Application of LC-MS In Supporting PK/PD Studies … · days, drug discovery revolved around...

Journal of Pharmacy Research Vol.5 Issue 5.May 2012

Tausif Ahmed et al. / Journal of Pharmacy Research 2012,5(5),2514-2526

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Research ArticleISSN: 0974-6943

Available online throughhttp://jprsolutions.info

*Corresponding author.Tausif AhmedAssistant Director, Department of Transla-tional Research (M&S), PiramalHealthcare Ltd, Goregaon (E), Mumbai,400063 India

Application of LC-MS In Supporting PK/PD Studies During Drug Discovery And DevelopmentTausif Ahmed* and Shashank Rohatagi

*Department of Translational Research (M&S), Piramal Life Sciences Ltd., Goregaon(E), Mumbai – 400063, India

Received on:11-01-2012; Revised on: 17-02-2012; Accepted on:19-04-2012

ABSTRACTMass spectrometry has made tremendous advancement during the last decade. This progress has led to the advent of entirely new instruments/technologiesand has revolutionized the analysis of small and large molecules. LC-MS due to its selectivity, low sample volume requirements, sensitivity and speed hasdiversity of applications. Today with the advent of high-throughput discovery programs across the industry, LC-MS has become an invaluable tool in drugdiscovery and drug development. LC-MS is being used during initial screening of compounds for ranking of compounds using vitro DMPK assays(solubility, plasma stability, metabolic stability, protein binding, CYP inhibition, Caco-2 permeability, tabolite profiling, blood partitioning, reactivemetabolite screening, in vivo PK etc). The late stage development applications include quantification of drug/metabolites in plasma samples (or othermatrices) obtained from clinical trials using validated ioanalytical methods. Other applications include biomarker and large molecule quantifications.Applying LC-MS and novel bioinformatics to sample analyses presents an opportunity to quantitatively evaluate thousands of endogenous molecules aswell as drug-derived metabolites. Further advances in the area of LC-MS instrumentation must continue and should provide many fruitful benefits for bothmass spectrometry and drug discovery and development. All this would ultimately lead to reduction in time & cost of drug development process.

Key words: LC-MS, drug discovery, development, PK-PD

INTRODUCTION:The drug development process involves several steps, from target identifica-tion and screening, lead generation and optimization, preclinical and clinicalstudies to final registration of a drug. In 2003 the average development timeof a new chemical entity (NCE) up to the registration was 12 years and itscost was approximately 800 million USD [1]. A study published in 2006estimates that costs vary from around 500 million to 2,000 million dollarsdepending on the therapy or the developing firm [2]. A study published in2010 in the journal Health Economics, including an author from the USFederal Trade Commission, was critical of the methods used by diMasi et al.but came up with a higher estimate of ~$1.2B [3]. In 2011, several drugs failedin phase 3 trials due lack of efficacy, although the results until phase 2 trialswere promising. During 2007-2010 [4] almost 90% of failed phase 3 trialswere either due to lack of efficacy (66%) or safety issues (21%). Amongthese failures about 28% drugs were for oncology targets and 18% for neuro-logical disorders. This is a very important factor to consider as many phar-maceutical companies ignored the complexity of biology in rational drugdesign and discovery. As Arrowsmith (2011) [5] points out in his article,“propensity to assume that success in one disease will translate into successin new and significantly different disease. This is particularly apparent inoncology, for which success in one tumor type has been assumed to translateinto success in variety of other tumors, without firm evidence that the mecha-nism of action remains relevant”. For example sunitinib (sutent, pfizer) wasapproved in 2006 for renal cell carcinoma and imanitib resistant gastro-intes-tinal stromal cell carcinoma and recently for rare pancreatic neuroendocrinecancers[6]. However, between April 2009 and May 2011 Pfizer has reportedunsuccessful late-stage trials with sunitinib in breast cancer, metastaticcolorectal cancer, advanced non-small-cell lung cancer, and castration-resis-tant prostate cancer. These failures clearly indicate mechanisms of tumor

development are different from one organ to another and forcing the com-pounds into expensive late stage clinical trials for different indication thaninitially sought after can contribute to poor success of the drugs to be ap-proved, due to lack of mechanistic mode of action of the drug candidate.Another point to consider is that predictive value of proof-of-concept (POC)trials in animal models is inadequate and in certain cases may not translate todesired clinical outcome [7]. Frye (2011) [8] points out that about 49% ofknown targets have not been validated for efficacy in humans thus wideningthe gap for discovering novel drugs for unmet medical needs. It has beenestimated that for every 5,000 new chemical entities (NCEs) evaluated in adiscovery program, only one is approved for market. Among the major rea-sons for NCE failure, other than poor clinical efficacy, are serious undesiredside effects, adverse drug reactions, and unfavorable drug metabolism andpharmacokinetics (DMPK) [9]. During the past years, pharmaceutical com-panies have invested and introduced a number of new approaches dedicatedto improve the rate of success of development of new drugs. In the earlydays, drug discovery revolved around structure activity relationships thatwere aimed to increase the efficacy and reduce toxicity of the drug. However,with increased number of candidates failing in the clinic due to ADME relatedproperties, evaluation and optimization of drug metabolism and pharmacoki-netic (PK) properties of NCEs have become essential parameters for consid-eration in the early phases of drug discovery. New approaches to medicinalchemistry such as parallel synthesis and combinatorial chemistry strategiesand refinement of high throughput screening in biology place drug discoveryat a crossroad. High compound numbers are essential whatever the eventualscenario to ensure that early lead matter is available. The size of compoundfiles of the future, millions of compounds, means informatics and automationare key ingredients for a successful drug discovery organization. Since thequantity of NCEs synthesized initially for in vitro screening is very limitedand due to lack of automation for every screen, an integrated approach isrequired to screen the compound at an early stage to derive maximum infor-mation and to rank order compounds.

The development of higher throughput approaches in absorption, distribu-tion, metabolism and excretion (ADME) studies is driven by the advances inhigh-speed chemistry, pharmacological screening and advances made in the



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NCE

In vitro binding against target receptor

Physicochemical characterization (Experimental or computational)

In vitro binding against selectivity receptor

Functional activity against target receptor

Metabolic stability plasma/microsomes

Membrane permeability (PAMPA/CaCo-2)

PPB

In vivo PK-PD evaluation

Figure 1: Workflow of activities during compound screening

Table 1: List of Different DMPK And Toxicity Studies Conducted Before First in Man Studies

mass spectrometry, a view of the future where many more compoundswould need to be screened, and the availability of the technology. Depart-ments of Drug Metabolism and Pharmacokinetics in the pharmaceutical in-

being followed in a drug discovery company is depicted in Figure 1. Higherthroughput could allow a parallel process, which collects large amounts of invitro pharmacology and ADME data as the primary stage. DMPK studies,supported by bioanalytical research, play a pivotal role in screening outcompounds early and minimize the risk of compound being failing late indevelopment. Typical DMPK attributes investigated during early discoveryinclude metabolic stability, cytochrome P450 (CYP)-reaction phenotyping,CYP inhibition and induction assays, detection of reactive metabolites, me-tabolite identification (Met ID), determination of in vitro permeability, andestimation of plasma protein binding. The data not only help in the screening

traditional sequence, provide more comprehensive data on a single com-pound, or just screen more compounds. The pre-ADME days of discoveryhad screening sequences based on an in vitro functional response often fol-lowed by an oral rodent pharmacodynamic model. A general flow of activities

dustry are organizing themselves for the rapid evaluation of large numbers ofcompounds [10-13]. Higher throughput can move a screening approach up the



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A. Drug Discovery:

Target identification and validation (Biomolecular MS)

-Peptide mapping LC-MS/MS -Proteome screening and imaging -MALDI/ESI-MS protein folding

Lead finding

-LC-MS compound libraries -Fast open access LC-MS -Prep. LC-MS

Lead optimization

-LC-MS compound libraries -Fast open access LC-MS prep LC-MS-Metabolite identification (qualitative)-Structure analyis MS -MS, -Bioanalytical method development and validation -Sample analysis

potential candidates, but also helps in establishing structural activity rela-tionships (SAR). Various types of studies being conducted at different stages

2. LC-MS SUPPORT TO DISCOVERY STUDIES:

2.1 In vitro ADME:Preclinical in vitro studies can be divided in four broad parts .e.g. absorption,distribution, metabolism and excretion. In vitro ADME profiling (solubility,stability (plasma and buffer), PAMPA, MET-ID, Caco-2, metabolic stabil-ity, protein binding, CYP reaction phenotyping, reactive metabolite screen-ing, CYP inhibition, CYP induction and blood to plasma portioning) ofNCE’s is routinely done in drug discovery to rank order the NCE’s and helpunderstand the compound liabilities. Evaluation and optimization of drugmetabolism and PK properties of NCEs is essential for success in drugdiscovery and development. In drug discovery, the NCEs are tested initiallyfor in vitro biological activity and the active hits are progressed for in vitroADME screening [18,19]. The scale and number of in vitro ADME screensconducted varies from one organization to another, due to differences inoperating philosophy and available resources. Some organizations use a cen-tralized and dedicated ADMET profiling service model that supports alldiscovery in high throughput screening mode (HTS), whereas in others onlyselected few assays are being done in-house or outsourced to contract re-search organizations (CRO’s) [20]. High throughput LC-MS/MS using triplequadrupole mass spectrometers with an atmospheric pressure ionization(API) source and operated under selected reaction monitoring (SRM) modehas emerged as an enabling technology for quantitative bioanalysis in drugdiscovery. Figure 2 gives a glimpse of role of LC-MS at different stages ofdrug discovery and development. Due to the large number of NCE’s beingscreened for ADMET assays, there is immense pressure on the bioanalyst toincrease throughput. The manual optimization of chromatographic and MS/MS conditions for each compound becomes a limiting factor. The sampleextraction procedure followed in ADMET bioanalysis is typically proteinprecipitation using a generic organic solvent in order to cut short the time forsample processing. Some of the recent technological advances made in thehigh throughput bioanalysis with reference to ADMET profiling will bediscussed briefly here. Some excellent reviews focusing on ADMETbioanalysis have already been published [21, 22]. The commonly conducted invitro ADMET assays are listed in Table 2. Some of the common in vitroassays are described below.

Bioanalysis is a sub-discipline of analytical chemistry covering the quantita-tive measurement of xenobiotics (drugs and their metabolites, and biologicalmolecules in unnatural locations or concentrations) and biotics (macromol-ecules, proteins, DNA, large molecule drugs, metabolites) in biological sys-tems. Bioanalysis based data is authentic, reproducible, precise and withspecificity. Bioanalysis supports both drug discovery and drug develop-ment. Bioanalysis can aid in laying a strong foundation of drug developmentin future. At present Bioanalysis is playing major role in microdose clinicalstudy, exploratory study, regulatory submission study, pre-clinical study,high throughput screening of bioactive constituents. Bioanalysis is support-ing in different sections of pharmaceutical industry like metabolomics,proteomics, clinical and preclinical studies. In recent times, liquid chroma-tography-mass spectrometry (LC-MS) has become the choice of scientistsfor both qualitative and quantitative analysis [17]. MS has revolutionizedanalytical chemistry. The recent, pervasive use of LC-MS is providing sen-sitive, selective, rapid, and information-rich analytical methodology to drugdiscovery research. With the possible exception of combinatorial chemistry,no single technology has revolutionized and streamlined the drug discoveryprocess to such a great extent. In the hopes of expediting the drug discoveryprocess, major pharmaceutical companies have invested considerable finan-cial and human resources in mass spectrometers. Current pharmaceuticaldevelopment has become more demanding, especially with the advent ofhigh-throughput discovery programs across the industry in the areas ofchemistry, pharmacology, and pharmacokinetics. In this regard, interdiscipli-nary rapid-screening protocols that allow for the examination of an increasednumber of new chemical entities (NCEs) have arisen. Many of these newscreening protocols have been successful due in large part to the ability of themass spectrometer to function as a sensitive and selective detector withminimal method development effort. Drug discovery has been the subject ofmany articles in peer-reviewed journals, but until now no concise treatise hadbeen written to bring together the numerous applications of mass spectrom-etry and drug discovery. Mass Spectrometry in drug discovery is intended tobring the knowledge gap that the widespread use of LC/MS has created.From a practical and application point of view, the focus of current reviewwill be to highlight the use of mass spectrometry as a bioanlytical tool insupporting different PK and PD studies during drug discovery and develop-ment. A brief description of different PK and PD studies will also be pro-vided.

these data may also be used to support the times at which efficacy studiesshould be most appropriately carried out. In addition, further PK studiesmay be carried out to develop a PK/pharmacodynamic (PD) model. The PDparameters can be physiological measurements such as blood pressure, tu-mor size or endogenous analytes such as cytokines [15]. PK is the study of thetime-course of the absorption, distribution, metabolism and excretion ofdrugs (i.e. what the body does to the drug). PD is the study of biologicaleffects of drugs and their mechanisms of action (i.e. what the drug does to thebody). PK-PD modeling is employed to establish a correlation of the concen-tration-time relationship (PK) with the effect-concentration relationship (PD)in order to provide a better understanding of the time course of an effect (PK/PD) after drug administration. By integrating PK-PD, it is possible to char-acterize the onset, intensity and duration of the pharmacological effects of adrug and to relate these to its mechanisms of action [16].

discovery phases rarely involve any definitive quantitative bioanalysis. This

fractions, plasma or blood. While at the in vivo ‘efficacy’ stage pharmacoki-netic (PK) studies may be conducted in rodents or non rodents. PK screeningstudies may be used to identify drug candidates with desired PK parameters,

is usually restricted to in vitro metabolic stability studies, using cellular

of drug discovery and development is depicted in Table 1 [14]. The early



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2.1.1 In Vitro metabolism and metabolite identification:The in vitro drug metabolism is one of the initial screening tools in drugdiscovery, where experiment for oxidation, reduction, glucuronide conjuga-tion (phase-I & phase-II metabolism) is performed through pooled liverenzyme in different species (Rat, dog monkey & human) and the metabolitegenerated are studied. Most common phase I and Phase II metabolites screenedfor identification is shown in Table 3. The metabolites can be further identi-fied through metabolite identification softwares or use of MS/MS or HRMS(high resolution mass spectrometry). The current approaches utilized for

metabolite identification during early discovery include isotopic-patternmatching, hydrogen/deuterium-exchange MS, data dependent analyses andmass defect filters [23].

Identification of metabolites and metabolic pathways is essential duringearly drug discovery process to understand potential liabilities and risksassociated with metabolites. Objectives of metabolite identification duringdrug discovery include:

a. Identification of metabolic soft and hot spots in an NCE and understandthe structure activity relationship (SAR). The soft spots include inactivepolar metabolites that may be rapidly eliminated from the body. The hotspots include reactive intermediates (electrophiles, free radicals) which maycovalently bind to tissue macromolecules (proteins/DNA) and/or cause oxi-dative stress resulting in toxicity.

b. Identification of potential metabolites that have pharmacological activity.c. Identification of species differences in metabolite profiles so as to exploreoccurrence of possible unique human metabolites and estimate their expo-sure in preclinical species. Recent FDA guidance [24] recommends identifyingpotential human metabolites as early possible so that expensive and time-consuming toxicology studies with metabolites can be avoided if the metabo-lite exposures are covered in toxicology studies. In vitro studies using humanliver microsomes and hepatocytes are often employed to identify potential

B) Drug Development:

Preclinical CMC

-Impurity profiling LC-MS/MS -Structure analysis MS/MS -Metabolite(quantitative) -Identification LC-MS/MS -Bioanalytical LC-MS/MS

Clinical CMC

-Impurity profiling LC-MS/MS. -Structure analysis MS/MS -Metabolite identification(quantitative) LC-MS/MS -Bioanalytical method validation (fully validated) -Sample analysis

Figure 2: Role of LC-MS/MS at Different Stages of Drug Discoveryand Development

Table 2: Commonly Conducted In vitro ADMET Assays:

human metabolites preclinically before initiation of clinical trials.

Elucidation of the structure of many metabolites is not straightforward espe-cially when complex rearrangement leading to a unique metabolite and/or theformation of metabolites with isobaric molecular ions [25-29]. Recent advancesin the LC-MS/MS technology allow detection of metabolites with high sen-sitivity up to femtomole levels. Liquid chromatography (LC) with diodearray detector is widely used to separate the mixture of metabolites anddrugs while mass spectrometry serves as an ideal detector to identify thestructure of metabolites [25]. In most cases chromatogram obtained from



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diode array detector complements the mass chromatogram. Mass spectro-metric detectors are more sensitive and robust compared to other detectorssuch as diode array detectors and nuclear magnetic resonance (NMR) spec-troscopy. However, final confirmation of metabolite structure needs to beestablished using NMR spectroscopy. Different types of ion sources suchas electro-spray ionization (ESI), nano electrospray ionization (Nano ESI)and atmospheric pressure chemical ionization (APCI) serves as interfacebetween liquid chromatography and mass spectrometry and assist in theionization of molecules.

Development of triple quadrupole mass spectrometers (tandem mass spec-trometers) coupled with LC has made a significant impact in the metaboliteidentification research. Triple quadrupole instruments provides MS2 frag-mentation patterns which are necessary to identify the metabolic soft spots[28, 29]. To date multiple reaction monitoring mode (MRM) remains to be themost sensitive method to confirm trace levels of circulating metabolites inbiological fluids derived from animal or human ADME studies.

Recent technology such as triple quadrupole ion trap mass spectrometerswhere trap technology is integrated with triple quadrupole mass spectrom-eters is both sensitive and qualitative [30, 31]. Triple quadrupole ion trap massspectrometer coupled with LC is one of the ideal equipment to characterizethe structures of metabolites. These instruments are more sensitive andprovide MS2, MS3 fragmentation spectra of metabolites formed even infemtomole quantities and help to characterize their structures. Apart fromMS2 and MS3 fragmentation spectra - neutral loss scans, precursor ion scansand enhanced resolution scans from the trap instruments are characteristic todetect the metabolites. For example neutral loss scan of 176 m/z is used toidentify phase II metabolites such as glucuronide conjugates. Other impor-tant and routinely screened glutathione adducts are identified by neutral lossscan of 129 m/z and negative precursor ion scans of 272 m/z. Enhancedresolution scans are helpful in identifying the isotopic pattern of the metabo-lite structures. Apart from the above, information dependent acquisition

scan modes such as Enhanced Mass triggered – Enhanced Product Ion (EMS– EPI) scan, Neutral loss triggered Enhanced Product Ion (NL-EPI) scan,Precursor Ion Scan triggered Enhanced Product Ion (EMS - PI) scan, En-hanced Resolution – Enhanced product ion Scan (ER – EPI), Multiple Reac-tion Monitoring triggered Enhanced Product Ion (MRM – EPI) scans havebeen effective tools in identifying the structure of metabolites. Recently wehave reported the importance of different scan modes in identifying thestructure of metabolites using triple quadrupole ion trap mass spectrometer(API4000 Q-trap) coupled with LC.

Accurate mass measurements (high resolution) in metabolite identificationexperiments can provide information which can distinguish isobaric molecu-lar ions and correct fragmentation pattern. Higher versions of ion trap instru-ments such as orbitrap [32, 33] are capable of providing high resolution / highmass accuracy measurements of precursors and product ions regardless ofrelative ion abundance. Orbitrap instruments also have the MSn capabilities(ie acquisition of MS10 spectrum is possible which are highly useful inassigning the metabolic soft spot in the molecule). Lim and co-workers haveevaluated Linear Ion Trap/Orbitrap to identify the metabolites of carvedilol.Mass accuracy of Linear Ion Trap/Orbitrap is up to fifth decimal and thisadvantage helps to differentiate isobaric hydroxylated metabolites with greaterconfidence. Orbitrap mass spectrometers are technologically capable of iden-tifying and characterizing the structure of metabolites. Metabolites ofNefazodone were characterized using both quadrupole ion trap and LinearIon Trap/Orbitrap. Excellent isotopic pattern from LTQ-Orbitrap detectedand confirmed twenty eight metabolites of Nefazodone while QuadrupoleIon Trap could detect all the metabolites but reduced quality of isotopicpattern hinders the confirmation [33].Triple Quadrupole – Time of flight mass spectrometers (Q-TOF) with liquidchromatography were also used for metabolite identification. In triple qua-drupole – time of flight mass spectrometers the ions of interest are massselected in first quadrupole, followed by fragmentation in a second quadru-pole or hexapole and analyzed by time of flight detector. Time of flightdetectors [34] has high resolution and the ions of interest can be separatedfrom isobaric molecular ions, thus facilitating the measurement of mass tocharge (m/z) value of ions with an accuracy of 2 parts-per-million (ppm).For example when a drug with ether functional group (R-CH2-O-CH3) ismetabolized (i.e. O- demethylation followed by oxidation of alcohol) to acid(R-COOH) the molecular weight remains same and if the fragmentationpattern of drug and metabolite is similar quadrupole resolution is not enoughto differentiate them. In such cases accurate mass measurements by time offlight analyzers can differentiate the drug from metabolite and structures canbe assigned. Time of flight analyzers can differentiate masses up to fourthdecimal with precision and provides extra dimension in identification of themetabolites.

Rapidly developing mass spectrometric imaging technology such as MALDI(matrix assisted laser desorption ionization), DESI (desorption electro sprayionization) and DART (direct analysis in real time) coupled to liquid chroma-tography are used to image the drug and metabolites. This approach can alsobe used for knowing the spatial distribution of drug and metabolites in ani-mals and tissues [35, 36].

The in vitro GF (Gastric fluid) & IF (intestinal fluid) stability studies ofNCE’s is performed by spiking the NCE in GF & IF media at simulated bodyconditions (i.e. incubation of sample at 37°C) for 0, 6, 12 and 24 hours. TheNCE is then analyzed at all time points and is compared with zero referencetime point, this experiment is conducted simultaneously for full scan modeand SIM (selected ion monitoring) in both positive and negative voltage inLC-MS/MS for comparing the drug stability with standard drug solution.

Table 3 List of Common Phase I and Phase II Metabolites:Biotransformation Mass Change (Dalton)

Phase I MetabolitesHydroxylation + 15.9949Epoxidation + 15.9949N/S Oxidation + 15.9949Dihydroxylation + 31.9898Demethylation - 14.0157Oxidative displacement of chlorine - 17.9662Oxidative displacement of fluorine - 1.9957Dehydrogenation - 2.0157Desethylation - 28.0312Nitro reduction - 29.9742Hydrogenation + 2.0157Dehydration -18.0106Decarboxylation - 43.9898Reductive displacement of chlorine - 33.9611Reductive displacement of fluorine - 17.9906Loss of nitro group - 44.9851Alcohol to carboxylic acid + 13.9792Ketone formation + 13.9792Hydration + 18.0106Methyl to carboxylic acid + 29.9741Phase II MetabolitesGlucuronide conjucation + 176.0321Sulfate conjugation + 79.9568Methylation + 14.0157Acetylation + 42.0106Glycine conjugation + 57.0215Taurine conjugation + 107.0041Cysteine conjugation + 119.0041Glutathione conjugation + 305.0682N- Acetylcysteine conjugation + 161.0147



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The solubility screening is done through UV-Visible detector in transmitmode, this experiment is helpful in bioanalysis method development, con-ducting other in vitro assays and intravenous (IV) formulation for preclinicalstudies.

2.1.2 CYP inhibition:Recently, FDA published draft guidance on drug interaction studies [37]. Itcovers both in vitro and in vivo drug interaction studies with a much moredetailed description of study design and data interpretation compared to theearlier published guidances. In addition, transporter-mediated drug interac-tions are included in the regulatory guidance for the first time. It is now verycommon to screen the compounds for their inhibition or induction potential.CYP inhibition assays can be carried out by incubating the compound withgenetically engineered microsomes (cDNA-expressed) or using human livermicrosomes[38, 39]. The screening of compounds for their CYP inhibitionpotential has been made easy by the development of rapid, selective andsensitive LC-MS assays. A method for the simultaneous evaluation of theactivities of seven major human drug-metabolizing cytochrome P450s(CYP3A4, CYP2D6, CYP2C9, CYP1A2, CYP2C19, CYP2A6, andCYP2C8) was developed by Dierks et al [40]. This method uses an in vitrococktail of specific substrates (midazolam, bufuralol, diclofenac,ethoxyresorufin, S-mephenytoin, coumarin, and paclitaxel) and fast gradientliquid chromatography tandem mass spectrometry. This cocktail methodoffered an efficient, robust way to determine the cytochrome P450 inhibitionprofile of large numbers of compounds. The enhanced throughput of thismethod greatly facilitates its use to assess CYP inhibition as a drug candidateselection criterion. Walsky and Obach developed and validated high-pressureliquid chromatography-tandem mass spectrometry analytical methods, us-ing stable isotope-labeled internal standards. This analytical approach, throughits high sensitivity and selectivity, permitted the use of very low incubationconcentrations of microsomal protein (0.01–0.2 mg/ml). Analytical assayaccuracy and precision values were excellent in this method. Enzyme kineticand inhibition parameters obtained using these methods demonstrated highprecision and were within the range of values previously reported in thescientific literature. This method would be useful for routine assessments ofthe potential for new drug candidates to elicit pharmacokinetic drug interac-tions via inhibition of cytochrome P450 activities [41].

2.1.3 Permeability:Oral route is the most desirable route of drug administration due to manyadvantages such as ease of administration and better patient compliance.Therefore it is important to develop drugs that can be absorbed effectivelythrough the intestinal epithelium. Intestinal absorption is a formidable bar-rier that restricts the bioavailability of many potential new drugs. To studyintestinal absorption, number of systems such as immobilized artificial mem-brane chromatography, PAMPA, in situ intestinal segments, everted sacs,isolated membrane vesicles etc have been used [42]. However, Caco-2 cellmonolayer assay has emerged as one of the standard in vitro tools to studyintestinal absorption [43]. With the advent of LC-MS/MS, the need for radio-labelled compounds in Caco-2 assays has been eliminated. Also, mass spec-trometry permits the simultaneous measurement of multiple compounds,thus reducing the number of incubations and increasing the throughput of theexperiment. It has been reported that Caco- 2 cells poorly express CYP3A4,but other enzymes such as hydrolases, carboxylesterases, uridine diphosphoglucuronosyltransferases, glutathione S-transferases, sulfotransferases andcatechol-O-methyl transferases are present and functional in Caco-2 cells.With the aid of LC-MS and LC-MS/MS, the metabolism of compounds byCaco-2 cells can be also be investigated [42]. Colleen et al have reported a fullyautomated nanoelectrospray tandem mass spectrometric method for analy-sis of Caco-2 samples. The method reported by this group had the advan-tages of reduced method development time, no carry-over effects, lower

sample consumption and ease of use as compared with conventional pulled-capillary nanoelectrospray. The permeability and recovery data results fromthis method and conventional methods were comparable [43, 44].

2.1.4. Protein binding:At present pharmaceutical scientist are using HPLC and LC-MS/MS forprotein binding assay through Human serum albumin column. In proteinbinding assay, the drug is spiked in blood, plasma or tissue homogenate isincubated at 37°C (simulated body conditions), then the drug is extractedfrom the biological matrix, then analyzed and compared with standard drugsolution. Other methods of protein binding include equilibrium dialysis,ultracentrifugation and ultrafiltration [45]. Some drugs binds with erythro-cytes, so the percentage binding can be checked through similar experimentsas conducted for plasma/serum. In such studies, compound is spiked in blankblood and then incubated at 37°C after which the sample is centrifuged forplasma and RBC separation, the separated RBC is collected for extractionand then analyzed for drug, which is compared to standard drug solutionresult.

3. LC-MS SUPPORT TO PRECLINICAL DEVELOPMENT STUDIES:

3.1 In vivo studies:Pre-clinical in vivo PK studies test the potential of drug candidates in ani-mals. The drug absorption and elimination can be evaluated with bioanalyticalassay via determination of PK profile in plasma, blood, serum, bile, urine,feces or any other biological matrix. Toxicokinetics and tissue distributionstudies investigate the distribution of drug in different tissues e.g. blood,kidney, lung, spleen, skeletal muscles, heart, adipose tissue, liver, brain,intestine, stomach, testes, ovary. The focus right from the initial drug dis-covery should be on the optimization of biomarkers using the pre dose studysamples and identifying the novel biomarkers, existing disease based & mu-tation based biomarkers. Pharmacodynamics related biomarker can be identi-fied through post dose samples. Then the relationship between disease pro-gression and biomarkers can be developed. Different types of in vivo PKstudies conducted during drug discovery and development where LC-MSplays a pivotal role are listed in Table 4. Figure 3 depicts different types ofADME studies performed at different stages of drug discovery. Some of thekey in vivo studies are described in detail below.

3.1.1. Single dose bioavailability study:Generally during initial discovery, if the compound has shown good in vitropotency and selectivity, it is advanced into the in vivo PK screening. Typi-cally the oral dose selected is 10 mg/kg and the intravenous dose is 1 mg/kg.This study is done in rodents (mice and rat). The formulation prepared inappropriate pharmaceutical vehicle e.g. 0.25% carboxyl methyl cellulose fororal; solution (simple deionized water if water soluble or use of cosolventssuch as PEG, solutol etc) formulation for intravenous dosing. It is veryimportant to focus on the preclinical formulation right from the discoveryphase so as to avoid any issues later when the toxicology studies are beingplanned. The formulation should be such so as to provide sufficient exposurein order to achieve good safety margins during the toxicology studies. Fol-lowing drug administration, blood samples are withdrawn at pre-dose and atmultiple time points post-dose through eye orbit vein or tail vein & collectedin EDTA coated vial. The obtained blood is centrifuged at 4000 rpm for 10minutes at 2-8°C and the separated plasma is stored at -70 °C until analysisby LC-MS/MS [46]. Typically the PK studies are performed in mice and rats(rodent toxicology species) and dogs (non rodent toxicology species), butmonkey PK studies are also conducted when it is the non rodent toxicologyspecies.

During initial discovery, when the number of compounds is more, it is not



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possible to conduct in vivo PK study for each of the compound. Hence oneapproach used for rank ordering the compounds is the cassette dosing. Cas-sette dosing involves simultaneous administration of several compounds to asingle animal followed by rapid sample analysis by LC-MS/MS. Although,this approach is advantageous in terms of resources and throughput, it hasalso has risks such as the potential of compound interactions. Cassettedosing can be analytically challenging. A highly selective method is needed todetect multiple compounds in a single sample simultaneously. Sensitivity isalso important because very low doses of compounds are administered tominimize the risk of drug interactions and toxicity. Sources of analyticalinterference should also be considered, including collision cell cross talk

between structurally related compoundswith similar fragmentation patterns, in-source fragmentation of metabolites. Ionsuppression due to competition between theanalyte and co-eluting analytes, residualmatrix components, or mobile phase com-ponents is another challenge [47].

One approach is to minimize this risk is tochoose very low doses of the compoundsand always use a standard compound ascontrol in the cassette dosing PK studies[47]. Due to the advances made in the arenaof LC-MS, several other approaches to re-duce the number of samples from in vivoPK studies have been tried. These include“sample-pooling”, which involves combin-ing equal aliquots of samples obtained at aparticular time point from several animalseach dosed with individually with a differ-ent compound. An alternative approach isto pool the samples collected at each con-secutive time point from a single animaldosed with an individual compound to pro-duce a single sample for analysis [48].Korfmacher et al developed the “cassette-accelerated rapid rat screen (CARRS)” ap-proach in which each compound is adminis-tered individually to two rats. Duplicatesamples collected at each time point are thenpooled and run alongside limited standardcurve [49].

3.1.2. Excretion mass balance study:Mass balance/excretion study provides fun-damental information about the rates androutes of elimination of new drug candidates.Mass balance studies are an important com-ponent of Investigational New Drug (IND)application submitted to regulatory agen-cies for seeking approval for first in human(FIH) studies. During initial drug discoverywhen the radiolabeled compound is not avail-able, these studies can be done using coldcompound. The cold compound is adminis-tered orally and intravenously to rodent andnon rodent species. Urine and feces samplesare collected at regular intervals (e.g. 0-24 h,24-48 h, 48-72 h, 72-96 h, 96-120 h and

Table 4: Different Types of In vivo ADME Studies:

120-144 h) depending on the half-life of the drug. The samples were stored atapproximately –70°C until analysis by a fit for purpose LC-MS/MS method.The amount of unchanged drug excreted in urine and feces samples are calcu-lated at each time interval. Cumulative amount and percentage excretion ofunchanged drug is calculated with respect to the amount of compound ad-ministered to each animal. Exploratory metabolite identification can also becarried out in these matrices. Once the compound has been declared as a lead,excretion/mass balance studies should be done using a radiolabeled com-pound, preferentially C14 labeled to give a confirmatory answer. Mass bal-ance excretion studies in laboratory animals and humans using radiolabeledcompounds represent a standard part of the development process for new



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drugs. From these studies, the total fate of drug-related material is obtained:mass balance, routes of excretion, and, with additional analyses, metabolicpathways. However, rarely does the mass balance in radiolabeled excretionstudies truly achieve 100% recovery [50]. The systemic pharmacokinetics oftotal radioactivity is measured, and, finally, the excreta and blood samplesgathered in such studies are used for identification and quantitative profilingof metabolites and parent drug. Complete mass balance, in theory, wouldrequire that 100% of the radioactivity administered is accounted for in theexcreta and, for animal species, in the cage wash. However, it is rarely thecase that exactly 100% recovery is obtained [50]. There are several studydesign elements and specifics on study execution that need to be adhered toin order to give the best chance possible to achieve a high recovery. Theseinclude selection of an appropriate position of the radionuclide in the mol-ecule, quantitative delivery of the dose, meticulous sample collection prac-tices, and sound radiometric measurement techniques. The concentration ofthe parent drug and its metabolites can be determined by analyzing theexcreta or target organs from the mass balance study using LC/flow scintilla-tion detection. Furthermore, the LC eluent can be split in parallel for metabo-lite profiling and structure identification [51]. Number of studies have beenreported in literature where excretion mass balance studies coupled with LC-MS/MS have provided useful information about the metabolism and dispo-sition of drugs such as gemopatrilat, dasatinib and PF-04971729 (Pfizercompound) in animals and humans [52-54].

3.1.3. Single Dose Toxicokinetics & Tissue Distribution study:These studies are done typically in rodents which are the toxicology studies.

In these studies, blood samples and different tissues are withdrawn at mul-tiple time points. Blood is collected in EDTA coated vials & centrifuged at4000 rpm for 5 minutes at 2-8 °C. The separated plasma is stored at -70 °Cuntil bioanalysis. The tissues are collected after euthanizing the animal,weighed, homogenized in phosphate buffer and stored -70°C until analysis.After this procedure can bioanalyze the sample. Generally during lead opti-mization, tissue distribution studies in animals are done using radiolabeledcompounds (C14 or H3). During the synthesis of radiolabel, it should beensured that the molecule is labeled in such a way that there is no loss of labeldue to exchange, fragmentation or via expiration. These studies are generallydone in pigmented rats such as Long-Evans and in one non rodent species.These studies involve single dose administration through intended clinicalroute, collecting different tissues at predetermined time points and measur-ing radioactivity using liquid scintillation counter (LSC). Alternatively, quan-titative whole body autoradiography (QWBA) may also be used, where thinsections of the whole animals are prepared and exposed to phosphor imagingscreen which is then scanned with a phosphor imagers [55]. These studies aredone to mimic the toxicity studies. Based on the data obtainedin animals,radioactivity exposure observed in rats is extrapolated to human tissues.Apart from this, mass balance studies, excretion of drug in milk and embryofetal studies are also conducted to support marketing approval of investiga-tional product [56]. The monitoring of metabolites in humans following ad-ministration of labeled compounds, help identify species differences in me-tabolism [57].

3.1.4. Specialized in vivo studies:To fully understand the reasons for poor PK of drug, specialized in vivostudies can be performed. Bile duct cannulated animals provide a usefulmodel to understand reasons of incomplete bioavailability. Determination ofparent and metabolites in bile help understand role of conjugative pathwaysin the metabolic clearance of drugs. For compounds suspected to have poordissolution or instability in GIT, the GIT can be removed at the end of theexperiment and analyzed for drug and metabolite. Other in vivo model whichhelps in sorting out problem of low bioavailability is portal vein cannulatedanimals. One can measure both systemic and portal levels following oraladministration and can determine the hepatic extraction [58].

4. LC-MS SUPPORT TO CLINICAL STUDIES:Application of mass spectroscopy (MS) to clinical analysis has evolvedconsiderably since it was used, both in research facilities and for routineanalysis in clinical biochemistry laboratories [59]. Much of the uptake of MSin the field of clinical research has been driven by the technological advanceswithin chromatography and MS. Brought together, the advances of LC, APIand MS/MS have been developed and a number of assays for clinical diagno-sis are now available. The analysis of steroid hormones by MS is verycommonly used. Other commonly encountered uses include newborn screen-ing for congenital metabolic diseases such as aminoacidopathies and fattyacid oxidation disorders, drugs of abuse and analysis of endogenous peptides[60]. The analysis of different steroids is particularly useful for the diagnosisand treatment of complex endocrine disorders such as primary hyperaldos-teronism, adrenal insufficiency, congenital adrenal hyperplasia, Cushing’ssyndrome and gonadal dysfunction. Multi-steroid analysis has been madepossible only by the use of multi-dimensional LC-MS/MS. Thirteen differ-ent steroids had been identified in protein precipitated serum using ESI-LC/MS/MS with a linear ion trap [61]. With the advent of MALDI ionization,blood spots can be analyzed by MS. The example where this technique isused clinically is during the newborn screening for phenyl ketonuria, which isa congenital disorder in which phenylalanine hydroxylase is dysfunctional[62]. One area of mass spectrometry which is still under utilized is highresolution MS (HRMS). The accurate mass determination provided by HRMSallows monitoring over defined mass range. This data permit calculation of

chemistry

Biology HTS

In vitro metabolic stability screen (microsomes,s9,

hepatocytes)

Caco-2/ MDCK-MDRI absorption screen

P450 enzyme inhibition screen and time dependent

Rapid rat (CARRS)PD/PK screen

Rat/Dog monkey IV/PO initial PK studies

Discovery metabolite ID studies

Special PKstudies and other pre-development tests

Development(includes safety and clinical)

Level 1

Level 2

Level 3

Level 4

Figure 3 : Types of ADME studies conducted at different stages of drugdiscovery and development



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accurate mass shifts of the metabolites, and also determination of their mo-lecular formulae. This can help in prediction of nature of metabolites. HRMSdata can also help in distinguishing isobaric molecular ions. It can also helpassign exact structures to fragments where two or more structures of thesame nominal mass are possible.

4.1 Role of LC-MS in biomarker estimation:Biomarkers are measurable characteristics of physiological or pathologicalstates. They aim to serve a variety of functions during drug development.Afinitor (generic name everolimus or RAD001) is an orally active mamma-lian target of rapamycin (mTOR) inhibitor anticancer agent recently ap-proved by FDA and EMEA (63- NDA 22334 Afinitor). This is an examplewhere use of biomarker and PK-PD modeling has been successfully em-ployed in predicting clinically useful treatment regimens for everolimus afterthe principle of determining optimal biologic regimens. From this example, itis highlighted that biomarkers aim to serve a variety of functions during drugdevelopment including determination of proof of principle, refinement ofpatient selection, assessment of respondents early in treatment and facilita-tion of optimal biologic dose determination [64-66]. Till date, biomarkers re-flecting PI3K (phosphatidylinositol 3-kinase) and mTOR pathway activityor inhibition have been investigated in both preclinical and clinical settings.Although they do not yet have a defined role in the clinic, emerging data areshowing promise. In a phase I study of mTOR inhibitor, RAD001(Everolimus), a strong correlation was noted between the degree of mTORinhibition in skin and tumor (through evaluation of pS6 and peIF-4G levels),indicating skin might serve as a valuable surrogate tissue than using tumorbiopsies from cancer patients [67]. Exelexis has presented some early datafrom their XL765 and XL147 (anti cancer PI3K inhibitors in clinical devel-opment) incorporating plasma glucose and insulin as biomarkers [68]. Singlebiomarkers for stratification of the population i.e. preselection of the pa-tients most likely to respond to chemotherapy are ideal but may not suffice,and hence it is a usual practice to use a combination of biomarkers. This canlead to increase in sensitivity and specificity of the prediction of therapeuticoutcome. Univariate correlation analysies demonstrated significant correla-tions between sensitivity to everolimus with the biomarkers such as pS6,pAKT, p4E-BP1 and Ki-67 [64]. In order to derive meaningful informationfrom the biomarker data, it is important that we have reliable methods fordetermination of biomarker levels. This has been made possible with thetechnological advancements made in the field of mass spectrometry. Tong etal developed a simpler method of preprocessing matrix-assisted laser des-orption ionization time-of-flight (MALDI-TOF) MS data for differentialbiomarker analysis in stem cell and melanoma cancer studies. They proposedan alternative yet simple solution to preprocess raw MALDI-TOF-MS datafor identification of candidate marker ions. Their model identified 10 candi-date marker ions for both data sets. These ion panels achieved over 90%classification accuracy on blind validation data. The algorithms were robustfor handling noisy data and cost effective for candidate marker selection [69].Alexandrov et al [70] proposed a new procedure of biomarker discovery inserum protein profiles based on: a) discrete wavelet transformation of thespectra; b) selection of discriminative wavelet coefficients by a statisticaltest and c) building and evaluating a support vector machine classifier bydouble cross-validation with attention to the generalizability of the results.In addition to the evaluation results (total recognition rate, sensitivity andspecificity), the procedure provides the biomarker patterns, i.e. the parts ofspectra which discriminate cancer and control individuals. The evaluationwas performed on MALDI-TOF serum protein profiles of 66 colorectalcancer patients and 50 controls [70]. Benkali et al [71] used dual decouplingbetween nano-LC, MS and MS/MS to reduce the overall analysis time,evaluate the quantitative performance and reproducibility of nano-LC-MALDIanalysis in biomarker discovery and evaluate the robustness of biomarkersselection. Profiling of proteins using Two-dimensional Liquid Chromatogra-

phy Mass Spectrometry (2-DLC) can also be performed using ion exchangechromatography or chromato focusing to separate proteins based on theircharge and Reversed-Phase Liquid Chromatography-Electrospray Ioniza-tion Mass spectrometer to separate proteins based on their hydrophobici-ties [72].

MALDI-TOF-MS is used in clinical chemistry for disease marker identifica-tion in combination with 1-D and 2-D gel electrophoresis separations usingeither peptide mass fingerprinting (PMF) or peptide sequence tag (PST)followed by data base searching. In microbiology, MALDI-TOF-MS can beutilized to analyze specific peptides or proteins directly desorbed fromintact viruses, bacteria and spores. The capability to register biomarker ionsin a broad m/z range, which are unique and representative for individualmicroorganisms, forms the basis of taxonomic identification of bacteria byMALDI-TOF-MS. Moreover, this technique can be applied to study eitherthe resistance of bacteria to antibiotics or the antimicrobial compounds se-creted by other bacterial species. More recently, the method was also suc-cessfully applied to DNA sequencing (genotyping) as well as screening formutations. High-throughput genotyping of single-nucleotide polymorphisms(SNP’s) has the potential to become a routine method for both laboratoryand clinical applications [73]

The pharmacologists, toxicologist, PK scientists and clinicians need to con-tinue a translational collaboration as we monitor for responses and toxiceffects, to ensure we seek explanation of what transpires, success or failure,expected or unexpected. Biomarker development needs to continue in orderto assist patient selection, monitor for responses and further understanding.Molecular characterization of patients should remain a goal. Trials of combi-nation therapy with chemotherapy, radiation, hormones and other biologicalagents are following closely behind. The product of several decades of workinvestigating inhibition of the critical PI3K cellular signalling pathway isfinally coming to fruition, with much reason for optimism. The same ap-proach can also be applied to other therapeutic areas for successful develop-ment [74, 75].

4.2 Role of LC-MS in clinical PK-PD studies:The development of PD biomarkers in oncology has implications for designof clinical protocols from preclinical data and for predicting clinical outcomesfrom early clinical data. Two classes of biomarker have received particularattention. Phosphoproteins in biopsy samples are markers of inhibition ofsignaling pathways, target sites for many novel agents. Biomarkers ofapoptosis in plasma can measure tumor cell killing by drugs in phase I clinicaltrials. Several examples have been reported in clinic where PK/PD modelinghas been utilized for optimization of dosage regimen. Few specific examplesare mentioned here.

Clinical PK/PD modeling was used to predict optimal clinical regimens ofeverolimus, a novel mTOR inhibitor, to carry forward to expanded phase Iwith tumor biopsy studies in cancer patients. Inhibition of S6 kinase 1(S6K1), a molecular marker of mTOR signaling, was selected for PD analysisin peripheral blood mononuclear cells (PBMCs) in a phase I clinical trial. PKand PD were measured up to 11 days after the fourth weekly dose. A PK/PDmodel was used to describe the relationship between everolimus concentra-tions and S6K1 inhibition in PBMCs of cancer patients and in PBMCs andtumors of everolimus-treated CA20948 pancreatic tumor–bearing rats. Thiswas made possible only because of the development of a rapid and sensitiveLC-MS method for quantitation of everolimus in blood [76]. Single dose PKand PD of RWJ 67657, a specific p38 mitogen-activated protein kinaseinhibitor was studied in a first-in-human study. This was a placebo con-trolled, double-blind, randomized trial in healthy male subjects. To evaluatethe PD of RWJ 67657, inhibition of cytokine production was monitoredfrom ex vivo–stimulated PBMCs. PK/PD modeling was used to characterize



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the inhibitory activity of RWJ 67657. RWJ 67657 inhibited TNF-a, IL-8,and IL-6 in a concentration-dependent manner with mean IC50 values of0.18 µM, 0.04 µM, and 0.43 µM, respectively. At 20 mg/kg, the medianinhibition was greater than 85%. There were no significant adverse effectsassociated with single doses of this drug. This study demonstrates that RWJ67657 has acceptable safety and pharmacokinetics to warrant further inves-tigation in a repeat-dose setting. Plasma samples were analyzed for RWJ67657 concentrations by a validated LC-MS/MS assay. The lower limit ofquantitation was 0.1 ng/mL. The precision and accuracy were 1.5% and2.4%, respectively [77]

Children differ from adults in their response to drugs. These differences maybe caused by changes in the PK and/or PD between children and adults andmay also vary among children of different ages. While a child grows, enzymepathways (involved in the PK),

function and expression of receptors and proteins (involved in the PD)mature, which can be referred to as “developmental changes” in childhood.Due to the advances made in the area of mass spectrometry, very smallvolume of plasma/blood can be quantified to define the PK of NCE’s whichultimately helps in understanding the PK in pediatrics. This ultimately helpsin the rational dose selection in pediatrics. Once a population PK model isdeveloped, internal and external validations should be performed. If the modelperforms well in these validation procedures, model simulations can be usedto define a dosing regimen, which in turn needs to be tested and challenged ina prospective clinical trial. This methodology will improve the efficacy/safety balance of dosing guidelines, which will be of benefit to the individualchild [78].

Despite decades of widespread use of sulfadoxine-pyrimethamine asantimalarials, there are few data to inform dose recommendations in children.The objective of the study conducted by Barnes and co workers was tocharacterize the PK properties of sulfadoxine-pyrimethamine in African adultsand children with acute falciparum malaria. They concluded that PK factorsmay contribute to the increased risk of sulfadoxine-pyrimethamine antima-larial treatment failure in young children. The current dose recommendationsneed revision. They proposed that children aged 2 to 5 years should betreated with 1 g sulfadoxine/50 mg pyrimethamine to achieve drug concentra-tions equivalent to those in adults. This recommendation was made possibleonly because of development of a sensitive and rapid LC-MS bioanalyticalmethod for quantification of blood concentrations of sulfadoxine-py-rimethamine [79].

A rapid, specific and robust assay based on protein precipitation and liquidchromatography-electrospray ionization tandem mass spectrometry (LC–ESI–MS–MS) was developed and validated for the quantitative analysis oflumefantrine, an antimalarial drug, in human plasma using piperazine bischloroquinoline as internal standard (ISTD) by Munjal and co workers. Theprecursor to product ion transitions of m/z 528.2 → 510.3 and m/z 409.2→ 205.1 were used to measure the analyte and the ISTD, respectively.Analysis was performed on C8 column (50 mm 9 4.6 mm, 5 lm) with 10 mMammonium acetate/acetonitrile/0.05% acetic acid (10/85/5, v/v) as mobilephase at a flow rate of 0.6 mL min-1. The assay exhibited a linear dynamicrange of 0.21–25.05 µg mL-1. The RSD% of intra-day and interday assaywas =15%. The application of this assay was demonstrated in abioequivalence study and will be ideal for PK studies in pediatric population[80].

With the emergence of robust and sophisticated modeling and simulationtools, biomarkers, and bioanalytical methods with quick turnaround times,adaptive designs are becoming instrumental in the acceleration of early clini-cal development. One example to highlight this approach is mentioned here.PK and exposure-response modeling of a selective sphingosine 1-phosphate

receptor-1 modulator (CS-0777) was conducted in an iterative process toguide early clinical development decisions by Rohatagi and co workers [81].Blood samples were analyzed for CS-0777 and M-1 (active metabolite)using a validated method involving LC-MS/MS. Analytes and internal stan-dards were extracted from 200 µL aliquots using a protein precipitationextraction procedure. Samples were extracted with acetonitrile, vortexed, andcentrifuged. The supernatant was transferred to a clean 96-well plate, dried,and reconstituted with 0.1% formic acid in water and methanol (70:30 v:v).Analytes were detected and quantified by tandem mass spectrometry inpositive ion mode on a Sciex API 4000 (MDS Analytical Technologies,Concord, Ontario, Canada) equipped with a turbo ion spray interface. ForCS-0777, the standard curve was linear over a concentration range of 0.05 to50 ng/mL, and for M-1, the standard curve was linear over a concentrationrange of 0.2 to 200 ng/mL. Based on the intra run and inter run precision(<10%) and accuracy (<10%), the method was deemed appropriate [81].

Tigecycline is a first in class glycylcycline antibiotic and possess an extendedspectrum of antimicrobial activity. It was approved by FDA and EMEA fortreatment of complicated skin structure infections and complicated intra-abdominal infections in adults. In march 2009, it was also approved forcommunity acquired pneumonia. It has the ability to overcome many knownmechanisms of resistance such as active efflux and ribosome protection mecha-nisms [82]. A highly sensitive human bone LC-MS assay for the quantitationof tigecycline in human serum, bones, skin and lungs [83] lead to the completeunderstanding of its PK. The PK data in combination with the PD data leadto its prescribing in an off-label setting and particularly in cases of nosocominalpneumonia. The utilization of mass spectrometry in development of highlysensitive, rapid and reproducible bioanalytical methods and their applicationto different types of clinical PK studies has been highlighted by number ofpublications [84-87]

5. ROLE OF LC-MS IN IMPURITY IDENTIFICATION:At many levels of the drug discovery, the identification and quantification ofimpurities in Active Pharmaceutical Ingredients (API) is a very demandingactivity. Regulatory authorities like ICH, USFDA, Canadian Drug and HealthAgency are emphasizing on the purity requirements and the identification ofimpurities in API’s. Qualification of the impurities is essential for evaluatingbiological safety of an individual impurity; thus, enlightening the need andscope of impurity identification in pharmaceutical research [88-89]. Syntheticimpurities are of particular concern during process research and safety evalu-ation activities. Impurities in active pharmaceutical product may be a start-ing material, by-product, breakdown product or polymorph. They can ap-pear at the synthesis level as well as during or after the formulation processand storage. According to ICH guidelines on impurities, in new drug prod-ucts, identification of impurities below 0.1% level, is not considered to becompulsory, unless impurities are expected to be potent or toxic. Accordingto ICH, the maximum daily dose qualification threshold is 0.1 % for 1- 2 g/day or 0.05% for >2 g per day intake. As the number of novel lead candidatesthat enter into preclinical development increases, significant resources areneeded to identify impurities. LC/MS-based approaches provide sensitivetool for structural analysis procedures for throughput analysis of impurities.Hybrid triple stage quadruple mass analyzers are proved to be best for thispurpose. Although LC/UV is still the most commonly used technique for thedetection and quantitative determination of degradants, however, when un-known degradants are discovered in stability or during stress studies, , LC/MS is often used to determine the molecular mass and MS/MS is used toprovide structural characterization [90, 91].

6. CONCLUSIONS AND FUTURE CHALLENGES:LC-MS analysis employed for the qualitative and quantitative determinationof drugs and their metabolites in biological fluids, plays a significant role in



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the evaluation and interpretation of bioequivalence, PK and toxicokineticstudies. The quality of these studies, which are often used to support regu-latory filings, is directly related to the quality of the underlying bioanalyticaldata. It is therefore important that guiding principles for the validation ofthese analytical methods be established and should be followed uniformlyacross the academia and pharmaceutical community.

Mass spectrometry can play a pivotal role in drug discovery and develop-ment in future. At present Bioanalysis is playing major role in microdoseclinical study, exploratory study, regulatory submission study, pre-clinicalstudy and high throughput screening of bioactive constituents. Bioanalysisis supporting different sections of pharmaceutical industry like metabolomics,proteomics, clinical study, preclinical study. The bioanalyst has at its dis-posal technologically advanced mass spectrometers (LC-MS/MS, MALDI-TOF, Surface Enhance Laser Desorption Ion Technique-TOF/TOF & LC-NMR/MS etc.), strong chromatographic separation system, high sensitivedetection system, strong validated xenobiotics based software, high puritysolvents and strong chromatographic supportive micro bore columns.

Future challenges:The science of mass spectrometry is more than a century old and has madeimmense advancement recently but still there is a room for improvement.Some of the challenging areas ahead where we could see further improve-ments in future are discussed below.

• New developments in metabolomics and proteomics• New and different approaches for high throughput screening, lead

optimization, lead screen, lead hit and lead validation of new chemi-cal entities

• Advanced and latest chromatographic separation theory• Regulatory compliance based bioanalytical data processing as per

21CFR part11• Bioanalytical method development, pre-validation and full valida-

tion report template• Advanced tandem technology based bioanalysis• Laboratory information management system (LIMS) through

bioanalytical data compilation and maintenance• Electronic based bioanalytical data system for submission to inter-

national regulatory agencies i.e electronic submissions• Application of LC-MS in diagnostics for future diseases in human

Modern technology is being used in drug discovery and development anddirectly and indirectly supporting bioanalysis in fields such as ADME pro-filing through computed tomography, positron emission tomography, mag-netic resonance image (MRI) and NMR. At present pharmaceutical compa-nies are using radiolabelled (e.g.C14 or H3) bioactive constituents for drug andmetabolite profiling in animals and humans through LC-MS/MS, LC-NMR/MS & functional positron emission tomography, MRI. At present somediagnostic companies are using mass spectrometry for optimization for newdisease maker, discovery of novel biomarker, mutation based induced biomarkerand other PD based biomarkers. Biochemical markers are gathering interestas early indicators of drug efficacy. Mass spectrometry techniques will con-tinue to be applied to the quantitation of those discrete small-molecule chemicalentities that can indicate disease or treatment states. The science of develop-ing suitable surrogates is developing rapidly and mass spectrometry will beincreasingly challenged by the imposing sensitivity requirements needed tomake these advances possible. Bioanalysis now covers not only drugs devel-oped for medicinal use, but also drugs used for illicit purposes, forensicinvestigations and environemental concerns. The technologies associated withthe measurement of small and large molecules (proteins and peptides) con-tinue ot expand and refined further. LC-MS is a staple of small molecule druganalysis. However, its development for the measurement of proteins and

peptides has seen exponential growth in recent times. Miniaturization andhigh throughput is also being applied to bioanalytical platforms and samplepreparation. Clearly the field is striving to accomplish more, faster with lessinput costs. It is beyond doubt that many new discoveries are yet to unsurfacein this ever growing and demanding area.

Our ability to study the physical and biological world has become closelylinked to the capabilities of our instrumentation. As instrumentation im-proves, so do our abilities to make observations. The evolution of massspectrometry instrumentation and drug discovery have become intertwined,so that many of the advances in the instrumentation have arisen because of ademand for throughput, sensitivity, or a need to perform chemical analysiswith respect to a given pharmacological problem. This symbiosis appearsdestined to continue and should provide many fruitful benefits for both massspectrometry instrumentation and drug discovery. Althogh many evolution-ary changes have been forecasted, it is possible, perhaps even likely, that themost important innovations have yet to be recognized or devised.

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