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SPECTROSCOPIC METHODS OF ANALYSISMASS SPECTROMETRY

Mike S. Lee

Milestone Development Services, Newtown, Pennsylvania

INTRODUCTION

The dramatically increased expenditures for both in-house

and outsourced pharmaceutical research and development

(R&D) have led to a greater dependence on technology.

New technologies are constantly introduced into drug

development to address throughput issues and improve

development cycles. The incorporation of new technol-

ogies has resulted in fundamental change in the drug

development paradigm. Recently, sample generating-

based technologies such as high throughput biomolecularscreening and automated parallel synthesis have shifted

the bottleneck to sample analysis-based technologies.

The current focus on analytical techniques in the

pharmaceutical industry emphasizes four primary figures

of merit: sensitivity; selectivity; speed; and high

throughput. Mass spectrometry (MS) provides each of

these key attributes, and therefore, has been benchmarked

an effective solution for pharmaceutical analysis in each

stage of drug development (1). Perhaps more enabling than

the MS-based technology itself is the diverse applications

of MS in conjunction with sample preparation, chromato-

graphic separation, and informatics. It is within thiscontext that MS has played an increasingly vital role in the

pharmaceutical industry and has become the preferred

analytical method for trace-mixture analysis (Fig. 1F1 ).

A variety of MS formats are widely accepted and

applied in the pharmaceutical industry. The specific MS

application is often defined by the sample introduction

technique. The pharmaceutical applications highlighted in

this article feature two types of sample introduction

techniques: dynamic and static. Dynamic sample intro-

duction involves the use of high-performance liquid

chromatography (HPLC) on-line with MS. The resulting

liquid chromatography/mass spectrometry (LC/MS) for-

mat provides unique and enabling capabilities for

pharmaceutical analysis. The electrospray ionization

(ESI) (2) and atmospheric pressure chemical ionization

(APCI) (3) modes are the most widely used. Static sample

introduction techniques primarily use matrix-assisted laser

desorption/ionization (MALDI) (4).

The advances in MS instrumentation (5) and role of MS

within the pharmaceutical industry (1) have been recently

reviewed. This article will focus on MS technologies with

regard to specific applications in drug development. The

intent of this article is to provide an overview of MS

applications and describe the significant integration of this

technology into drug development. A detailed and in-

depth overview of current MS technologies and appli-

cations can be obtained from the recent proceedings of the

American Society for Mass Spectrometry Conference on

Mass Spectrometry and Allied Topics (www.asms.org)

and the Association of Biomolecular Resource Facilities

(www.abrf.org).

GENOMICS

Though the contributions of MS have been somewhat

limited in the field of genomics, there has been increased

participation and interest (6). Certainly, the worldwide

recognition received from the Human Genome Project

created a sense of urgency toward determining genetic

variation.

Genomics refers to the study of genetic data to draw

correlation between individual genetic inheritance andmedically or biologically important parameters. For

example, these parameters may involve a patients

response to a specific drug. Knowledge of the genetic

basis of individual drug response may provide under-

standing of the observed variability in drug response

arising as a result of genetically determined differences in

drug absorption, disposition, metabolism, or excretion (7).

Furthermore, knowledge of genetic variation may be

useful during the target selection process when multiple

targets are available within a specific disease state. Thus,

the pharmaceutical industry has great interest in

determining the genetic variation in patient populations.

Due to the existence of sequence variations, or

polymorphisms, no two human genomes are identical.

Single nucleotide polymorphisms (SNPs) are the most

abundant genetic variation with an estimated frequency of

1 SNP per 500 basepairs. Since SNPs are so prevalent in

the genome, they can act as markers that are linked with a

phenotype to provide a comprehensive measure of

interaction with a specific drug. The validation of a

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particular SNP represents an important stage for

establishing SNPs as a routine clinical diagnostic marker.

The validation of SNPs via MALDI-TOF MS is emergingas a valuable genotyping tool (8,9). A schematic of a

MALDI-TOF MS instrument is shown in Fig. 2F2 .

A recent study performed by Stroh et al. (10) compared

the performance of MALDI-TOF MS with restriction

fragment length polymorphism (RFLP) and fluorescence

polarization (FP). The study involved the analysis of

known mutations of the IL-1b gene. The procedure

involved amplification of patient DNA samples using

standard PCR techniques followed by a primer extension

step where a separate post-PCR primer is hybridized

directly adjacent to the SNP site. The resulting MALDI-

TOF MS data provided a direct confirmation of molecular

weight for fast analysis of polymorphisms.

Knowledge of the DNA sequence flanking the SNP site

allows for the optimum choice of post-PCR primer size,

primer location, and dideoxy-nucleotide(s). Thus, theassay can be designed to extract complete information

about the SNP regardless of its state. This emerging MS-

based approach for SNP genotyping has potential to be

highly automated without the requirement for fluorescent

tags. Furthermore, multiplexing can be attained by

selecting post-PCR primers of varying lengths to dedicate

predetermined regions of the mass spectrum to specific

SNPs.

PROTEOMICS

The study of protein structure, function, quantity, and

interactions during maturation and progression of disease

is referred to as proteomics. Analytical approaches that use

a combination of two-dimensional (2-D) gel electrophor-

esis for protein separation and MS analysis for protein

identification followed by database searches is a widely

practiced proteomics strategy (11). The tryptic peptides

extracted from gels are analyzed by MALDI-TOF MS and

microcolumn or capillary LC tandem mass spectrometry

Fig. 2 Schematic of a MALDI-TOF MS instrument. MALDI-TOF samples areprepared with a matrix that contains a small organic molecule capable of absorbing

ultraviolet light. A laser is used to desorb ions from the sample plate and the resulting

ions are forced into the flight tube by application of the acceleration voltage from

extraction grids. All ions leave the source with the same kinetic energy and travel

down the flight tube toward an ion reflector. Separation is based on mass with lighter

ions traveling faster than heavier ions. The ion reflector is used to correct for small

kinetic energy differences between ions of the same mass resulting in improved

resolution and mass accuracy. (Courtesy of Applied Biosystems, Framingham, MA.)

Fig. 1 Structure analysis matrix that illustrates pharmaceutical

analysis preferences for four specific sample types: nontrace/-

pure; nontrace/mixture; trace/pure; and trace/mixture. (Courtesy

of Milestone Development Services, Newtown, PA.)

X-ray

NMR

MS

UV

IR

HPLC/UV

LC/MS

LC/NMR

Trace

Non-

Trace

Pure Mixture

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(MS/MS) techniques. Typically, the MALDI-TOF MS

techniques are used to quickly identify peptide fragments

and confirm the presence of known proteins. Nano-scale

capillary LC/MS/MS techniques (using 50 100 mmdiameter columns, operating at flow rates of 20 500

nL/min) are used to further interrogate the complex protein

mixture at the low femtomole level. These techniques

require the use of a specialized ESI source shown in Fig. 3F3 .

The combination of MALDI-TOF MS and capillary

LC/MS/MS was recently described for the identification of

disease state markers in human urine (12). In this study,

urine proteins obtained from emphysema patients were

separated on 2-D gels and selected spots were digested

with trypsin and analyzed by MALDI-TOF. A database

search using Protein Prospector identified a potential

biomarker for emphysema as human alpha-1-antitrypsin

(A1AT). The corresponding MALDI spectrum contained

nine out of 18 peptides with masses that match the

expected tryptic digest fragments for A1AT.

The same tryptic digest protein sample was analyzed

by capillary LC/MS/MS using an ion trap mass

spectrometer followed by a database search with

SEQUEST.F4 Fig. 4 illustrates the components of an ion

trap mass spectrometer. The highly automated data-

dependent MS/MS analysis provided excellent

sequence coverage for 11 tryptic peptides related to

A1AT in a single LC/MS run. A tryptic peptide that

corresponds to the A1AT sequence SVLGQLGITKobserved at retention time (rt) 30.5 min. was observed

in the spectrum. The LC/MS data also provided

sequence information on unmatched MALDI peaks.

The need to detect lower concentration of protein and

peptide mixtures has resulted in the increased use of

hybrid quadrupole/orthogonal TOF (QTOF) MS/MS

instruments (13) in conjunction with microcolumn LC.

Fig. 5F5 shows a schematic of a QTOF instrument. This

LC/MS approach provides a resolution of ca. 0.1 mass

units allowing for the analysis of complex product ion

spectra (14,15). A recent publication by Chalmers and

Gaskell highlights the current challenges in proteome

analysis with regard to MS instrumentation (16).

NATURAL PRODUCTS DEREPLICATION

Historically, an excellent source of novel lead drug

compounds is natural products. Natural product screening

activities typically occur during drug discovery and

Fig. 3 Schematic of a nano-scale capillary ESI interface. This specialized LC/MS

interface, operating at flow rates from 20 to 500 nL/min and using 50 to 100 mm ID

columns, typically provides low femtomole sensitivity. Fully automated sample

handling and preparation procedures (i.e., desalting and preconcentration) combined

with specialized devices for high separation and variable nL gradient flow rates

provide unique capabilities for high-throughput analysis of proteins. (Courtesy of

New Objective, Cambridge, MA.)

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involve the testing of crude extracts obtained from

microbial fermentation broths, plants, or marine organ-

isms. When activity above a certain level is detected,

active components are isolated and purified for identifi-

cation. This process is often time-consuming, where the

physicochemical characteristics of the active components

are determined, known compounds are identified (dere-plication), and the novel compounds are scaled-up for

more detailed investigation.

Analysis strategies that use on-line ESI-LC/MS

approaches provide an integrated format for natural

product dereplication by combining traditional fraction

collection, sample preparation, and multi-component

analysis into a single step. In this way, crude extracts are

screened without extensive purification and chemical

analysis. Furthermore, less material is required due to the

sensitivity of the technique and chromatographic resol-

ution is retained.

The key to natural products analysis using this approach

is dependable molecular weight determination. This

information is used with existing natural product databases

that contain information on the bioactive compounds, the

physical descriptions of the microorganisms from which

they come, their spectrum of activity, the method of

extraction and isolation, and physical data (i.e., molecular

weight, UV absorption maxima). Molecular weight is the

Fig. 4 Schematic of an ion trap MS instrument. This device

consists of two endcap electrodes (entrance and exit) and a ring

electrode. An ion trap MS separates ions based on mass-to-chargeratio (m/z). Once ions are introduced into the ion trap MS, the

radiofrequency (rf) amplitude is increased so that ions are

sequentially ejected (by increasing mass) and detected. This type

of MS provides a routine (i.e., benchtop) and sensitive detector

using either GC and LC interfaces. Furthermore, this instrument

provides a unique format for multiple stages of MS analysis

(MSn). (Courtesy of ThermoFinnigan, San Jose, CA.)

Fig. 5 Schematic of a QTOF MS instrument. Ions formed in the source region are

introduced into a quadrupole mass filter (see Fig. 7) that separates ions based on

mass-to-charge ratio (m/z). Selected ions are then transferred into the TOF MS for

detailed analysis (i.e., high resolution capabilities). (Courtesy of Micromass,

Manchester, UK.)

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most critical information for initial searches because of its

link to structural specificity. This information is used to

make pivotal decisions on whether or not to proceed to

more time-consuming isolation steps based on novelty of

the compound. In recently reported studies (17,18),

LC/MS is used to increase sensitivity and accelerate

analysis. These features serve to significantly reduce labor.A recent review described LC/MS-based approaches for

the characterization of natural product mixtures in

conjunction with high-throughput screening (19).

The instrumental configuration of the LC/MS system

developed by Ackermann et al. (17) features an HPLC, UV

detector, fraction collector, ESI-tandem quadrupole

(MS/MS), and MALDI-TOF MS. Filtered fermentation

broths were extracted with butanol or ethyl acetate, and

eluted on a gradient C18 reversed-phase HPLC separation.

The eluent was split 1:10 between a tandem quadrupole

MS/MS instrument (scanning 2502000 amu/3 sec in the

full scan mode) and a single wavelength UV detector (254

or 230 nm). One-min fractions were collected after the UV

detector. Of these fractions, 2050 mL is used for

MALDI-TOF analysis, and the remainder is concentrated

for microbiological testing. The LC/UV chromatogram

was compared to the bioactivity assay histogram to

highlight the peaks that contain activity. The molecular

weights of the active peaks were obtained for novelty

assessment of the compounds.

Similar approaches that use on-line LC/MS and

LC/MS/MS techniques have been recently described for

natural products dereplication (20,21). Approximately,000

natural product extracts can be screened annually for in

vivo and in vitro activity using LC/MS-based systems. Astandard approach for dereplication involves a comparison

of retention time, full scan mass spectra (i.e., molecular

weight information), and MS/MS spectra with those from

known biologically active standards. Thus, previously

identified components are rapidly eliminated and do not

require time-consuming structure elucidation studies. The

savings of effort allow researchers to focus efforts on

novel chemistries. Samples of novel compounds can then

be infused into an ion trap mass spectrometer, and a

multiple stage mass analysis (MSn) fragmentation map is

generated.

COMBINATORIAL CHEMISTRY

Recent reviews (22,,25) describe MS-based methods

ranging from the analysis of complex molecular libraries

(26) to open-access formats for drug discovery and

development (27). High throughput criteria were central to

each application.

An important development in the quest for high

throughput combinatorial library analysis was the multiple

ESI interface described by Wang et al. (28). This novel

ESI interface enabled effluent flow streams from an array

of four HPLC columns to be sampled independently and

sequentially using a quadrupole MS instrument. The

interface featured a stepping motor and rotating plateassembly. The effluent flow from the HPLC columns was

connected to a parallel arrangement of electrospray

needles co-axial to the mass spectrometer entrance

aperture. The individual spray tips were positioned 908

relative to one another in a circular array. Each spray

position was sampled multiple times per second by precise

control of the stepping motor assembly.

The parallel sample analysis format using a multiplexed

LC/MS interface with an orthogonal time-of-flight (TOF)

MS was described by de Biasi et al. (29). This approach

illustrated the high-throughput capabilities of a multi-

plexed ESI interface in combination with an MS format

that accommodates fast chromatography methodologies.

The system featured a four-way multiplexed electrospray

interface attached directly with the existing source of the

TOF-MS instrument. A rotating aperture driven by a

variable speed stepper motor permitted the sampling of the

spray from each electrospray probe tip. The data files were

synchronized with the corresponding spray.

As the preparation of large libraries for lead discovery

became routine, the burden placed on analysis techniques

focused mainly on throughput and quality (30). However,

biological assay requirements typically required pure

compounds. Thus, the focus shifted toward the use of

automated high throughput purification methods applied tolibraries of discrete compounds (31). Reverse-phase

analytical and preparative HPLC formats in conjunction

with MS techniques have been critical for the high

throughput purification approaches for parallel synthesis

libraries. A variety of approaches that featured the use of

gradient methods, short columns, and high flow rates have

been described (32). Highly automated LC/MS approaches

for purification at the multimilligram level were described

for a quadrupole system by Zeng et al. (33). These

methods featured the use of short columns that were

operated at ultra high flow rates. Preparative columns were

operated at flow rates in excess of 70 ml/min to match the

linear velocity of the short analytical columns (4.0

ml/min). Analytical LC/MS analyses of compound

libraries were achieved in 5 min for chromatographically,

well-behaved compounds. Slightly longer preparative

LC/MS analysis times (810 min/sample) were required

for compounds that exhibited poor chromatographic peak

shapes and/or for compound mixtures the required higher

resolution separations. The fraction collection process is

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initiated in real-time once the reconstructed ion current is

observed for a specific m/z value that corresponds to the

compound of interest. This design permitted the collection

of one sample per fraction. Thus, the need for very large

fraction collector beds and postpurification screening and

pooling was eliminated. Unattended and automated

operation of this system led to the purification ofovercompounds (mg quantities) per day.

BIOAFFINITY SCREENING

With the integration of highly automated parallel synthesis

techniques into drug discovery programs, hundreds of

thousands of compounds are now screened against a

particular biological target. Once activity is determined for

a mixture, the identification of the active component(s) is

necessary. A recent study described the use of bioaffinity

selection LC/MS methods for the identification of active

mixture component(s) (34). This approach features an

integrated bioaffinity-based LC/MS screening method to

separate and identify compounds from mixtures.

A mixture of compounds is incubated with the target

protein and the components bound to the protein are

selected by using a size exclusion chromatography (SEC)

spin column. In this experiment, the unbound com-

pounds are retained on the column. The bound components

are eluted and identified with LC/MS. Increased specificity

is obtained by dissociating the bound compounds and

performing a second equilibration incubation with the

protein. This procedure preferentially selects for the

compounds with higher affinity, and results in anenhancement of the quantitative LC/MS response.

Iterative stages of incubation, size-exclusion, and

LC/MS allow the tighter binding components to be

enriched relative to weaker binding components.

In this study, the peroxisome proliferator-activated

receptor (PPARg), which is a target for anti-diabetic drugs

(construct molecular weight of 32,537 Da), is incubated

with 10 ligands that range in molecular weight from 283 to

587 units. A spin column of 6000 Da cutoff is used for

SEC purposes. The retained mixture of components is

analyzed by fast perfusive chromatography (35,36), using

a standard full-scan LC/MS strategy. This analysis

procedure allows for the identification and quantitation

of the protein and the ligands, compared to their responses

prior to incubation. The ligand-protein complex that

dissociated under the reversed-phase chromatographic

conditions is selectively detected.

This analysis scheme provided a quick measurement of

binding affinity, and serves as a screening tool during drug

candidate selection. Spreadsheets were constructed and

used to calculate the binding affinity of the components. In

the example described above, two incubation cycles

followed by the SEC separation provided an enhancement

of strong binders to weak binders. This LC/MS-based

method provides a unique approach to obtain information

in situations when lower concentrations of tighter binding

ligands are present in the same mixture with higherconcentrations of weaker binding ligands. Furthermore,

this method is more efficient than synthetic deconvolution

procedures and does not require the use of radioligands.

Combinatorial chemistry initiatives have created a

tremendous challenge for activities that deal with the

screening of these mixtures for activity against a specified

target (37). MS-based approaches that use affinity

selection (38), encoding methodologies (3941), pulsed

ultrafiltration (42), and anti-aggregatory approaches (43)

have been described.

The use of MS formats that provide accurate mass

capabilities have been recently illustrated for screening

combinatorial libraries (4447). The unambiguous con-

firmation/identification of combinatorial library com-

ponents from small quantities of material have been

illustrated using a hybrid quadrupole/orthogonal TOF

(QTOF) (44,45) and Fourier transform ion cyclotron

resonance (FTICR) (47) mass spectrometry. A schematic

of a FTICR-MS system is shown in Fig. 6F6 . Accurate

isotope patterns or isotopic signature and unique mass

differences between isobaric compounds can be obtained

using these two MS formats.

OPEN-ACCESS SYSTEMS

Chemists now routinely use open-access MS systems in

the same way that they previously used thin-layer

chromatography (TLC) to monitor reaction mixtures for

the desired product and to optimize reaction conditions. In

practice, medicinal chemists require only molecular

weight data, and are comfortable with a variety of MS

ionization methods to obtain this information. However,

confidence in the actual method and procedure is a

requisite. Today, molecular mass measurement has

quickly become a preferred means of structure confir-

mation over NMR and IR during the early stages of

synthetic chemistry activities (i.e., drug discovery), where

sample quantities are limited.

In the open-access LC/MS procedure described by

Pullen et al. (48), the samples are directly introduced from

solution for ease of automation and sample preparation.

Chemists prepare samples in solvent to a suggested

concentration range, then log the samples into the system.

The sample log-in is done at any time during the

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continuous automated queue. Autosampler vials are used

to hold the samples, and autosamplers are used to directly

deliver samples in solution to the mass spectrometer. The

system uses a standard method to analyze the samples in

queue, average spectra according to a preset scheme, and

print out a spectrum for the chemist. Fail-safe procedures

for untrained users and instrument self-maintenance at

start-up and shutdown were also developed.

Taylor et al. (49) further demonstrated the value of

open-access LC/MS systems for generating a widened

scope of pharmaceutical analysis applications, including

1) characterization of synthetic intermediates and target

compounds; 2) reaction monitoring; 3) reaction optimiz-

ation; 4) analysis of preparative HPLC fractions; and 5)

analysis of TLC plate spots. The availability of these

methods led to the increased use of LC/MS for structural

analysis. The short analysis time and reliable structure

confirmation resulted in the use of LC/MS as a first choice

for structure characterization for synthetic chemistry

applications.

Open-access LC/MS formats have spawned new

dimensions in access and data management. The use of a

direct exposure probe (DEP) for automated sample

introduction has been developed for quick (ca. 3 min)

Fig. 6 Schematic of a FTICR MS instrument. This type of MS consists of an ion cyclotron resonance (ICR) analyzer cell that is situated

in the homogeneous region of a large magnet. The ions introduced into the ICR analyzer are constrained (trapped) by the magnetic field to

move in circular orbits with a specific frequency that corresponds to a specific mass-to-charge ratio (m/z). Mass analysis occurs when

radiofrequency (rf) potential is applied (pulsed) to the ICR analyzer so that all ions are accelerated to a larger orbit radius. After the pulse

is turned off, the transient image current is acquired and a Fourier transform separates the individual cyclotron frequencies. Repeating this

pulsing process to accumulate several transients is used to improve the signal-to-noise ratio. (Courtesy of Bruker Daltonics, Billerica,

MA.)

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IN VIVO DRUG SCREENING

The application of APCI-LC/MS techniques for the rapid

determination of protein binding and pharmacokinetics in

drug discovery were recently described by Allen et al.

using a single quadrupole instrument (54). A cocktail

approach consisted of four experimental compounds and acontrol compound dosed orally at 1 mg/kg with plasma

samples obtained at 0.5, 1, 2, 4, and 8 h post dose. To

insure reproducibility, the control compound was tested

with each cocktail. This approach generated timely

systemic exposure (AUC and Cmax) data on 44 test

compounds in three work days, using two laboratory

scientists.

The use of LC tandem quadrupole MS/MS-based

screening approaches for quantitative bioanalytical

measurements allow a large, chemically diverse, range

of potential drug candidates to be analyzed quickly and

confidently. A schematic of a tandem quadrupole MS/MSinstrument is shown in Fig. 8F8 . The development of unique

LC/MS-based systems for in vivo pharmacokinetic

screening reduces the analysis to a manageable number

of samples, and results in a cost-effective approach to

evaluate new lead compounds. Approaches to this type of

methodology will likely vary, according to the behavior of

the molecules of interest, standard operating procedures

(SOPs), performance capabilities of the mass spec-

trometer, and integration of automated sample preparation,

and data analysis procedures. Success will likely be

dependent on the above parameters, as well as on the

degree of tolerance to which the specific screen is set.

The simultaneous pharmacokinetic assessment of

multiple drug candidates in one animal has been termed

n-in-one or cassette dosing. As discussed for the

previous example, this parallel approach results in an

increased productivity for bioanalysis during drugdiscovery. Beaudry et al. (55) recently investigated the

extension of this methodology to study larger numbers of

compounds in each mixture, and to integrate sample

preparation with the LC/MS/MS system for increased

efficiency.

The number of analytes studied in parallel was extended

to 63 plus an internal standard. The increased number of

analytes was made possible due to improvements to the

collision region of the MS/MS system that provide

increased sensitivity and reduced memory effects. In

addition, robotic systems for sample handling and on-line

(solid phase extraction) SPE of plasma samples were

integrated with the LC/MS/MS system. An isocratic

reversed-phase HPLC method provided a cycle time of 4.5

min per sample. The on-line sample preparation and short

analysis resulted in an increased sample throughput that

required less time from the scientist. The method produced

good performance, in terms of extraction efficiency,

linearity, and limit of detection (LOD), and has the

capability of analyzing 320 960 samples per day. The

strategic emphasis of this approach is on providing high

throughput LC/MS methods for evaluating large numbers

of drug candidates during drug discovery to eliminate poor

pharmacokinetic performers.

METABOLIC STABILITY SCREENING

The use of fast gradient elution LC/MS techniques on a

single quadrupole instrument was described for high

throughput metabolic stability screening (56). The method

uses as HPLC column-switching apparatus to desalt and

analyze lead candidates incubated with human liver

microsomes. Substrates were selected whose in vivo

clearance is controlled predominantly by phase I oxidative

metabolism as opposed to phase II metabolism or renal

clearance. In this way, the resulting data could be resolved

into four categories of metabolic stability: high ($60%);

moderate ($3059%); low ($1029%); and very low

(,10%).

The rapid structure identification of metabolites is a

powerful complement to previously described quantitative

approaches. The utility of an automated metabolite

identification approach, using LC/MS/MS with an ion

trap mass spectrometer has been demonstrated (57). In this

Fig. 8 Schematic of a tandem quadrupole MS/MS instrument.

A tandem quadrupole MS/MS instrument consists of two

quadrupole MS filters, MS1 and MS2, separated by a collision

cell. Each quadrupole MS filter consists of four cylindrical orhyperbolic shaped rods. A unique combination of direct current

(dc) potential and radiofrequency (rf) potential is applied to each

pair of rods (one pair 1808 out of phase with the other). A mass

spectrum results by varying the voltages at a constant rf/dc ratio.

A variety of scan modes (e.g., full scan, product ion, precursor

ion, neutral loss) provide unique capabilities for quantitative and

qualitative structure analysis. (Courtesy of Micromass, Manche-

ster, UK.)

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study, MSn analysis is automated to provide maximum

structural information in combination with predictive

strategies for biotransformation. Automated data-depen-

dent scan functions are used to generate full scan, MS/MS,

and MSn mass spectra of metabolites within a single

chromatographic analysis. This feature is unique and

avoids the multiple (2 4) injections that are necessarywith other MS/MS configurations (e.g., tandem quadru-

pole). Along with the significant savings in time, detailed

structure information is generated, which enables a

comprehensive analysis of substructure relationship to be

constructed for each metabolite. These automated studies

provide unique advantages during drug discovery, and

provide an early perspective on the metabolically labile

sites, or soft spots of a drug candidate. This knowledge

is useful during lead optimization activities, and can lead

to the initiation of proactive research efforts that deal with

metabolism-guided structural modification and toxicity.

METABOLITE PROFILING AND IDENTIFICATION

The application of LC/MS-based techniques for the

structure identification of drug metabolites has played a

significant role in drug development. The early identifi-

cation of drug metabolites provides valuable insights into

the pathways of metabolism and biotransformation. Once

metabolites are identified/confirmed, metabolism-guided

structural modification during the drug discovery stage is

initiated to facilitate the selection of drug candidates for

subsequent development.The identification of metabolite structures with LC/MS

and LC/MS/MS techniques using quadrupole-based MS

instruments are an effective approach due to their ability to

analyze trace mixtures from complex samples of urine,

bile, and plasma. The key to structure identification

approaches is based on the fact that metabolites generally

retain most of the core structure of the parent drug (58,59).

Therefore, the parent drug and its corresponding

metabolites would be expected to undergo similar

fragmentation and to produce mass spectra that indicate

major substructures.

Kerns et al. demonstrated the application of LC/MS and

LC/MS/MS standard method approaches in preclinical

development for the metabolite identification of buspir-

one, a widely used anxiolytic drug (60). The success of this

method relies on the performance of the LC/MS interface

and the ability to generate abundant ions that correspond to

the molecular weight of the drug and drug metabolites.

The production of abundant molecular ions is an ideal

situation for molecular weight confirmation because

virtually all the ion current is consolidated into an adduct

of the molecular ion (i.e., [M 1 H]1, [M 1 NH3]1).

Full-scan mass spectra generally contain an abundant

[M 1 H]1 ion signal with little detectable fragmentation.

Product-ion spectra are obtained to reveal product ions and

neutral losses that are associated with diagnostic

substructures of the buspirone molecule. To assist withthe MS/MS structure identification, the gross substructure

of buspirone is categorized into profile groups (61). Profile

groups directly correlate specific product ions and neutral

losses with the presence, absence, substitution, and

molecular connectivity (62) of specific buspirone sub-

structures and their modifications. The profile groups of

buspirone are identified with abbreviations that correspond

to the three specific substructures: azaspirone decane

dione (A), butyl piperazine (B), and pyrimidine (P).

Substituted substructures are designated with a subscript

(s), and a dash ( ) denotes substructure connectivity.

Thus, the buspirone molecule is represented by ABP.

The AsBP designation refers to metabolite structures

that contain the azaspirone decane dione, butyl piperazine,

and pyrimidine substructures with substitution on the

azaspirone decane dione substructure. The profile group

categorization within a corresponding database allows the

rapid visual recognition of primary substructures affected

by metabolism.

Metabolite structure databases can be easily constructed

and contain information on the structure, molecular

weight, UV characteristics, RRT, and product ions of

metabolites obtained from rat bile, urine, and liver S9

samples. Using this format, Kerns et al. reported the

predominant buspirone metabolite profile groups as AsB P, A B Ps, and As B Ps. These profile groups

indicate azaspirone decane dione and pyrimidine as

metabolically active sites of attack and the presence of

multiple substitution sites on each of these substructures.

IMPURITY PROFILING AND IDENTIFICATION

Synthetic impurities are of particular concern during

process research and safety evaluation activities. Often,

impurities are the result of synthetic by-products or

starting materials of the scale-up process. Impurities

provide a comprehensive indicator of the chemical process

and are diagnostic of overall quality. Process chemists use

this information to guide process optimization. Knowledge

of the identity and relative amount of impurities is used to

diagnose process reactions so that changes in reagents and

reaction conditions leads to better yields and higher quality

material.

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With an increasing number of novel lead candidates that

enter into preclinical development, considerable resources

are needed to identify impurities. LC/MS-based

approaches provide integrated sample clean-up and

structure analysis procedures for the rapid analysis of

impurities. This advantage was demonstrated during the

preclinical development of TAXOLw

(18). LC/MS playedan important role for the identification of impurities

contained in extracts and process intermediates from Taxus

brevifolia and T. baccata biomass. Because drugs derived

from natural sources often have a very diverse set of

structural analogs, it is important to determine which

analogs are carried through the purification process and

ultimately appear as impurities. This task presents a unique

challenge during the early stages of drug development due

to the highly complex nature of the samples.

Kerns and coworkers described a structure identification

strategy that incorporates LC/MS and LC/MS/MS

techniques using quadrupole-based instruments for rapid,

sensitive, and high-throughput impurity analysis (18). This

approach integrates traditional steps of sample prep-

aration, separation, analysis, and data management into a

single instrumental method. The resulting multidimen-

sional data include retention time, molecular weight, UV,

and substructure information. A structure database is

developed for each candidate and is used to rapidly

identify the same impurities in new samples. Structures are

proposed based on using the drug candidate as a structural

template and, with the use of a standard method approach,

consistency for comparison of results throughout the

preclinical development process is ensured.

Nearly all of the impurities contained the characteristicpaclitaxel core substructure as indicated by the character-

istic product ion at m/z 509 with variations due to

modifications. Many of these taxanes contained a side-

chain similar to paclitaxel, with variations occurring on the

terminal amide of the side chain. The product ions that

differed from the characteristic side-chain ions of

paclitaxel (m/z 286) by values indicative of specific

substructures were used to identify these terminal amide

variations. A comparison with the paclitaxel substructural

template indicated structural differences beyond the

position of the amide group in the side-chain substructure.

When a new impurity was encountered during chemical

process research, retention time and molecular weight

information were compared to the database for rapid

identification. This approach is similar to the procedure

described for natural product dereplication. If the

compound is not contained in the structure database,

then the corresponding LC/MS/MS analysis is performed

to obtain substructural detail and the proposal of a new

structure.

A standard reversed-phase HPLC method was used for

all the samples that are associated with a drug candidate to

reduce time-consuming method development/method

refinement procedures. Standard reversed-phase methods

typically involve a 2030 min cycle time and provide

information for a wide range of compounds. The

incorporation of a standard method strategy allows theuse of autosampling procedures and standard system

software for data analysis.

During the development of TAXOLw, 90 taxane

impurities were rapidly identified and added to the

structure database. This MS/MS information was routinely

obtained for impurities down to the 100 ng level (injected),

and required approximately 23 h for the analysis of each

sample. The compounds are structurally categorized with

profile group terminology. The LC/MS-based methods

were significantly faster than the previously used

analytical methods based on scale-up, isolation, fraction-

ation, and individual structural analysis. Software tools

capable of sample tracking, interpretation, and data

storage facilitate the structure profiling of impurities,

degradants, and metabolites (63). Key pharmaceutical

analysis elements that deal with sample preparation, real-

time analysis decisions, databasing, distribution/visualiza-

tion of results (Fig. 9F9 ) and prediction of fragmentation are

now highly integrated.

DEGRADANT PROFILING AND IDENTIFICATION

During the course of drug development, the bulk drug and

drug formulation are studied under a variety of stressconditions such as temperature, humidity, acidity, basicity,

oxidization, and light. Qin et al. described the utilization of

stressing conditions that may cause degradation (64). The

resulting samples may be used to validate analytical

monitoring methods and to serve as predictive tools for

future formulation and packaging studies.

A traditional approach to study degradant formation

involves similar time-consuming scale-up and preparation

steps as described for metabolite and impurity analysis.

Similarly, this area of pharmaceutical analysis has

experienced the issues associated with faster drug

development cycles. Rourick and coworkers recently

described proactive approaches to obtain degradant

information with quadrupole LC/MS methods during the

preclinical development stage (65). The corresponding

structural information provides insight for decisions on

which leads to further develop for clinical testing. The

early structural information on degradants of a drug

candidate offers a unique capability for synthetic

modification to minimize degradation. Structural infor-

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mation can also facilitate planning of preclinical drugdevelopment in process research, formulation develop-

ment, and safety assessment.

The strategy for impurity and degradant identification

described by Rourick et al. subjects lead candidates to

various development conditions followed by LC/MS and

LC/MS/MS analysis protocols. A structure database is

constructed from the corresponding results and is used to

reveal unstable regions within the drug structure as well as

to ascertain which candidate or homologous series of drug

candidates may be the most favorable for further

development. High capacity and throughput speed are

necessary so that many lead candidates may be evaluated.

Applicability of the method to a wide range of compound

classes is desirable. Once the drug candidate enters clinical

development and manufacturing, the structure database is

useful for the rapid identification of impurities and

degradants in samples generated during these stages of

development.

The method exposes drug candidates to forced

degradation conditions, (e.g., acid, base, heat, and

moisture) as a predictive profile. The coordinated use ofLC/MS and LC/MS/MS provide structure identification

for speed, sensitivity, and high throughput. Standard

methods, useful for 80% of the compounds, are applied.

Various types of structural data are obtained for

elucidation purposes (e.g., retention time, molecular

weight, MS/MS), and unknown compounds are elucidated

with the candidate drug as a structural template. The

LC/MS analysis provides retention time and molecular

weight data, whereas LC/MS/MS provides substructural

detail for structure identification. Drug candidates are

incubated under drug processing, storage, and physiologi-

cal conditions that were expected to occur throughout drug

lifetime.

Using this approach, 10 degradants of cefadroxil, an

orally effective semisynthetic cephalosporin antibiotic,

were elucidated in a 2-day study. The use of standard

LC/MS methods provided consistency from sample to

sample throughout the development process, and allowed

for the construction and use of a structural database for the

rapid identification of impurities and degradants during

Fig. 9 Visualization of molecular fragments using a lasso tool application. The lasso tool is used to identify a particular fragment and,

if a signal corresponding to its mass is present in the spectrum, the fragment is highlighted and the corresponding assignment is added to

an assignment table. (Courtesy of Advanced Chemistry Development, Toronto, Ontario, Canada.)

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development. The reversed-phase HPLC conditions

provided a general measure of the polarity of each

compound, useful for interpretation of substructural

differences between related compounds. Due to the

mass-resolving capability of the mass spectrometer,

chromatographic resolution of co-eluting or unresolved

components was not required. Abundant protonatedmolecule ions, [M 1 H]1, provided reliable molecular

weight information, and product-ion spectra generated

valuable substructure information for each degradant. The

product-ion spectrum of cefadroxil was used as a template

for interpretation; specific product ions and neutral losses

were compared to the spectra obtained from the unknown

degradants. Product ions common to each spectrum

provided evidence of substructures unchanged by the

degradation conditions and differences were indicative of

structural variations.

QUANTITATIVE BIOANALYSISSELECTED IONMONITORING

The quantitative analysis of targeted components in

physiological fluids is a major requirement in clinical

development. In 1991, Fouda et al. (66) pioneered the use

of APCI-LC/MS on a single quadrupole instrument for the

quantitative determination of the renin inhibitor, CP-

80,794, in human serum. Because the pharmacological

action is below 200 pg/ml, a quantitative assay in the low

pg/ml range was required to monitor the drugs

pharmacokinetic and pharmacodynamic properties. Also,

the structure of the CP-80,794 molecule lacked asignificant chromophore for UV detection with conven-

tional HPLC methods. Furthermore, the low volatility and

thermal instability precluded analyses with GC/MS

methods.

Quantitative LC/MS assays in clinical development

generally involve four intensive steps: sample prep-

aration; assay calibration; sample analysis; and data

management. In the method developed by Fouda and

coworkers, human serum samples were prepared with a

liquid liquid extraction procedure. Assay calibration

involved the use of human serum samples fortified with

CP-80,794 at 11 concentrations (6 replicates per

concentration) ranging from 0.05 to 10 ng/ml. The

LC/MS analysis involved the use of the SIM mode to

monitor the molecular ions[MH]2 that correspond to the

drug (m/z 619) and internal standard (m/z 633). In this

particular LC/MS application, the negative ion mode was

highly sensitive for this class of compound. Samples were

loaded onto an HPLC autosampler and 80 mL aliquots are

injected onto the column at 4-min intervals. The elution

times of the drug and internal standard were 3.1 and 3.4

min, respectively.

At the time, this application provided a powerful

benchmark for the use of quadrupole LC/MS-based

methods in the pharmaceutical industry and paved the

way for the tremendous growth of MS-based applications

in support of clinical development. This particular assaysuccessfully supported several clinical studies with

sensitive and reliable results. This performance was

benchmarked on more than 4000 clinical samples, and led

to a widened scope of MS application for quantitative

bioanalysis (6771).

QUANTITATIVE BIOANALYSISSELECTED

REACTION MONITORING

The use of selected reaction monitoring (SRM) methods

for quantitative bioanalysis represents increased dimen-

sions of mass spectrometry analysis. A SRM method that

features a tandem quadrupole MS/MS instrument for the

quantitative analysis of an antipsychotic agent, clozapine,

in human plasma was recently described by Dear et al.

(72). Preclinical development studies of clozapine in rats

and dogs used HPLC with fluorescence detection (FLD).

With this method, a better limit of quantitation (LOQ) of 1

ng/ml was obtained. As the compound moved into the

clinical stages of development, a more sensitive method of

analysis was required to obtain rapid metabolic infor-

mation in support of drug safety evaluation studies. As a

result, a standard LC/MS/MS method was developed forthe quantitative analysis of clozapine (I) and four

metabolites (II-V) in human plasma.

The LC/MS/MS strategy deployed is similar to

previously described approaches for protein, natural

products, metabolite, and impurity identification. An

ionization technique that generates abundant molecular

ion species with very little fragmentation is desirable. The

product-ion spectrum is obtained to generate the

substructural template of the molecule. Abundant and

structurally unique transitions (molecular ion ! product

ion) are identified from the spectrum, and are used in the

corresponding SRM experiment for quantitation. The

SRM experiment provides a high degree of selectivity and

better LOD than full-scan or SIM experiments for the

analysis of complex mixtures (73,74). The selectivity of

MS/MS reduces the requirements for complete chromato-

graphic resolution of each component. Therefore,

LC/MS/MS experiments for quantitation typically

emphasize short analytical run times to provide high

sample throughput.

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15/18

expand current approaches as well as create low-flow,

miniaturized formats (7779) are envisioned. Continued

advances in integrated approaches that feature MS will

generate significant interest with regard to pharmaceutical

analysis. For example, the use of integrated systems that

feature NMR/MS have been described for applications that

involve LC/NMR/MS (80,81) and MS/NMR (82). A

schematic of an LC/NMR/MS system is shown in Fig. 10F10 .

In these configurations, NMR is used to provide structural

and stereochemical information (80,81) or to verify

binding/interaction (82) while MS is used to provide

molecular weight information. New challenges that deal

specifically with probing mechanism of action will likely

generate the need for a broader application of MS as well

as spur the development of novel technologies for sample

preparation, chromatography, and information

management.

REFERENCES

1. Lee, M.S.; Kerns, E.H. Mass Spectrom. Rev. 1999, 18,187279.

2. Fenn, J.B.; Mann, M.; Meng, C.K.; Wong, S.F.; White-house, C.M. Science 1989, 246, 6471.

3. Bruins, A.P.; Covey, T.R.; Henion, J.D. Anal. Chem. 1987,59, 26422646.

4. Hillenkamp, F.; Karas, M.; Beavis, R.C.; Chait, B.T. Anal.

Chem. 1991, 63, 1193A 1203A.5. Burlingame, A.L.; Boyd, R.K.; Gaskell, S.J. Anal. Chem.

1998, 70, 647R 716R.

6. Kelleher, N.L. Chem. Biol. 2000, 7, R37R45.7. Wolf, C.R.; Smith, G. Pharmacogenetics. Br. Medical Bull.

1999, 55, 366386.

8. Ross, P.; Hall, L.; Smirnov, I.; Haff, L. Nat. Biotechnol.

1998, 18, 13471351.9. Haff, L.A.; Smirnov, I.P. Genome Res. 1997, 7, 378388.

10. Pezzullo, L.II.; Hall, S.K.; Affourtit, J.P.; Seymour, A.B.;Stroh, J.G. Proceedings of the 48th ASMS Conference on

Mass Spectrometry and Allied Topics, Long BeachCalifornia, 2000; 1552.

11. Yates, J.R. J. Mass Spectrom. 1998, 33, 1 19.

12. Gale, B.L.; Bleibaum, J.L.; MacKenzie, R.T.; Martin, R.L.;Straub, K.M. Proceedings 48th ASMS Conference MassSpectrometry and Allied Topics, Long Beach, California,

2000; 62.Q113. Morris, H.R.; Paxton, T.; Dell, A.; Langhorne, J.; Berg, M.;

Bordoli, R.S.; Hoyes, J.; Bateman, R.H. J. Rapid Commun.

Mass Spectrom. 1996, 10, 889896.14. Stevenson, T.I.; Loo, J.A. Proceedings of the 47th ASMS

Conference on Mass Spectrometry and Allied Topics,

Dallas, Texas, 1999; 215.Q1

15. Buko, A.; Tang, Q.; Trevillyan, J.; Chiou, G. Proceedingsof the 47th ASMS Conference on Mass Spectrometry and

Allied Topics, Dallas, Texas, 1999; 248.

Fig. 10 Schematic of an LC/NMR/MS system. In this configuration, NMR is used to provide structural, stereochemical, or ligand

binding information while MS is used to provide molecular weight information. (Courtesy of Bruker Daltonics, Billerica, MA.)

ENCL 1002000128/10/200100012



16/18

16. Chalmers, M.J.; Gaskell, S.J. Curr. Opin. Biotechnol. 2000,11, 384390.

17. Ackermann, B.L.; Regg, B.T.; Colombo, L.; Stella, S.;Coutant, J.E. J. Am. Soc. Mass Spectrom. 1996, 7,12271237.

18. Kerns, E.H.; Volk, K.J.; Hill, S.E.; Lee, M.S. J. Nat. Prod.1994, 57, 13911403.

19. Strege, M.A. J. Chromatogr. B 1999, 725, 6778.20. Gilbert, J.R.; Lewer, P. Proceedings of the 46th ASMS

Conference on Mass Spectrometry and Allied Topics,Orlando, Florida, 1998; 21.

21. Janota, K.; Carter, G.T. Proceedings of the 46th ASMSConference on Mass Spectrometry and Allied Topics,Orlando, Florida, 1998; 557.

22. Burdick, D.J.; Stults, J.T. Methods Enzymol. 1997, 289,499519.

23. Fitch, W.L. Mol. Divers. 1998-99, 4, 3945.24. Submuth, R.D.; Jung, G. J. Chromatogr. B Biomed. Sci.

Appl. 1999, 725, 4965.25. Swali, V.; Langley, G.J.; Bradley, M. Curr. Opin. Chem.

Biol. 1999, 3, 337341.26. Dunayevskiy, Y.; Vouros, P.; Carell, T.; Wintner, E.A.;

Rebek, J., Jr. Anal. Chem. 1995, 67, 29062915.27. Cepa, S.; Searle, P. Proceedings of the 46th ASMS

Conference on Mass Spectrometry and Allied Topics,Orlando, Florida, 1998; 283.

28. Wang, T.; Cohen, J.; Kassel, D.B.; Zeng, L. Comb. Chem.High Throughput Screen 1999, 2, 327334.

29. de Biasi, V.; Haskins, N.; Organ, A.; Bateman, R.; Giles,K.; Jarvis, S. Rapid Commun. Mass Spectrom. 1999, 13,11651168.

30. Van Hijfte, L.; Marciniak, G.; Froloff, N. J. Chromatogr. BBiomed. Sci. Appl. 1999, 725, 3 15.

31. Weller, H.N. Mol. Divers. 1998-99, 4, 4752.32. Weller, H.N.; Young, M.G.; Michalczyk, S.J.; Reitnauer,

G.H.; Cooley, R.S.; Rahn, P.C.; Loyd, D.J.; Fiore, D.;Fischman, S.J. Mol. Divers. 1997, 3, 6170.

33. Zeng, L.; Wang, X.; Wang, T.; Kassel, D.B. Comb. Chem.High Throughput Screen 1998, 1, 101111.

34. Davis, R.G.; Anderegg, R.J.; Blanchard, S.G. Tetrahedron

1999, 55, 16531667.35. Regnier, F.E. Nature 1991, 350, 634635.36. Afeyan, N.B.; Fulton, S.P.; Regnier, F.E. J. Chromatogr.

1991, 544, 267279.37. Schriemer, D.C.; Hindsgaul, O. Comb. Chem. High

Throughput Screen 1998, 1, 155170.38. Kaur, S.; McGuire, L.; Tang, D.; Dollinger, G.; Huebner, V.

J. Prot. Chem. 1997, 16, 505511.39. Geysen, H.M.; Wagner, C.D.; Bodnar, W.M.; Markworth,

C.J.; Parke, G.J.; Schoenen, F.J.; Wagner, D.S.; Kinder,D.S. Chem. Biol. 1996, 3, 679688.

40. Hughes, I. J. Med. Chem. 1998, 41, 3804 3811.41. Wagner, D.S.; Markworth, C.J.; Wagner, C.D.; Schoenen,

F.J.; Rewerts, C.E.; Kay, B.K.; Geysen, H.M. Comb. Chem.High Throughput Screen 1998, 1, 143153.

42. van Breeman, R.B.; Huang, C.-R.; Nikolic, D.; Woodbury,C.P.; Zhao, Y.-Z.; Venton, D.L. Anal. Chem. 1997, 69,21592164.

43. Park, S.; Wanna, L.; Johnson, M.E.; Venton, D.L. J. Comb.Chem. 2000, 2, 314317.

44. Blom, K.F.; Combs, A.P.; Rockwell, A.L.; Oldenburg,K.R.; Zhang, J.H.; Chen, T. Rapid Commun. MassSpectrom. 1998, 12, 11921198.

45. Lane, S.J.; Pipe, A. Rapid Commun. Mass Spectrom. 1999,13, 798814.

46. Fang, A.S.; Vouros, P.; Stacey, C.C.; Kruppa, G.H.;Laukien, F.H.; Wintner, E.A.; Carell, T.; Rebek, J., Jr.Comb. Chem. High Throughput Screen 1998, 1, 2333.

47. Speir, J.P.; Perkins, G.; Berg, C.; Pullen, F. RapidCommun. Mass Spectrom. 2000, 14, 19371942.

48. Pullen, F.S.; Kerkins, G.L.; Burton, K.I.; Ware, R.S.;Teague, M.S.; Kiplinger, J.P. J. Am. Soc. Mass Spectrom.1995, 6, 394399.

49. Taylor, L.C.E.; Johnson, R.L.; Raso, R. J. Am. Soc. MassSpectrom. 1995, 6, 387393.

50. Scientific Instrument Services. AutoProbeTM ApplicationNote; Ringoes: New Jersey, 2000.

51. Whitney, J.L.; Kerns, E.H.; Rourick, R.A.; Hail, M.E.;Volk, K.J.; Fink, S.W.; Lee, M.S. Pharm. Tech. 1998, May,7682.

52. Tong, H.; Bell, D.; Keiko, T.; Siegel, M.M. J. Am. Soc.Mass Spectrom. 1999, 10, 11741187.

53. Richmond, R.; Gorlach, E.; Seifert, J.M. J. Chromatogr. A

1999, 835, 2939.54. Allen, M.C.; Shah, T.S.; Day, W.W. Pharm. Res. 1998, 15,

9397.55. Beaudry, F.; Yves Le Blanc, J.C.; Coutu, M.; Brown, N.K.

Rapid Commun. Mass Spectrom. 1998, 12, 12161222.56. Ackermann B.L.; Ruterbories K.J.; Hanssen B.R.; Lind-

strom, T.D. Proceedings of the 46th ASMS Conference onMass Spectrometry and Allied Topics, Orlando, Florida,1998; 16.

57. Lopez, L.L.; Yu, X.; Cui, D.; Davis, M.R. Rapid Commun.Mass Spectrom. 1998, 12, 17561760.

58. Perchalski, R.J.; Yost, R.A.; Wilder, B.J. Anal. Chem.

1982, 54, 14661471.59. Lee, M.S.; Yost, R.A.; Perchalski, R.J. Annu. Rep. Med.

Chem. 1986, 21, 313321.60. Kerns, E.H.; Rourick, R.A.; Volk, K.J.; Lee, M.S.

J. Chromatogr. B 1997, 698, 133145.61. Kerns, E.H.; Volk, K.J.; Hill, S.E.; Lee, M.S. J. Rapid

Commun. Mass Spectrom. 1995, 9, 15391545.62. Lee, M.S.; Klohr, S.E.; Kerns, E.H.; Volk, K.J.; Leet, J.E.;

Schroeder, D.R.; Rosenberg, I.E. J. Mass Spectrom. 1996,31, 12531260.

63. Williams, A.; Lee, M.S.; Lashin, V. Spectroscopy 2001, 16,3849.

64. Qin, X.Z.; Ip, D.P.; Chang, K.H.; Dradransky, P.M.;Brooks, M.A.; Sakuma, T. J. Pharm. Biomed. Anal. 1994,12, 221233.

65. Rourick, R.A.; Volk, K.J.; Kerns, E.K.; Lee, M.S. J. Pharm.Biomed. Anal. 1996, 14, 17431752.

66. Fouda, H.; Nocerini, M.; Schneider, R.; Gedutis, C. J. Am.Soc. Mass Spectrom. 1991, 2, 164167.

67. Wang-Iverson, D.; Arnold, M.E.; Jemal, M.; Cohen, A.I.Biol. Mass Spectrom. 1992, 21, 189194.

ENCL 1002000128/10/200100012



17/18

68. Ayrton, J.; Dear, G.J.; Leavens, W.J.; Mallett, D.N.; Plumb,R.S. Rapid Commun. Mass Spectrom. 1997, 11,19531958.

69. Kaye, B.; Herron, W.J.; Macrae, P.V.; Robinson, S.;Stopher, D.A.; Venn, R.F.; Wild, W. Anal. Chem. 1996, 68,16581660.

70. Olah, T.V.; McLoughlin, D.A.; Gilbert, J.D. RapidCommun. Mass Spectrom. 1997, 11, 1723.

71. Ayrton, J.; Plumb, R.; Leavens, W.J.; Mallett, D.; Dickins,M.; Dear, G.J. Rapid Commun. Mass Spectrom. 1998, 12,217224.

72. Dear, G.J.; Fraser, T.J.; Patel, D.K.; Long, J.; Pleasance, S.J. Chromatogr. A 1998, 794, 2736.

73. Johnson, J.V.; Yost, R.A. Anal. Chem. 1985, 57,758A768A.

74. Kusmierz, J.J.; Sumrada, R.; Desiderio, D.M. Anal. Chem.1990, 62, 23952400.

75. Chang, J.P.; Kiehl, D.E.; Kennington, A. Rapid Commun.Mass Spectrom. 1997, 11, 12661270.

76. Tiller, P.R.; El Fallah, Z.; Wilson, V.; Juysman, J.; Patel, D.Rapid Commun. Mass Spectrom. 1997a, 11, 15701574.

77. Schultz, G.A.; Corso, T.N.; Prosser, S.J.; Zhang, S. Anal.Chem. 2000, 72, 40584063.

78. Li, J.; Wang, C.; Kelly, J.F.; Harrison, D.J.; Thibault, P.Electrophoresis 2000, 21, 198210.

79. Li, J.; Kelly, J.F.; Chernushevich, I.; Harrison, D.J.;Thibault, P. Anal. Chem. 2000, 72, 599609.

80. Holt, R.M.; Newman, M.J.; Pullen, F.S.; Richards, D.S.;Swanson, A.G. J. Mass Spectrom. 1997, 32, 6470.

81. Shockcor, J.P.; Unger, S.E.; Savina, P.; Nicholson, J.K.;Lindon, J.C. J. Chromatogr. B Biomed. Sci. Appl. 2000,748, 269279.

82. Moy, F.J.; Haraki, K.; Mobilio, D.; Walker, G.; Powers, R.;Tabei, K.; Tong, H.; Siegel, M.M. Anal. Chem. in press.

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Author QueriesJOB NUMBER: 100200012

JOURNAL: 2820-3

Q1 Reference No.12 and 14 not in MSS. Author

please check.

ENCL 1002000128/10/200100012


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