evolution of an open-access quantitative bioanalytical mass spectrometry service in a drug discovery...

Copyright © 2006 John Wiley & Sons, Ltd. Biomed. Chromatogr. 20: 585–596 (2006)DOI: 10.1002/bmc

Evolution of an open-access ms service in a drug discovery environment 585ORIGINAL RESEARCHORIGINAL RESEARCH

Copyright © 2006 John Wiley & Sons, Ltd.

BIOMEDICAL CHROMATOGRAPHYBiomed. Chromatogr. 20: 585–596 (2006)Published online in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/bmc.668

Evolution of an open-access quantitative bioanalyticalmass spectrometry service in a drug discoveryenvironment

Patricia Wright,* Christophe Chassaing, Nigel Cussans, Drew Gibson, Caroline Green, Michelle Gleave,Russell Jones, Paul Macrae and Kenneth Saunders

Pfizer Global Research and Development, Department of Pharmacokinetics, Dynamics and Drug Metabolism, Ramsgate Road, Sandwich, CT139NJ, UK

Received 13 March 2006; accepted 15 March 2006

ABSTRACT: Increased demand for assays for compounds at the early stages of drug discovery within the pharmaceutical indus-try has led to the need for open-access mass spectrometry systems for performing quantitative analysis in a variety of biologicalmatrices. The open-access mass spectrometers described here are LC/MS/MS systems operated in ‘multiple reaction monitoring’(MRM) mode to obtain the sensitivity and specificity required to quantitate low levels of pharmaceutical compounds in an excessof biological matrix. Instigation of these open-access systems has resulted in mass spectrometers becoming the detectors of choicefor non-expert users, drastically reducing analytical method development time and allowing drug discovery scientists to concen-trate on their core expertise of pharmacokinetics and drug metabolism. Setting up an open-access facility that effectively allows auser with minimal mass spectral knowledge to exploit the MS/MS capability of triple quadrupole mass spectrometers presents asignificantly different challenge from setting up qualitative single stage mass spectrometry systems. Evolution of quantitative openaccess mass spectrometry within a pharmaceutical drug metabolism and pharmacokinetics group, from its beginnings as a singlegeneric system to a series of specialist fully integrated walk-up facilities, is described. Copyright © 2006 John Wiley & Sons, Ltd.

KEYWORDS: open-access; LC/MS; quantitation; pharmacokinetics; bioanalysis; MS/MS

INTRODUCTION

The aim of pharmaceutical drug discovery is to identifynew chemical entities that are safe and effective inaddressing a medical need, and taking them from thechemist’s bench into drug development in clinical trials.Key to the efficiency of this process is to identify asearly as possible those compounds that have the poten-tial to become medicines, whilst weeding out com-pounds that do not exhibit the necessary characteristics.When assessing the potential of a compound to becomea drug candidate, the physicochemical characteristicstogether with how the molecule behaves with in vitrostudies (e.g. metabolic stability in microsomes andabsorption across a Caco-2 layer) are determined. Thisdata is then used to predict how the compound is likelyto behave in animal studies which in turn are usedto predict the pharmacokinetics in man. Quantitativebioanalysis is central to each stage in the process of

drug discovery, from logD determinations to ascertain-ing the levels of circulating compound in plasma.

In this context, LC/MS/MS has been established as acore technology in bioanalysis (Fouda et al., 1991; Kayeet al., 1992; Dear et al., 1998). The sensitivity of triplequadrupole mass spectrometers in MRM mode makesthem ideal for quantitation of low levels of analyte inthe presence of endogenous material.

All the quantitative mass spectrometric work under-taken within our department is performed on triplequadrupoles in MRM (multiple reaction monitoring)mode. These assays are developed in support of bothdiscovery projects and candidates in development, usu-ally first in human clinical samples. Previous experienceof using selected ion monitoring (SIM) on single quadr-upole instruments (Sciex API 100 and MicromassZMD) to assay microsomal incubates and Caco-2 cellmedia, has shown that SIM is neither specific nor sensi-tive enough to meet departmental needs.

There are major differences between the assays re-quired for compounds in early discovery comparedwith those in development (Table 1). In spite of thesedifferences, the approach taken for assay developmentwas the same for both discovery and developmentassays in the early 1990s; that is the chromatographyand mass spectrometer settings were fully optimized for

*Correspondence to: P. A. Wright, Pfizer Global Research andDevelopment, Department of Pharmacokinetics, Dynamics and DrugMetabolism, Ramsgate Road, Sandwich, UK, CT13 9NJ.E-amil: [email protected]

Abbreviations used: MRM, multiple reaction monitoring; SIM,selected ion monitoring.


586 P. Wright et al.ORIGINAL RESEARCH

each analyte by a mass spectrometrist. This approachwas satisfactory at the time as only a few (five or sixper year) requests for mass spectrometric discoveryassays were being submitted. This was due to UVabsorption being the detection technique of choice forthe majority of compounds. By the mid 1990s, however,the requests for mass spectral assays for discoverycompounds were increasing rapidly. There were threereasons for this:

• Increasing potency of the potential drug candidatesmade the required limits of quantitation difficult toattain using UV or electrochemical detection.

• Discovery scientists were spending an increasingamount of time developing extraction techniques andoptimizing chromatography conditions to get samplesof the quality required for sensitive UV, electro-chemical or fluorescence assays.

• A drive to further improve the efficiency of the drugdiscovery/development process meant that discoveryscientists were expected to report back to the pro-jects in days rather than weeks.

Triple quadrupole mass spectrometers in MRM modeoffered the sensitivity needed and also allowed the ana-lyst to adopt a more generic approach to extraction, asthe endogenous background is often rendered invisibleby the specificity of this technique.

It became no longer feasible for a small massspectrometry group to support discovery assays in theway they had been whilst maintaining the samplethroughput required. It was decided to meet theincreasing mass spectrometric needs of discovery byintroducing a facility for self-service quantitation.

Open access mass spectrometry has been introducedin other laboratories (Brown et al., 1994; Pullen et al.,1995; Taylor et al., 1995; Wood and Hachey, 2000;Mallis et al., 2002; Greaves, 2002). These systems, how-ever, were designed to obtain structural informationrather than for quantitative assays.

The approach that would assure maximum successrate for this self-service system would be to continue to

utilize the full capability of the triple quadrupole byoperating in MRM mode. This meant that not only themolecular ion had to be determined for each analytebut also a suitable MS/MS experiment (i.e. voltagesdetermined, product ion selected) generated. The firstself-service facility was instigated in 1996 on a SciexAPI III Plus. This instrument did not have ‘autotune’capability, so an approach had to be determined whichallowed ease of use by a non-specialist and was capableof robust, high-throughput analyses. The system neededto be generic as far as possible to remove the need forindividual optimization of LC/MS/MS conditions.

Evolution of this system from its beginnings as asingle generic system on a Sciex API III Plus massspectrometer in 1996 to a series of specialist fullyintegrated walk-up facilities on multiple Sciex instru-ments (API 365, 2000s, 3000s and 4000s) is described.

Within our drug metabolism and pharmacokineticdepartment, 10 mass spectrometers are available inopen-access mode (Fig. 1); six API 2000 massspectrometers (one with a turbulent flow chromatogra-phy system, two with a monolithic column gradientHPLC system, two set up with a switching valve fordual column fast analysis and one set up for on-line de-salting of Caco-2 derived samples); two API 3000 massspectrometers (both with a monolithic column gradientHPLC system); and one API 4000 and one API 365with the original generic isocratic HPLC system.

EXPERIMENTAL

Chemicals

The mobile phase is obtained premixed from Romil (Cam-bridge, UK). The composition of mobile phase B was 90:10(v/v) methanol (Super Purity grade)–water containing 2 mMammonium acetate and 0.027% formic acid. The compositionof mobile phase A was 10:90 (v/v) methanol (Super Puritygrade)–water containing 2 mM ammonium acetate and0.027% formic acid. The methanol for the reconstitutionsolvent was also of Super Purity grade and purchased from

Table 1. Comparison of discovery and development assays with a mass spectrometric end point run in our Drug MetabolismLaboratories in 2001

Limit of Typical number Number of Assay validation toquantitation of samples compounds GLP guidelines Data archiving

Assay type Typical matrices required (pg/mL) per assay assayed in 2001 required? required?

Discovery Microsomes, 100–1000 6–300 1486 No Nohepatocytes,liver slices, bile,urine, plasma,bile, buffer

Development Plasma, urine 10–100 60–2000 17 Yes Yes(preclinicaland clinical)


Evolution of an open-access ms service in a drug discovery environment 587ORIGINAL RESEARCH

Figure 1. Diagram summarising the evolution of open-access mass spectrometry within Drug Metabolism in Pfizer,Sandwich from a single instrument in 1996 to seven instruments in 2005.

Figure 2. Mixture of Pfizer compounds used to validate system.

Romil (Cambridge, UK). The water used was deionized witha Milli-Q plus deionizer (Millipore, Bedford, MA, USA).Compounds used to test the system (Fig. 2) were chosento represent the typical spread of molecular weights and

functional groups of compounds submitted for assay develop-ment. They were also chosen because several exhibitedproblematic chromatography when assayed previously. Allthe compounds in Fig. 2 were obtained from the Compound



Control Unit of Pfizer Global Research and Development,Pfizer UK.

Instrumentation

The API III Plus, 365, 2000, 3000 and 4000 mass spectro-meters were supplied by Sciex (Toronto, Canada). The APIIII Plus, API 365, API 2000 and 3000 systems were purchasedwith Macintosh G3 computers for instrument control. TheAPI 4000 was supplied with a Dell Optiplex GX 400 PC. Theorifice voltage was set to 35 V and the interface was operatedat 100°C on all instruments (except for the monolithic columngradient and turbulent flow chromatography systems wherethe interface was operated at 450°C because of the higherflow rate entering the source). Only positive ion assays wereoffered on a self-service basis as the majority of Pfizer com-pounds respond well in positive ion mode. The other para-meters are discussed in the Results and Discussion section.

The autosamplers were CTC HTS PALs (Presearch,Hitchin, UK) fitted with one stack capable of holding up tosix 96-well development blocks. These stacks were cooled toapproximately 10°C to improve sample stability and reduceevaporation. If all six blocks were full of samples, the totalanalysis time would be over 38 h (for 3 min run time/3 min35 s total cycle time). The injection volume was set to 180 µL(out of 200 µL sample volume). The autosamplers injectedfrom approximately 2 mm above the bottom of the well toavoid picking up any particulate matter (each block was cen-trifuged to reduce the likelihood of blockages). In order tosample 180 µL without drawing up any particulate matter, itwas necessary to use the 96-well blocks with tapered wellsrather than the round-bottom wells.

The HPLC systems Agilent 1100 binary pumps were usedon all but one of the open access systems (supplied byAgilent Technologies, Stockport, UK). The Hewlett-Packard1100 series isocratic pump and Hewlett-Packard 1100 seriesbinary pump used for turbulent flow chromatography weresupplied by Cohesive Technologies, Franklin, MA, USA.

HPLC conditions

Typical chromatograms for the various chromatographysystems are shown in Fig. 3. Good peak shape is observedfor the majority of compounds analysed on these open accesssystems.

Generic isocratic HPLC system. The pump was operatedisocratically at 1 mL/min mobile phase B. The HPLC columnused was a Hypersil HS C18 50 × 4.6 mm, 5 µm (ThermoHypersil, Runcorn, UK). The HPLC eluent was split 50:1post column using an Acurate flow splitter (Presearch,Hitchin, UK), with 20 µL/min entering the mass spectrometerand the rest flowing to waste. The 50:1 split was employedto improve the robustness of the system by ensuring that98% of endogenous material was diverted to waste ratherthan contaminating the mass spectrometer. At a flow rate of20 µL/min, the mass spectrometer behaves as a concentrationdependent detector and so discarding 98% of the analytesdoes not seriously affect sensitivity (Bruins, 1991; Wrightet al., 1995). The acquisition time was 3 min (total cycle time

3 min 35 s). The high organic content of this mobile phase(90%) ensures rapid elution of the majority of analytes.

Divert valve HPLC system for Caco-2 cell mediumanalysis. A stepped gradient and a Rheodyne six-portswitching valve were used to desalt the samples on-line. Thecolumn was washed with 100% mobile phase A for the firstminute after injection with a post column switching valve dir-ecting the flow to waste (see Fig. 4). At the end of 1 min themobile phase was switched to 100% mobile phase B for thenext 3 min. The flow rate was maintained at 1 mL/minutethroughout. The HPLC column used was a Hypersil HS C18

50 × 4.6 mm 5 µm (Thermo Hypersil, Runcorn, UK). TheHPLC eluent was split 50:1 post column using an Acurateflow splitter, with 20 µL/min entering the mass spectrometerand the rest flowing to waste. The acquisition time was 4 min(total cycle time 4 min 35 s).

Monolithic HPLC system. Fast gradient HPLC with aRheodyne six-port switching valve was used to achieve someon-line sample clean up. The column was washed with 100%mobile phase A for the first 30 s after injection with a postcolumn switching valve directing the flow to waste. At theend of 30 s the mobile phase ramped to 100% mobile phaseB (which contained 90% methanol) over the next 30 s. Thecomposition was maintained at 100% mobile phase B for afurther 30 s before being stepped down to 100% mobile

Figure 3. Typical chromatograms from three of the open ac-cess systems. Chromatogram A: The generic isocratic HPLCsystem (Hypersil HS C18 50 × 4.6 mm 5µ with mobile phase).Chromatogram B: The turbulent flow chromatography system(on-line extraction with 50 × 1 mm I.D. HTLC Cyclonecolumn followed by analysis on 33 × 2.1 mm I.D. HTLCCyclone). Chromatogram C: The monolithic HPLC system.



logies, Oregon, USA) that offer minimal chromatographybut are highly compatible with high-throughput use.

Sample preparation

The Caco-2 cell medium buffer was analysed directly withoutany sample preparation. For the logD samples, 80 µL of

Figure 4. Flow diagram showing the divert valve set up usedfor both the Caco-2 cell medium analysis and the fast gradi-ent monolithic gradient HPLC systems. Position A is held for1 minute with the HPLC eluant being directed to waste; after1 minute the valve is switched to position B to direct theeluant into the mass spectrometer via a flow splitter.

phase A. The column was re-equilibrated at 100% mobilephase B for a further 30 s. The flow rate was maintained at3 mL/min throughout. The HPLC column used was aChromolith SpeedRod RP-18e18 50 × 4.6 mm, 5 µm (MerckKGaA, Germany). The HPLC eluant was split 5:1 post col-umn using an acurate flow splitter with 600 µL/min enteringthe mass spectrometer and the rest flowing to waste. The ac-quisition time was 2 min (total cycle time 2 min 35 s).

Turbulent flow HPLC system. The turbulent flow configura-tion is described in Fig. 5.

Dual column fast analysis system. The dual column fastanalysis system shown in Fig. 6 was used for high-throughputanalysis of lipophilicity (logD) and metabolic lability (bothmicrosomes and hepatocytes) samples. This parallel systemallowed sample analysis to be completed within 30 s persample. The HPLC columns used were Opti-Lynk guardcolumns (2.1 × 15 mm C18, porous 40 µm; Optimize Techno-

Figure 5. Flow diagram for the turbulent flow extractionand the analysis. Plasma (200 µl diluted 1:1) was loaded onthe extraction column with solvent A (0.01% aqueous TFA),while the analytical column was equilibrated with solvent C(acetonitrile/10 mM ammoniums acetate/methanol 45/45/10v/v). After back-flushing the extraction column with solventA, the analytes were eluted to the mass spectrometer withsolvent C. Both analytical and extraction columns werewashed after wash injection with solvents B (0.1% aqueousammonia) and D (acetonitrile/tetrahydrofuran 50/50 v/v).

Figure 6. Flow diagram for the dual column fast analysissystem. Pump 1 delivers 100% mobile phase A (low organic)at 1.5 mL/min to one HPLC column 1, whilst the pump 2delivers 100% mobile phase B (high organic) at 1.2 mL/minto column 2. Samples are loaded onto column 1 in the loworganic phase with the flow going to waste. After 12 seconds,the valve switched to divert the high organic flow tocolumn 1, eluting the analytes into the mass spectrometer,and the low organic to column 2. The next injection loadssample onto column 2, at the same time as the analytes elutefrom column 1.



the buffer was analysed neat but the octanol was diluted1:10 with methanol and 10 µL taken for analysis. Themicrosomes and hepatocytes analysed on the dual columnhigh-throughput system were diluted 1:2 with acetonitrile,centrifuged and 80 µL of the supernatant injected.

For all other samples, because of the wide variety of bothanalytes and biological matrices submitted, the method ofsample preparation was left to the submitting scientist whohas extensive experience of extraction procedures suitable fora particular series of compounds. It was strongly recom-mended, however, that solid-phase extraction (SPE) be usedwherever possible for the generic isocratic HPLC system andthe dual column fast analysis system, as SPE extracts causefew problems with blocking/contaminating the system.Liquid–liquid extraction was also widely used. Protein precip-itation was avoided for samples that are analysed on thegeneric isocratic HPLC method as this mode of preparationhas resulted in instrument downtime. Protein precipitation(with 3 vols of acetonitrile) was the sample preparationmethod of choice, however, for the turbulent flow and mono-lithic gradient HPLC systems. Both these systems have an ini-tial divert to waste period that was successful in preventingendogenous material building up in the source.

Submitting scientists had been instructed not to add strongacids, bases or involatile salts (e.g. borate buffer) during sam-ple preparation. Sample extracts were taken to dryness undernitrogen at 37°C and re suspended in 200 µL of an appropri-ate solution.

For the generic isocratic method the re-suspended solutionwas 70:30 v/v methanol–water containing 2 mm ammoniumacetate. Samples to be analysed on the monolithic gradientsystem were re-suspended in mobile phase A. The acetonitrilesupernatant was analysed directly by turbulent flow chromato-graphy. The autosamplers were set up to inject from tapered96-well blocks only (Porvair Sciences Ltd, Shepperton, UK)and these blocks were centrifuged for 30 min at 1,500 × g toremove any particulates prior to analysis.

RESULTS AND DISCUSSION

Establishing mass spectrometry conditions

Selecting appropriate MRM conditions was critical toobtaining a selective, sensitive assay. It was necessary,therefore, to establish an approach that would provideleast pitfalls for an inexperienced user.

Various methods for obtaining MRM conditions forthe open access systems were considered. An autotunefacility, where the software is set to automaticallyoptimize the instrument voltages for a particular com-pound, is available on most modern mass spectro-meters, including Sciex API 2000s, API 3000s and API4000s. This is achieved by infusing authentic standardof the compound of interest to obtain a constant signal,then ramping the various lens voltages until maximumresponse is obtained for the specified ion. This optimi-zation may be performed for both MS and MS/MSexperiments.

Autotune, although a useful option, is not used onour open access system for several reasons:

• Autotune works more reproducibly when the stand-ards are infused and this requires a greater degree ofuser intervention than just putting a sample block inthe autosampler.

• Electronic spikes during the voltage ramp may resultin an unsuitable voltage being selected.

• A state file is created for each compound. This statefile has to be then used to generate a method file,which means, again, more user intervention. Also,hundreds of state files and method files would begenerated very quickly making system administrationdifficult.

An alternative approach to rapidly establishingappropriate MRM transitions and collision energiesinvolves obtaining Q3 CID (third quadrupole collision-induced dissociation) spectra rather than true production spectra for each analyte (Hiller et al., 1997). In thisapproach the sample is introduced by flow injection,passes through Q1 (that is not used as a mass analyser),then through Q2 which is filled with collision gas toinduce fragmentation. Q3 is scanned and spectra ob-tained with five different collision energies appliedto Q2. The resulting spectra show varying degrees offragmentation of the analyte, with the spectrum fromthe lowest collision energy setting containing mainly themolecular ion. A suitable collision energy and production for the MRM transition is chosen from one of thefive Q3 spectra obtained. This approach is rapid andinnovative but does have the major disadvantage thatit lacks the specificity of MS/MS spectra. Fragmentions from any impurities in the sample will contributeto Q3 spectra and may mislead the user into selectinga product ion which is not derived from the analyte ofinterest.

Figure 7 shows the effect of ramping collision energyon peak intensity of the seven compounds (shown inFig. 2). Selection of the correct collision energy wascrucial in obtaining good sensitivity.

The method eventually chosen used three genericstate files that set three different collision energies. Thevalues of the three collision energies were chosen basedon experience with a wide range of compounds. Forsimplicity these state files were called ‘low’ collision en-ergy (25 eV), ‘medium’ collision energy (40 eV), and‘high’ collision energy (55 eV). The validity of usingthese values was checked using the compounds shownin Fig. 2. The MRM peak areas obtained for each com-pound using the generic state file that gave the best re-sponse, compared to the peak areas obtained whenboth the orifice voltage and collision energy were indi-vidually optimized for each compound, are listed inTable 2. This data showed that one of the three genericstate files gave a response within 30% of the fully



Figure 7. Effect of ramping collision energy (collision energy= Q0-R02; ST3 and R03 are linked to R02 as recommendedin the operators’ manual) on the peak intensity of the sevencompounds listed in Figure 1. A 0.2 µg/mL mixture of theseseven compounds in 50/50 (v/v) methanol/water was infusedat 10 µL/min. The compounds were monitored in MRMmode with following transitions: m/z307 to m/z110 (Fluco-nazole), m/z366 to m/z110 (Revatropate), m/z416 to m/z167(Zamifenacin), m/z442 to m/z198 Dofetilide), m/z427 tom/z147 (Compound X), m/z516 to m/z306 (Candoxatril),m/z730 to m/z630 (UK-141, 495).

Table 2. Comparison of the peak area obtained for seven compounds (Figure 2) when analysed in MRM mode using state files inwhich the orifice and collision energy voltages are fully optimized for each analyte and those areas obtained when the most suit-able of the three generic state files is used

Percentage area withPeak area (n = 2) generic state fileOptimized compared to area with

Compound MRM transition conditions Generic conditions Generic state file optimized state file

Fluconazole m/z307 → m/z220 4,591 3,259 Low 71UK-112,166 m/z366 → m/z302 6,795 5,142 Medium 76Zamifenacin m/z416 → m/z167 67,506 48,955 Medium 73Dofetilide m/z442 → m/z198 5,052 5,162 Medium 102Compound X m/z427 → m/z147 32,843 33,529 Medium 102Candoxatril m/z516 → m/z306 14,733 16,130 Low 109UK-141,495 m/z730 → m/z630 5,367 5,243 High 98

optimized response for all seven compounds, which wassufficient to obtain the sensitivity that was required fordiscovery assays.

Peak intensity of the seven test compounds (Fig. 2)was not significantly dependent on the orifice voltage

selected in the range 10–110 V and so an averageorifice voltage/declustering potential of 60 V was foundto be suitable for most compounds. The orifice voltage(declustering potential) is set lower for the ‘low’ statefile because compounds which fragment optimally atlow collision energies are often prone to in source CIDfragmentation and give more intense protonatedmolecular ions at lower orifice voltages.

Evolution of the self-service system

Generic open-access systems on Sciex API III Plus,365 and API 2000 (1996–1999). The starting point fordeveloping the original open-access mass spectrometrysystem was to review the conditions for all the quanti-tative assays performed on the API III Plus instru-ments in our laboratory over the preceding three years,1993–1996. Certain trends became apparent from thisassay review:

• Short (50 mm) HLPC columns with highly organicmobile phases were being used to obtain short acqui-sition times (3 min or less). With suitable samplepreparation, the specificity of MRM means that it isnot usually necessary to separate the analyte fromendogenous material (unless the level of ionizationsuppression is unacceptable) and so shorter run timescan be achieved with a mass spectrometric end pointthan with other detectors (UV, fluorescence orelectrochemical). It is possible to quantitate usingflow injection rather than having an HPLC columnin-line at all, but sharper peak shapes are obtainedby focussing on an HPLC column by injecting theanalyte in a weaker solvent than is present in themobile phase.

• A fixed orifice voltage of 60 V and ion spray voltageof 4500 V would give acceptable sensitivity for mostcompounds.

• The choice of collision energy was critical and settinga fixed collision energy would result in a significantfailure rate for a system that was expected to analyse



thousands of compounds of diverse structure overthe coming years.

On the basis of this review conditions were selectedfor the open access system on a single Sciex API IIIPlus that is likely to be suitable for the majority ofcompounds. Three state files were created for the user.These state files contained identical parameters withthe exception that each contained a different collisionenergy (denoted as ‘low’, 15 eV, ‘medium’, 40 eV and‘high’, 55 eV). A mass spectrometrist obtained production spectra of the analyte at each of the three collisionenergies and fedback to the open access user which ofthe three state files they should use and what the MRMtransition should be.

In addition, it was decided that the open-access sys-tem should utilize the IonSpray interface rather thanAPCI to avoid thermal decomposition of labileanalytes. Owing to the limited capacity of the ion spraysource, the flow from the HPLC column was split 50:1such that only 20 µL/min entered the mass spectro-meter. This flow rate lay within the range of flow ratesconsidered optimal for ion spray ionization.

This original system on a single API III Plus provedvery successful but had several limitations:

• It was not true open-access. Sciex’s data acquisitionsoftware, RAD, would not allow sample batches tobe added during data acquisition.

• The instrument, due to the cryogenic pumpingsystem, had to be recycled regularly to maintainperformance, limiting usage to approximately 15 hper day (maximum 1100 samples per week).

• The system would sometimes fail to recycle after anovernight run because the gate valve design wouldfail to seal due to a build-up of involatile material onthe curtain plate. Thus the instrument was unable tobe used for about 5 h the next day whilst it waspumped down.

• There was no system suitability between samplebatches so any deterioration in performance of themass spectrometer was not detected before it becameproblematic.

In order to address some these problems the self-service assays were transferred to a Sciex API 365.Also, this mass spectromter’s more refined opticsallowed a resolution setting of 0.7 amu peak widthat half height (PWHH), compared with 1.3 PWHHresolution on the Sciex API III plus, without decreasein response.

The API 365 was capable of 24 h operation, increas-ing the maximum sample capacity to 1800 samples perweek. It was also compatible with the new Sciex openaccess software MS Express, which transformed thefacility into a true walk-up service. The new Sciex soft-ware had full control over the Agilent 1100 HPLC

pump and autosampler (CTC HTS PAL). In addition,because of the ease of use of MS Express, the openaccess users themselves determined which of thesethree state files was suitable for their analyte.

This new open access system was so successful that itbecame saturated with samples within a few months ofinstallation. More mass spectrometers were, therefore,required to meet demand. In order to increase capacity,Sciex API 2000s were purchased for open access use.These instruments had superseded the API 365 becausethey are smaller and cheaper, but retained good sensi-tivity. The API 365 continued to be used on a self-service basis in addition to four API 2000s.

The huge increase in demand for mass spectralassays during this period marks the transition from UVassays to MRM assays on a triple quadrupole being thedetection technique of choice for bioanalysis.

Specialist open-access systems on Sciex API 365,2000s, 3000s and 4000s (1999–2003)

Over the past 3 years it had become apparent that us-ing all the open access mass spectrometers in a singlegeneric mode was not the most efficient use ofresource. A need for a series of open-access systemsaimed at particular type of analyses was identified.There were several drivers for this.

Requirement for higher sensitivity assays. Previously,a limit of quantitation of 0.5 –1.0 ng/mL was sufficientto obtain pharmacokinetic data for compounds at thisearly stage of drug discovery. Increasingly, however,discovery scientists required terminal phase pharmaco-kinetics (i.e. concentrations at later time points afterdosing) in order to improve their predictions of thepharmacokinetics parameters that the drug will exhibitin the first in human study. A limit of quantitation of0.1 ng/mL (or lower) may be required to determine thisterminal phase. In addition, low dose studies (e.g. forinhaled compounds) and serial sampling studies wheresample volume is limited, require a lower limit of quan-tification. Making higher sensitivity instruments (SciexAPI 3000 and an API 4000) available for use when thesensitivity of an API 2000 is not sufficient has ad-dressed this issue. The API 3000s and 4000 are oper-ated under similar generic conditions as the API 2000and API 3000 (Table 3).

The analytes are in a problematic matrix. The Caco-2 cell mono layer culture is used as a model of humansmall intestinal absorptive cells (Cogburn et al., 1991).The ability of the drug candidate to cross the cellmonolayer from one buffer filled chamber to anotheris used as an indicator of the oral absorption to beexpected in humans. The buffers from both sides of themembrane need to be analysed to determine the extent



of passage of the compound. The Caco-2 cell buffercontains high levels of inorganic salts that build upin the generic isocratic open-access system leading torapid decrease in sensitivity. Fitting one of the API2000 generic systems with a switching valve (see Fig. 4)eliminated this problem; the sample is loaded onto thecolumn in a mainly aqueous mobile phase with the flowgoing to waste for the first minute of each analysis todesalt the samples on-line; the mobile phase composi-tion is then stepped up to 90% organic to elute theanalyte from the column.

Improvements in efficiency can be achieved by reduc-ing the amount of sample preparation time. Theideal for the discovery scientist would be to do little orno sample preparation prior to analysis. The need forhigh quality sample extraction has been reduced oreliminated by using column switching based techniqueson the open-access systems; turbulent flow chromato-graphy (Ayrton et al., 1999; Chassaing et al., 2001; seeFig. 5) and fast gradient HPLC on monolithic analyticalcolumns with a divert to waste period (see Fig. 4). Boththe monolithic gradient HPLC and the turbulent flowchromatography systems allow for analysis of proteinprecipitated samples (usually plasma, hepatocytes ormicrosomes). Both these approaches have been so suc-cessful that they have replaced the original genericisocratic HPLC method, such that four general open-access systems are running the monolithic gradientHPLC system and one open-access system is runningthe Turbulent Flow chromatography system (this isused for protein precipitated microsomal incubates).

Need to determine properties of a large number ofcompounds in early drug discovery. In order toreduce attrition of compounds when they reach phar-maceutical development, it is necessary to understandmore about the properties of the potential drug candi-dates at the drug discovery stage. In this way chemistscan tailor their syntheses to increase the potential forthe compounds they produce to be efficacious andnon-toxic. To this end, an API 4000 was dedicated toperforming high through-put analyses to determinemetabolic lability and compound lipophilicity using thedual column fast analysis system.

Software

Data acquisition (RAD and MS express). The SciexAPI III plus, 365, 2000s and API 3000s were controlledby the Macintosh-based software. The data acquisitionsoftware for the Sciex API III plus was ‘RAD’ and hadno open access functionality. Batches could not beadded while data acquisition was taking place.

The data acquisition software on the Sciex 365, 2000sand API 3000 was ‘Sample Control’. The open access

acquisition software, MS Express, was commerciallyavailable from Sciex (Toronto, Canada) and it ran ontop of Sample Control. MS Express had a dedicatedlog-on screen with drop down windows that lock outthe user from the mass spectrometer control functions.The log-on screen only allowed users to add samples orbatches of samples to a sample list. This list controlledthe mass spectrometer operation using the pre-defined‘template’ methods.

Data acquisition was a two-stage process. Initially,the discovery scientist was involved in logging in astandard solution to determine which of the three statefiles were suitable for their analyte. This involvedlogging in a 1 µg/mL standard solution of the analyte toundergo four experiments. A Q1 scan was performedto confirm the presence of the protonated molecularion. Three product ion scans at low, medium and highcollision energies were then obtained. These runs tooka total of 16 min and the user was not required to bepresent. From these experiments the scientist chosea relevant set of MRM conditions, that is, the MH+

m/z value (from the Q1 scan) and a suitable pro-duct ion m/z value, together with the collision energyat which the product ion is most intense. Discoveryscientists were advised to select a product ion from themiddle of the mass range if possible, i.e. m/z above 100and a loss of greater than 70 amu. For example, if acommon loss such as 44 amu (CO2) was used, thespecificity of the assay may be reduced. The user re-quired no specialist mass spectral knowledge to operatethe system and obtain the parameters. Once selected,the MRM values and state file were used to analysesamples from in vivo and in vitro studies.

Once the discovery scientist had determined theMRM conditions, the assay was set up and run, MSExpress asked the user to enter their name, sampleidentification and state file required (i.e. low, mediumor high). The scientist was then prompted to enter thenumber of samples in the batch and the MRM transi-tions. Up to four MRM channels could be monitoredto allow for co-analysis of several analytes and/orinternal standard and/or metabolites. At this stage asystem suitability check may be requested. The finaldialogue box asked for the location of data storage. MSExpress was set up to acquire the data to a temporaryfile on the local hard disk and, once acquisition wascomplete, automatically send the file across the net-work to an NT file server. This ensured all data wascentrally located and could be processed on a desktopMacintosh.

MS Express allowed the user to add samples to anincomplete 96-well block or to select a new block. Upto six 96-well blocks could be logged into the systemand the software is sophisticated enough to expect anew block to be placed in the next free position in thestack.



Data processing (MacQuan). Data is acquired andtransferred to a server on a sample-by-sample basis,allowing instant access to the data from a remote dataprocessing station. MacQuan is used to process thedata. As the system does very little chromatographymost compounds have similar peak shapes and can bequantified with a default set of integration parameters.The users can change these parameters if necessary.

Error detection and system suitability. In the eventof an error the open access system is shut down auto-matically and safely. The sample control mass spectro-meter software has selectable options, which initiateshut-down of the mass spectrometer and all auxiliaryequipment if an error is detected (i.e. HPLC pump,autosampler and mass spectrometer status are moni-tored). In practice the main errors generated by thesystem are associated with the chromatography. Maxi-mum and minimum pressures on the HPLC system areset within the sample control method to shut the sys-tem down safely in event of a column blockage or lackof mobile phase. CTC PAL autosampler generatederrors will also result in shutdown.

At the beginning of each batch, a system suitabilitystandard is logged in and run by the discovery scientist.The MS Express software refers to this system suitabil-ity standard as a ‘validation sample’. The user has onlyto enter the well position of the ‘validation sample’when logging in the batch. A solution of fluconazole(0.1 µg/mL on API 2000 and API 3000; 0.01 µg/mL onan API 4000), which is put in a spare well in the 96-well block, is used to check system performance.Fluconazole was chosen because it is particularly sensi-tive to ionization suppression and is stable at roomtemperature in a methanol–water (70:30 or 10:90 v/v)solution for several months. A short programme (anApple Script) is run post acquisition to check retentiontime and peak height. If the results fall outside pre-defined limits, the system is halted and an error mes-sage displayed on the screen. Once the fault has beenrectified the run can be restarted as the sample list isstored in a file on the hard disk.

The mass spectrometer will shutdown the electro-nics and gas flows and the HPLC pump will switch offafter 30 min if no samples are added to the samplequeue. If shutdown, the action of logging in a batch ofsamples will start up the mass spectrometer, switch onthe gas flows and source heater, then start the HPLCpump, equilibrate the pump and, when ready, run thesamples.

API 4000 software (Analyst). The data acquisition andprocessing software for the API 4000 is the PC-based‘Analyst’. Analyst does not currently offer open accesssoftware of a comparable standard to the Macintosh-based software MS Express. Although it is not true

open access software, Analyst does allow samples to beadded to the sample list whilst acquiring data.

Limitations of the systems. Assays for certain com-pounds fail for a variety of reasons such as poor massspectral sensitivity, poor chromatography, or inappro-priate sample preparation (e.g. the analyte has not ex-tracted). In the event of an initial failure, success maybe achieved by discussing with a mass spectrometrist analternative approach. For example, a series of com-pounds gave a strong response in negative ion but anextremely poor response in positive ion mode. With anumber of open access mass spectrometers available,one was set up for negative ion assays specifically forthe analysis of samples from this compound series.Negative ion methods were not made available con-comitant with positive ion methods on the same instru-ments because negative ion assays are particularlyprone to ionization suppression and so it is importantto keep the instrument free from contamination. Inaddition, a different mobile phase would be required asthe formic acid in the generic mobile phase can causeionization suppression negative ion mode.

The minimal chromatography used in order to obtainhigh sample throughput may be viewed as a sourceof potential problems. The lack of separation of theanalyte from endogenous matrix, particularly with thegeneric isocratic HPLC system, occasionally resulted inionization suppression. When only moderate sensitivityis required, the desired limit of quantitation may beachieved in spite of ionization suppression effects. Useof the fast gradient monolithic column gradient systemhas generally reduced ionization suppression; the switchto waste period results in less polar endogenousmaterial entering the mass spectrometer and the fastgradient achieves at a least partial separation from theremaining endogenous material. Similarly, the turbulentflow chromatography and the Caco-2 cell system withthe divert valve achieve some on-line sample clean-upand suffer less from ionization suppression than theoriginal generic isocratic method. The monolithic gradi-ent system has an advantage over the turbulent flowchromatography system in that the chromatographyset-up is simpler, requiring only a binary HPLC pumpand a single switching valve.

Other potential problems arise from co-eluting drug-related material, which may affect assay specificity. Theassays are specific in the sense that the use of MRMmakes it unlikely (but has been observed on occasion)that an endogenous component undergoes the sameMRM transition. It is possible, however, that a closelyrelated metabolite may generate the same parent toproduct transition. In particular, glucuronides and N-oxides may decompose in the mass spectrometer toregenerate the original compound. This decompositionis usually thermally induced (Ramanathan et al., 2000),



but also may result from in source CID (Jemal and Xia,1999). The possibility of thermal decomposition of co-eluting metabolites is reduced by operating the inter-face at minimum temperature required for adequatevolatilization of the HPLC eluant (100°C; except forthe monolithic gradient system where 450°C was re-quired because of the higher flow into the massspectrometer) and setting the orifice voltage to lowestvalue that will give good sensitivity for the majority ofcompounds. As the compounds being assayed are usu-ally from very early drug discovery, little is knownabout their metabolism at the time of analysis. If, at alater stage of the compound’s development, it is foundthat potentially labile metabolites are being formed, theoriginal assay data can be re-evaluated and furtherwork performed as necessary.

Where there is some knowledge of metabolism, thereis an increasing tendency of the discovery scientists tomonitor for the presence of metabolites using MRMtransitions obtained from metabolite identification stud-ies. Caution should be exercised in doing this as, with-out an authentic standard of the metabolite to establishrelative ionization efficiency, the amount of metabolitepresent cannot be estimated with any degree of cer-tainty. Also, the limited chromatography may not sepa-rate isomers so, for example, a peak attributed to amono oxidized metabolite may in fact be due to two ormore co-eluting mono-oxidized metabolites. Discoveryscientists have been warned of the possibility of gener-ating misleading information using this approach andare aware that further work will need to be done toconfirm their observations. Usually this further work isundertaken if the discovery compound exhibits promis-ing efficacy and pharmacokinetic parameters and islikely to progress to early development.

CONCLUSIONS

LC/MS/MS is now the core analytical technique in ourdrug metabolism and pharmacokinetics facility. It isused for a wide variety of compounds exhibiting arange of physical chemical properties. The compoundsmay be present in a variety of biological matrices(microsomes, hepatocytes, plasma, urine, CSF, bile).The limit of quantitation for these assays ranges from50 pg/mL to 10 ng/mL. An internal standard is notalways used. This may be because a suitable internalstandard is not available or because the quality ofresults obtained are satisfactory without one. Where aninternal standard is used it is invariably a structuralanalogue as deuterated standards are not available forcompounds at this early stage of drug discovery.

The open access quantitative mass spectrometry sys-tems have revolutionized bioanalysis within the Depart-ment of Drug Metabolism at Pfizer (Sandwich, Kent,

UK). Mass spectrometry has replaced other detectiontechniques as the method of choice for the discoverymetabolism scientist. These systems are high-throughput(2–4 min acquisition time per sample), sensitive (LOQtypically 0.05–1 ng/mL) and specific (extraction andchromatography do not have to be fully optimized). Itis very easy to use with most operators requiring onlyan hour’s training. The ten mass spectrometers now usedin open access mode are capable of running over 10,000samples per week. These open access systems are heavilyused, particularly for overnight analyses, but are rarelyoperated to full capacity. The reasoning behind offering10 mass spectrometers in open access mode when theyare not operated to capacity is to ensure that massspectrometer availability is not rate-limiting. The dis-covery scientists have the freedom to schedule theirexperiments around the needs of the therapeuticproject and not the availability of analytical equipment.

The mass spectrometers have remarkably little down-time, mainly due to simple precautions taken with sam-ple preparation such as not using involatile salts andcentrifuging the samples. The system is self-policing inthat any instrument down-time due to careless practiceon the part of an individual inconveniences colleagueswith whom that individual shares offices/laboratories.When down-time does occur, the problem is usually as-sociated with the chromatography system, i.e. blockedfilter or deteriorating HPLC column.

Other laboratories have also adopted a three colli-sion energy approach to optimizing MS/MS param-eters. For example, one facility has written software forautomatically determining mass spectrometry condi-tions for bioanalysis (Whalen, 2000) on a Sciex API2000. This is also based on acquiring product ion spec-tra at three collision energies (low, medium and high).This is not an open access system, however, but hasbeen highly successful and is in routine use.

These open access systems have relieved the discov-ery scientists of the need to do much of their analyticalmethod development and allowed them to concentrateon their core expertise of pharmacokinetics and drugmetabolism. The next logical step in the evolution ofthe open access system would be miniaturized theHPLC systems to reduce both solvent and sample con-sumption. To this end, micro-turbulent flow chromato-graphy is being investigated (0.5 mm i.d. turbulent flowcolumn). Using a narrower bore monolithic columnis also of considerable interest, but at the moment a2 mm i.d. Chromolith column is not commercially avail-able due to manufacturing limitations.

Acknowledgements

Barry Kaye, former head of the Clinical Assay Group,is acknowledged for his ideas and support at the earlystages of this project.



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