13 THE ROLE OF LIQUID CHROMA TOGRAPHY–MASS SPECTROMETRY IN PHARMACOKINETICS AND DRUG METABOLISM Ray Bakhtiar, Tapan K. Majumdar, and Francis L. S. Tse 13.1 INTRODUCTION Recent advances in mass spectrometry have rendered it an attractive and ver- satile tool in industrial and academic research laboratories. As a part of this rapid growth, a considerable body of literature has been devoted to the appli- cation of mass spectrometry in clinical studies. In concert with separation tech- niques such as liquid chromatography, mass spectrometry allows the rapid characterization and quantitative determination of a large array of molecules in complex mixtures. Herein, we present an overview of the above techniques accompanied with several examples of the use of liquid chromatography– tandem mass spectrometry in pharmacokinetics/drug metabolism assessment during drug development. Since the evolution of pharmaceutical research [1, 2], the stages of drug dis- covery and development have followed three predominant patterns: (i) the systematic and methodical approach by chemists to rationally design and syn- thesize a molecule to target a specific molecular system (e.g., ion channels, receptors, enzymes, DNA); (ii) the isolation and purification of the active ingredients of medicinal plants or microorganisms to screen their spectrum of 605 HPLC for Pharmaceutical Scientists, Edited by Yuri Kazakevich and Rosario LoBrutto Copyright © 2007 by John Wiley & Sons, Inc. activity using in vitro models; or (iii) the serendipitous discovery of a com- pound with a novel pharmacological action (e.g., the accidental discovery of antidepressants). Today, one of the increasingly popular and complementary approaches for drug discovery in the pharmaceutical industry is to perform massive parallel synthesis in solution or on a solid support. In addition, with the advent of functional genomics and proteomics, cell-based assays, and mol- ecular biology, a multitude of therapeutic targets have been validated [3]. With an increasing number of potential molecular targets identified through the science of functional proteomics and genomics, diverse libraries of new chemical entities (NCEs) have to be generated and evaluated. Consequently, the rapid growth of combinatorial libraries has posed a need for faster, accurate, and sensitive analytical techniques capable of large-scale high- throughput screening (HTS). Although in vitro assays do not necessarily reflect the complexity of the in vivo interactions, the speed and simplicity of the former have rendered them an integral part of the screening process. In recent years, the in silico and experimental modeling of pharmacokinetic/ pharmacodynamic (PK/PD) relationship have become increasingly popular [4, 5].The integration of PK (i.e., drug dose and biological fluid concentration) and PD (i.e., pharmacologic effect) provides a key determinant in under- standing the dosing regimen and therapeutic effect of a potential drug com- pound. To this end, analytical assays also play a pivotal role in defining the PK/PD relation of NCEs. In many cases, both the drug concentration and PD biomarkers (vide infra) can be directly measured in peripheral fluids using specific analytical techniques. Furthermore, samples generated from large-scale clinical trials along with the ambitious development timelines to get safe and efficacious drugs to market warrant the use of HT bioanalysis. Numerous improvements in speed, sensitivity, and accuracy, augmented with innovations in automation in con- junction with mass spectrometry (MS) detection, have allowed for versatile and multifaceted platforms [6–8]. 13.2 IONIZATION PROCESSES Mass spectrometry (MS) is playing an increasingly visible role in the molecu- lar characterization of combinatorial libraries, natural products, drug meta- bolism and pharmacokinetics, toxicology and forensic investigations, and proteomics. Toward this end, electrospray ionization (ESI), atmospheric pres- sure chemical ionization (APCI), and atmospheric pressure photo-ionization (APPI) have proven valuable for both qualitative and quantitative screening of small molecules (e.g., pharmaceutical products) [9–14]. The utility of ESI (Figure 13-1) lies in its ability to generate ions directly from the solution phase into the gas phase. The ions are produced by appli- cation of a strong electric field to a very fine spray of the solution containing the analyte. The electric field creates highly charged droplets whose subse- 606 THE ROLE OF LC–MS IN PK AND DRUG METABOLISM quent vaporization (or desolvation) results in the production of gaseous ions. T he fact that ions are formed from solution has established the technique as a convenient mass detector for liquid chromatography (LC/MS) and for automated sample analysis. In addition, ESI-MS offers many tangible benefits over other mass spectrometric methods including the ability to qualitatively analyze low-molecular-weight compounds, inherent soft-ionization, excellent quantitation and reproducibility, high sensitivity, and its amenability to automation. Analogous to the ESI interface, APCI (Figure 13-2), also referred to as the heated nebulizer (HN), induces little or no fragmentation to the analyte. IONIZATION PROCESSES 607 Figure 13-1. A simplified schematic of the ESI process. (Courtesy of Dr. P. Tiller.) Figure 13-2. A simplified schematic of the APCI process. (Courtesy of Dr. P. Tiller.) Therefore, the APCI spectrum also tends to be simpler in interpretation than the traditional electron ionization (EI), which results in extensive fragmenta- tion of the precursor ion. As a result, APCI and ESI are referred to as “soft- ionizations,” while EI is considered a “hard-ionization” technique. Generally, volatile and thermally stable compounds can be subjected to LC/APCI/MS analysis. In quantitative analysis,APCI provides a greater (i.e., in terms of lin- earity) dynamic range than ESI and it is considered rugged, easy to operate, and relatively tolerant of higher buffer concentrations (i.e., fewer matrix effects). In ESI, at about 10 –5 M and higher, the ion signal becomes fixed and independent of sample concentration (plateauing effect) and may exhibit non- linearity at higher concentrations. In contrast, APCI can offer a wider linear dynamic range. For example, in our laboratory (data not shown) we have rou- tinely developed reversed-phase LC/APCI/MS/MS assays ranging from 1.0ng/mL to 10,000ng/mL with a correlation coefficient of >0.996. Further- more, APCI can accommodate flow rates of up to 2.0mL/min and is effective in the analysis of medium- and low-polarity compounds [12]. In qualitative drug metabolism studies, a combination of APCI and ESI experiments can prove valuable in distinguishing certain oxidative biotransformations (e.g., N-oxidation versus hydroxylation) [15, 16]. In contrast to ESI, APCI is not suited for the analysis of biopolymers, proteins, peptides, and thermally labile species. In the APCI process, electrons originating from a corona discharge needle ionize the analyte via a series of gas-phase ion-molecule reactions. For example, in the positive-ion mode, the energetic electrons start a sequence of reactions with the nebulizing gas (typically nitrogen), giving rise to nitrogen molecular ions. Using APCI, depending on the composition of the HPLC mobile phase, ions such as [H 2 O + H] + , [CH 3 OH + H] + , [NH 3 + H] + , and/or [CH 3 CN + H] + are formed via series of ion-molecule reactions with the nitro- gen molecular ions. Subsequently, additional ionization is initiated by exother- mic proton transfers from the protonated solvent ions to the neutral analyte molecules yielding [analyte + H] + , [analyte + CH 3 OH + H] + , [analyte + NH 3 + H] + ions, and so on. In general, metal adduct ions are observed less commonly in APCI as opposed to ESI, where they are more prevalent. Greater sensitiv- ity is attained if the solvent is polar and contains ions through the addition of an electrolyte.The desolvation process is then further enhanced by the heating element within the APCI assembly, which is maintained at 300–550°C. One of the drawbacks of APCI is its lack of compatibility with low eluent flow rates. The stability of the ionization response may be poor at low rates (i.e., less than 50µL/min). In contrast, ESI is compatible with miniaturized columns and amenable to sample-limited scenarios such as biochemical and biotechnological applica- tions. ESI can be considered a flow-sensitive technique. The dimension of the primary droplets is dependent on the flow rate. Therefore, by using columns with a smaller internal diameter (i.d.) and consequently lower flow rates, the concentration of the analytes in the spray solution can vary and it can be 608 THE ROLE OF LC–MS IN PK AND DRUG METABOLISM considered a concentration-dependent ionization process. It is concentration- dependent in the sense that the surface charge density of the droplets in the gas phase is higher due to more effective desolvation of the droplets since lower flow rates are used. The use of solvent-buffer post-column addition also allows optimization for improved analyte ion current response. Increasing the flow rate increases droplet size, which decreases the yield of gas-phase ions from the charged droplets. Recently, atmospheric pressure photo-ionization (APPI) [17–19] was intro- duced as a complementary ionization technique to ESI and APCI. APPI (Figure 13-3) is now commercially available by several MS vendors such as Agilent Technologies, Applied Biosystems (Sciex), Waters (Micromass), and Thermo Electron (Finnigan) Corporations. This technique can be used to ionize an analyte that otherwise is not easily ionizable using either APCI or ESI. In APPI, to increase ionization efficiency, a high-intensity UV radiation source is used (i.e., a 10-eV krypton discharge lamp) in a direct or an indirect mode. In the direct mode, often a molecular ion is generated by irradiation; while in the indirect mode, a dopant is used in conjunction with the analysis. A photoionizable dopant such as acetone or toluene is employed to mediate (as dopant photo-ions) the production of ions by proton or electron transfer. The dopant is introduced to the APPI ionization chamber by a separate pump at an optimized steady flow rate during analysis (e.g., 10–15% of the mobile phase flow rate, post-column). A number of excellent articles have recently been published on the applicability of APPI for the analysis of small mole- cules [17–19]. IONIZATION PROCESSES 609 UV Lamp Heater Curtain Plate Orifice Curtain Gas Primary Ionization Region PhotoSpray tm Source Block Dopant Quartz Tube Nebulizer Gas (Gas1) LC Effluent Figure 13-3. A simplified schematic of the APPI process. (Courtesy of Sciex/Applied Biosystems Corporation.) 13.3 TANDEM-MASS SPECTROMETRY (MS/MS) F or purposes of quantitative analysis, selected ion monitoring (SIM) and selected reaction monitoring (SRM) are two commonly utilized approaches. The latter is also referred to as multiple reaction monitoring (MRM). In both modes, considerable structural information is lost; nonetheless, these tech- niques are extremely powerful for target compound quantification in biolog- ical matrices, if the compound of interest is known. In the SIM mode, the MS is tuned to a particular m/z window (preferably at unit resolution), which corresponds to the ion of interest (i.e., [M + H] + ,or a stable adduct such as [M + X] + , where X = Na,K, NH 4 , etc.). SIM may require a more elaborate chromatographic separation in order to minimize interfer- ence from endogenous species. However, in the SRM approach, higher selec- tivity and sensitivity are realized. Thus, shorter chromatographic runs (faster injection cycles) and limited sample pretreatment could be tolerated without significant loss in sensitivity. In addition, due to lack of MS/MS capability,SIM has been more commonly performed on single quadrupole MS, while SRM has been broadly adapted on triple quadrupole (Figure 13-4) and ion-trap mass spectrometers.The increase in sensitivity and selectivity of SRM stem from the ion-chromatogram (i.e., LC-MS/MS) obtained by specific precursor-to-product ion transition for an analyte of interest (Figure 13-4). Conversely, in an SIM mode, the relative background noise due to the presence of other isobaric species (i.e., ions with a same m/z as the analyte of interest) can result in a lower signal-to-noise ratio for the analyte. Due to the widespread acceptance of SRM in quantitative analysis, the remaining part of this section focuses on a description of tandem- mass spectrometry (MS/MS), which is utilized in SRM (or MRM) experiments. Tandem-mass spectrometry or collision-induced dissociation (CID) is one of the most widely used techniques for probing the structure of ions in the gas phase [20].To this end, ease of application to various instrumental types, along with its experimental simplicity, account for the wide popularity of CID. In a typical CID experiment, a beam of ions with a specific m/z (denoted as the precursor or parent ion) is selected and collided with a neutral and nonreac- tive gas-phase target (e.g., argon, xenon, helium, nitrogen). These collisions result in subsequent fragmentation and product ions that are a direct conse- quence of dissociation of the precursor ion. Generally, the resulting fragmen- tation pattern is unique to a particular ion structure. The various CID techniques can be subdivided into categories based on the translational or collision energy of the precursor ion prior to collision with the target gas. The two main categories include low-energy CID, in the range of 1–300eV (i.e., used in triple quadrupole and ion-trap instruments), and high-energy CID at approximately 1–25KeV (i.e., used in guided-ion beam or sector instruments). Currently, one of the most common approaches is to perform MS/MS exper- iments on a triple quadrupole instrument.Tandem-MS experiments have been particularly popular for the qualitative and quantitative analysis of small mol- 610 THE ROLE OF LC–MS IN PK AND DRUG METABOLISM ecules such as pharmaceutical products in biological fluids [21–23]. In recent years the sensitivity and selectivity of MS/MS analysis of xenobiotics have been put to use in toxicokinetics , pharmacokinetics, metabolic, formulation, and early drug discovery studies. 13.4 SAMPLE PREPARATION USING AN OFF-LINE APPROACH One of the critical steps in qualitative and quantitative analysis is the sample preparation procedure. Sample preparation step can affect specificity, sensi- tivity,accuracy, precision, and throughput of a bioanalytical procedure. In addi- tion to development and optimization of the chemistry involved in sample processing, the use of semiautomated or fully automated protocols has been SAMPLE PREPARATION USING AN OFF-LINE APPROACH 611 Figure 13-4. Representative MRM scans (plasma extract of a proprietary compound) using an API 5000 triple quadrupole unit (Sciex). Each panel contains a distinct MRM transition for the same compound: m/z 1021.6 → 1003.5 (left panel) and m/z 1021.6 → 971.5 (right panel). Signal-to-noise ratio is designated as S/N. Experimental conditions: ESI, positive ion mode, protein precipitation was used for sample preparation, injec- tion volume was 10µL, the column was a C 18 , and dimension was 20 × 2.1 mm, using a linear gradient elution: 0min (20% B)–6min (90% B)–8min (90% B), where B was 0.2% formic acid in acetonitrile and A was 0.2% formic acid in water; separation was performed at room temperature. implemented in recent years [24, 25]. The popularity of off-line sample pro- cessing in batch-mode has dramatically improved the throughput of this rate- limiting step . Generally, there are three commonly used approaches for off-line sample processing: SPE (solid-phase extraction), LLE (liquid–liquid extraction), and protein precipitation (PPT).These three methods have been successfully used in conjunction with robotics for achieving an increase in sample preparation throughput. For example, Figure 13-5 is the photograph of a Beckman’s Biomek 2000 (other models such as Biomek 3000 and Biomek FX are also applicable) for semi-automated sample preparation that can accommodate SPE, LLE, and PPT procedures.This scheme has been established for use with SPE, LLE, or PPT in a 96-well plate format to analyze pharmaceutical prod- ucts in biological matrices (e.g., whole blood, plasma, serum, and cerebral spinal fluid (CSF)) in our laboratories (unpublished data). 13.4.1 SPE In the 96-well SPE format, similar to the traditional manual procedure, issues such as the nature of the bonded-phase (e.g., ion exchange, C 2 ,C 8 ,C 18 , cyano, phenyl, polymeric, strong or weak cation exchange, strong or weak anion exchange, mixed phases, etc.), solvent strength (for conditioning/washing of the phases, target analyte elution), and chemical characteristics (e.g., solubil- ity, presence of the key functional groups) of the analyte(s) need to be addressed.A general scheme for initial development of an SPE method is out- 612 THE ROLE OF LC–MS IN PK AND DRUG METABOLISM Figure 13-5. Photograph of a Biomek 2000 setup for semiautomated PPT, LLE, or SPE process in the authors’ laboratory (also see www.beckmancoulter.com and reference 99). lined below. Depending on the structure of the compound (hydrophobicity and ionizable functionalities), specific steps to optimize sample recovery are needed. • Condition sample for optimum retention • Condition SPE bed with methanol • Equilibrate SPE bed with water • Load sample onto SPE bed (a) Cation exchange: wash with 2% formic acid [low pH (3)] (b) Anion exchange: wash with 50mM NaOAc buffer [high pH (8–10)] • Wash bed with 5% methanol • Elute retained materials with an organic solvent (i.e., CH 3 OH, CH 3 CN, isopropyl alcohol, or a combination thereof) (a) Cation exchange: add 5% NH 4 OH to eluent (b) Anion exchange: add 2% formic acid to eluent Some of the most commonly utilized robotic modules for the 96-well SPE procedure are Tomtec Quadra (Tomtec, Hamden, CT, USA), Packard Multi- Probe (Packard Instruments, Meriden,CT,USA), Biomek (Beckman–Coulter, Fullerton, CA), and Tecan (Durham, NC, USA) units. For example, we have successfully and routinely adopted the Tomtec Quadra technology in the development and validation of several off-line SPE assays in whole blood, plasma, and urine followed by MS detection. The Packard Multi-Probe liquid handling workstation (Figure 13-6) has also shown promise for off-line SPE procedures involving plasma and serum [26–28]. In addition, this unit as well as the Tecan and Biomek systems can be pro- grammed for the initial sample (e.g., plasma) transfer step from vials to the 96-well blocks, buffer addition (if applicable), and to aliquot internal standard. The advantage of the above capabilities is a significant reduction in time and labor for the entire sample processing procedure. Possible technical problems such as carry-over by fixed-tip pipettes used to aliquot the biological fluid can be alleviated by incorporation of several wash cycles or their replacement with disposable pipette tips. In addition, possible inaccurate transfer of samples from the collection tubes to the 96-well blocks due to pipette tip clogging by endogenous protein clots or lipid layers should also be considered. Specific steps such as storage of the plasma samples at −80°C and/or centrifugation at 14,000 rpm prior to sample transfer can be considered for precluding fibrino- gen clot formation. 13.4.2 PPT Due to its ease of use and speed, PPT is one of the most common approaches in sample preparation in early drug discovery [25]. While PPT is fast, easy-to- apply, and applicable to a broad class of small molecules, it also suffers from SAMPLE PREPARATION USING AN OFF-LINE APPROACH 613 several disadvantages. Briefly, in a PPT procedure, often an equal or higher volume (e .g.,1:3) of acetonitrile (or sometimes methanol) is added to a sample of plasma, which contains the test article as well as an internal standard. The sample is mixed and centrifuged, resulting in the formation of a protein pellet and its corresponding supernatant. The supernatant is transferred, dried, reconstituted, or directly injected onto a LC column. Clearly, this procedure is easily amenable to automation and is applicable to a host of structurally 614 THE ROLE OF LC–MS IN PK AND DRUG METABOLISM Figure 13-6. Photograph (top panel) of a Packard Multi-Probe (www.perkinelmer. com) platform for semiautomated PPT, LLE, or SPE process in the authors’ labora- tory. The bottom panel shows a typical layout using the corresponding operating software package. [...]... cartridge, into a disk, or in a 96-well plate format, and it performs essentially the same function as the organic solvent in LLE This is particularly critical in minimizing ion suppression by co-eluting matrix components, when an ESI interface is used for the LC/MS analysis Due to a different mechanism of operation, ion suppression is not a major determinant for signal loss in APCI [37–40]) The ion suppression... examines the system to perform over long periods of time within the predetermined accepted tolerance In the PQ phase, often the individual components of the system are not tested; instead, the system is treated as a whole (for more details see www.fda.gov; General Principles of Software Validation; Final Guidance for Industry and FDA Staff, January 11, 2002) The PQ is usually performed prior to the analysis... submitted for worldwide regulatory filing 13.9 RITALIN®: AN APPLICATION OF ENANTIOSELECTIVE LC-MS/MS Currently there is a trend toward the synthesis and large-scale production of a single active enantiomer in the pharmaceutical industry [61–63] In addition, in some cases a racemic drug formulation may contain an enantiomer that will be more potent (pharmacologically active) than the other enantiomer(s) For. .. validated for the determination of MPH enantiomers in rat, rabbit, dog, and human plasma [58] For example, a validated LC-MS/MS method with a lower limit of quantification of 87 pg/mL in human plasma was reported [58] Figure 13-16 depicts a representative plasma concentration–time profile, obtained using LC-MS/MS, for a child with ADHD subsequent to an oral administration of 17.5 mg of racemic form of... internal standard Arrows indicate that the product-ion that was selected for the multiple-reaction monitoring (MRM) experiment [59, 60] For complete metabolic profile and disposition of STI571 (imatinib, GleevecTM) in humans, see reference 103 a 96-well plate format were utilized A 3M Empore octyl (C8)-standard density 96-well plate was used for plasma sample extraction A Sciex API 3000 triple quadrupole mass... quantitative HPLC- MS bioanalysis, J Chromatogr B 830 (2006), 293–300 F Beaudry and P Vachon, Electrospray ionization suppression, a physical or a chemical phenomenon, Biomed Chromatogr 20 (2006), 200–205 X, Xu, H Mei, S Wang, Q Zhou, G Wang, L Broske, A Pena, and W A Korfmacher, A study of common discovery dosing formulation components and their potential for causing time-dependent matrix effects in high-performance... Critical review of development, validation, and transfer for high throughput bioanalytical LC-MS/MS methods, Curr Pharm Anal 1 (2005), 3–14 54 D H Wilson, D Sepe, and G Barnes, Inter-laboratory differences in sirolimus results from six sirolimus testing centers using HPLC tandem mass spectrometry (LC/MS/MS), Clin Chim Acta 355 (2005), 211–213 55 For more information, please see WWW.FDA.GOV and WWW.ICH.ORG... uncapping and re-capping steps for each individual vial In this regard, the Tomtec Corporation is in the process of final testing and commercialization of the “Formatter” (Figure 13-7), which is designed to alleviate the above bottleneck According to the vendor (and tests during a demo by one 616 THE ROLE OF LC–MS IN PK AND DRUG METABOLISM Figure 13-7 Photograph of a Tomtec “Formatter” (a prototype) designed... degasser, and SPE capability (for more details see www.sparkholland.com) MATRIX EFFECT AND ION SUPPRESSION 619 A caveat for all direct sample injection assays is an understanding of the analyte chemical stability in the biological fluid during the analysis period Nonetheless, an increasingly growing body of literature is suggestive that direct injection of post-dose biological fluids for quantification purposes... in vivo chiral inversion—that is, prochiral → chiral, chiral → nonchiral, chiral → diastereoisomer, and chiral → chiral transformations—could create critical issues in the interpretation of the metabolism and pharmacokinetics of the drug Therefore, selective analytical methods for separations of enantionmers and diastereomers, where applicable, are inherently important RITALIN®: AN APPLICATION OF ENANTIOSELECTIVE . screen their spectrum of 605 HPLC for Pharmaceutical Scientists, Edited by Yuri Kazakevich and Rosario LoBrutto Copyright © 2007 by John Wiley & Sons,. increasingly popular and complementary approaches for drug discovery in the pharmaceutical industry is to perform massive parallel synthesis in solution or