Burger's Medicinal Chemistry and Drug Discovery || Mass Spectrometry and Drug Discovery

MASS SPECTROMETRY AND DRUGDISCOVERY

RICHARD B. VAN BREEMEN

ANDREW G. NEWSOME

JEFFREY H. DAHL

Department of Medicinal Chemistryand Pharmacognosy, Universityof Illinois, Chicago, IL

1. INTRODUCTION

At the beginning of the twentieth century,mass spectrometers were invented to helpphysicists and physical chemists to prove theexistence of isotopes of the elements. As radio-activity and nuclear physics were explored,specialized mass spectrometers were used tocharacterize the fission products of radioac-tive elements as they were created or discov-ered. In addition, mass spectrometers wereused for the measurement of isotopic enrich-ment of radioactive elements, their inorganicderivatives, and even the isotopic purificationof radioactive elements as inorganic com-pounds. As this era of mass spectrometryreached maturity by the 1940s, some physi-cists announced that there would no longer beany need for mass spectrometry since vir-tually all of the elements had been discoveredand characterized. Of course, these prognos-ticators were wrong because the entire field oforganic mass spectrometry was about tobegin.

1.1. Electron Impact (EI)

Whilemass spectrometerswere being used forthe purification of fissionable material foratomic weapons as part of the ManhattanProject ofWorldWar II, organic mass spectro-metry was being invented for the analysis andquality control of petroleum distillates andpetroleum-based fuels. By 1945, the applica-tion of mass spectrometry to organic chemis-try had emerged as a productive new area ofresearch and discovery. Commercial produc-tion of organic mass spectrometers began dur-ing the 1940s, and petroleum companies be-came the first customers for these new analy-tical instruments. Early commercial mass

spectrometers used electron impact ionization(see Eqs 1 and 2) to generate ions from gas-phasemolecules that were separated by accel-eration through an electromagnetic field pro-vided by either a fixed magnet or an electro-magnet. After separation, the ions were de-tectedusinga simple impact detector suchas aFaraday cup. This basic design is still in usetoday for the identification and quantitativeanalysis of volatile organic compounds.

Mþ e�ð70 eVÞ!Mþ . þ 2e� formation of

positive molecular ions using EI ionization

ð1Þ

Mþ e�ð2--10 eVÞ!M�.

electron capture EI ionization ð2ÞToward the late 1950s, organic mass spec-

trometers began to beused for the analysis of awider variety of organic molecules and even-tually became a fundamental analytical toolfor the characterization of synthetic organiccompounds. Today, mass spectrometers areused routinely to confirm the molecularmasses of organic compounds, to determineelemental compositions and to verify theirstructures based on fragmentation patterns.Fragmentation results from the cleavage ofchemical bonds within an ion resulting in theformation of a product ion of lower mass andone or more neutral products. Of course, onlythe fragment ions and not the neutral speciesare detected in a mass spectrometer becausethis instrument measures the mass-to-chargeratio (m/z) of ions in the gas phase. The energyfor fragmentation is either the result of excessenergy imparted to the molecular ion duringionization or during a process known as colli-sion-induced dissociation (CID) which will bediscussed along with tandem mass spectro-metry (MS–MS) below. Since the fragmenta-tion pattern reflects the relative strengths ofchemical bonds in a compound, mass spectra(a plot of ion relative abundance versus m/z)provide structurally significant fragment ionsfor compound identification. Rules for struc-ture elucidation of chemical structuresthrough the interpretation of mass spectrahave been developed. (For a review of EI and

97

Burger’s Medicinal Chemistry, Drug Discovery, and Development, Seventh Edition,edited by Donald J. Abraham and David P. RotellaCopyright � 2010 John Wiley & Sons, Inc.

ion fragmentation pathways, see McLaffertyet al., 1993 provided in Section 4.)

In many cases, EI imparts so much excessenergy into amolecule that only fragment ionsand no molecular ions are observed. There-fore, “softer” ionization techniques were de-veloped to enhance molecular mass informa-tion. The first of these ionizationmethods waschemical ionization (CI). Developed by re-searchers in the petroleum industry [1], CIbecame another standard ionization techni-que for organicmass spectrometry. DuringCI,high-energy electrons (as in EI) are used toionize a gas called a reagent gas at a constantpressure (usually �1mbar) in the mass spec-trometer ionization source. The reagent gas inturn ionizes the sample molecules throughion-molecule reactions that usually involvethe exchange of protons. Less frequently, sam-plemolecule ionizationmight involve a chargeexchange. Two of the most common ionizationmechanisms in CI are summarized in Equa-tions 3 and 4.

MþRHþ !MHþ þRCI through

proton transfer; R ¼ reagent gas ð3Þ

MþRþ . !Mþ . þR

CI through charge exchange ð4Þ

1.2. Types of Mass Analyzers

During the 1960s, high-resolution double-fo-cusing magnetic sector instruments becamestandard tools for the determination of ele-mental compositions using a type of analysis

called accurate mass measurement. In massspectrometry, resolving power is defined asM/DM (resolution is the inverse of this term,DM/M), where M is the m/z value of a singlycharged ion, and DM is the difference (mea-sured inm/z) betweenMand the next highestion.Alternatively,DMmaybedefined in termsof the width of the peak. High resolution istypically regarded as a resolving power of atleast 10,000 such that the molecular ions ofmost drug-like molecules (that is, compoundswith molecular masses less than�500) can beresolved from each other. After resolving asample ion from others in a mass spectrum,an accurate mass measurement may becarried out by comparing the m/z value of theunknown to that of a calibration standard.

Since the 1960s, other types of mass spec-trometers capable of high-resolution accuratemassmeasurementshavebecomeavailable ascommercial products including Fourier trans-form ion cyclotron resonance (FTICR) massspectrometers, reflectron TOF instruments,quadrupole time-of-flight hybrid (QqTOF)mass spectrometers, and recently, ion trap-TOF hybrid mass spectrometers (see Table 1for a listing of types of organic mass spectro-meters and a comparison of their performancecharacteristics). By the early 2000s, FTICRand QqTOF instruments became more popu-lar than magnetic sector mass spectrometersfor accurate mass measurements, high-reso-lution measurements, and drug discovery ap-plications. As will be discussed below, accu-rate mass measurements are essential tomany types of mass spectrometry-basedscreening and drug discovery today.

Table 1. Types of Mass Spectrometers and Tandem Mass Spectrometers

Instrument Resolving Power m/z Range Tandem MS

Magnetic sector 100,000 12,000 Low resolutionQuadrupole <4,000 4,000 NoneTriple quadrupole <4,000 4,000 Low resolutionTime-of-flight (TOF) 15,000 >200,000 NoneFTICR >200,000 <10,000 MSn, high resolutionOrbitrap <200,000 6,000 MSn, high resolutionIon trap <4,000 <10,000 MSn, low resolutionQqTOF >14,000 4,000 High resolutionIon trap-TOF >10,000 4,000 High resolutionTOF–TOF 15,000 >10,000 High resolution

98 MASS SPECTROMETRY AND DRUG DISCOVERY

1.3. Gas Chromatography-MassSpectrometry

Biomedical applications of mass spectrometrybegan during the 1960s both at academic in-stitutions and at pharmaceutical companies.These applications depended upon the volati-lization (usually by heating) of pharmaceuti-cal compounds and biochemicals prior to theirgas-phase ionizationusingEI orCI. In order toincrease the thermal stability and volatility ofthese compounds, a variety of derivatizationmethods were developed to mask polar func-tional groups and reduce hydrogen bondingbetween molecules. These methods were par-ticularly useful for use with gas chromatogra-phy-mass spectrometry (GC-MS) which wasintroduced during the 1960s as a practical andpowerful tool for qualitative and quantitativeanalysis of compounds in mixtures. Both EIand CI were immediately useful for GC-MS,since both of these ionizationmethods requirethat the analytes be in the gas phase. Whencapillary GC was incorporated into GC-MS,this technique reached maturity. GC-MSmaybeused to select, identify and quantify organiccompounds in complex mixtures at the femto-mole level. The speed of GC-MS is determinedby the chromatography step, which typicallyrequires several minutes up to one hour peranalysis. By the 1970s, some organic chemistswere announcing that organic mass spectro-metry had reached maturity and that no newapplicationswere possible. Like the physicistsand physical chemists who had pronouncedthe end of mass spectrometry a generationearlier, this group would soon be provedwrong.

Although GC-MS remains important forthe analysis of many organic compounds, thistechnique is limited to volatile and thermallystable compounds that comprise only a smallfraction of all organic compounds and evenfewer biomedically important molecules.Therefore, thermally unstable compounds in-cluding many pharmaceutical compoundssuch as nucleic acid analogs and biomoleculessuch as proteins, carbohydrates, and nucleicacids cannot be analyzed in their native formsusing GC-MS. (For more details regardingGC-MS and its applications, see Watson,2007 provided in Section 4) Although

derivatization facilitates the GC-MS analysisof many of these compounds, alternative ioni-zation techniques were needed for the analy-sis of the vastmajority of polar andnonvolatilecompounds of interest to drug discovery.

1.4. Desorption Ionization Techniques

During the 1970s and the early 1980s, deso-rption ionization techniques such as field des-orption (FD), desorption EI, desorption CI(DCI), and laser desorption were developed toextend the utility of mass spectrometry to-ward the analysis of more polar and less vo-latile compounds (See Watson, 2007 providedin Section 4) for more information regardingdesorption ionization techniques includingDCI and FD). Although these techniqueshelped extend themass range ofmass spectro-metry beyond a general limit of m/z 1000 andtoward ions ofm/z 5000, a breakthrough in theanalysis of polar, nonvolatile compounds oc-curred in 1982 with the invention of fast atombombardment (FAB) [2]. FAB and its counter-part liquid secondary ion mass spectrometry(LSIMS) facilitate the formation of abundantmolecular ions, protonated molecules, and de-protonated molecules of nonvolatile and ther-mally labile compounds such as peptides,chlorophylls, and complex lipids up to approxi-mately m/z 12,000. FAB and LSIMS use en-ergetic particle bombardment (fast atoms orions from 3–30,000V of energy) to ionize com-pounds dissolved in nonvolatile matrices suchas glycerol or 3-nitrobenzyl alcohol and desorbthem from this condensed phase into the gasphase for mass spectrometric analysis. Proto-nated or deprotonated molecules are usuallyabundant and fragmentation is minimal.Although still in use, FAB and LSIMS havebeen replaced by electrospray and matrix-as-sisted laser desorption ionization (MALDI) formost biomedical applications.

Introduced in the late 1980s, MALDI hashelped solve the mass limit barriers of laserdesorption mass spectrometry so that singlycharged ions may be obtained up to m/z500,000 and sometimes higher [3]. For mostcommercially available MALDI mass spectro-meters, ions up to m/z 200,000 are readilyobtained. Like FAB and LSIMS, MALDI sam-ples aremixedwith amatrix to form a solution

INTRODUCTION 99

that is loaded onto the sample stage for ana-lysis. Unlike the other matrix-mediated tech-niques, the solvent is evaporatedprior toMAL-DI analysis leaving sample molecules trappedin crystals of solid-phase matrix. The MALDImatrix is selected to absorb the pulse of laserlight directed at the sample. Most MALDImass spectrometers are equipped with apulsed UV laser, although IR lasers are avail-able as an option on some commercial instru-ments. Therefore, matrices are often substi-tutedbenzenes orbenzoicacidswithstrongUVabsorption properties. DuringMALDI, the en-ergy of the short but intense UV laser pulseobliterates the matrix and in the process des-orbs and ionizes the sample (see Fig. 1). LikeFAB and LSIMS, MALDI typically producesabundant protonated or deprotonated mole-cules with little fragmentation.

1.5. Liquid Chromatography-MassSpectrometry (LC-MS)

By the time that GC-MS had become a stan-dard technique in the late 1960s, LC-MS wasstill in the developmental stages. Producinggas-phase sample ions for analysis in a

vacuum system while removing the HPLCmobile phase proved to be a challenging task.Early LC-MS techniques included a movingbelt interface to desolvate and transport theHPLC eluate into a CI or EI ion source or adirect inlet system in which the eluate waspumped at a low flow rate (1–3mL/min) into aCI source. However, neither of these systemswas robust enough or suitable for a broadenough range of samples to gain widespreadacceptance. Since FAB and LSIMS requirethat the analyte be dissolved in a liquid ma-trix, this ionization technique was easilyadapted for use with HPLC in an approachknown as continuous-flow FAB [4]. Becausethe matrix often interfered with HPLC andrequired postcolumnaddition and because theLC-MS interface required frequent mainte-nance and cleaning, continuous-flow FAB israrely used today.

Like continuous-flow FAB, the popularityof particle beam interfaces has diminished.During particle beam LC-MS, the HPLCeluate is sprayed into a heated chamberconnected to a vacuum pump. As the dropletsevaporate, aggregates of analyte (particles)form and pass through a momentum

Figure1. Scheme showing the process ofMALDI froma solidmatrix absorbing strongly at thewavelength ofthe incident laser pulse.


separator that removes the lower molecularmass solvent molecules. Finally, the particlebeamenters themass spectrometer ion sourcewhere the aggregates strike a heated platefrom which the analyte molecules evaporateand are ionized using conventional EI or CI.Particle beamLC-MS is limited to theanalysisof volatile and thermally stable compoundsthat are amenable to flash evaporation andEI or CI mass spectrometry.

Thermospray became the first widely uti-lized LC-MS technique (during the late 1970sand the early 1980s). During thermospray, theHPLC eluate is sprayed through a heatedcapillary into a heated desolvation chamberat reduced pressure. Gas-phase ions remain-ing after desolvation of the droplets are ex-tracted through a skimmer into the massspectrometer for analysis. The sensitivity ofthermospray is poor since there is no mechan-ism or driving force to enhance the number ofsample ions entering the gas phase from thespray during desolvation. Also, thermally la-bile compounds tend to decompose in theheated source. These problems were solvedwhen thermospray was replaced byelectrospray.

Electrospray and atmospheric pressurechemical ionization (APCI) have become themost widely used ionization sources andHPLC-interfaces for drug discovery usingmass spectrometry. Unlike thermospray, par-ticle beam or continuous-flow FAB, electro-spray and APCI interfaces operate at atmo-spheric pressure, do not depend upon vacuumpumps to remove solvent vapor, and are com-patible with awider range ofHPLC flow rates.Also, no matrix is required. Both APCI andelectrospray are compatible with awide rangeof HPLC columns and solvent systems. Likeall LC-MS systems, the solvent system shouldcontain only volatile solvents, buffers or ionpair agents to reduce fouling of the massspectrometer ion source. In general, APCI andelectrospray form abundant molecular ionspecies. When fragment ions are formed, theyare usuallymore abundant in APCI than elec-trospray mass spectra.

The APCI ion source and HPLC interface(see Fig. 2) uses a heated nebulizer to form afine spray of the HPLC eluate, which is muchfiner than the particle beam system but simi-lar to that formed during thermospray.Heated nitrogen gas is used to facilitate the

Figure 2. During APCI, eluate from a HPLC system is sprayed through a heated capillary and desolvatedusing heated nitrogen. Functioning as a CI reagent gas, solvent molecules are ionized by a corona dischargeand then ionize sample molecules through proton transfer or charge exchange.

INTRODUCTION 101

evaporation of solvent from the droplets. Theresulting gas-phase sample molecules are io-nized by collisions with solvents ions, whichare formed by a corona discharge in the atmo-spheric pressure chamber. Molecular ions,Mþ . orM-., and/or protonated or deprotonatedmolecules can be formed. The relative abun-dance of each type of ion depends upon thesample itself, the HPLC solvent, and the ionsource parameters. Next, ions are drawn intothe mass spectrometer analyzer for measure-ment through a narrow opening or skimmerthat helps the vacuum pumps to maintainvery low pressure inside the analyzer whilethe APCI source remains at atmospheric pres-sure. For example, the positive ion APCImassspectrum of lycopene is shown in Fig. 3. Thecarotenoid lycopene is the red pigment of ripetomatoes and is under clinical investigationfor the prevention of prostate cancer [5].

During electrospray, the HPLC eluate issprayed through a capillary electrode at highpotential (usually 2000–7000V) to form a finemist of charged droplets at atmospheric pres-sure. As the charged droplets migrate towardthe opening of the mass spectrometer due toelectrostatic attraction, they encounter across-flow of heated nitrogen that increasessolvent evaporation and prevents most of thesolvent molecules from entering themass spectrometer (see Fig. 4). Molecularions, protonated or deprotonated molecules,and cationized species such as [M þ Na]þ and

[M þ K]þ can be formed. (For additional in-formation on electrospray ionization, seeCole,1997 provided in Section 4). In addition tosingly charged ions, electrospray is unique asan ionization technique in that multiplycharged species are common and often consti-tute themajority of the sample ion abundance.The relative abundance of each of these spe-cies depends upon the chemistry of the ana-lyte, the pH, the presence of proton donatingor accepting species, and the levels of traceamounts of sodium or potassium salts in themobile phase. In contrast, APCI, APPI, MAL-DI, EI, CI, and FAB/LSIMS usually producesingly charged species. A consequence of form-ing multiply charged ions is that they aredetected at lower m/z values (i.e., z >1) thanthe corresponding singly charged species. Thishas the benefit of allowing mass spectro-meters with modest m/z ranges to detect andmeasure ions of molecules with very highmasses. For example, electrospray has beenused tomeasure ionswithmolecularmasses ofhundreds of thousands or even millions ofdaltons on mass spectrometers with m/zranges of only a few thousands. (For a reviewof LC-MS techniques, see Niessen, 2006 pro-vided in Section 4.)

An example of the C18 reversed phaseHPLC-negative ion electrospray mass spec-trometric (LC-MS) analysis of an extract ofthebotanicalTrifoliumpratenseL. (red clover)is shown in Fig. 5. Extracts of red clover are

100 200 300 400 500

157

119

[M+H]+

537

100

m/z

444 467

Rel

ativ

e ab

unda

nce

Figure 3. Positive ion APCImass spectrum of the red carotenoid lycopene in a solution ofmethanol and tert-butyl methyl ether (1 : 1; v/v). In this analysis, lycopene formed a protonated molecule detected atm/z 537 asthe base peak of the mass spectrum instead of a molecular ion, Mþ ..


used as dietary supplements by menopausaland postmenopausal women and are underinvestigation as alternatives to estrogen re-placement therapy [6]. The two-dimensionalmap illustrates the amount of informationthat may be acquired using hyphenated tech-niques such as LC-MS. In the time dimension,chromatograms are obtained, and a samplecomputer-reconstructed mass chromatogramis shown for the signal at m/z 269. An intensechromatographic peakwas detected eluting at12.4min. In the m/z dimension, the negativeion electrospray mass spectrum recorded at

12.4min shows a base peak atm/z 269. Basedon comparison to authentic standards (datanot shown), the ion of m/z 269 was found tocorrespond to the deprotonated molecule ofgenistein that is an estrogenic isoflavone [6].Since almost no fragmentation of the genis-tein ion was observed, additional characteri-zation would require CID and MS–MS as dis-cussed in the next section.

When analyzing complex mixtures such asthe botanical extract shown inFig. 5, theuse ofchromatographic separation prior to massspectrometric ionization and analysis is

Figure 4. Schematic of an electrospray source. During LC-MS with electrospray, nitrogen is used as anebulization and drying gas to facilitate the formation of small droplets that become charged as they emergefromaTaylor coneandelectrosprayneedle at highpotential.As the solvent evaporates, analytes becomes gas-phase ions that are accelerated into the mass spectrometer through a potential gradient. In some designs,heatednitrogen is directed across the entrance to themass spectrometer as a curtain gas to provide additionaldesolvation and to prevent the solvent from entering the mass spectrometer.

INTRODUCTION 103

essential in order to distinguish between iso-meric compounds. Even simple mixtures ofsynthetic compounds might contain isomersthat would require LC-MS for adequate char-acterization. Another problem overcome byutilizing a chromatography step prior to massspectrometric analysis is ion suppression. Nomatter what ionization technique is used, thepresence of multiple compounds in the ionsource might enhance the ionization of onecompound while suppressing the ionization ofanother. Usually, only some of the compoundsin a complex mixture can be detected bymass spectrometry without chromatographic

separation. The presence of salts and buffersin a sample can also suppress sample ioniza-tion. Therefore, LC-MShas become apowerfultool for analyzing natural products, syntheticorganic compounds, and pharmaceuticalagents and their metabolites.

In general, APCI facilitates the ionizationof nonpolar and low molecular mass speciesand electrospray is more useful for the ioniza-tion of polar and high molecular mass com-pounds. In this sense, APCI and electrosprayare often complementary ionization techni-ques. However, during the analysis of largeor diverse combinatorial libraries, both polar

Figure 5. Two-dimensional map showing the LC-MS analysis of an extract of red clover under investigationfor the management of menopause. Reversed phase separation was carried out using a C18 HPLC column inthe time dimension and negative ion electrospray mass spectrometry was used for compound detection andmolecular mass determination in the second dimension.


and nonpolar compounds are usually present.As a result, no one set of ionization conditionsusing APCI or electrospray is adequate todetect all the compounds contained the libraryof compounds. Therefore, a UV ionizationtechnique called atmospheric pressure photo-ionization (APPI) has been developed for usewith combinatorial libraries and LC-MS [7].DuringAPPI, a liquid solution orHPLCeluateis sprayed at atmospheric pressure as inAPCI. Instead of using a corona discharge asin APCI, ionization occurs during APPI due toirradiation of the analyte molecules by anintenseUV light source.Obviously, the carriersolvent must not absorb UV light at the samewavelengths or interference would preventsample ionization and detection.

1.6. Tandem Mass Spectrometry (MS–MS)

Desorption ionization techniques such asFAB, MALDI, and electrospray facilitate themolecularmass determination of awide rangeof polar, nonpolar, low, and high molecularmass compounds including drugs and drugtargets such as proteins. However, the “soft”ionization character of these techniquesmeans that most of the ion current is concen-trated in molecular ions and few structurallysignificant fragment ions are formed. Inorder to enhance the amount of structural

information in these mass spectra, CID maybe used to produce more abundant fragmentions from molecular ion precursors formedand isolated during the first stage of massspectrometry. Then, a second mass spectro-metric analysis may be used to characterizethe resulting product ions. This process iscalled tandem mass spectrometry or MS–MSand is illustrated in Fig. 6.

Another advantage of the use of tandemmass spectrometry is the ability to isolate aparticular ion such as the molecular ion of theanalyte of interest during the first mass spec-trometry stage. This precursor ion is essen-tially purified in the gas phase and free ofimpurities such as solvent ions, matrix ions,or other analytes. Finally, the selected ion isfragmented using CID and analyzed using asecond mass spectrometry stage. In this man-ner, the resulting tandem spectrum containsexclusively analyte ions without impuritiesthat might interfere with the interpretationof the fragmentation patterns. In summary,CID may be used with LC-MS-MS or deso-rption ionization and MS-MS to obtain struc-tural information such as amino acid se-quences of peptides, sites of alkylation of nu-cleic acids, or to distinguish structural isomerssuch as b-carotene and lycopene.

The types of tandem mass spectrometersused for biomedical research are shown in

300 340 380 420 460 500 540 580m/z

0

100

50

467

536

Rel

ativ

e ab

unda

nce

M–.

[M-C5 H9]–

Figure 6. Negative ion electrospray tandemmass spectrum of lycopene. CIDwas used to induce fragmenta-tion of themolecular ion ofm/z536.As a result, the fragment ion ofm/z467was formedby the loss of a terminalisopreneunit. This fragment ionmaybe used to distinguish lycopene from isomerica-caroteneandb-carotenethat lack terminal isoprene groups.

INTRODUCTION 105

Table 1 and include triple quadrupole instru-ments (ideal for quantitative analysis), iontrap and FTICR (high resolution) mass spec-trometers which are capable of multiplestages of tandem mass spectrometry, and hy-brid instruments such asQqTOFand ion trap-TOF mass spectrometers (capable of high-re-solution tandem mass spectrometry). Exceptfor the TOF–TOF mass spectrometers, all theinstruments listed in Table 1 are compatiblewith electrospray, APPI, and APCI and maybe interfaced with HPLC systems. The TOF–TOF mass spectrometer is ideally suited forusewith a pulsed ionization technique such asMALDI.

Over the course of the last century, massspectrometry has become an essential analy-tical tool for a wide variety of biomedical ap-plications including drug discovery and devel-opment. By combining mass spectrometrywith chromatography as in LC-MS or by add-ing another stage of mass spectrometry as inMS–MS, the selectivity of the technique in-creases considerably. As a result, mass spec-trometry offers all of the analytical elementsthat are essential to modern drug discoverynamely speed, sensitivity, and selectivity.

2. CURRENT TRENDS AND RECENTDEVELOPMENTS

Since the early 1990s, drug discovery researchhas focused on combinatorial chemistry [8,9]and high-throughput screening [10] in an ef-fort to accelerate the pace of drug discovery.The goal has been to produce in a short timelarge numbers of synthetic organic com-pounds representing a great diversity of che-mical structures through aprocess called com-binatorial chemistry and then quickly screenthem in vitro against pharmacologically sig-nificant targets such as enzymes or receptors.The “hits” identified through these high-throughput screens may then be optimized byquickly and efficiently synthesizing and thenscreening large numbers of analogs calledtargeted or directed libraries. As a result,lead compoundsmight emerge from such com-binatorial chemistry drug discovery programsin a few weeks instead of several years.Furthermore, a single organic chemist using

combinatorial synthetic methods mightsynthesize thousands of compounds or morein a single week instead of less than five in thesame timeusing conventional techniques, anda singlemedicinal chemistmight identifyhun-dreds of lead compounds per month instead ofjust one or two in the same period of time.

Accompanying this new drug discoveryparadigm, new scientific journals have beenestablished such as Combinatorial Chemistry& High Throughput Screening, Journal ofCombinatorial Chemistry, Journal of Biomo-lecular Screening, and Molecular Diversity(see the list of journal Web sites provided inSection 4). The variety of topics published inthese journals reflects the multidisciplinarynature of the current drug discovery processand ranges from organic chemistry, medicinalchemistry, molecular modeling, molecularbiology, andpharmacology to analytical chem-istry. As described below, the most significantanalytical component of drug discovery hasbecome mass spectrometry. Only mass spec-trometry has become an essential element atall stages of the drug discovery and develop-ment process.

Although a variety of spectroscopic andchromatographic techniques including infra-red spectroscopy, nuclear magnetic resonancespectroscopy, fluorescence spectroscopy, gaschromatography, high-performance liquidchromatography (HPLC) and mass spectro-metry are being used to support drug discov-ery in various capacities, some of them such asgas chromatographyand fluorescence spectro-scopy are not applicable to most new chemicalentities, some are not specific enough for che-mical identification (e.g., infrared spectro-scopy), and other techniques suffer from lowthroughput (e.g., nuclear magnetic resonancespectroscopy). Unlike gas chromatography,HPLC is compatible with virtually all drug-like molecules without the need for chemicalderivatization to increase thermal stability orvolatility. In addition, mass spectrometry pro-vides a universal means to characterize anddistinguish drugs based on both molecularmass and structural features while at thesame time providing high throughput. Withthe development of routine LC-MS interfacesand ionization techniques such as electro-spray and APCI, mass spectrometry has also


become an ideal HPLC detector for the ana-lysis of combinatorial libraries [11], and LC-MS, MS-MS, and LC-MS-MS have becomefundamental tools in the analysis of combina-torial libraries and subsequent drug develop-ment studies [12–14].

The application of combinatorial chemistryand high-throughput screening to drug dis-covery has altered the traditional serial pro-cess of lead identification and optimizationthat previously required years of human ef-fort. Consequently, neither the synthesis ofnew chemical entities nor their screening islimiting the pace of drug discovery. Instead, anew bottleneck is the verification of the struc-ture and purity of each compound in a combi-natorial library or of each lead compoundobtained from an uncharacterized libraryusing high-throughput screening. Since thenumber of lead compounds entering the drugdevelopment process has increased in partbecause compounds are entering developmentat earlier stages than in the past, the tradi-tional drug development investigations con-cerning absorption, distribution, metabolism,and excretion (ADME) and even toxicologyevaluations of new drug entities have becomeadditional bottlenecks. As a solution to thedrug development bottlenecks, high-through-put assays to assess the metabolism, bioavail-ability, and toxicity of lead compounds arebeing developed and applied earlier than everduring the drug discovery process so that onlythose compounds most likely to become suc-cessful drugs enter the more expensive andslower preclinical pharmacology and toxicol-ogy studies. In support of these new combina-torial chemistry synthetic programs and newhigh-throughput assays, mass spectrometryhas emerged as the only analytical techniquewith sufficient throughput, sensitivity, selec-tivity, and robustness to address all of thesebottlenecks.

2.1. LC-MS Purification of CombinatorialLibraries

Although combinatorial libraries were origin-ally synthesized as mixtures, today most li-braries are prepared in parallel as discretecompounds and then screened individuallyin microtiter plates of 96-well, 384-well, or

1536-well formats. In order to facilitate sub-sequent structure–activity analyses and toassure the validity of the screening results,many laboratories verify the structure andpurity of each compound prior to high-throughput screening. SemipreparativeHPLChas become themost popular techniquefor the purification of combinatorial librarieson the milligram scale because of highthroughput and the ease of automation. Typi-cally during semipreparative HPLC, fractioncollection is initiated whenever a UV signal isobserved above a predetermined threshold.This procedure usually results in the collec-tion of several fractionsper analysis andhencecreates additional issues such as the need forlarge fraction collector beds and the need forsecondary analysis using flow injection massspectrometry, LC-MS, or LC-MS-MS to iden-tify the appropriate fractions. When purifica-tion of large numbers of combinatorial li-braries is required, this approach can becomeprohibitively time consuming and expensive.

To enhance the efficiently of this purifica-tion procedure, the steps ofHPLCpurificationand mass spectrometric analysis may be com-bined into automated mass-directed fractio-nation [15–17]. Any sizeHPLC columnmay beused, and only a small fraction of the eluant(�mL/min) is diverted to the mass spectro-meter equipped for APCI or electrospray ioni-zation. Since all of the components includingautosampler, injector,HPLC, switching valve,mass spectrometer, and fraction collector arecontrolled by computer, the procedure may befully automated. For greatest efficiency, thesystem may be programmed to collect onlythose peaks displaying the desired molecularions or alternatively, all peaks displayingabundant ions within a specified mass range.An example of theMS-guided purification of acompound synthesized during the parallelsynthesis of a combinatorial library of discretecompounds is shown in Fig. 7. Although thecrude yield of the reaction product was only30% (Fig. 7a), the desired product was de-tected based on its molecular ion (Fig. 7b).After MS-guided fractionation, reanalysisusing LC-MS showed that the desired productwas >90% pure (Fig. 7c).

The use of MS-guided purification ofcombinatorial libraries provides a means for

CURRENT TRENDS AND RECENT DEVELOPMENTS 107

reducing the number of HPLC fractions col-lected per sample and eliminates the need forpostpurification analysis to further character-ize and identify each compound as would benecessary when using UV-based fractiona-tion. The ionization technique (i.e., electro-spray, APCI, or APPI), and ionization mode(positive or negative) must be suitable for thecombinatorial compound, so that molecularion species are formed. Also, a suitable mobilephase and HPLC columnmust be selected. Asan alternative to HPLC, supercritical fluidchromatography-mass spectrometry (SFC-MS) has been used for the high-throughputanalysis of combinatorial libraries [18,19].The advantages of SFC-MS relative to conven-tional LC-MS for the purification of combina-torial libraries of compounds are the lowerviscosities and higher diffusivities of con-densed CO2 compared to HPLC mobilephases, and the ease of solvent removal anddisposal after analysis. However, SFC instru-mentation remains more expensive and less

widely available than conventionalHPLC sys-tems. Furthermore,many drug-likemoleculesare poorly soluble in condensed CO2.

2.2. Confirmation of Structure and Purityof Combinatorial Compounds

The determination of molecular masses, ele-mental compositions, and structures of com-pounds used for high-throughput screening,whether discrete compounds or combinatoriallibrarymixtures, is typically carried out usingmass spectrometry, since traditional spectro-scopic and gravimetric techniques are too slowto keep pace with combinatorial chemicalsynthesis. In addition, mass spectrometrymay be used to assess the purity of compoundsbeing used for high-throughput screening.The highest throughput technique for con-firming molecular masses and structures ofdrug candidates is flow injection analysis ofsample solutions using electrospray, APCI, orAPPI mass spectrometry. Typically, no sam-ple preparation is necessary.

(a) (c)

(b)

PrepLCMS analysis8e7

6e7

4e7

2e7

6e6

4e6

2e6

0.534.36

50 mg inj. Desiredproduct

Analytical purity assessmentfollowing prepLCMS

Inte

nsity

(cp

s)In

tens

ity (

cps)

Purity = 90.7%

3.81

2 4

35

30

25

20

15

10

5

Time (min)Time (min)

Threshold forfraction collection

RIC of desired peak from PrepLCMS

6.49 min

5

Time (min)5

Figure7. Mass-directedpurificationof a combinatorial library.Chromatographic separationwas carried outusing gradient elution of 10–90%acetonitrile inwater for 7min following an initial hold at 10%acetonitrile for1min. (a) Total ion chromatogram showing desired product and impurities. (b) Computer-reconstructed ionchromatogram (RIC) corresponding to the expected product. (c) Postpurification analysis of the isolatedcomponent with a purity >90%. (Reproduced from Ref. [15] by permission of Elsevier Science.)


Although any organic mass spectrometermaybeused to confirm themolecularmass of acompound, tandem mass spectrometers pro-vide additional structural informationthrough the use of CID to produce fragmentions. As discussed above (see also Table 1),tandem mass spectrometers include triplequadrupole instruments, QqTOF mass spec-trometers, ion trap-TOF mass spectrometers,ion trap mass spectrometers, multiple sectormagnetic sector instruments, FTICR instru-ments, and TOF–TOF mass spectrometers,and the new orbitrapmass spectrometers. Formost medicinal chemistry applications, APCIor electrospray ionization is used.

In addition to molecular mass and frag-mentation patterns, high-precision andhigh-resolution mass spectrometers such asQqTOF instruments, ion trap-TOFmass spec-trometers, reflectron TOF mass spectro-meters, double focusing magnetic sector massspectrometers, and FTICR instruments arenecessary for the measurement of exactmasses of drugs and drug candidates for thedetermination of elemental compositions. Thecombination of high-resolution and high-pre-cision is especially useful for determining theelemental compositions of compounds in com-binatorial library mixtures without having toisolate each compound using chromatographyor some other separation technique. SinceFTICR instruments, orbitrap mass spectro-meters, and hybrid ion trap-TOF and QqTOFmass spectrometers are capable of accuratemass measurements at high resolving powerof both molecular ions and fragment ions gen-erated during MS–MS, these instruments arebecoming extremely popular within drug dis-covery programs.

Although accurate mass measurements ofcompounds in combinatorial libraries canusually be measured using electrospray orAPCI mass spectrometry with infusion, on-line HPLC separation is sometimes requiredto overcome ion suppression problems due tothe presence of buffer, contaminants or reac-tion by-products. However, LC-MS is a rela-tively slow process due to the slow chromato-graphic separation step. Several approacheshave emerged to increase the throughput ofthis technique; parallel LC-MS, fast LC-MSand ultrahigh pressure liquid chromatogra-

phy (UHPLC)-MS. One approach to increas-ing throughput of the rate-limiting chromato-graphic separation has been to simulta-neously interface multiple HPLC columns toa single mass spectrometer. This approach iscalled parallel LC-MS. Commercial parallelelectrospray interfaces andHPLCsystemsarenow available that can accommodate up to 8HPLC columns simultaneously [20–22].Although the multiple sprays are introducedto the ion source simultaneously, thesestreams may be sampled in a time-dependentmanner to minimize cross-contamination be-tween channels.

Another solution to increasing the through-put of LC-MS has been to minimize the timerequired for HPLC separation through anapproach called fast HPLC. HPLC separa-tions are accelerated byusing shorter columnsand higher mobile phase flow rates. Sincecoelution of some species is likely to occurduring fast chromatographic separations,the selectivity of the mass spectrometeris essential for the characterization and/orquantitative analysis of the target compound.However, samples prepared using combina-torial chemistry are usually simple mixturesof reagents, by-products, and product thatrequire only partial chromatographic purifi-cation to prevent ion suppression effects dur-ing mass spectrometric analysis. A variety ofHPLC columns are used for fast LC-MS thatinclude narrow bore (2mm) and analyticalbore (4.6mm) columns with length typicallyfrom 0.5–5 cm. The mobile phase flow rate forthese fast LC-MS analyses is usually from1.5–5mL/min.

Similar stationary phase chemistries areused for UHPLC, but the particle size of thepacking material is reduced to <2mM forgreater column efficiency. If the UHPLC col-umns are shortened compared to conventionalcolumns, similar separation efficiencies maybe obtained while the analysis times and sol-vent consumption can be reduced to two- tothreefold. Alternatively, UHPLC may be op-erated at higher flow rates to obtain goodseparations in just seconds instead ofminutes.However, the small particle size of UHPLCcolumns produce high back pressures of up to800 bar,which is twice themaximumpressureof conventional HPLC systems. Therefore,


UHPLC systems are engineered to operate atpressures up to at least 800 bar.

In addition to molecular mass determina-tion using conventionalMS or high-resolutionaccurate mass measurement and structuralconfirmation using MS–MS, fast LC-MS, andUHPLC-MS are also used to assess the purityand yield of combinatorial products [15,23].Prior to high-throughput screening, many re-searchers analyze combinatorial libraries forboth purity and structural identity usingmass spectrometry so as to assure the validityof structure–activity relationships that mightbe derived from the screening data. Fast LC-MS, UHPLC-MS, and LC-MS–MS may becarried out to satisfy this requirement usinggradients (usually a step gradient with a re-verse phase HPLC or UHPLC column) with atotal cycle time of <3min [24] for fast HPLC or<1min per analysis using UHPLC or isocraticsystems.

2.3. Encoding and Identification ofCompounds in Combinatorial Librariesand Natural Product Extracts

The utility of mass spectrometric identifica-tion in combinatorial chemistry is limited notonly to the analysis of synthetic products as ameans of quality control but also for the iden-tification of active compounds or “hits” duringhigh-throughput screening. Although thesynthesis and screening of discrete com-pounds [25] enables them to be followedthrough the entire process by using partialencoding or bar coding, it is sometimes advan-tageous to screen libraries prepared as mix-tures [26] and use a technique such as massspectrometry to rapidly identify the hit(s) inthemixture. One approach to the rapid decon-volution of combinatorial library mixtures isto prepare libraries containing compounds ofunique molecular mass, and then identifythem using mass spectrometry. However,such libraries are necessarily small since themolecular mass of most drug-like molecules isbetween 150–400Da. Because of the molecu-lar mass degeneracy of larger combinatoriallibraries, several encoding strategies havebeen devised to rapidly identify active com-pounds in these mixtures [27–29].

Since most combinatorial libraries containcompounds with degenerate molecularmasses, various tagging strategies have beendevised to uniquely identify library com-pounds bound to beads. Most of these taggingapproaches are based on the synthesis of en-codingmolecules. For example, peptide [30] oroligonucleotide [31] labels have been synthe-sized on the beads in parallel to the targetmolecules and then sequenced for bead decod-ing. Alternatively, haloarene tags have beenincorporated during synthesis and then iden-tified with high sensitivity using electron-cap-ture gas chromatography detection [32]. Inaddition to the increased time and cost for thesynthesis of a library containing tagging moi-eties, the tagging groups themselves mightinterfere with screening giving false-positiveor -negative results.

For peptide libraries, one solution to thisproblem uses MALDI mass spectrometry todirectly desorb and identify peptides frombeads that were screened and found to behits [33]. This technique is called the termina-tion synthesis approach. Since the peptidelibrary compounds are analyzed directly, pro-ducts with amino acid deletions or substitu-tions, side-reaction products, or incompletedeprotection are readily observed. Also, sincethere are no extramolecules used for chemicaltagging, this source of interference is avoided.However, this approach is specific to peptidelibraries and not necessarily applicable toother types of combinatorial libraries.

Another approach that eliminates possibleinterference from the chemical tags, “ratioencoding,” has been developed for the massspectrometric identification of bioactive leadsusing stable isotopes incorporated into thelibrary compounds [29,34]. Within the liganditself, the code might be a single labeled atomthat is conveniently inserted whenever a com-mon reagent transfers at least one atom to thetarget compound or ligand. The code consistsof an isotopic mixture having one of the manypredetermined ratios of stable isotopes andcan be incorporated in the linker or addedthrough a reagent used during the synthesis.The mass spectrum of the compound shows amolecular ion with a unique isotope ratio thatcodes for a particular library compound. Forexample, Wagner et al. [29] used isotope ratio


encoding during the synthesis of 1000 com-pound peptoid library and was able to identifyuniquely all the components based on theirisotopic patterns andmolecularmasses. Sinceisotope ratio codes are contained within eachcombinatorial compound, a chemical tag is notrequired. The speed of MS-based decodingoutperforms most other decoding technolo-gies, which are time consuming and decodea restricted set of active compounds.

Although combinatorial synthesis providesrapid access to large numbers of compoundsfor screening during drug discovery and leadoptimization, these libraries areusually basedon a small number of common structures orscaffolds. There is a constant need for increas-ing the molecular diversity of combinatoriallibraries and finding new scaffolds, and nat-ural products have always been a rich sourceof chemical diversity for drug discovery. Thetraditional approach to screening natural pro-ducts for drug leads utilizes bioassays to testorganic solvent extracts for activity. If strongactivity is detected, then activity-guided frac-tionation of the crude extract is used to isolatethe active compound(s), which are identifiedusing mass spectrometry (including tandemmass spectrometry and accurate mass mea-surements), IR, UV/VIS spectrometry, andNMR. Recently, a variety of mass spectrome-try-based affinity screening methods havebeen developed to streamline the tedious pro-cess of activity-guided fractionation. Theseapproaches are discussed in Section 2.4.

Whether lead compounds in natural pro-duct extracts are isolated using bioassay-guided fractionation or mass spectrometry-based screening, there is a high probabilitythat the structure of the active compound(s)has already been reported in the natural pro-duct literature. In such cases, the tediousprocess of complete structure elucidationusing a battery of spectrometric tools shouldbe unnecessary. Instead, mass spectrometryalone may be used to quickly “dereplicate” oridentify the known compounds based on mo-lecular mass, fragmentation patterns and ele-mental composition in combination with nat-ural product database searching [35–39].Commercially available natural products da-tabases include NAPRALERT [40], Scientific& Technical information Network (STN) [41],

and the Dictionary of Natural Products [42].Since some of these databases also containUV–vis absorbance data, it is also advanta-geous to use a photodiode array detector be-tween the HPLC and mass spectrometer toobtain additional spectrometric data duringLC-UV-MS dereplication [36,37].

2.4. Mass Spectrometry-Based Screening

The earliest approaches to combinatorialsynthesis used portioning andmixing [26] andenabled the synthesis of combinatorial li-braries containing hundreds of thousands tomillions of compounds. However, efficientscreening techniques did not exist to rapidlyidentify the “hits” within large combinatorialmixtures. Therefore, chemistsweremotivatedto develop ways to prepare large numbers ofdiscreet compounds using massively parallelsynthesis, which could be assayed quickly forpharmacological activity using high-through-put screening one compound at a time.Although high-throughput screening of dis-creet compounds has become the standardapproach to drugdiscovery inmedicinal chem-istry, there is a shortage of chemically diversenovel structures to serve as lead compoundsfor combinatorial synthesis and lead optimi-zation. A traditional source of molecular di-versity for drug discovery, natural productsare receiving renewed interest in drug discov-ery programs. However, the complexity ofnatural products sources such as botanical ormicrobial extracts requires screening techni-ques that are suitable formixtures. Toaddressthis requirement, mass spectrometry-basedscreening assays have been developed thatare suitable for screening complex mixturesincluding natural product extracts. All of themass spectrometry-based screening methodsuse receptor binding of ligands as the basis foridentification of lead compounds.

2.4.1. Affinity Chromatography-Mass Spectro-metry Since the introduction of affinity chro-matography approximately 40 years ago, thistechnique has become a standard biochemicaltool for the isolation and identification of newbinding partners to specific target molecules.Therefore, the coupling of affinity chromato-graphy to mass spectrometry is a logical ex-


tensionof this approach, and theapplication ofaffinity LC-MS to the screening of combina-torial libraries has been demonstrated by sev-eral groups [43,44]. During affinity LC-MSscreening, a receptor molecule such as a bind-ingprotein or enzyme is immobilized ona solidsupport within a chromatography column.The library mixture is pumped through theaffinity column in a suitable binding buffer sothat any ligands in the mixture with affinityfor the receptor would be able to bind. Then,unbound material is washed away. Finally,the specifically bound ligands are eluted usinga destabilizing mobile phase and identifiedusing mass spectrometry. This affinity-col-umn LC-MS assay is summarized in Fig. 8.

In some applications [43], ligands areeluted from the affinity column and thentrapped on a second column such as a reversephase HPLC column. LC-MS or LC-MS–MSidentification of the ligands (hits) is then car-ried out using the trapping column. In othersystems, ligands are identified directly fromthe affinity column using mass spectrometry.For example, Kelly et al. [44] prepared anaffinity column containing immobilized phos-phatidylinositol-3-kinase andused it for directLC-MS screening of a 361-component peptidelibrary. Electrospray mass spectrometry andtandemmass spectrometry were used to iden-tify the ligands released from the affinity col-umn using pH gradient elution.

Advantages of affinity chromatography-mass spectrometry for screening during drugdiscovery include versatility and reuse of thecolumn. Both combinatorial libraries and nat-ural product extracts can be screened usingthis approach, and a wide range of bindingbuffers may be used. Mass spectrometry-com-patible mobile phases are only required dur-ing the final LC-MS detection step. Further-more, a single column may be used multipletimes to screen different samples for ligandsunless the destabilization solution irreversi-bly denatures, releases, or inhibits thereceptor.

Despite these advantages, affinity chroma-tography has numerous drawbacks that haveprompted the development of alternativemass spectrometer screening tools. For exam-ple, immobilization of the receptor mightchange its affinity characteristics causingfalse-negative or false-positive hits. This isparticularly problematic for receptors thatare solution-phase in their native state. Also,developing and then implementing an immo-bilization scheme is often a slow, tedious, andeven expensive process, which is unique foreach new receptor. Finally, false-positive hitsare often obtained when screening large, mo-lecularly diverse libraries, since there areusually compounds in suchmixtures thathaveaffinity for the stationary phase or linker mo-lecule instead of the receptor.

Isolation

LC-MS–MSidentification

m/z

Binding

Library +

Affinity Column

Wash unbound library compounds to waste

Elute bound ligands using pH change

Trap ligands on C18

column

Elute trapped ligandsonto HPLC column

LR LR LRR R R

Figure8. Affinity chromatography combinedwithLC-MS–MS for screening combinatorial librarymixtures.


2.4.2. Gel Permeation Chromatography-MassSpectrometry Another type of chromatogra-phy that has been combined with mass spec-trometry as a screening system for drug dis-covery is gel permeation chromatography(GPC) [45,46]. Also called size exclusion chro-matography, GPC separates molecules ac-cording to size as they pass through a station-ary phase containing particles with a definedpore size. During GPC-based screening, a li-brary mixture is preincubated with a macro-molecular receptor to allow any ligands in thelibrary to bind, and then GPC is used to se-parate the large receptor–ligand complexesfrom the unbound low molecular mass com-pounds in the mixture. Finally, ligands arereleased from the receptor during reversedphase HPLC and identified either on-line oroff-lineusing tandemmass spectrometry.Thisscreening method is illustrated in Fig. 9.

During the preincubation and GPC steps,any binding buffer may be used, since thebinding buffer will be removed during reversephase LC-MS analysis. However, the GPCseparation step must be carried out quickly,since ligands begin to dissociate from the re-ceptor immediately and can become lost intothe size exclusion gel. Despite this disadvan-tage, this approach allows both receptor andligand to be screened in solution, which avoidssome of the problems associated with the use

of affinity columns for screening. TheGPCLC-MS-MS screening method is suitable forscreening natural product extracts as well ascombinatorial library mixtures.

2.4.3. Affinity Capillary Electrophoresis-MassSpectrometry Affinity capillary electrophor-esis was originally used for the determinationof the binding constants of small molecules toproteins [47–49]. This solution-based techni-que is rapid and requires only small amountsof ligands. Affinity constants are measuredbased on the mobility change of the ligandupon interaction with the receptor present inthe electrophoretic buffer [50]. By combiningaffinity capillary electrophoresis with on-linemass spectrometric detection and identifica-tion, affinity constants for multiple com-pounds can be measured in a single analy-sis [51]. Recognizing that on-line mass spec-trometric detection was helpful for the identi-fication of each ligand,Chuet al. [52] extendedthis approach to include the screening of com-binatorial libraries as a means of drug discov-ery. The data in Fig. 10 show the results ofscreening a 100-tetrapeptide library for affi-nity to vancomycin using affinity capillaryelectrophoresis-mass spectrometry. Withoutvancomycin in the electrophoresis buffer, allthe peptides eluted within 3min. When van-comycin was present, the peptides eluted in

GPC isolation

LC–MS–MSidentification

m/z

Binding

+ RL

OO

O O

OO

O

O OO

O O

OO

O

O

Reversed phase desalting/denaturation

L–R

L

L–R +

Figure 9. GPC followed by LC-MS–MS for screeningmixtures of combinatorial libraries. After incubation ofa receptor with a library of compounds, the ligand–receptor complexes (L–R) are separated from the lowmolecular mass unbound library compounds using GPC. Next, the L–R complexes are denatured duringreversed phase HPLC to release the ligands for MS–MS identification.


order of affinity with the highest affinity com-pounds being detected between 4.5 and 5min.Positive ion electrospray tandem mass spec-trometry was used to identify the highestaffinity ligands (see Fig. 10B).

Note that the some peptide ligands such asFmoc-DDFA were detected as adducts withTris that was used in the electrophoresis buf-fer. Although the identification of this peptidewas not prevented by the formation of thisadduct, some buffers used during electrophor-esis might interfere with mass spectrometricionization and detection. Also, the types oflibraries that have been screened using thisapproach have contained modest numbers ofsynthetic analogs such as peptides. Librariesexceeding 400 members required preliminarypurification using affinity chromatography toreduce the number of compounds [52]. As aresult, this approach is probably not ideal for

screening libraries containing molecularly di-verse compounds or for screening natural pro-duct extracts. However, affinity capillary elec-trophoresis-mass spectrometry is fast witheach analysis requiring less than 10min.Also,it may be used to measure affinity constantsfor ligand–receptor interactions.

2.4.4. Frontal Affinity Chromatography-MassSpectrometry Like affinity chromatogra-phy-mass spectrometric screening (seeSection 2.4.1), frontal affinity chromatographyutilizes an affinity column containing immobi-lized receptor molecules [53]. The differencebetween the two screeningmethods is that theligands are continuously infused into the col-umn during frontal affinity chromatographyand detected using mass spectrometry. Com-pounds with no affinity for the immobilizedreceptor elute immediately in the void volume,

Figure 10. Affinity capillary electrophoresis-UV-mass spectrometry of a 100-tetrapeptide library screenedfor binding to vancomycin (104mM in the electrophoresis buffer). (a) The elution of peptides was monitoredwith UV absorbance during capillary electrophoresis, and the elution time increased with increasing affinityfor vancomycin. (b) Positive ion electrospray mass spectrum with CID of the Tris adduct of the protonatedpeptide detected at�5min in the electropherogram shown in A. (Reproduced from Ref. [52] by permission ofthe American Chemical Society.)


but the elution of the ligands is delayed. Ascompounds compete for binding sites on theaffinity column, these sites become saturateduntil ligands begin to elute from the column attheir infusion concentration (see Fig. 11). Inthis manner, frontal affinity chromatographymay be used to measure affinity constants forligands, and by using a mass spectrometer foron-line identification of ligands, this techniquebecomes a screening method [54,55].

During frontal affinity chromatography-mass spectrometry, signals for compoundseluting from the affinity column are recordedon-line by a mass spectrometer, and the lastcompounds to elute at their infusion concen-trations represent the highest affinity com-pounds or “hits.” Since frontal affinity chro-matography utilizes a conventional affinitycolumn, this technique would be most acces-sible to investigators already using affinity-mass spectrometry (see Section 2.4.1). How-ever, the same limitations and disadvantagesof using immobilized receptors still apply suchas nonspecific binding to the stationary phase,the development time and cost of preparingthe affinity columns, and the possibility thatimmobilizing the receptormight alter its bind-ing characteristics and specificity. In addition,mass spectrometric detection createssome additional limitations. Since all library

compounds must be monitored simulta-neously, the compounds must be selected sothat theyhaveuniquemolecularmasses. Also,one compound in the mixture should not sup-press the ionization of another. Therefore, thisapproach is probably restricted to the screen-ing of small combinatorial libraries that aresimilar in chemical structure and ionizationefficiencies. Finally, the binding buffer usedfor affinity chromatography must be compa-tible with on-line APCI or electrospray massspectrometry. This means that the mobilephasemust be volatile and usually of low ionicstrength (i.e., typically <40mM for electro-spray ionization). However, the mobile phaselimitations may be overcome by collectingfractions from the affinity column and theanalyzing them off-line using LC-MS as indi-cated in Fig. 11 [55].

2.4.5. Solid-Phase Mass Spectrometric Screen-ing Since drugs are usually in a soluble formin order to be transported to the active sites incells and tissues, it is logical that most massspectrometry-based screeningmethodsutilizesolution-phase analysis of these compounds,and it is no surprise thatmost successfulmassspectrometry screening assays use electro-spray ionization or APCI. However, solid-phase ionization techniques such as MALDI

Time

MS

res

pons

e

Time

MS

res

pons

e

Time

MS

res

pons

eTime

Det

ect

or

resp

onse Control Affinity

interaction

Figure 11. Frontal affinity chromatography screening using mass spectrometry. The control represents thevoid volume of the column and the elution profile of compounds with no affinity for the immobilized receptor.Compounds eluting later than those in the control elute in increasing order of affinity for the immobilized receptor.When the binding buffer is not compatiblewithmass spectrometry, fractions can be collected and analyzed off-lineusingLC-MStodeterminetherelativeamountsofeachpotential ligandeluting fromthecolumn.BasedonRef. [55].


might be effective also provided that ligand–receptor interactions are allowed to take placein an environment similar to in vivo conditionsand provided that a suitable separation step iscarried out prior to the preparation of theMALDI sample.

In order to utilize MALDI mass spectro-metry for screening, several research groupshave developed immobilized receptors onMALDI targets or on solid supports that canbe placed on a MALDI target for use in theaffinity purification of potential drugs fromtest solutions. Following procedures origin-ally developed for affinity chromatography,the preparation of affinity surfaces forMALDImass spectrometry has been achieved quiteeasily. However, the utility of these affinityMALDI chips for screening mixtures of smallmolecules during drug discovery has beenunproductive. One of the problems has beenthe high background noise at low m/z valuescaused by the matrix used for MALDI. Thisproblem may be mitigated by eliminating thematrix or using alternative sample stagessuch as porous silicon chips [56,57]. However,noise persists due to the affinity support andimmobilized receptor molecules. Another pro-blem to be overcome is to eliminate the highbackground noise caused by nonspecific bind-ing of test compounds to the affinity target.Although this problem is similar to the false-positive results and nonspecific binding thatoccurs during affinity chromatography-massspectrometry (see Section 2.4.1), the signalsfor nonspecific binding ismagnified by the factthat the actual affinity surface is being irra-diated and sampledby theMALDI laser beam.As a result, affinity-based screening coupledwith MALDI mass spectrometry has not beena successful drug discovery approach.

A recent method termed self-assembledmonolayers for MALDI (SAMDI) offers a solu-tion to the problems associated with MALDIscreening [58]. In an anthrax lethal factorinhibition assay, self-assembled monolayersof an anthrax protein were constructedagainst a background of triethylene glycolgroups on a glass plate. The self-assembledmonolayers provided well-defined arrays ofexposed receptor sites for ligand interaction,and the background noise and nonspecificbinding problems pervasive in previous MAL-

DI screening appear to have been largely sub-dued. Next, MALDI-TOF mass spectrometrywas used successfully to screen a library of10,000molecules for inhibitors of the receptor.TheSAMDI techniquewas evenused to derivequantitative values (IC50 andZ-factor) in goodagreement with those obtained by conven-tional screening methods.

Progress is beingmade in the use of affinityprobes for the capture of proteins and othermacromolecules from biological solutions fol-lowed by MALDI mass spectrometric detec-tion and identification [59,60]. One affinityMALDI mass spectrometry method has beenpaired with affinity probes using surface plas-mon resonance systems [61]. These affinity-based MALDI mass spectrometry screeningassays are promising approaches for testingblood or other biological fluids for the presenceof specific proteins or other macromolecules.As a result, these have the potential to becomeclinical diagnostic tools or might even lead tothe identification of new therapeutic targets.Recently,MALDImass spectrometryhasbeenused for molecular imaging of drug com-pounds, metabolites, biomarkers, proteins,and other chemicals in tissue sections [62].

2.4.6. PulsedUltrafiltration-Mass SpectrometryA versatile approach to screening solution-phase combinatorial libraries andnatural pro-duct extracts is pulsed ultrafltration-massspectrometry [63,64], which utilizes a stan-dard LC-MS system with an ultrafiltrationchamber substituted for the HPLC column.The principle of pulsed ultrafiltration screen-ing of combinatorial libraries is shown inFig. 12. During pulsed ultrafiltration, ligan-d–receptor complexes remain in solution inthe ultrafiltration chamber while unboundlibrary compounds and buffers are washedaway.Afterunboundcompoundsare removed,the hits from the library are eluted from thechamber by destabilizing the ligand–receptorcomplex using an organic solvent, a pHchange, or a combination of both. The releasedligands are identified on-line using APCI orelectrospray mass spectrometry [63] or col-lected and analyzed off-line using mass spec-trometry, LC-MS, or LC-MS–MS [65].

An example of pulsed ultrafiltration massspectrometry for the screening of a library of


20 adenosine analogs for ligands to adenosinedeaminase is shown in Fig. 13. After a 15minpreincubation of the library compounds(17.5mM each except for EHNA, which waspresent at 1.75mM) with 2.1mM adenosinedeaminase in 50mM phosphate buffer, an

aliquot containing 420pmol of the receptorwas injected into the ultrafiltration andwashed for 8min at 50mL/min with water toremove the phosphate buffer and unbound orweakly binding library compounds. Methanolwas introduced into the mobile phase to dis-

Wash unbound compounds to waste

LC-MS identification

m/z

Elute bound ligands into a trapping column

or directly into MS

Ligand–receptor complexes

Unbound compounds

Elute desalted ligands into MS

Pulse containing extract or library of compounds

Ultrafiltration separation

+

+

+

+ + +

+ +

+

HP L C

+

+ +

Figure 12. Combinatorial library or natural product extract screening using pulsed ultrafiltration massspectrometry. During the loading step (left), ligands are bound to the receptor either on-line (top) using a flow-through approach or off-line (bottom two incubations).Unbound compoundsand binding buffer, cofactors, etc.are washed out of the ultrafiltration chamber to waste during a separation step (middle). Bound ligands aredissociated from the receptor molecules and eluted from the chamber by introducing a destabilizing solutionsuch as methanol, pH change, etc. Finally, released ligands are identified using mass spectrometry, tandemmass spectrometry, or LC-MS (right). (Reproduced from Ref. [66] by permission of John Wiley & Sons.)

100

50

020 30 40 50

200 500300 4000

400

800

1200

1600

0

Library without adenosine

m/z

278

267 129

207 354 375

410

299 142 172 236

Library with adenosine

EHNA [M + H]+

Pos

itive

ion

mas

s sp

ectr

omet

er r

espo

nse

Figure 13. Identification of EHNAas the highest affinity ligand for adenosine deaminase in a combinatoriallibrary of 20 adenosine analogs using ultrafiltration electrospray mass spectrometry. (Reproduced fromRef. [63] by permission of the American Chemical Society.)


sociate the enzyme–ligand complex and re-lease bound ligands for identification by elec-trospray mass spectrometry. During metha-nol elution, only EHNA (erythro-9-(2-hydro-xy-3-nonyl) adenine) was detected as the[M þ H]þ ion of m/z 278 (Fig. 13). In controlexperiments using the library without en-zyme, no library compounds were detectedduringmethanol elution (Fig. 13 control). Des-pite being present at a 10-fold lower concen-tration than the natural substrate adenosineanalogs, EHNA was easily identified since ithad the highest affinity among the librarycompounds (Kd¼ 1.9nM). This demonstratesthe utility of ultrafiltration electrospray massspectrometry for identifying a high-affinityligand among a set of analogs that bind to aspecific receptor. In a follow-up lead optimiza-tion study using pulsed ultrafiltrationmass spectrometry, a synthetic combinatoriallibrary of EHNA analogs was screenedfor binding to adenosine deaminase, andstructure–activity relationships for EHNAbinding were identified [67].

As an illustration of the versatility ofpulsed ultrafiltration-mass spectrometry,binding assays for a variety of receptors havebeen reported including dihydrofolate reduc-tase [65], cyclooxygenase-2 [64], serum albu-min [68,69] and estrogen receptors [70].Pulsed ultrafiltration is not only useful foridentifying ligands to different receptors butalso a wide range of combinatorial librariesand natural product extracts in any suitablebinding buffer may be screened. In addition tocombinatorial libraries, complex natural pro-duct extracts have been screened [70], andneither plant nor fermentation brothmatriceswere found to interfere with screening [64]. Asanother example of the flexibility of thisscreening system, a centrifuge tube equippedwith an ultrafiltration membrane [71] hasbeen used instead of an on-line ultrafiltrationchamber. Other applications of pulsed ultra-filtration-mass spectrometry include screen-ing drugs and drug candidates for metabolicstability [72], metabolic activation toreactive metabolites [73] and the measure-ment of affinity constants for ligand–receptorinteractions [68,69].

Metabolism and toxicity screening applica-tions of pulsed ultrafiltration use hepatic

microsomes in the ultrafiltration chamber.For metabolic screening, drugs and the cofac-tor NADPH are flow injected through theultrafiltration chamber (oxygen is dissolvedin the mobile phase), and the metabolitesformed by microsomal cytochrome P450 andany unreacted compounds flow out of thechamber formass spectrometric identificationand/or quantitative analysis [72]. On-line ap-plications require the use of volatile buffers,but LC-MS and LC-MS-MS may be used off-line to analyze the ultrafiltrate no matterwhat buffer had been used. Screening drugsfor metabolic activation using pulsed ultrafil-tration-mass spectrometry is carried out in asimilar manner, except that glutathione iscoinjected along with NADPH and the drugsubstrate [73]. MS-MS may be used on-line orLC-MS-MS used off-line to screen for glu-tathione adducts as an indication that thedrug wasmetabolized to a reactive intermedi-ate(s) that was trapped by reaction withglutathione. Finally, pulsed ultrafiltrationmay be used with UV or mass spectrometricdetection to measure affinity constants ofindividual compounds [68].

In order to measure affinity constants andother physicochemical properties of bindingsuch as the number of binding sites, twopulsed ultrafiltration measurements are car-ried out. First, an aliquot or pulse of a liquid isinjected through the chamber, and the elutionprofile is recorded. Then, the chamber isloaded with a receptor, and the ligand is re-injected. If binding occurs, the elution profilewill be delayed in proportion to the affinityconstant. The control injection is used to con-trol for nonspecific binding to the apparatus.Since the concentration of receptor and totalamount of liquid are known, and since theconcentration of free ligand is measured as itelutes from the chamber over a wide range ofconcentrations, theaffinity constant andotherbinding parameters may be calculated.

In most of the applications of pulsed ultra-filtration to date, serial analyses were carriedout with a throughput of approximately one ortwo assays per hour. Since the purpose ofthese assays was to screen complex mixturesor to obtain metabolism data for new drugentities, the throughput of these analyses wasacceptable but not high throughput. The rate


limiting step in these analyses is the ultrafil-tration separation and not the mass spectro-metric detection. Several solutions have beenreported to increase the throughput of pulsedultrafiltration mass spectrometry. van Bree-men et al. [72] used amultiplex ultrafiltrationsystem with ultrafiltration chambers ar-ranged in parallel and interfaced to a singlemass spectrometer. An off-line variation ofthis approach is to use centrifuge tubes fittedwith ultrafiltration membranes to obtain ul-trafiltrates of multiple samples simulta-neously [74]. Then, each ultrafiltrate can beanalyzed using LC-MS. Another solution toincreasing the throughput of pulsed ultrafil-tration mass spectrometry has been to minia-turize the ultrafiltration chamber volumewhile maintaining the flow rate and chamberpressure. Since the ultrafiltration membranecannot withstand high pressure without rup-turing, the ultrafiltration process cannot beaccelerated simply by increasing the flow ratethrough the chamber. For example, Beverlyet al. [75] fabricated a 35mL ultrafiltrationchamber which was approximately threefoldlower in volume than previously reported ver-sions [63]. As a result, ultrafiltration massspectrometric analyses could be carried outat the rate of at least three per hour, whichcorresponded to a threefold enhancement ofthroughput. This study suggests that chip-based ultrafiltration mass spectrometrywould have the potential to result in a trulyhigh-throughput system.

The advantages of pulsed ultrafiltration-mass spectrometry include the variety of dif-ferent applications that may be carried out,the convenience of on-line screening, solution-phase screening, the ability to screen combi-natorial libraries and natural product ex-tracts, the diversity of receptors that may bescreened, and the freedom to use either vola-tile or nonvolatile binding buffers. For meta-bolic and toxicity screening, flow injectionanalyses have the additional advantages thatproduct feed-back inhibition is prevented sothat themetabolic profilemore closely approx-imates the in vivo system [72]. Finally, thedisadvantages of pulsed ultrafiltrationscreening for drug discovery include thewash-ing step, during which dissociation and loss ofweakly bound ligands might occur, and the

slow speed of each experiment, which can takeup to one hour.

2.4.7. DrugDevelopment Assays Based onMassSpectrometry Often, lead compounds identi-fied during high-throughput screening haveundesirable properties that preclude theiruse as drugs, such as poor oral bioavailabil-ity, unacceptably rapid metabolism, or theformation of toxic metabolites [76]. Identifi-cation of these problems early in the drugdevelopment process can save time and re-duce the costs of drug development by guid-ing medicinal chemists toward lead com-pounds more likely to become drugs. Theapplication of mass spectrometry to drug de-velopment assays such as the high-through-put estimation of lipophilicity, metabolic sta-bility, and the detection of reactive metabo-lites are just a few examples of how LC-MSand LC-MS-MS are being used to expeditedrug development. Many drug developmentapplications such as screening for metabolicstability, in vitro metabolism and the forma-tion of reactivemetabolites can be carried outusing pulsed ultrafiltation mass spectrome-try as described in Section 2.4.6. Some otherdrug development assays utilizing massspectrometry are described below.Determination of log P Using LC-MS The li-pophilicity of a drug candidate is usually es-timated by its log P, where P is the partitioncoefficient between water and octanol. Be-cause most compounds contain ionizablegroups that significantly affect solubility, thepartition coefficient is often measured at aspecific pH in which case the value is knownas the log D. Traditionally, log P and log Dmeasurements have been carried out usingthe shake-flask method, in which the testcompound is added to water and octanol, agi-tated and the phases allowed to separate. Theconcentration of the analyte is thenmeasuredin both solvents by a suitable technique suchas LC-UV or LC-MS. The shake-flask methodis the most accurate method for log P or log Destimation; however, it is the most costly andtime consuming. Faster and less expensivealternatives to the shake-flask method in-clude computer-based in silico prediction,LC-MS retention time-based estimation, anda recent 96-well plate format coupled to


LC-MS [77]. In the microtiter plate method, adeep 96-well plate is loaded with aqueous andorganic phases at the appropriate pH alongwith the compound of interest, and then vor-tex mixed. The phases are separated by cen-trifugation, and the plate is loaded into anautosampler for quantitative analysis usingLC-MS. The needle depth of the autosamplercan be controlled to sample from either theaqueous or organic layers (see Fig. 14). Aneedle wash is performed to eliminate carry-over. Quantitation of the analytes in eachphase is carried out using LC-MS. This ap-proach facilitates the rapid and accurate de-termination of logP or logDwhile eliminatingtedious liquid handling operations.Drug Metabolism and Metabolic ActivationMetabolism studies of lead compounds areusually initiated in vitro by incubating thelead compound with liver microsomes andenzymatic cofactors such as NADPH or hepa-tocytes. The products are analyzed by usingLC-MS and compared to control reactions tofind new peaks, which correspond to drugmetabolites. To detect metabolites of leadcompounds formed in vivo, laboratory animalsare administered the investigational com-pound followed by LC-MS analysis of bodyfluids and tissues. To enhance the throughputof metabolite identification and quantifica-tion, several LC-MS and LC-MS–MS ap-proaches have been developed [78].

The use of LC-MS with electrospray orAPCI facilitates the detection and character-ization of metabolites resulting from commonbiotransformations, such as metabolic oxida-tion or dealkylation. Accurate mass measure-ments recorded using high-resolution massspectrometers such as TOF, QqTOF, orbitrap,or IT-TOF mass spectrometers (see Table 1)facilitate the discrimination of drug metabo-lites from endogenous compounds by provid-ing molecular formulas of metabolites. Data-dependent product ion tandem mass spectro-metry combinedwith accuratemassmeasure-ments on QqTOF, orbitrap, or IT-TOF massspectrometers further enhance metaboliteidentification by providing structural infor-mation as well as elemental composition data.Commercially available software is availablefrom several vendors, which enables auto-matic data analysis and metabolite detectionbased on elemental composition, mass defectfiltering and isotope pattern-matching func-tions [79]. Mass defect filtering relies on theunique difference between the nominal massof a compound (an integer value) and its exactmass (value including the fractional mass),which is specific to its elemental composition.The mass defects and isotope patterns of drugmetabolites are usually similar to those ofthe precursor drug. For example, Batemanet al. [79] investigated the metabolism of theantiretroviral agent indinavir by using

Figure 14. Determination of log D and log D using LC-MS in a 96-well plate format.


UHPLC-MS on a QqTOF mass spectrometer.By applying a mass defect filter, the back-ground was dramatically reduced and severalnew metabolites were detected that were nototherwise apparent.

Electrophilic metabolites of lead com-pounds that might cause toxicity may be de-tected by trapping the metabolite with glu-tathione (GSH) or other water-soluble nucleo-phile and then detecting the GSH conjugateusing LC-MS–MS. Since GSH is an endogen-ousnucleophile, LC-MS–MSscreeningmaybeused to detect GSH conjugates formed in vitroor in vivo. Glutathione conjugates formedin vivo are either excreted in bile or as mer-capturic acids in the urine. The potential ofLC-MS–MS to be used as a screening tool todetect GSH conjugates with selectivity wasreported in the 1990s by Nikolic et al. [73].Initially described as an application of pulsedultrafiltration LC-MS–MS, screening forreactive drug metabolites trapped as GSHconjugates using tandem mass spectrometryhas become widely accepted, althoughsample cleanup may involve protein precipi-tation or solid-phase extraction instead ofultrafiltration.

During LC-MS–MS with CID, GSH conju-gates form characteristic GSH fragment ionsthat are useful for their selective detection.For example, during positive ion electrospraytandem mass spectrometry, GSH conjugatesfragment to eliminate a neutral molecule ofpyroglutamic acid (129 mass units) [80]. Thetandem mass spectrometry technique of con-stant neutral loss scanning may be used oninstruments such as a triple quadrupole massspectrometer to detect GSH conjugates due totheir characteristic loss of 129 units. Sincesome GSH conjugates do form this type offragment ion in high abundance, negative ionelectrospray may be used instead to detectanother characteristicGSH ion ofm/z 272 thatis formed by cleavage at the sulfur linkagebetween the drug metabolite and theGSH moiety [81]. Whether used for the char-acterization of drug metabolites or the detec-tion of reactive metabolites after trappingwith nucleophiles such as GSH, LC-MS, andLC-MS-MS are unsurpassed for their sensi-tivity, speed and selectivity. By applying LC-MS, LC-MS-MS and drug metabolite analysis

software, information about the types ofmetabolites, their structures, and rates of for-mationmay be obtained and used to select thebest lead compounds to be advanced in thedrug discovery and development process.Other Drug Development Assays High-throughput LC-MS assays to measure otherimportant pharmacokinetic parameters suchas serum protein binding have been reported.Serum protein binding can be measured byonline ultrafiltrationMS [68] orwith a 96-wellfiltrate assembly [82]. Intestinal permeabilitymeasurements are usually carried out usingthe Caco-2 cell model in a multi-well formatthat can be analyzed by LC-MS [83]. It shouldbe noted that LC-MS or LC-MS–MS quanti-tative analysis to support drug discovery anddrug development assays may be acceleratedusing UHPLC. These UHPLC-MS systemsreduce the runtime per sample to below 5minwhile maintaining or even improving analy-tical sensitivity and reproducibility [84].

3. THINGS TO COME

Mass spectrometry has become an essentialanalytical tool at every stage of the drug dis-covery and development process. In this chap-ter, the various applications of mass spectro-metry to combinatorial chemistry, drugdiscovery, and lead optimization have beenhighlighted. In the past decade, advancesin instrument technology have progressed ra-pidly. In particular, UHPLC, high-resolutionmass spectrometers, and tandem mass spec-trometers are becomingmorewidely availableandarebeingused tomakedrugdiscovery anddevelopment more rapid than ever before.

Intense competition between instrumentvendors and the demands of biomedical massspectrometrists (the consumers) continue todrive technological innovation. For example,innovations in ion sources and ion optics im-prove detection limits by an order of magni-tude with each generation instruments,although this trend may not be able to con-tinue indefinitely. New combinations of iontraps with TOF and orbitrap mass spectro-meters make it possible to explore MSn frag-mentation patterns with accurate mass mea-surements without the high cost of FT-ICR

THINGS TO COME 121

mass spectrometers. A full exploration of theapplications of these hybrid instruments hasonly begun.

Among themost promising of the newmassspectrometry technologies just becoming com-mercially available is ion mobility spectrome-try (IMS). Specialized IMS instruments arealready in use by security teams for the detec-tion of drugs, explosives, and toxins in thefield. Recently, IMS has been interfacedwithMS to add a new dimension of separationin an instrument termed ion mobility massspectrometry (IMMS). These instruments di-rect ions along a high-pressure path con-strained by an electric field. The ion packetundergoesmultiple collisions with a drift tubegas, which retards the motion of moleculeswith larger cross-sectional areas. Distinctfrom both gas chromatography and time-of-flight, ion mobility spectrometry separatesions based on their shape and charge withina few tens of milliseconds. After exiting thedrift tube, the ions can be separated and de-tected by conventional mass spectrometeranalyzers. IMMS is ideal for the separationand analysis of large biomolecules in the gasphase, such as studies of protein conformationand binding. Smallmolecule separations, par-ticularly for complex natural products ex-tracts, might also benefit from the ability ofIMMS to separate and characterize isomers orother compounds with similar structureswithin just a few microseconds instead of theminutes required for HPLC or even the sec-onds needed for UHPLC.

To match the increased analytical perfor-mance of LC-MS technology, data analysissoftware must be developed that is bothuser-friendly and highly functional. Toolssuchasautomatic featurepicking,massdefector isotope pattern filtering, and metaboliteidentification are only just becoming avail-able. These tools will become more sophisti-cated, and will become better integrated intothe data analysis workflow. Evenmore power-ful will be software that allows rapid profilingof large sample sets such as is needed formetabolomics. As the quality and quantity ofmass spectrometry data increases, robuststrategies tomanage and archive the datawillneed to be implemented to allow efficient data

mining and interchange of data between ven-dor platforms.

In conclusion, mass spectrometry providesrapid, reliable, sensitive, and selective analy-sis of combinatorial libraries for structureconfirmation, purity analysis, and library de-convolution. In addition, mass spectrometricscreening methods have been developed andapplied to drug discovery and development.High-throughput LC-MS methods have be-come routine for solubility and bioavailabilitytesting and metabolite screening to ensurethat only high-quality lead compounds areadvanced to more expensive clinical testing.In the case of natural products, mass spectro-metry facilitates the rapid and accurate activ-ity screening, dereplication, and characteriza-tion of complex extracts.

At different timesduring the last 100years,first physicists and physical chemists andthen organic chemists pronounced that massspectrometry had run out of new applicationsand had no future. Gladly, they were wrong.Today, medicinal chemists recognize the po-tential of mass spectrometry to contribute toall facets of drug discovery and development.Mass spectrometryhasbecomea fundamentalanalytical tool for drug discovery, and this roleshould continue to grow in the future.

4. WEB SITE ADDRESSES ANDRECOMMENDED READING FOR FURTHERINFORMATION

http://www.asms.org Homepage of theAmerican Society forMass Spectrometry.

This web site contains additional information aboutmass spectrometry and links to a variety ofeference materials regarding biomedical massspectrometry.

http://benthamscience.com\cchts

Combinatorial Chemistry& High ThroughputScreening

http://pubs.acs.org/journals/jcchff/

Journal of CombinatorialChemistry

http://www.5z.com/moldiv/

Molecular Diversity

http://www.liebertpub.com/BSC/default1.asp

Journal of BiomolecularScreening


R. B. Cole, editor. Elecrospray Ionization MassSpectrometry. New York: John Wiley & Sons;1997.

F.W.McLafferty, F. Turecek. Interpretation ofMassSpectra, 4th ed. Mill Valley, CA: UniversityScience Books; 1993.

W. M. A. Niessen. Liquid Chromatography-MassSpectrometry. 3rd ed.BocaRaton, FL:CRCPress;2006.

J. T. Watson. Introduction to Mass Spectrometry,4thed.Philadelphia,PA:Lippincott-Raven; 2007.

ACKNOWLEDGMENTS

The authors would like to thank Young GeunShin, Benjamin Johnson, and Jennifer Moselfor assistance with the preparation of themanuscript. In addition, the authors acknowl-edge the support from NIH grants S10RR10485, R01 CA101052, and P01 CA48112.

REFERENCES

1. Field F. J Am Soc Mass Spectrom1990;1:277–283.

2. BarberM, Bordoli RS, Elliott GJ, Sedgwick RD,Tyler AN. Anal Chem 1982;54:645A–657A.

3. Hillenkamp F, Karas M, Beavis RC, Chait BT.Anal Chem 1991;63:1193A–1203A.

4. ItoY, TakeuchiT, IshiiD,GotoM. JChromatogr1985;346:161–166.

5. Chen L, Stacewicz-Sapuntzakis M, Duncan C,Sharifi R, Ghosh L, van Breemen R, Ashton D,Bowen PE. J Natl Cancer Inst2001;93:1872–1879.

6. Liu J, Burdette JE, Xu H, Gu C, van BreemenRB, Bhat KPL, Booth N, Constantinou AI, Pez-zuto JM,FongHHS,FarnsworthNR,BoltonJL.J Agric Food Chem 2001;49:2472–2479.

7. Syage JA, Evans MD. Spectroscopy2001;16:14–21.

8. Gordon EM, Gallop MA, Patel DV. Acc ChemRes 1996;29:144–154.

9. Thompson LA, Ellman JA. Chem Rev1996;29:132–143.

10. Loo JA. Eur Mass Spectrom 1997;3:93–104.

11. Kyranos JN, Hogan JC. Anal Chem1998;70:389A–395A.

12. Dunayevskiy Y, Vouros P, Carell T, WintnerEA, Rebek J. Anal Chem 1995;67:2906–2915.

13. Dunayevskiy Y, Lyubarskaya YV, Chu YH,Vouros P, Karger BL. J Med Chem1998;41:1201–1204.

14. Demirev PA, Zubarev RA. Anal Chem1997;69:2893–2900.

15. Zeng L, Burton L, Yung K, Shushan B, KasselDB. J Chromatogr A 1998;794:3–13.

16. Zeng L, Kassel DB. Anal Chem1998;70:4380–4388.

17. Kiplinger JP, Cole RO, Robinson S, RoskampEJ, Ware RS, O’Connell HJ, Brailsford A. J.Batt, Rap Commun Mass Spectrom1998;12:658–664.

18. Ventura MC, Farrell WP, Aurigemma CM,Greig MJ. Anal Chem 1999;71:2410–2416.

19. Ventura MC, Farrell WP, Aurigemma CM,Greig MJ. Anal Chem 1999;71:4223–4231.

20. de Biasi V, Haskins N, Organ A, Bateman R,Giles K, Jarvis S. Rap CommunMass Spectrom1999;13:1165–1168.

21. Wang T, Zeng L, Cohen J, Kassel DB. CombChem High Throughput Screen1999;2:327–334.

22. Yang L, Wu N, Clement RP, Rudewicz PJ.In: Proceedings of the 48th ASMS ConferenceMass Spectrometry and Allied Topics. 2000;p 861–862.

23. Weller HN, Young MG, Michalczyk SJ, Reit-nauer GH, Cooley RS, Rahn PC, Loyd DJ, FioreD, Fischman SJ. Mol Diversity 1997;3:61–70.

24. Fang AS, Vouros V, Stacey CC, CC Kruppa GH,Laukien FH, Wintner EA, Carell T, Rebek J Jr.Comb Chem High Throughput Screen1998;1:23–33.

25. Powers DG, Coffen DL. Drug Discov Today1999;4:377–383.

26. Furka A, Bennett WD. Comb Chem HighThroughput Screen 1999;2:105–122.

27. Janda KD. Proc Natl Acad Sci USA1994;91:10779–10785.

28. Czarnik AW. Proc Natl Acad Sci USA1997;94:12738–12739.

29. WagnerDS,MarkworthCJ,WagnerCD, Schoe-nen FJ, Rewerts CE, Kay BK, Geysen HM.Comb Chem High Throughput Screen1998;1:143–153.

30. Kerr JM, Banville SC, Zuckermann RN. J AmChem Soc 1993;115:2529–2531.

31. Brenner S, Lerner RA. Proc Natl Acad Sci USA1992;89:5381–5383.

32. Ohlmeyer MHJ, Swanson RN, Dillard LW,Reader JC, Asouline G, Kobayashi R, Wigler

REFERENCES 123

M, Still WC. Proc Natl Acad Sci USA1993;90:10922–10926.

33. YoungquistRS, FuentesGR,LaceyMP,KeoughT. Rap Commun Mass Spectrom 1994;8:77–81.

34. Karet G. Drug Discov Develop 1999;1:32–38.

35. Potterat O,WagnerK, HaagH. J Chromatogr A2000;872:85–90.

36. Cordell GA, Shin YG. Pure Appl Chem1999;71:1089–1094.

37. Shin YG, Cordell GA, Dong Y, Pezzuto JM, RaoAVNA,RameshM,KumarBR,RadhakishanM.Phytochem Anal 1999;10:208–212.

38. Zani CL, Alves TMA, Queiroz R, Chaves MAL,Fontes ES, Shin YG, Cordell GA. Phytochem-istry 2000;53:877–880.

39. Constant HL, HLBeecher CWW.Nat Prod Lett1995;6:193–196.

40. Corley DG, Durley RC, RC J Nat Prod1994;57:1484–1490.

41. Stinson S. Chem Eng News 1994;72:18.

42. Running WE. J Chem Info Comp Sci1993;33:934–935.

43. Nedved ML, Habibi-Goudarzi S, Ganem B, He-nion JD. Anal Chem 1996;68:4228–4236.

44. Kelly MA, Liang HB, Sytwu II, Vlattas I, LyonsNL, Bowen BR, Wennogle LP. Biochemistry1996;35:11747–11755.

45. Kaur S, McGuire L, Tang D, Dollinger G, Hueb-ner V. J Protein Chem 1997;16:505–511.

46. Siegel MM, Tabei K, Bebernitz GA, BaumEZ. JMass Spectrom 1998;33:264–273.

47. Chu YH, Avila LZ, Biebuyck HA, WhitesidesGM. J Med Chem 1992;35:2915–2917.

48. Chu YH, Whitesides GM. J Org Chem1992;57:3524–3525.

49. Rundlett KL, Armstrong DW. J Chromatogr1996;721:173–186.

50. Avila LZ, Chu YH, Blossey EC,Whitesides GM.J Med Chem 1993;36:126–133.

51. Karger BL. J Med Chem 1998;41:1201–1204.

52. Chu YH,Dunayevskiy YM,Kirby DP, Vouros P,Karger BL. J Am Chem Soc1996;118:7827–7835.

53. Kasai K, Oda Y. J Chromatogr 1986;376:33–47.

54. Schriemer DC, Bundle DR, Li L, Hindsgaul O.Angew Chem Int Ed 1998;37:3383–3387.

55. Ng ESM, Yang F, Kameyama A, Palcic MM,Hindsgaul O, Schriemer DC. Anal Chem2005;77:6125–6133.

56. Shen Z, Thomas JJ, Averbuj C, Broo KM, En-gelhard M, Crowell JE, Finn MG, Siuzdak G.Anal Chem 2001;73:612–619.

57. Wei J, Buriak JM, Siuzdak G. Nature1999;399:243–246.

58. Mrksich M, Min DH, Tang W. Nat Biotechnol2004;22:717–723.

59. Nelson RW. Mass Spectrom Rev1997;16:353–376.

60. Nelson RW, NedelkovD, Tubbs KA. Anal Chem2000;72:404A–411A.

61. Nelson RW, Krone JR. J Mol Recognit1999;12:77–93.

62. McDonnell LA,HeerenRM.MassSpectromRev2007;26:60643.

63. van Breemen RB, Huang CR, Nikolic D, Wood-bury CP, Zhao YZ, Venton DL. Pulsed ultrafil-tration mass spectrometry: a new method forscreening combinatorial libraries. Anal Chem1997;69:2159–2164.

64. Nikolic D, Habibi-Goudarzi S, Corley DG, Gaf-ner S, Pezzuto JM, van Breemen RB. AnalChem 2000;72:3853–3859.

65. Nikolic D, van Breemen RB. Comb Chem HighThroughput Screen 1998;1:47–55.

66. Shin YG, van Breemen RB. Biopharm DrugDispos 2001;22:353–372.

67. Zhao YZ, van Breemen RB, Nikolic D, HuangCR, Woodbury CP, Schilling A, Venton DL. JMed Chem 1997;40:4006–4012.

68. Gu C, Nikolic D, Lai J, Xu X, van Breemen RB.Comb Chem High Throughput Screen1999;2:353–359.

69. vanBreemenRB,WoodburyCP, VentonDL. In:Larsen BS,McEwenCN, editors.Mass Spectro-metry ofBiologicalMaterials.NewYork:MarcelDekker; 1998; p 99–113.

70. Liu J, Burdette JE, Xu H, Gu C, vanBreemen RB, Bhat KP, Booth N, Constanti-nou AI, Pezzuto JM, Fong HH, FarnsworthNR, Bolton JL. J Agric Food Chem 2001;49:2472–2479.

71. Wieboldt R, Zweigenbaum J, Henion J. AnalChem 1997;69:1683–1691.

72. van Breemen RB, Nikolic D, Bolton JL. DrugMetab Dispos 1998;26:85–90.

73. NikolicD,FanPW,BoltonJL, vanBreemenRB.Comb Chem High Throughput Screen1999;2:165–175.

74. Liu D, Guo J, Luo Y, Broderick DJ, SchimerlikMI, Pezzuto JM, van Breemen RB. Anal Chem2007;79:9398–9402.

75. Beverly MB, West P, Julian RK. Comb ChemHigh Throughput Screen 2002;5:65–73.

76. van deWaterbeemdH, Gifford E. Nat Rev DrugDiscov 2003;2:192–204.


77. Chiang P, Hu Y. Comb ChemHigh ThroughputScreen 2009;12:250–257.

78. Prakash C, Shaffer C, Nedderman A. MassSpectrom Rev 2007;26:340–369.

79. BatemanK,Castro-Perez J,WronaM,ShockcorJ, Yu K, Oballa R, Nicoll-Griffith D. Rap Com-mun Mass Spectrom 2007;21:1485–1496.

80. MaL,WenB, RuanQ, ZhuM.ChemResToxicol2008;21:1477–1483.

81. MahajanM, Evans C. RapCommunMass Spec-trom 2008;22:1032.

82. Fung E, Chen Y, Lau Y. J Chromatogr B2003;795:187–194.

83. vanBreemenRB,LiY.ExpertOpinDrugMetabToxicol 2005;1:175–185.

84. Xu R, Fan L, Rieser M, El-Shourbagy T. JPharm Biomed Anal 2007;44:342–355.

REFERENCES 125

Documents

Burger's Medicinal Chemistry and Drug Discovery || Mass Spectrometry and Drug Discovery