commercial ion trap using mass-selective

instability mode,51 there have been several advances in the specific operation

of the ion trap which have expanded its capabilities as a mass spectrometer.

One of the most powerful abilities of the quadrupole ion trap is its

ability to selectively fragment ions to produce a characteristic 'daughter' ion

spectrum, called MS/MS. The produced daughter ion spectrum can be used

to unambiguously identify a compound even when the parent ion peak is

indistinguishable from peaks produced by other compounds only one or a few









m/z units away. This ability is accomplished by the application of an

alternating current (AC) voltage to the end-cap electrodes and is called

collisionally activated dissociation (CAD).52 In much the same way as the

earliest detection method used with the ion trap (mass-selective detection), in

CAD a voltage is applied to the end-cap electrodes of the ion trap at a

selected frequency. Ions within the trap which have a secular frequency equal

to the applied voltage will gain kinetic energy through resonant absorption.

The gain in energy will result in increased translational motion along the axis

of the applied voltage. Since in normal operation of the ion trap there is a

background pressure of a light buffer gas (generally helium at a pressure of

1x10"3 torr), the increase in translational motion results in increased and more

energetic collisions between the selected ion and the buffer gas. These

collisions deposit energy in the ion and increase its internal energy. If the

collisions are of sufficient energy for a sufficient amount of time, the internal

energy of the ion is increased to the point that bonds within the ion are broken.

Since the mass spectrometer detects ions, only those fragments of the ion

which retain the charge are detected.

To be able to distinguish the daughter ion spectrum produced by CAD

from the normal background ions present in most samples, it is necessary to

first isolated the parent ion so that the daughter ions (of lower m/z than the

parent) are easily discernible. To accomplish this, there have been several

methods employed. Among the proposed, and used, methods for the isolation









of the parent ion are: (1) the two-step isolation method,53 where two separate

sets of RF and DC voltages are applied to the ring electrode in sequential

fashion to first remove the ions of lower m/z than the parent ion and then to

remove the ions of higher m/z than the parent ion; (2) the apex isolation

method,54 where the RF and DC voltages are selected such that the desired

m/z of the parent ion is moved to the apex region of the stability diagram

where ions of both higher and lower m/z than the selected parent ion become

unstable (and so are ejected from the trap) at the same time; (3) the forward-

reverse isolation method,55 where an appropriate RF voltage is applied to the

end-cap electrodes so that ramping the RF drive amplitude first up and then

down causes the ejection of first the ions of lower m/z than the selected m/z

and then the ions of higher m/z than the selected m/z; and the stored

waveform inverse Fourier transform (SWIFT) isolation method,56 where a

broadband waveform with a missing notch of selected frequencies is produced

by SWIFT and applied to the end-caps electrodes such that all ions except

those within the frequencies of the notch are excited by the applied voltage

and are ejected from the trap. Each of these methods has advantages and

disadvantages related to their ability to effectively isolated the selected m/z

with minimum loss. For most of the studies presented in this dissertation, the

SWIFT isolation method was found to be the preferred method since it

produced the best isolation with a minimum loss of parent ion signal intensity.









Ion Trap Theory


A quadrupolar ion traps consist of a three-electrode arrangement

consisting of two end-cap electrodes and a ring electrode (Figure 1.4). In the

mode of operation most commonly used an RF and DC potential is applied to

the ring electrode while the end-caps are grounded. In this mode of operation,

the applied potential produces a time dependent quadrupolar electric field in

the ion trap. The potential 0 at any point within the ion trap can be described

by the equation:

S0o (r -2z2)+ 1.1
2

where o is the potential applied to the ring electrode, ro is the inside radius of

the ring electrode, and z,, is one half the distance between the end-cap

electrodes. The factor of two applied to the z' term is the result of the

historical physical shape of commercial ion traps where ro2 = 2z2.

The potential applied to the ring electrode can be a combination of RF

potential (V) and direct current (DC) potential (U) such that:

0o =U-VcosQt 1.2

where Q is the angular frequency of the RF potential in radians s1 and t is the

time in seconds. The field is uncoupled so that the force experienced in one

direction is independent of the force experienced in the other perpendicular



































Figure 1.4. Schematic diagram of a three-dimensional quadrupole ion trap.57




110









direction. The force impressed on the ions by the electric field is described by

applying Newton's equation of force (F = ma) in the form:

d2u 9__ 1.3
m-- = -e-a u.
dt2 Qu

where u can be either r or z, m is the mass of the ion, and e is the charge

on the ion (in coulombs). Combining equation 1.1 and equation 1.2 into

equation 1.3 for 0, then differentiating with respect to the individual axes and

rearranging, the equations for the motion of ions within the quadrupolar field

become:

d2r + 2e (U-Vcos Qt)r = 0 1.4
dt2 mr +2z)

d2z 4e (U V cosQt)z=0 1.5
6dt2 mr.

Notice that equations 1.4 and 1.5 do not have cross terms, indicating that the

ion motion along one axis is independent of the motion along the other.

These equations can be put into the form of a known second-order

linear differential equation called the Mathieu equation with the following

substitutions:

-, -16eU .
a= =-20r m(r + 2Zo )2 1.6

8e V1.
q = -2q= m(r] + 2zA e 2 1.7

O~t
Q- 1.8
2









Substituting equations 1.6, 1.7, and 1.8 into expressions 1.4 and 1.5

transforms both expressions into a Mathieu equation with the form:

d2U+ (a. -2q cos 2)u =0 1.9


Stable trajectories for ions along both the r and z axis are obtained

only at certain a, and q. values. It is easy to see from equations 1.6 and 1.7

that all parameters other than U and V are constant for an ion of a certain

Ue Ve
charge and mass. Because of this, a. and q, (rather than mie,
m m

traditionally mass spectrometrists have used m/z to indicate the mass to

charge ratio of an ion where m represents the mass of the ions and z

represents the number of fundamental charges on the ion). The values of

au and qu which result in stable trajectories along both the r and z coordinates

can be plotted to produce a diagram which shows stable and unstable regions

in au and q. space. This type of diagram is called a stability diagram and

provides a convenient method of visualizing ion trajectories within the ion trap

(Figure 1.5).

The lines which crisscross the region of stable trajectories in both the

r and z direction are iso-P3 lines and indicate a, q values that produce identical

secular frequency of motion for ions of a given m/z. Current ion traps operate

with no DC potential applied between the ring and end-cap electrodes. For

this mode of operation, the ions line up along the a, = 0 line in the stability














































Figure 1.5. A stability diagram indicating the a, and qz values which produce
stable trajectories for ions held within the ion trap. The lines which crisscross
the region in which ions are stable in both the r and z directions are iso-P3 lines.
These lines indicate values that produce identical secular frequency of ion
motion.









diagram. Since q, is inversely proportional to the m/z of an ion, the ions line

up from left to right along the a, = 0 line in order of decreasing m/z. Also, as

indicated by equations 1.4 and 1.5, the time-dependent motion (or secular

frequency) of the ions is inversely related to the m/z of the ion, such that, at a

given RF potential (V) ions of lower m/z values will have higher secular

frequencies. When the RF potential is increased to the point that an ion has a

secular frequency greater than 13=1 along the z coordinate (the right hand

edge of the stability diagram), the ion becomes unstable in the z direction

(along the end-cap axis) and is ejected from the trap out one or the other of

the end-caps (when holes in the end-caps are present). Note that at this point

the ion will still be stable in the r direction as indicated in the stability diagram.

This is the theoretical basis of the mass-selective instability mode of operation

of the ion trap which is used today for most commercial ion trap instruments.50

The equations given to describe the motion of ions in a quadrupolar

electric field (equations 1.4 and 1.5) are strictly defined as applying only to a

single ion. When several ions are present, the resulting interaction of the ions'

electric field with the applied electric field causes the trajectories of the ions to

be disturbed. This perturbation of the applied electric field is called the 'space

charge' effect.47 The result of the space charge effect is that some of the ions

are shielded from the applied electric field. This shielding results in the

secular frequencies of the ions being reduced and broadened.58 The resulting









mass spectrum peaks are also broadened (a loss of resolution) and shifted so

that they appear at higher m/z's (loss of mass accuracy).

Several methods have been developed to reduce the space charge

effect on the resulting mass spectrum. One such method (which is currently

employed on commercial ion traps) is to apply a supplementary AC potential

to the end-caps (1800 out of phase) at a frequency close to the secular

frequency of the ions at pz = 1 (the right-hand edge of the stability diagram).59

This application of a supplementary potential is called resonant ejection.

Resonant ejection of the ions has been found to reduce the broadening of the

ion peaks and reduce the mass shifts associated with the space charge effect.

Another method (which can be used in conjunction with resonant

ejection) is automatic gain control (AGO), which controls the number of ions

injected into the trap and so reduces the space charge effect.60 AGC is a

simple process. Immediately prior to the analytical scan of the ion trap, ions

are injected into, or formed inside of, the ion trap for a set period of time. The

ions formed during this set period of time are quickly scanned out of the ion

trap and detected. The total ion current produced by the ions is measured.

Based on the amount of total ion current measured in this pre-analytical scan,

the ion formation time used for the analytical scan is set so that the total ion

current (which is related to the total number of ions present in the trap) is such

that the space charge effect is reduced. Both resonant ejection and AGC, in

conjunction with the mass-selective instability mode of operation, have









improved the analytical capabilities of the ion trap such that it rivals or exceeds

other forms of mass analyzers.

One result of the application of a supplementary AC voltage for

resonant ejection of ions from the ion trap is the ability to extend the m/z range

of the trap. The quadrupole ion trap has a theoretically unlimited m/z range

only if the RF potential can be increased to infinity (this assumes that the size

of the trap and the frequency of the RF remains constant during scan-out, both

of which are good assumptions). However, the RF potential can only be

increased to a certain point before the voltage will begin to arc across the

spacers between the ring and end-cap electrodes, or through the gas present

in the trap, to ground. This maximum allowable voltage sets the upper mass

limit during the normal operation of the ion trap. Using the values in Table 1.1

for the ITS-40 ion trap (which was used in all of the research performed in this

dissertation) and equation 1.7, the m/z range is restricted to a maximum of

approximately 650 Daltons. The qjec, value of 0.908 comes from the stability

diagram and coincides with /8=1 along the a, = 0 line (the point at which the

ion's trajectory becomes unstable and the ion is ejected from the trap). This

restriction on the maximum m/z is one of the most serious drawbacks to the

quadrupole ion trap. However, that restriction on the maximum m/z is only

applicable when the qj,,, used is at, or near, a value of 0.908. Since the

application of a supplementary AC potential to the end-caps of the trap will

cause ions of the correct secular frequency to gain translation motion in the




















Table 1.10 rating Parameters for the ITS-40 Ion Trap
f 1.0485 x105 Hz

Q (f2r) 6.5879 x106 radians sec"1

r 0.01 m

Z. 0.007811 m

mas 7500 V

qejecl 0.908

mass = Daltons (kg/6.022x1 026)









direction of the applied potential (along the z axis-toward the end-cap

electrodes), by reducing the frequency of the applied potential, ions of larger

m/z values can be ejected from the trap at a lower RF potential. The RF is

increased normally during the scan-out event, yet ions are ejected from the

trap at a lower qej,, which is related to the frequency of the supplementary

applied AC potential. This method of extending the mass range of the ion trap

is referred to as being extended by use of axial modulation.61 The m/z limit of

the ion trap can also be extended by reducing the diameter of the ion trap or

by reducing the frequency of the RF; however, both of these solutions can

have a negative impact on the mass spectrum due to an increase in the space

charge effect. For ions with a m/z greater than 650 Daltons investigated for

this dissertation, axial modulation was used to extent the mass range of the

instrument.


Overview of Dissertation


The focus of this dissertation is on the development of a quadrupole

ion trap laser microprobe instrument which is capable of mapping

pharmaceutical compounds in intact tissue. Chapter 2 reviews the previous

work done in this laboratory by Christopher D. Reddick who initially

constructed the instrument (called Tubby, due to the large tub of a vacuum

chamber in which it was constructed) used in these studies and the results of

those initial studies which led to the current research. Christopher Reddick's









research centered on the initial construction of Tubby and the use of matrix-

assisted laser desorption and ionization (MALDI) as a means of detecting

pharmaceutical compounds in intact tissue. Those studies showed that the

instrument was capable of detecting pharmaceutical compounds at levels

which were compatible with dosing levels of most pharmaceutical compounds

(approximately 50 ng/mg). The ability to spatially resolve the location of those

compounds in the intact tissue mass was not accomplished during the initial

studies with the instrument because of analyte migration during the MALDI

preparation step. The ability to spatially resolve the location of pharmaceutical

compounds in intact tissue formed the focus of my research.

Chapter 3 describes the first of two methods investigated to preserve

the spatial distribution of pharmaceutical compounds in intact tissue: laser

desorption coupled to chemical ionization (LD/CI). The chapter starts with an

overview of the laser desorption process and gives a brief description of

chemical ionization. Initial experiments were performed to demonstrate the

viability of LD/CI for the detection of trace level compounds in intact tissue. A

micro-manipulation stage was constructed so that selected locations on the

surface of an intact tissue mass could be sampled. Final experiments were

conducted which demonstrated the ability of LD/CI to preserve the spatial

location of pharmaceutical compounds in intact tissue.

Chapter 4 describes the second method investigated to preserve

spatial location information: the electrospraying of a MALDI matrix onto the









surface of tissue. The traditional method of MALDI is described (the drop

method) and the blurring of spatial information is verified. An electrospray

apparatus was constructed which was capable of electrospraying the MALDI

matrix solution onto intact tissue. The method of electrospraying the MALDI

matrix solution is demonstrated as being capable of preserving the spatial

location information of pharmaceutical compounds in intact tissue.

Chapter 5 describes experiments preformed on both model and actual

tissue samples to determine the location of drug compounds in intact tissue

masses. The drug spiperone, at a level of 25 ng/mg of tissue, was mapped

with a lateral resolution of 150 pm in a tissue sample by both LD/CI and the

MALDI matrix electrospray method. An experimental drug which had been

intravenously dosed to a level of 58.5 ng/mg in an animal, was mapped in a

sample of tissue with a resolution of 150 tm by both methods.

In chapter 6, the final conclusions of the dissertation are presented

and a brief description of future work which would be of interest to continuing

the project is described.













CHAPTER 2
INITIAL STUDIES

Overview of Instrument


All experimental results presented here were obtained on a

quadrupole ion trap instrument initially constructed at the University of Florida

specifically for the analysis of pharmaceutical compounds in tissue by

Christopher D. Reddick. A complete description of the instrument can be

found in Chris' dissertation.62

The instrument consists of a Finnigan 4500 EI/Cl ion source from

Finnigan MAT (San Jose, CA) (Figure 2.1). One of the advantages of this ion

source is that the ion volume can be changed with each sample to minimize

carry-over effects. The only modification to the ion source was the blocking of

the GC transfer line inlet hole so that a sufficient pressure of Cl reagent gas

(methane, ammonia, and isobutane were used where indicated for this

dissertation) could be obtained for the LD/CI studies. The ion source and

probe lock were mounted 900 in relation to the ion trap to allow the laser beam

to be introduced into the source orthogonal to the probe face (Figure 2.2).

This design assured that a small laser spot size with high power density could

be obtained and that the ions would be desorbed in the direction of the ion

extraction and focusing lenses. Additionally, the 900 design allows for future










Mounting Flange
(6 N


pulp
,- ? '


4 lYl


H) I


Nil
I I II


Ion Source


Figure 2.1. Schematic drawing of Finnigan EI/Cl ion source used in instrument.


7.



































Figure 2.2. Schematic drawing of instrument Tubby.62









modifications to the instrument that will permit the viewing of the sample for

laser desorption position selection.

To direct the ions from the ion source to the ion trap, a DC quadrupole

was used as a 900 ion deflector as described by Pedder and Yost.63 The

deflector consisted of four quarter-round stainless steel rods with a radius of

0.5" and a length of 3.25". The rods were held in position by two anodized

aluminum caps and were secured to the inside of the caps by Teflon screws to

prevent grounding. Opposing rods were connected electrically so that a

voltage could be applied to each pair of rods to turn the ions. A complete

characterization of the DC turning quad has been given by Pedder." A

stainless steel tube lens was mounted to two of the quarter-round steel rods of

the turning quad and served as the entrance lens into the ion trap.

The ion source and DC turning quad were housed in a differentially

pumped, cradle-type vacuum chamber. The chamber was pumped by two

TPH 300 LUs Balzers (Hudson, NH) turbomolecular pumps. The

turbomolecular pumps were mounted directly to the stainless steel vacuum

chamber (one in the source region and one in the analyzer region) through two

4.0" connection ports machined into the bottom of the vacuum chamber. Each

turbo pump was individually backed by a 300 L/min mechanical pump (Alcatel

Corporation, Hingham, MA). The pressure in each region was monitored by

separate Bayard-Alpert type ion gauges (Granville-Phillips, Boulder, CO).









To minimize ion-molecule reactions between chemical ionization (Cl)

reagent gas introduced into the ion source during Cl experiments and ions in

the analyzer region, an aluminum dividing wall was added to the instrument

between the source and analyzer regions as shown in Figure 2.2. With the

installed wall, a pressure differential of approximately two orders of magnitude

(3x104 torr in the source region and 3x10.6 torr in the analyzer region) could

be maintained during Cl experiments.


MALDI


The initial studies which Chris Reddick performed on Tubby were

designed to characterize the instrument's performance for use as a laser

microprobe mass spectrometer with matrix-assisted laser desorption/ionization

(MALDI). Since the results of those experiments led directly to my research,

and because my research also involved the use of MALDI as a desorption and

ionization method, a brief overview of the history and theory of MALDI is

included here.


History


In the early 1980s, the desorption of bio-organic molecules for mass

spectrometry above 10,000 Daltons was performed almost exclusively by

plasma desorption.6566 Then in 1988 both Tanaka et al. in Japan and Karas

and Hillenkamp in Germany published articles which described a method of









sample preparation that allowed the analysis by laser desorption mass

spectrometry of bio-organic molecules of up 100,000 Daltons and 67,000

Daltons, respectively.6768 Both of the preparation methods involved the

addition of a matrix to the sample which enhanced the desorption and

ionization of large bio-molecules.

The method which Tanaka et al. described involved the dispersion of an

ultra fine (about 300 A diameter) cobalt power in glycerol which was dissolved

in an organic solvent. This matrix solution was mixed with the sample (also in

solution) before being applied to the sample holder. The matrix/analyte

solution was allowed to dry before analysis. A nitrogen laser (337 nm

wavelength) was used to desorb and ionize the sample, with the ions mass

analyzed with a time-of-flight mass analyzer. The technique was called, "ultra

fine metal plus liquid matrix method" and was able to demonstrate the

detection of the [7M+cation]+ ion of lysozyme from chicken egg white

lysozymee mw = 14306 Daltons). The metal powder served as a chromophore

for absorbing the irradiating laser light and the glycerol served to disperse

local heating and provide a renewable surface area for multiple laser shots.

The method developed by Karas and Hillenkamp was born out of an

earlier observation that laser desorption of amino acids which had strong

absorbance near the wavelength of laser light used (a Nd:YAG, frequency-

tripled to 355nm or frequency-quadrupled to 266 nm) required a lower

threshold irradiance (defined as the minimum laser power required to









produced ions from the sample) and produced a greater ratio of molecular-

type ions to fragments than amino acids which had a lower absorbance at the

selected wavelength.69 As a test, the authors mixed a strongly absorbing

amino acid (tryptophan) with a weakly absorbing one (alanine). The resulting

spectrum showed alanine [M+H]*, along with the tryptophan [M+H]*, even

though the laser power used was approximately one tenth of that necessary to

produce ions from a sample of alanine alone. Based on these observations,

Hillenkamp and Karas chose the UV absorbing compound nicotinic acid

dissolved in water (at a concentration of 10-3 M) as their matrix and mixed an

equal amount of matrix solution with sample solution (bovine albumin, mw =

67,000 in water at a concentration of 10-5 M). The mixture was air dried on a

probe tip and desorbed and ionized by the frequency-quadrupled output of a

Nd:YAG laser (at 266 nm) and the ions analyzed by time-of-flight mass

spectrometry. Their method was called, "matrix-UVLD" where the UV stands

for ultraviolet and the LD stands for laser desorption. After several name

changes, today the most widely accepted name for this sample preparation

method is MALDI (matrix-assisted laser desorption/ionization).


Theory of MALDI


Desorption


The desorption process of MALDI has been extensively researched and

is fairly well understood qualitatively.70 Most of the proposed models for









desorption of large bio-molecules by UV MALDI are variations of the same

general theory.71 That general, qualitative theory for the desorption of large

bio-molecules by MALDI is well represented by the hydrodynamicc model"

proposed by Vertes, Irinyi, and Gijbels.72 The basics of the hydrodynamic

model are that the incident laser light causes rapid heating of the solid (both

matrix and analyte). Once the sublimation temperature of the matrix is

achieved, rapid evaporation of both matrix and analyte produces an expanding

plume of material into the gas phase by direct sublimation. The temperature,

density, and velocity of the solid and expanding vapor can be calculated by

application of a set of hydrodynamic equations (Figure 2.3). Figure 2.3A

shows the calculated effect of a 10-ns laser pulse (Nd:YAG, frequency-

quadrupled to 266nm) on temperature at the surface-vapor interface of a

nicotinic acid sample. Note that after the end of the laser pulse (50 and 100

ns traces), both the surface of the sample and the vapor created during the

desorption event are significantly cooled. The surface is cooled by the

sublimation process and the vapor is cooled by the rapid expansion into the

gas phase. Figure 2.3B shows the density distribution of the expanding vapor

into the gas phase. The initial vapor density (10Ons trace) is relatively thick and

located, as expected, primarily within 4 4im of the surface. Once the irradiation

stops (50 and 100 ns traces), the rate of evaporation quickly subsides due to

the rapid cooling of the surface and the plume detaches from the surface and

expands.














SOUO VAPO A
380



200

Ion
4)
F180 I
o S m










VII .440 b
z (micrometer)

B

50

E
4.o0


20 iOn
I
.... ... .---
-o 0 6 .. .40 "
z (micrometer)









OFigure 2.3. The calculated temperature(A), density(B), and velocities ofR










particles desorbed (C) from a matrix of nicotinic acid by a 10 ns laser pulse
(Nd:YAG, 266 nm wavelength with 10~7 W/CM2 power) using the hydrodynamic
model. Dashed vertical line indicates matrix/vacuum interface. The dashed
h a i n 3




^72
horizonta line in ( n t

0^_ ,.. .. . .2o,....... .I ~~r^ T T .T .. .. .
z (micrometer)


(N:A,26n aeeghwt 10 W/m oe) sn h hdoyai
moe.Dsedvria in niae mti/vcu intrfce Th ase
horizonta lin in (A)^ inicte 30.









Using the same process, the velocity, density, and temperature were

calculated for sinapinic acid desorbed by a 10 ns laser pulse (at 308 nm

wavelength) (Figure 2.4). In Figure 2.4A, the density of desorbed molecules in

the expanding plume was calculated and plotted for laser irradiances

increasing from 2 xl05 to 4 x106 W/cm2 (the letters f through a indicate a

regular increase of laser power by five steps of 7.6 x105 W/cm2). The areas

under the curves of Figure 2.4A were integrated to determine the total flux of

molecules desorbed at each laser irradiance level; these were then plotted to

show the increase of material ejected with increasing laser power (Figure

2.4B). The calculated desorption flux vs. irradiance was compared to

experimental data collected on the MALDI of bovine insulin with sinapinic acid

as the matrix and an excimer laser operating with 10 ns pulse at 308 nm

wavelength (Figure 2.5).73 The hydrodynamic model fits well with the

experimentally calculated ion flux vs. irradiance; however, there were

differences. In the experimental data, the threshold of ion production was

found to be 2 x106 W/cm2 while the threshold calculated from the

hydrodynamic model was found to be 2 x105 W/cm2. The authors suggest that

the cause for this deviation may well be the optical constants that were used in

their calculations. The absorbance coefficient used for sinapinic acid was from

the solution-phase data, since that was the only value available. Another

difference between the calculated values and the experimentally determined

values was the slope of the flux vs. irradiance. The measured ion yield shows







45




1010 A
100*
10 "










z(micrometer)







o b
10
%..e C
C 10" d








10 "
13" f










10il .. ..... . ...... 8


10" 1
z (micrometer)



10 B













(SoIrrdo W/m/
Z~h10 17 . .. ... .

acid matrix at 40 ns after 10 ns laser pulse (excimer laser, 308 nm







wavelength). Letters f through a indicate varying laser irradiances
(explanation in text). (B) shows plot of calculated desorbed matrix flux vs.
laser irradiance.72
E 10 I


10 10
4)
L_10 14
010

10"-3
10 02 0
Irrodiance (W/CM2)

Figure 2.4. (A) shows calculated plume density profiles of matrix sinapinic
acid matrix at 40 ns after 10 ns laser pulse (excimer laser, 308 nm
wavelength). Letters f through a indicate varying laser irradiances
(explanation in text). (B) shows plot of calculated desorbed matrix flux vs.
laser irradiance.72












10


A





')




'3)
(:

9
>..

(9


a 3


10'







10-1




10-4
10


,2 x106 W/cm2


Ir II I I I I I I


Power Density (W/cm2)

Figure 2.5. Plot of the dependence of molecular-ion yield, Y1, of bovine insulin
from sinapinic acid vs. laser irradiance level for excimer laser (at 308 nm
wavelength with 10 ns pulse). The different symbols indicate different
diameters of an iris placed in front of the microchannel plate detector to
prevent saturation.7









a power law dependence with laser intensity of approximately Y, oc 16, while the

exponent derived from the hydrodynamic model was somewhat higher (6.8).

The source of deviation between the two data sets can only be guessed;

however, the authors suggest several possibilities: statistical error in the

experimental data (no single power law exponent was given by the authors of

the experimental measurements), that the measured ion yield may not be

proportional to total desorbed molecule yield, and the assumption of a

Gaussian fluence distribution on the sample surface in the hydrodynamic

calculations. It is worth noting that both the experimental evidence and the

hydrodynamic model predict a saturation-like behavior at high irradiance

levels.

Velocity distributions were calculated for sinapinic acid and ferulic

acid and then compared to experimental data gathered on both matrices. The

calculated drift velocity (250 m/s) for ferulic acid neutrals compared well with

the maximum measured neutral particle velocity of 300 to 400 m/s, while the

calculated value for sinapinic acid neutrals (250 m/s) was found to be

substantially lower than the experimentally measured ion velocity (1140 m/s)

for sinapinic acid. The authors suggested that this difference might be the

result of the high extraction field present in the experimental data. Even with

the reported differences between the calculated and the experimental

evidence, there is enough agreement to make the model important for a

qualitative understanding of the MALDI desorption process.









One consideration worth noting is the fact that MALDI produces large,

thermally labile, bio-molecule ions with little or no fragmentation, while the

matrix itself often shows substantial fragmentation. This observation is not

completely consistent with the simple heating of the matrix offered by the

hydrodynamic model, since the analyte molecules would also be heated in the

process. The authors suggest that this observation is the result of the energy-

transfer pathways of the system. A model which offers an explanation for the

observed intact bio-molecules desorbed during UV MALDI called, "The

Homogeneous Bottleneck" model has been presented.74 As with the

hydrodynamic model, it is of value to understand the model for a clearer

understanding of the MALDI process, and so it will be briefly presented here.

During solvent evaporation, the analyte molecules (called the guest in

the homogeneous bottleneck model) are entrapped in the crystal lattice

structure formed by the matrix molecules (called the host). The lattice bonds

formed between host molecules will have a different vibrational frequency (and

energy transfer coefficient) than internal bonds of the host molecules, and both

of these bonds will be different than the bonds formed between host and

guest. Figure 2.6 is a schematic representation of the bonds involved in the

host/lattice and guest incorporation into a MALDI matrix. Several assumptions

are made to produce the kinetic equations used to solve the energy transfer

bottleneck in the model. One assumption is that the two most effective

mechanisms by which the excited volume can be cooled is by evaporation and

















I 4
* *
* I



it
N-
* I
I I
A


Figure 2.6. A schematic representation of the vibrational modes of the guest
molecule (G) incorporated into lattice of host molecules (H). Solid lines
represent chemical bonds and broken lines represent physical bonds. The
springs represent the coupling modes of energy transfer. VHB represents the
hydrogen bonds formed between host and guest and is the bond broken
during release of the guest molecule.74









sublimation. The possible mechanism of cooling by heat conduction was

ignored in the model because of the short timescale (10 ns) generally involved

during UV laser irradiation of the solid. The possible cooling by volume

evaporation was also ignored in the model because it has been shown to be

important only with matrices which have low coefficients of absorbance (this

process of cooling was included in a later publication which included IR

MALDI).75 Additionally, the laser pulse was assumed to have a purely

Gaussian distribution across the surface.

The authors presented and solved a set of kinetic equations which

were taken to be representative of the energy redistribution process during

laser irradiation of the host/guest solid. The physical processes which were

assumed in deriving the equations were as follows: the laser energy deposited

on the solid primarily excited (electronically, because it was UV radiation) the

host molecules. Since in typical MALDI conditions the host molecules are

present at a ratio of 1,000:1 to 100,000:1 compared to the guest molecules,

this seems to be a valid assumption. Through an extremely fast

(approximately in the ps timescale) internal conversion processes, this

deposited energy is converted to internal vibrational energy. The internal

vibrational energy of the host is then transferred to lattice vibrational energy at

a rate dependent on the specific kinetic rate constant. This vibrational energy

is also transferred, via direct coupling, to the guest molecule vibrational

energy, again at a rate dependent on the specific kinetic rate constant. The









lattice is cooled by phase transformations and by the transference of energy to

the guest. The heating rate of the guest molecule is determined by the direct

absorbance of energy from the laser light and by the transference of energy

from both the lattice and directly from the host. In addition, both the guest and

host molecules can be subjected to energy loss through irreversible

fragmentation. It should be noted that this model deals with the volume

energy density of the guest and not the energy content of the guest.

An energy bottleneck can form at any of these energy transference

points if the transfer rate coefficient is extremely low in relation to the others.

Solving the equations presented by the authors, there is found to be extremely

efficient transference of energy between host and lattice due to the large

number of couplings present in the system. Furthermore, the transference of

energy would be expected to be efficient in the direct coupling between host

and guest since the vibrational frequencies of the bonds can fall within the

same range. There would be expected to be a bottleneck in energy

transference at the direct host/guest bond based on the low concentration of

the guest in the matrix (generally 104 to 10.6 for traditional MALDI). There

would also develop a bottleneck at the bonds between the lattice and the

guest because of the vibrational difference between these bonds. Figure 2.7

shows the time history plot of the energy deposition during MALDI of nicotinic

acid with a frequency-quadrupled Nd:YAG. Figure 2.7(b) represents a pictorial

summation of the proposed theory. The lattice and host molecules are in










1000- (a)


U,r
C
500- p
.0 I


Pout

0-1



--
0 50 16o
t (ns)
1000- (b)







!---- - - g
Ise


I

0o 50 1 "6 o
t (ns)

Figure 2.7. Calculated time history plots of energy deposition into matrix and
guest. Plot (a) shows energy flux in (through laser irradiation) and out
(through sublimation) of system. Note the slight time lag (indicated by the
vertical dashed lines) between the maximum energy flux into the system and
the maximum energy removed from the system. Plot (b) shows energy
density of the host/lattice (H,L), guest (G), and thermally fragmented guest
molecules (B). The dashed horizontal line shows room temperature across
time of calculation. Note that for the timescale indicated, the lattice remains in
equilibrium with host.74









thermal equilibrium during the rapid energy deposition by the laser irradiation

due to efficient energy transference from host to lattice, while at the same time

the guest molecules are thermally cooler. This reduction in temperature of the

guest molecules compared to the host and lattice is a result of the bottleneck

in vibrational energy transference due to the difference between the bonds

formed between the matrix lattice and the bonds formed between the lattice

and the guest.

Using the same equations, but with different initial conditions, the

authors predicted the effects on the system for an increase in the

concentration of the guest molecules in the matrix, a higher sublimation

temperature for the host, and a lower power density with longer pulse length

for the laser irradiation of the system (Figure 2.8). In Figure 2.8(a) the effect of

increasing the fraction volume concentration of the guest molecules from 10 4

to 10-2 is shown. The increase in concentration of the guest molecule results in

a greater efficiency of transfer of energy between the host and the guest. The

greater efficiency results in an increase of guest temperature and an increase

in the amount of thermal degradation (indicated by B in Figure 2.8).

Additionally, since more of the available energy is transferred to the guest,

there is less energy transferred into the lattice and so the rate of sublimation is

reduced, which results in further heating of the guest and more thermal

degradation.










1000




500
F--




0




1000


F-


500


0


1000




- 500-


I0-


Figure 2.8. Homogeneous bottleneck model calculations for atypical MALDI
conditions. Plot (a) demonstrates predicted behavior of system when
concentration of guest is increased. Plot (b) shows predicted behavior when
sublimation temperature of matrix is increased. Plot (c) shows predicted
behavior when laser pulse is increased in duration while intensity of irradiance
is decreased.74


50
t (ns)









Figure 2.8(b) demonstrates the effect of an increase in sublimation

temperature for the host from 315 K to 400 K. The result of the increase of the

sublimation temperature is what would be expected. The lattice and host are

able to achieve higher temperatures before the onset of sublimation begins the

cool the system. Because of this increase in temperature, more energy is

transferred to the guest molecules and greater thermal degradation results.

The authors also point out the advantage of a sublimating matrix as apposed

to a melting matrix. If the matrix were to melt and evaporate, after melting the

energy transfer process would be enhanced between the host and the guest.

This increase in energy transfer might, if the melting temperature were high

enough, result in sufficient transference of energy to the guest to bring about

thermal degradation; however, this effect would be temperature dependent

and is not a guarantee of increased degradation. Figure 2.8(c) demonstrates

the effect of a longer laser pulse (increasing from 10 ns to 50 ns) along with a

reduction in power (from 1 x107 W/cm2 to 2 x106 W/cm2). This change

represents a 5-fold increase in pulse length and a 5-fold decrease in laser

power. The net result is that the same energy is deposited in the sample, but

the time over which the energy is delivered has been increased. This has a 2-

fold effect on the embedded guest molecules. First, the cooling of the matrix

by sublimation has been slowed due to the less rapid heating of the system

from the reduced power. Secondly, increasing the pulse length increases the

coupling time over which the host molecules can pump energy directly into the









guest. The combination of the two results, as expected, in an increase in

fragmentation of the guest molecule.

While neither the hydrodynamic model nor the homogeneous

bottleneck model can be taken as the only model to explain desorption during

MALDI, like most chemical models, they offer a useful explanation of the

process and summarize the current level of understanding of the process.


Ionization


While the desorption of neutrals and ions during MALDI is at least

fairly well understood, the ionization process is not. There seem to be almost

as many theories about the actual ionization process during MALDI as there

are papers written about the process. One fact is fairly clear from the

evidence presented so far, and that is that the ionization process for MALDI is

a collective one, not a single process.26 An excellent review has recently been

published on the current theories for ionization during MALDI, and readers

wishing for a more in-depth coverage of the process are referred there.70

While the factors affecting the desorption event during MALDI are germane to

this dissertation, the actual process of ionization is not. It is worthwhile,

however, to briefly cover some of the currently postulated processes for a

better understanding of MALDI. The following explanations were adapted

from the previously cited review article.









The ionization process during MALDI can be split into two categories:

primary ion formation and secondary ion formation. Primary ion formation

covers those ions formed before (pre-formed ions in the matrix) or formed first

during the ionization/desorption event (which would generally be matrix ions).

Secondary ion formation covers those mechanisms which lead to those ions

not generated during the primary ion formation process (which usually would

be the analyte ions).


Primary ion formation


One of the ways by which the MALDI process helps to create ions is

by reducing the energy necessary to produce the ions. This reduction is

brought about by screening of the Coulombic charge on ions by salvation in

the matrix. Transferring pre-formed ions from the solid-state to the gas phase,

along with the removal of the matrix solvation shell, would negate any benefit

gained by the screening of the Coulombic charges in the matrix; however,

collisions during the plume expansion could serve to add the necessary

energy needed to maintain separation of the pre-formed ions and reduce the

incidence of charge recombination in the gas phase. Again, this is presumably

not the major process of ion formation, only one of a collection of processes

which could contribute to the overall production of ions during MALDI.









The mechanism of multiphoton ionization has been proposed to be

one of the major mechanisms leading to ion formation during the MALDI

process.76 The mechanism is simple and can be presented as:

M ,(hv) M.++e- 2.1

where M represents the chromophore (matrix molecule) and e- represents

an ejected electron. This proposal is consistent with the observation that

efficiency of MALDI is dependent on the absorbance coefficient of the matrix

used.70 The ionization energy of most MALDI matrices in bulk crystal form is

still not known; however, the ionization energy of the individual matrix

molecules appears to be greater than the 7.36 eV energy of two photons for

the nitrogen (337 nm) lasers typically used in MALDI. Because of the low

irradances typically used in MALDI (1 x106 W/cm2) the absorbance of more

than two photons is unlikely. One possibility which has recently been

proposed is that matrix molecules absorb two photons of laser energy and

reach an excited state just below the threshold necessary for electron

ejection.77 During the plume expansion, where matrix/matrix collisions are

numerous, a thermal distribution of vibrational energy is established which can

then lead to the thermal emission of an electron from the electronically excited

matrix molecules. This mechanism has been shown to be in good agreement

with experimental observations.

Another proposed mechanism for achieving electron ejection without

absorbance of more than two photons of energy is when two electronically









excited matrix molecules pool their energy to produce an ion.78 The basis for

this theory stems from the observation of matrix ion suppression when the

analyte concentration is increased above normal (normal being w 1:10,000 vs.

matrix). The authors note that this cannot be simple proton affinity competition

since, when the analyte preferentially cationizes, the matrix ions (of the [M+H]*

form) are still suppressed. Also, when the primary form of analyte ion is

[M+H]*, this can suppress the formation of matrix ions of the [M+Cation] form

(Figure 2.9). The authors proposed a mechanism which involves two excited

state matrix ions:

mH+hvu-->m*H 2.2

m*H+A )m- +AH+ 2.3
m*H

for protonation and a similar mechanism, where the proton (H) is replaced by a

cation, for cationization. Both mechanisms predict an abundance of matrix [M-

H] ions during matrix suppression, even in the absence of [M+H]* matrix ions,

and that prediction has been supported by experiment.

A similar proposed mechanism is simple excited-state proton transfer.

This mechanism has been proposed since the seminal paper of Karas,

Bachman, and Hillenkamp which led to MALDI as it is practiced today.70 The

proposed mechanism is the same as shown in equations 2.2 and 2.3, but

instead of a second excited state matrix ion being required, the acidity of the

excited state matrix ion formed in equation 2.2 is increased to the point that

proton donation happens without the need for additional energy. The proton











































0 200 400 600 800 1000 1200 1400
m/X

Figure 2.9. Spectrum (a) shows MALDI of substance P in matrix of 2,5-
dihydroxy benzoic acid (DHB) where matrix-to-analyte ratio was 1000. The
spectrum shows strong signals for matrix [M+H] ion and cationization.
Spectrum (b) shows MALDI with matrix-to-analyte ratio of 100. The spectrum
shows strong signal for analyte ion of [M+H]*. Note that all matrix signal was
suppressed, as well as the [M+Na].78









exchange between the excited state molecule (with increased acidity) and the

analyte molecule could happen either in the solid state just prior to ejection, or

in the expanding plume after desorption while the plume is still fairly dense.

The possible contribution to MALDI ions from pre-formed ions in the

matrix prior to desorption has been proposed. It has been shown that ions are

produced (in the cationized form) even when the laser beam impinged on the

backside of a 200 nm thick gold foil (thus removing the possibility of photo-

excited molecules), leading to the belief that these were thermally desorbed

pre-formed ions.79 It is difficult of course, to distinguish between pre-formed

ions and those ions formed during early thick-plume formation.

For UV MALDI, the matrix is generally a strongly absorbing

chromophore of the incident light; in IR MALDI, however, this is not generally

the case. Because of the similar spectra produced by both IR and UV MALDI,

mechanisms which do not require photo-excitation have been proposed to

contribute to both forms of MALDI. Early work with simple laser desorption

demonstrated the formation of the [M+Na] ion of the sugar stachyose from a

sugar and Nal mixture (at a ratio of 5:1) when the laser light was focused on

the backside of a thin formvar film (with the mixture deposited on the opposite

side).80 When the formvar film and sample were not perforated (laser intensity

of 1 x1011 W/cm2, sample 20 pm thick), the [M+Na] ion was produced almost

exclusively with very little fragmentation. This observation led to the proposed

"shock wave" theory for desorption of ions. This proposal rules out the









possibility of simple thermal desorption of ions, since the large increase in

temperature at those laser intensities would have produced extensive thermal

degradation and fragmentation. The theory proposes that a thermally induced

shock wave propagates through the solid leading to the desorption of pre-

formed ions from the opposite side. This shock wave desorption is very

similar to process developed for secondary ion mass spectrometry (SIMS).

This model of desorption led to the proposal of the pressure pulse model for

desorption and ion formation.81 In the proposed model, two-center excited

annihilation (the contribution of two excited state matrix molecules) may lead

to ion formation with desorption the result of the pressure-driven shock wave.

Another model, at least superficially similar to the pressure-driven model

above, is the spallation theory proposed by Cramer, Haglund, and

Hillenkamp.82 Spallation is a process by which layers of material next to the

free surface area are ablated by thermally induced stress which can build-up

at rates faster than can be dissipated by acoustic waves, even though the

energy densities are too low for the process to be simple vaporization. The

proposed ionization occurs as a result of the bond-breaking (possibly a

piezoelectric process) that occurs during spallation which allows free charge to

develop at the fracture site. It should be noted that this ionization proposal

was only mentioned as a possibility and no serious investigation has been

conducted to prove or disprove the likelihood of it.









Secondary ion formation


Secondary ion formation would result from reactions within the

expanding plume formed during MALDI. Since the plume above the irradiation

site is considered to be dense enough for multiple molecule-molecule and ion-

molecule collisions, one proposed theory is that of gas-phase proton transfer.

Hydrogen atoms have been found in quantities which are considered to be

significant in MALDI plumes.83 This opens the door for a proposed

mechanism similar to that in fast atom bombardment:84

M+e-- (M H) +H 2.4

where the matrix captures a free electron and then produces a hydrogen

radical which is available for donation to analyte or matrix. Again the theory of

photo-induced increased acidicty of matrix ions could produce proton transfer

reactions in the expanding plume and result in protonated matrix and analyte

ions.70 Finally, in early work with laser desorption without MALDI matrices, it

was shown that cationization of molecules occurred readily in the gas phase.85

There is no reason to assume that a similar process would not also occur in

MALDI.

It must be emphasized that none of the proposed mechanisms can be

assumed to be the only mechanism by which ionization occurs in MALDI, but

they all may be important to a greater or lesser degree for ionization

depending on experimental conditions (i.e. matrix, solvent, analyte,

wavelength, irradiation intensity, etc.).









Practical Aspects of MALDI on Tissue


In traditional MALDI, the analyte and matrix are co-dissolved in a

solvent and pre-mixed before being deposited on the sample probe. The

solvent is then allowed to evaporate, incorporating the analyte molecule into

the crystal lattice structure formed by the matrix during drying. Because

MALDI experiments performed in this dissertation involved with mapping of

pharmaceutical compounds present in intact tissue, the matrix and analyte

could not be pre-mixed before application of the MALDI matrix and solvent.

This change in the MALDI preparation procedure had a direct effect on the

results and methods presented in this dissertation and should be considered.

For the MALDI experiments presented in this dissertation, the MALDI matrix

was dissolved in a solvent which was capable of dissolving both the matrix

and the analyte. A thin slice of tissue was placed on the probe tip and allowed

to air dry. It was necessary to allow the tissue to thoroughly dry before

analysis in the mass spectrometer because once introduced into the vacuum

of the mass spectrometer solvent from the sample would evaporate at an

elevated rate. This increase in the rate of evaporation of the solvent from the

sample, beneath the MALDI matrix coating, would loosen or remove the

MALDI matrix from the surface of the sample. Additionally, the evaporating

solvent would disturb the surface of the sample and would be a source of gas-

phase neutrals which could produce unwanted ion molecule reactions. Once

the tissue had dried (usually one to two hours), the solvent and matrix were









then pipetted (or electrosprayed where indicated) onto the surface of the

tissue and allowed to air dry (usually within one minute). Figure 2.10 shows a

representative drawing of the MALDI preparation method used for tissue

samples in this dissertation. The applied solvent (with matrix) served to extract

the analyte from the tissue. The extracted analyte was then incorporated into

the crystal lattice formed by the matrix molecules when the solvent

evaporated.62 The implications of the differences between this method and

the traditional method used in MALDI preparation on spatial resolution will be

made clear in chapter 3.


MALDI of Paclitaxel in Tissue


Paclitaxel is an anticancer drug from the Taxus alkaloid family of

products found in the Pacific yew tree bark.86 Paclitaxel functions as an anti-

cancer drug by stabilizing microtubules (composed of tubulin) during cell

division (mitosis).87 While the function of paclitaxel is well known, there is a

great deal of interest in how paclitaxel is delivered to and how it attacks the

living tumor. Clinical studies indicate that paclitaxel first binds (reversibly, with

low affinity) to plasma proteins.88 The paclitaxel is then cleared from the blood

and delivered to the tissue (including the tumor). This suggests the possibility

that paclitaxel may be primarily delivered to the outer layers of a hard tumor.

One of the long term goals of this research was to elucidate the delivery

location of paclitaxel in a human tumor.












Solution


Figure 2.10. Drawing of MALDI process for tissue. Top drawing shows the MALDI matrix in solution deposited on
top of tissue sample. The solvent extracts the analyte from the tissue sample into the solvent phase. Bottom
drawing shows analyte incorporated into MALDI matrix after evaporation of solvent.


96%!94-










An ovarian tumor was obtained from Bristol-Myers Squibb Oncology

Division (Princeton, NJ). The tumor was human in origin but had been

implanted and grown to size (approximately 10 mm in diameter) in an

immunodeficient, nude mouse. The mouse was administered a single dose of

paclitaxel in a 10% cremphor, 10% ethanol, 80% saline solution intravenously.

The concentration of paclitaxel in the tumor was reported by Bristol-Myers

Squibb to be 10-50 ng/ mg of tumor. Approximately one hour after

administration of the drug solution, the mouse was sacrificed and the tumor

was excised. The tumor was then snap frozen and sent to the University of

Florida packed in dry ice. The tumor was sliced thin (approximately 0.5 mm)

while frozen and 5.0 [tL of a 0.1 M 2,5-dihydroxy benzoic acid (DHB) in

methanol solution was placed on top of the tumor section and allowed to air

dry completely before analysis.

A UV laser was used for the MALDI experiments. The UV laser was a

Laser Science Inc. (Cambridge, MA) model VSL-337ND pulsed nitrogen laser

with a wavelength of 337.1 nm and a 3 ns pulse width (full width at half

maximum). The laser is near-diffraction limited, which allowed the beam to be

more easily focused to a tight spot. The maximum energy output was >250

p.J/ pulse with a peak power of 85 kW. The laser beam's intensity was

adjusted between 106_107 W/cm2 using a wheel attenuator (Newport Corp.,

Irvine, CA). The position of the laser beam was adjusted manually with an

x,y,z-micromanipulator attached to the beam deflector (Newport Corp.).









The top panel (A) of Figure 2.11 shows the MALDI MS spectrum (an

average of 30 laser shots) of the ovarian tumor after forward/reverse scan

isolation of a 50-dalton-wide window around the [M+H] ion of paclitaxel (m/z

854). Because of the wide window used (to maximize sensitivity) both the

[M+H]* ion (m/z 854) and the [M+Na] (m/z 876) can be seen. The [M+Na]*

ion was chosen for fragmentation because it typically represented the most

abundant paclitaxel ion (although the [M+H]* is the most abundant ion in panel

(A), this was not typical for other locations on the tissue sample). The laser

beam was moved to a new location on the sample and a daughter ion spectra

of the [M+Na]* ions from another 30 laser shots were acquired and averaged.

The center panel (B) of Figure 2.11 shows the MS/MS daughter ion spectrum

of the [M+Na] ion of paclitaxel from the tumor; this spectrum was reproducible

and so could be used for the detection of paclitaxel in tissue. The daughter

ion spectrum of the paclitaxel [M+Na]* ion from the ovarian tumor shows an

almost identical pattern to that of the daughter ion spectrum of the [M+Na]* ion

of a paclitaxel standard, as shown in panel (C) of Figure 2.11 and in Figure

2.12. In both cases the [M+Na]* ion intensity was reduced in the daughter ion

spectrum because it was selectively fragmented by CAD. Figure 2.13 shows

the structure of paclitaxel and the proposed fragmentation pathways of the

daughter ions observed in Figure 2.12. Visual inspection of the tumor after

analysis showed that the laser had burned completely through the tissue (a

depth of 0.5 mm). The holes blasted in the tissue were measured to be











3UU -

400 -

S300 -

I 200 -

100 -


[M+H] +
854


S ., 111.. ,,1


I I I I I I I I I I
100 200 300 400


I . . .


k IiI..I I&


+
[M+Na]
876


a1,;,ui


I I I I I I I I I I I I I I I I I I I


500
m/z


600 700 800 900


350
300
250
200
150
100
50
0




600
500
400
300
200
100
0


100 200 300 400 500 600 700 800 900
m/z


100 200 300 400 500 600 700 800 900
m/z

Figure 2.11. Spectrum A shows the MALDI spectum for paclitaxel in an
ovarian cancer tumor after isolation of the region around the [M+H]* ion at m/z
854. Spectrum B is the daughter ion spectrum of the [M+Na]* ion from a
different location on the same sample. Spectrum C is the daughter ion
spectrum of the [M+Na] of a paclitaxel standard.62


.4. 1,11 I












600 -

268

500 240 286

308
400 [M+H]
591 854
509
(nF
"300



200
531


100 [M+Na]+
S876


0 4 1;411JYO,4^A^^
100 200 300 400 500 600 700 800 900
m/z

Figure 2.12. Daughter ion spectrum of [M+Na] standard of paclitaxel by
MALDI.62
















/_ + Na

N/H H
O^N"/0 A<_.
QOH

0


286+ Na = 308
569 + Na = 591
-H2O
H2 -CH3CO2H
268
509 +Na = 531


Figure 2.13. Proposed fragmentation pathways of paclitaxel that lead to
observed daughter ion spectrum seen in Figure 2.12.









approximately 100 pam in diameter; thus the ablated tissue was estimated to

be approximately 0.09% of the total tissue mass on the probe (6.5 mg). Based

on the reported amount of paclitaxel in the tissue (50 ng/mg), the spectra in

Figure 2.11 (A) and (B) each represent approximately 290 pg of paclitaxel

ablated. The detection of paclitaxel at physiological trace levels satisfied the

first goal for the ion trap laser microprobe instrument; however, the second

goal, the identification of the location of paclitaxel within the tissue

mass, was not achieved. The thick (DHB) crystal matrix coating on top of the

tissue slice, coupled with the inability to visually inspect the tissue during

analysis, prevented any correlation of acquired spectra with a specific region

of the tissue.













CHAPTER 3
LASER DESORPTION / CHEMICAL IONIZATION


The results of the initial experiments performed on the instrument,

demonstrated the ability of the instrument for the detection of pharmaceutical

compounds at trace levels in intact tissue; however, the ability to map the

location of the compounds was not accomplished. The application of the

MALDI matrix onto the surface of the tissue during sample preparation

obscured visualization (after solvent evaporation and crystal formation) of the

tissue surface below. Additionally, experiments performed in our lab, as well

as experiments in other labs, indicated that the spatial location of a compound

extracted from a surface into the solvent and matrix mixture during MALDI

preparation allowed the analyte to migrate before crystallization of the

matrix.37'62'89 The primary cause of the migration of the analyte has been

given as convection currents induced in the solvent during the crystallization

process.37 The theory is that the growth of the crystals in the solvent creates

currents. The currents caused by crystal formation, as well as currents due to

evaporative cooling of the solvent surface, move the analyte and blur the

spatial location after crystallization is complete.

Because of the need for an accurate representation of the

pharmaceutical compound's spatial location within the intact tissue mass









under investigation, two alternative methods to the simple pipetting of the

MALDI matrix onto the surface of the tissue were investigated. The first of

these alternative methods was laser desorption coupled to chemical ionization

(LD/CI), which is described in this chapter. The second method was the

electrospraying of the MALDI matrix solution onto the surface of the tissue,

which is described in chapter 4.


LD/CI


In Chapter 2, the theory behind MALDI was separated into two parts:

desorption and ionization. The two events were treated as separate but inter-

linked events. In LD/CI the process of desorption is truly separated from the

process of ionization. Each will be treated separately in this chapter.


Laser Desorption


Much of the early work with laser desorption mass spectrometry was

done on inorganic samples (looking for elemental information), where

fragmentation was not an issue.90 In 1968, Vastola and Pirone used a pulsed

ruby laser to desorb and ionize polycyclic aromatic hydrocarbons (PAHs), alkyl

compounds, and the amino acid leucine.91 The amino acid was extensively

fragmented by the desorption and ionization process, while the PAHs showed

good results by forming radical molecular ions, M-. Two years later, the same

group showed the laser desorption/ionization of the alkali salts of 1-hexyl









sulfonate.92 This time the major ions produced from a series of four different

alkali salts was the alkali-cation form of the molecular ion, [M+cation]*. Almost

ten years after the original work of Vastola and Pirone, Posthumus et al.

presented the laser desorption/ionization mass analysis of digitonin (a large

thermally labile, non-volatile, organic molecule) which was detected as the

[M+Na]* ion.93 It is this work by Posthumus et al. which has been credited with

creating interest in the laser desorption/ionization of organic compounds.90

Since that time, all manner of lasers and mass analyzers have been put

together to do laser desorption/ionization mass spectrometry.

The desorption of large, thermally labile organic compounds by laser

desorption can be broken into three different types of processes (as with

MALDI, the contribution of these processes is dependent on experimental

conditions). For low power density conditions (< lx10 8 W/cm2), where the

substrate beneath the sample has a higher absorbance than the sample, a

simple thermal process is assumed to dominate.94 Thermal desorption of

thermally labile compounds without extensive fragmentation would seem to be

contradictory; however, it is the rate at which the sample is heated and the

high temperatures attained by the laser beam irradiance which allows this

process to produce intact molecular-type ions. Figure 3.1 shows an Arrhenius

plot of the temperature-dependent nature of vaporization and decomposition

of thermally labile compounds.95 For a compound to be non-volatile and

thermally labile, the activation energy for desorption would have to be higher





























Higher temperatures,
vaporization favored


Lower temperatures,
decomposition favored

S.Decomposition
reaction




Vaporization


Figure 3.1. The relationship between the temperature-dependent nature of
vaporization and decomposition. The two processes are represented by
Arrhenius plots of the rates of vaporization and decomposition. At a
sufficiently high temperature (the left side of the plot) the rate of vaporization
proceeds faster than decomposition. Laser heating produces extremely high
temperatures at a rate fast enough to move the sample to the left side of the
plot (where vaporization is preferred) before extensive fragmentation can
occur. The desorbed compounds are then cooled by expansion into the gas
phase.95


&nK









than the activation energy for decomposition. However, If the temperature is

raised high enough, fast enough, the rate of vaporization can exceed the rate

of decomposition and the compound can be evaporated into the gas phase

without decomposition. The vaporized material is then cooled during

expansion into the gas phase in much the same way that the matrix molecules

and analyte were cooled by expansion for MALDI. The major form of ions

formed during thermal desorption of organic compounds has been seen to be

the [M+Cation]* form.96 It has been proposed that this form of ion is produced

by the thermal emission of alkali-metal ions from the hot center of the laser

beam spot (which interacts mainly with the substrate beneath the sample).

The alkali-metal ions then combine in the gas phase with thermally emitted

neutral organic molecules produced mainly from the cooler outer edges of the

laser spot on the sample.97

When the laser power is increased (>1 x109 W/cm2) and the sample is

thick (which reduces the influence of the substrate beneath the sample) a

different desorption process has been observed, one which has been called

pressure-wave-driven desorption.98 The proposed mechanism is similar to

that which was previously covered in the MALDI section of the last chapter of

this dissertation. Basically, the rapid rise in temperature of the substance

under investigation produces an explosive decomposition of material, which in

turn produces a compression wave that is driven through the material. At the

surface, this pressure wave causes the ejection of particles (including intact









molecular-type ions) into the gas phase. The experimental evidence for this

proposed theory came in a series of experiments where thin layers of

carbolfuchsin were electrosprayed onto a thin gold film (5 Pm thick).98 The

laser (a frequency-quadrupled Nd:YAG, 266 nm wavelength) impinged upon

the film from the backside (the side opposite that where the sample was

deposited). When the power density was high (~ xl 09 W/cm2) and the film and

sample layers were perforated by the laser beam, numerous fragment ions of

carbolfuchsin were observed along with ions from the gold foil. However,

when the foil was not perforated( power density ~ x108 W/cm2) the primary

ion desorbed was the [M+H]* ion of carbolfuchsin and there was no

fragmentation. This was taken to be indicative of a pressure-wave-driven

desorption mechanism.

The third type of desorption process seen in laser desorption is the

MALDI process, where the absorbance of the laser light by the substance

plays a major role in the desorption process. As in MALDI, this process is a

collective process where thermal desorption and many other process play a

part, depending on experimental conditions. The original research by Karas et

al. demonstrated the importance of the laser wavelength in laser

desorption/ionization. Since this subject was already covered in chapter two, it

will not be duplicated here.









Chemical Ionization


It was noticed early on that during laser desorption the ionization

process appeared to happen primarily during the time when the laser beam

interacted with the substance, but that neutral molecules continued to be

desorbed from the surface for some time after the laser irradiation ceased.

During the first organic application of laser desorption/ionization by Vastola

and Pirone, it was noticed that ions were emitted for a 200-400 ps period,

which seemed to correspond to the peak of the laser pulse (800 ps).91 Neutral

molecules (ionized by an electron beam) were observed to be produced for

several hundred microseconds after the end of the laser pulse. Van Breeman,

Snow, and Cotter in 1983 showed that a 40 ns laser pulse (a C02 laser was

used) produced neutral molecules for 4-10 ms after the laser pulse ended

(Figure 3.2).99 The investigators demonstrated that under high pressure

source conditions (a Cl reagent gas of isobutane was maintained at a

pressure of 0.5 torr in the source region), the time over which ions left the

source was extended from 1 gs to 4-10 ms. This extension in time allowed for

better mass analysis by a scanning magnetic sector instrument since an

extended time period is required for the analyzer to scan the mass range. This

experiment also showed that chemical ionization could be used to increase the

number of analyte ions produced by the desorption event which could be used

to lower the limit-of-detection of the method. Figure 3.3 shows the results of

laser desorption of tetramethylammonium chloride during the first 500 Hs










LASER DESORPTION METHODS (schematic)

I DESORPTION FROM THE SURFACE


LASER PULSE


--* I*- 40 S


lIONS DESORBED


[NEUTRALS DESORBED|


T IONS LEAVING THE SOURCE

A LOW SOURCE PRESSURE (no reagent gas)


(1)ELECTRON BEAM OFF

A


[LASER IONIZATION]


(2) ELECTRON BEAM ON


LASER El|


B. HIGH SOURCE PRESSURE

(I) ELECTRON BEAM OFF


ISOBUTANEE)


[LASER ONIZATION


(2) ELECTRON BEAM ON


LASER CI


Figure 3.2. A schematic representation of the ions and neutrals produced by a
40 ns CO2 laser pulse. The diagram shows the detected ion as a function of
time under different conditions. A scanning magnetic sector instrument was
used to produce the results.99


- A + jT a. -^ - Tlur irir & S^^ - ;lT ,m I I--iij- : -


"'> A ( "~lj!S









140W


1200

1000


LASER [ESORPTION


(0LR)4n+
LASER DESORiPTION


-- LA E.SERIE .1 .
rt
40 L-osm^^ ^ E. 1.^- ,

200-

LASER, El
of
0 100 200 300 400
MICSECMN *fTER LUSER MAM

Figure 3.3. The intensity of ions detected during the first 500 ps after laser pulse in the desorption of
tetramethylammonium chloride.99


0(4) 0.









following a CO2 laser pulse.99 A TOF mass analyzer was used for these

experiments and the ions were extracted from the ion source at different times

by means of a drawout pulse which was delayed for a variable period after the

laser pulse event. An electron beam was used to ionize the neutrals desorbed

after the laser pulse. The interesting thing about Figure 3.3 is the intensity of

the ions produced. The intensity of the ions produced by the electron beam

ionization of neutral molecules desorbed after the laser pulse is approximately

three times that of the laser ionization alone. In our own lab, Robert

Perchalski studied the use of chemical ionization for extending the ion

production lifetime of several compounds sampled by laser desorption.100 He

concluded that there were two processes underlying the extended lifetime of

ion production noted in LD/CI. The first processes was the chemical ionization

of laser produced molecules, simple LD/CI. The second process was the

vaporization of the analyte from the sample surface by the action of the

reagent gas (termed desorption chemical ionization-DCI). Even when the

reagent gas entering the source was blocked from direct interaction with the

sample, the process of DCI could be initiated by the input of thermal energy in

the form of the laser beam. The DCI process was dependent on the ion source

temperature, and the signal lifetime was dependent on the melting point of the

compound and the size of the sample.

Because we were interested in mapping the location of trace level

pharmaceutical compounds in intact tissue, it was decided to try chemical









ionization in conjunction with laser desorption. Laser desorption of the intact

tissue meant that no matrix needed to be added to the tissue surface (which

was the cause of analyte migration). Chemical ionization was used so that

desorbed neutral molecules would be ionized for a lower limit-of-detection.

Chemical ionization is a general term used to describe the ionization

of molecules by ion-molecule reactions in the gas phase. Harrison offers a

comprehensive review of chemical ionization mass spectrometry; those

wishing a more detailed description of the process are refereed to his book.101

The basic concept of chemical ionization is as follows: a reagent gas is

introduced into the ion source at sufficient pressure (generally between 0.5

and 1 torr) to assure that an adequate number of collisions result between

reagent ions (produced by electron ionization) and the neutral reagent gas

molecules. Since the reagent gas partial pressure in the source is in large

excess of that of the analyte (usually 1,000 to 10,000 times greater) the

reagent gas preferentially undergoes electron ionization. Once ionized,

reagent gas ions react with the large number of reagent gas neutrals present

to form a steady state of reagent ions (for methane the major reagent ions are

CH5 and C2H5+). If a collision between one of the reagent ions and an analyte

neutral occurs, and the gas-phase proton affinity (PA) of the analyte is greater

than the proton affinity (PA) of the conjugate base of the reagent ion, then a

proton exchange reaction can occur to from an [M+H] ion of the analyte:

[X+H] + M -- [M+H] + X 3.1









where [X+H]* is the reagent ion, X is the conjugate base of the reagent ion,

and M is the analyte molecule. The difference in gas-phase proton affinity of

M and X determines the amount of energy deposited into M by the proton

transfer process. By selection of the proper reagent gas (to form ions whose

conjugate base PA is close to the PA of the analyte), the primary ion produced

by the Cl reaction is the [M+H]* ion and the number of fragment ions can be

reduced. In MS/MS, the molecular-type ion is preferred because it is more

structurally characteristic of the intact analyte. Furthermore, selecting an

ionization method which produces only molecular-type ions, with no

fragmentation, concentrates the ion current in a single ion. This production of

a single-type ion reduces the limit-of-detection for MS/MS.


LD/CI of Spiperone in Intact Tissue


To demonstrate the ability of LD/CI to detect pharmaceutical

compounds in intact tissue, a laser of sufficient power to desorb intact tissue

was needed. The laser used in the previous MALDI experiments (a VSL-

337ND pulsed nitrogen laser from Laser Science Inc., Cambridge, MA) had a

maximum energy output of approximately 250 gJ per pulse with a 3 ns pulse

length. This gave a power output of approximately 83 kW per pulse.

Focussed down to a spot size of 100 jtm diameter, the power density on the

tissue was 1 x109 W/cm2 which is considered to be sufficient to produce

vaporization of some material.90 However, since the sample was thick









(approximately 0.5 mm) interaction of the laser beam with the substrate (the

probe tip beneath the tissue) was not significant. This meant that the tissue

itself needed to have sufficient absorbance to produce desorption. The

absorbance of the tissue at 337 nm was not sufficient to desorb enough

analyte neutrals or ions from the surface of the tissue for detection. As was

pointed out earlier, in laser desorption/ionization where thick samples are used

a power density of greater than 1 x109 W/cm2 is generally used. While the

power density for the desorption of neutrals is considered to be lower than that

of laser desorption/ionization, the inability of the UV laser to produce sufficient

neutrals made it necessary to find a laser which would produce a consistent

desorption process. For that reason, a Lumonics (Ontario, Canada) series

TE-860-4 excimer CO2 IR laser with a wavelength of 10.6 iim and a maximum

energy output of 6 J per pulse (here typically 2.6 J per pulse) and a pulse

duration of 2 ns to 1.5 lis was used for the initial LD/CI experiments. The

beam size of the CO2 laser was reduced by physically blocking it from a 25 x

13 mm rectangular spot down to a round spot with a diameter of 4 mm before

focusing. The physical blocking reduced the energy per pulse used in these

experiments to approximately 115 mJ (assuming an even energy distribution

across the beam and simple reduction in beam size). It was necessary to

reduce the energy per pulse from the CO2 laser because early experiments

indicated that so much tissue was ablated at full laser power that the inside of

the ion volume was coated by the ablated material after a single laser pulse.









The coating of the inside of the ion volume allowed it to become electrically

charged (by the electron beam used in Cl), dramatically reducing ion

transmission after a single laser shot. The spot size after focusing had a

diameter of 750 gim, making the power density approximately 3 x 107 W/cm2.

Although this power density was less than that of the UV laser, the CO2 laser

produced consistent desorption of the tissue samples.

The laser was focused into the mass spectrometer chamber by a

single zinc-selenide focusing lens (Melles-Griot, Irvine, CA) with a focal length

of 25.4 cm. The laser was externally triggered after a 1 ms delay by a custom

built electronic circuit. The delay of the laser trigger was necessary to assure

that the ion gate (the electrostatic lens in the ion source just before the turning

quad) had sufficient time to switch from +170 V to -25V (the typical voltage

applied to the lens during ion transmission. The software (Gatorware) used to

control the scan and acquisition of the ITS-40 was written by Tim Griffin and

Nathan Yates at the University of Florida.102'103 Methane gas was introduced

into the ion source of the instrument for chemical ionization. The pressure

reading (uncorrected) in the ion source side of the vacuum housing was

4 x104 torr. A capacitance manometer, connected to the ion source by a

hollow probe shaft, showed that this chamber pressure produced a Cl ion

source pressure of 0.5 torr. This Cl source pressure was high enough to

produce good chemical ionization spectra of perfluorotributylamine (PFTBA,

mw = 671), a calibration compound commonly used for El and Cl in mass









spectrometers. The ionization was considered good chemical ionization when

the PFTBA fragment ion at m/z 502 (a common El fragment ion of PFTBA)

was less than 10% of the PFTBA ion at m/z 652 (a common Cl fragment ion of

PFTBA).

For the initial LD/CI studies, the drug spiperone was chosen as the

target compound. Spiperone, developed as an antipsychotic drug, belongs to

a class of compounds known as azipirones which are similar to serotonin in

structure and so bind to the 5-HT1A receptors in the central nervous system.104

In vivo neural recording studies of spiperone have shown that it binds not only

to the 5-HT1A receptors in the brain, but also to 5-HT2 receptors.104 Because

these receptors are present at various densities throughout the brain, there is

considerable interest in determining the concentration of spiperone in different

cerebral regions. Spiperone was chosen as a model compound because in

previous studies in our lab it had been demonstrated to be very stable over

time (solutions of spiperone did not significantly degrade over more than a

year) and it had been shown to be easily incubated into tissue samples.62

Additionally, spiperone has been the subject of previous MS studies in our lab

and so has been well characterized.105'106 Figure 3.4 shows the LD/Cl MS/MS

daughter ion spectrum of the [M+H] ion (at m/z 396) of a spiperone standard.

After isolation (a 5-dalton-wide window by the forward/reverse scan method),

the [M+H] ion was fragmented by CAD to produce the daughter ion spectrum

shown. The major daughter ions produced are at m/z 165 and 291; daughter













Spiperone MW 395


291


123


232 262


, J i i


1 I 1 1 I I j I i I I i I P I 1 a II


300 I
300


400


200


Figure 3.4. The LD/CI MS/MS spectrum of the [M+H] ion (m/z 396) of the
drug spiperone. The peak at m/z 397 is the 13C isotope of the [M+H] ion of
spiperone left after resonant excitation of the m/z 396 ion.


12000 -









8000



2-1
C




4000


I I I I


. == ....









ion peaks also appear at m/z 123, 232, and 262. The drawing above the

spectrum in figure 3.4 shows the molecular structure of spiperone and the

proposed fragmentation pathway which produced the observed daughter ions.

A whole liver was obtained from a male Sprague-Dawley rat after

sacrifice. The liver tissue was directly transferred to storage in a -20C

freezer. A 7.9 mg piece of frozen liver tissue (a slice of approximately 0.5 mm

thickness) was placed in a shallow stainless steel well. 4 ptL of a 100 ng/lIL

solution of spiperone in 1% aqueous acetic acid (HOAc) was placed on top of

the tissue. The tissue was allowed to incubate for one hour and was then

washed with several aliquots of 1% HOAc solution followed by several aliquots

of deionized water. The liver tissue was then blotted dry, placed on the probe

tip, and allowed to dry completely (approximately 1.5-2 hours) before analysis.

To determine the amount of spiperone absorbed by the tissue, the 1 % HOAc

washings of similar liver samples incubated in spiperone were collected and

analyzed by LC/MS. The same liver tissue samples (after washing) were

extracted with a solvent (with sonication), and the extract was analyzed. The

results of duplicate washing and extraction experiments indicated that 50% of

the collected spiperone was found in the washings and 50% was found in the

tissue extracts. For this reason, all concentration values for spiperone in

spiked tissue in this dissertation are given as 50% of the amount of spiperone

applied to the tissue. For all experiments, blank tissue samples were analyzed

to assure that there was no carry-over of analyte.









Figure 3.5 shows the LD/CI spectrum (5 laser shots) of a rat liver

tissue slice which was spiked to a level of approximately 25 ng/mg of tissue

with spiperone. Note that the peak at m/z 396, which corresponds to the

[M+H] ion, is completely buried in the tissue matrix noise (see inset for close-

up view of region around m/z 396). The ion injection time used during the

acquisition of this spectrum was only 90 [is (in contrast to the 4 ms used in the

other LD/Cl spectra presented here) to preserve close to unit resolution.

When the tissue was desorbed with an ionization time of 4 ms without the

isolation of a narrow m/z range, space charging shifted and broadened the

peaks, rendering the spectrum meaningless. It is obvious from Figure 3.5 that

without the capability for mass isolation and MS/MS available with the

quadrupole ion trap, the target drug could not be positively detected at such

low levels. In fact, we found that LD/CI of tissue produced such intense

interference ions that the standard forward/reverse scan isolation would not

adequately isolate the m/z region of interest. The tissue matrix ions

apparently continued to fragment during the forward and reverse stages of

isolation (the low m/z ions are ejected during the first (forward) step of the two-

step isolation), leaving ions at m/z values below the isolation region (data not

shown). Switching to SWIFT isolation waveforms, which provide axial

modulation at all frequencies around a notched set of frequencies selected by

the operator, overcame these problems. In this way the higher and lower m/z

interfering ions are ejected out of the trap at approximately the same time. If a










40000-





30000 -





c20000





10000





0 0
100


I iJL


200


4000-

3000-

2000

1000

0
3


lILI ,l


300


400


85 390 395 400 40


500


600


m/z
Figure 3.5. LD/CI MS spectrum of rat liver tissue spiked with the drug
spiperone to a level of 25 ng/mg. The inset shows a close up of the region
around the spiperone [M+H]* ion at m/z 396. The arrow indicates the peak at
m/z 396 which corresponds to the [M+H]* ion of spiperone.


I J ,R *


I "!









higher m/z interferent ion did fragment during isolation, the fragment ions

produced would still be given sufficient energy to be ejected from the trap. A

Stanford Research Systems (Sunnyvale, CA) model DS345 synthesized

function generator was used to generate the SWIFT waveforms used for

waveform isolation of ions in the ion trap during LD/CI experiments. The

waveforms were created with a computer program which was written at the

University of Florida by Peter Palmer which created isolation waveforms

similar to those described in a paper by Chen et al.107 Each SWIFT waveform

isolation burst took approximately 8 ms (7 bursts per scan were used for a

total isolation time of 56 ms), whereas the high mass ejection step of the two-

step isolation technique took 50 ms. This isolation technique produced

significantly better results.

Using the same tissue sample as above (with the probe tip rotated to

present a new area on the tissue to the laser beam), another LD/CI mass

spectrum of the spiked rat liver tissue was obtained (Figure 3.6). This time,

however, a 10-dalton-wide window around m/z 396 was isolated by SWIFT

waveform isolation following a 4 ms ion injection time. Once again, the peak

at m/z 396, which corresponds to the [M+H] ion of spiperone, is not the most

intense peak in the isolated region of the spectrum (see inset for close-up of

area). Note that the isolation of the region around the m/z of the ion of interest

leaves the lower m/z region relatively empty so that daughter ions produce by

CAD during MSA/MS can be easily identified.