Current Drug Metabolism - Volume 7, Issue 5, 2006

The impetus for this special edition stems from the realization that mass spectrometry is now an indispensable toolthat is needed for all the phases of new drug discovery and development. Mass spectrometry is first used for thestructural characterization of all new compounds and then for their pharmaceutical profiling. The new compoundsthat show activity in a high throughput screening assay are then brought to a discovery drug metabolism group forfurther evaluation. It can be stated that at least one mass spectrometry group is an essential requirement for asuccessful drug metabolism department. Mass spectrometry has become the premier analytical tool for the multiplein vitro and in vivo absorption, distribution, metabolism and excretion (ADME) assays. These assays use massspectrometry for both quantitative and qualitative applications. One of the most common uses of HPLC-MS/MSsystems is for bioanalytical assays of both preclinical and clinical pharmacokinetic (PK) samples. Anotherimportant application area is the identification of drug metabolites; new software tools and new MS instrumentationhas been developed to improve our ability to both detect and identify these metabolites. In addition, the newapplication area of biomarker discovery and measurement is becoming a very important part of the new drugdevelopment process. Furthermore, mass spectrometry is continuing to evolve as evidenced by new ionizationsources (nano-ESI) and new types of hybrid mass spectrometers (e.g., the Q-Trap MS and the LTQ-FTMS systems)that have become available in the last few years. I have selected experts in various fields of mass spectrometry towrite the reviews and mini-reviews that you will find in this special edition. I want to thank Chandra Prakash forsuggesting this special edition and thank the authors of these reviews and mini-reviews for their excellentmanuscripts. Together these papers and their references provide a good overview of the use of mass spectrometry incurrent drug metabolism applications.

MS has great utility for pharmaceutical profiling, the measurement of physicochemical and metabolic proper-ties that are crucial to the discovery and development of new drug candidates. An evaluation of the capabilities of MS toimprove the speed, specificity, sensitivity and cost per compound of method in development indicates when MS technolo-gies have utility compared to other analytical techniques. MS has been used successfully for methods that profile the criti-cal properties: permeability, lipophilicity, plasma and solution stability, solubility, plasma protein binding and integrity. Ingeneral, MS has utility in these methods using analytical strategies involving unique MS technologies (e.g., parallel mul-tiplexed interfaces, trap-and-elute), orthogonal detection to UV, high sensitivity for low LOQs, low concentration studies,highly specific MS/MS SRM, combinatorial analysis, use of internal standards, providing initial structural data in additionto quantitative and facile integration with HPLC autosamplers and other hardware that allow enhanced on-line experi-ments. Ultimately, it is important to evaluate the appropriateness of any technique that is being considered for use in amethod, to insure that it best meets all of the criteria for the organization's needs.

A balance between pharmacological activity, safety and drug metabolism and pharmacokinetics (DMPK) at-tributes determines the fate of a new chemical entity (NCE) in drug discovery. Because of the increased number of NCEsrequiring DMPK evaluation, several in vitro higher-throughput screens and counter screens designed to evaluate DMPKattributes have been introduced in drug discovery. The DMPK screens evaluate NCEs for potential absorption, metabo-lism, drug-drug interactions, brain penetration, protein binding and pharmacokinetics. Higher-throughput analytical meth-odologies for the determination of either a common end product of a screen or the parent compound (and/or possible me-tabolites) are essential for successful DMPK screens. Because of its speed, sensitivity and specificity, liquid chromatogra-phy-tandem mass spectrometry (LC-MS/MS) has become the technology of choice for sample analysis. In this review,several in vitro screening assays that we employ in drug discovery are discussed with an emphasis on LC-MS/MS role inaccelerating them.

Both combinatorial chemistry and parallel synthesis provide a valuable means for the production of large num-bers of compounds with diverse molecular architectures that become available for various drug discovery experiments. Inboth the lead optimization and lead selection stages, one requirement that is common for many processes is the need forbioanalytical support. This review summarizes current high throughput strategies and efficient methodologies that are em-ployed for drug metabolism and pharmacokinetic (DMPK) screens for a series of drug discovery compounds. For thesetypes of assays, high performance liquid chromatography coupled to a tandem mass spectrometer (HPLC-MS/MS) hasnow become the technique of choice. The major high throughput strategies including sample reduction and cassette dosingare discussed. The methods for increasing the speed of HPLC-MS/MS-based analyses, such as fast chromatography, di-rect sample injection, parallel technologies and combined ionization interfaces are also presented in this review. In addi-tion, the special challenges when performing HPLC-MS/MS bioanalysis, such as the choice of ionization sources, matrixionization suppression and the potential for endogenous interferences, are addressed.

Although quantitative bioanalysis using liquid chromatography in conjunction with atmospheric pressure ioni-zation tandem mass spectrometry (LC-MS/MS) has been in use for approximately fifteen years, new concepts and tech-nologies are continuously being introduced to enhance the multiple steps of quantitative LC-MS/MS bioanalysis. In thisreview article, we have focused on concepts and technologies that have recently been introduced to achieve further im-provements in biological sample collection/storage and extraction, chromatography and mass spectrometric detection.Under these major headings, a number of specific topics are presented, summarizing the most recent findings in these ar-eas. Included among the topics discussed are: off-line plasma extraction, on-line plasma extraction, enhanced mass reso-lution, atmospheric pressure photoionization, high-field asymmetric waveform ion mobility spectrometry, electron captureatmospheric pressure chemical ionization, enhancing MS detection via formation of anionic and cationic adducts, chemi-cal derivatization, ultra-performance chromatography, hydrophilic interaction chromatography, and MS-friendly ion-pairreversed-phase chromatography. In the end, we discuss potential pitfalls in LC-MS/MS bioanalysis and the means toavoid them. Such pitfalls may occur due to mass spectral interference from metabolites or prodrugs, due to the use of in-appropriate calibration standard and quality control samples for analysis involving unstable drugs or metabolites, and dueto the wild card phenomenon commonly known as the matrix effect.

Metabolism studies play a pivotal role in drug discovery and development. Characterization of metabolic "hot-spots" as well as reactive and pharmacologically active metabolites is critical to designing new drug candidates with im-proved metabolic stability, toxicological profile and efficacy. Metabolite identification in the preclinical species used forsafety evaluation is required in order to determine whether human metabolites have been adequately tested during non-clinical safety assessment. From an instrumental standpoint, high performance liquid chromatography (HPLC) coupledwith mass spectrometry (MS) dominates all analytical tools used for metabolite identification. The general strategies em-ployed for metabolite identification in both drug discovery and drug development settings together with sample prepara-tion techniques are reviewed herein. These include a discussion of the various ionization methods, mass analyzers, andtandem mass spectrometry (MS/MS) techniques that are used for structural characterization in a modern drug metabolismlaboratory. Mass spectrometry-based techniques, such as stable isotope labeling, on-line H/D exchange, accurate massmeasurement to enhance metabolite identification and recent improvements in data acquisition and processing for acceler-ating metabolite identification are also described. Rounding out this review, we offer additional thoughts about the poten-tial of alternative and less frequently used techniques such as LC-NMR/MS, CRIMS and ICPMS.

Recent advances in the biological and analytical sciences have led to unprecedented interest in the discoveryand quantitation of endogenous molecules that serve as indicators of drug safety, mechanism of action, efficacy, and dis-ease state progression. By allowing for improved decision-making, these indicators, referred to as biomarkers, can dra-matically improve the efficiency of drug discovery and development. Mass spectrometry has been a key part of biomarkerdiscovery and evaluation owing to several important attributes, which include sensitive and selective detection, multi-analyte analysis, and the ability to provide structural information. Because of these capabilities, mass spectrometry hasbeen widely deployed in search for new markers both through the analysis of large molecules (proteomics) and smallmolecules (metabonomics). In addition, mass spectrometry is increasingly being used to support quantitative measurementto assist in the evaluation and validation of biomarker leads. In this review, the dual role of mass spectrometry for bio-marker discovery and measurement is explored for both large and small molecules by examining the key technologies andmethods used along the continuum from drug discovery through clinical development.

Advances in mass spectrometry continue to bring new and exciting capabilities to the study of drug metabo-lism. This review covers the hybrid linear ion trap - triple quadrupole mass spectrometer, the QTrap. While still a recentaddition to the arsenal of mass spectrometry techniques available to the metabolism scientist, reports in the literaturehighlight the advantages of the system for metabolite identification. The system combines the selective scans of the triplequadrupole with the high speed, high sensitivity of the ion trap allowing metabolites to be found and characterized in asingle scan. Additionally, the system has MS3 and time delayed fragmentation scans that aid in structure elucidation.Since the fragmentation occurs in the collision cell of the triple quadrupole, the traditionally rich fragmentation of the col-lision cell fragmentation is preserved. In addition to helping to make traditional processes more efficient, work has been done that shows the potential of the in-strument to change traditional DMPK approaches. Researchers have reported methods that allow for both qualitativeanalysis of circulating metabolites and quantification of parent drug within the same analysis. The approaches reportedshow how the instrument can be used to collect more information from every sample and potentially streamline typicaldrug metabolism assays.

Fourier Transform Ion Cyclotron Mass Spectrometry (FTMS) provides the highest mass accuracy and mass re-solving power of the currently available mass spectrometers. One of the main drawbacks in its use for absorption, distri-bution, metabolism and excretion (ADME) applications has been its incompatibility with standard HPLC columns andflow rates. Hybrid instruments, such as the LTQ-FT, provide the much needed bridge between the excellent performanceand capabilities of the FT mass spectrometers and the well-established, tested and validated features of quadrupoles andion traps. The hybrid instruments are compatible with standard HPLC flow rates, have high-throughput and automationcompatibility, and also provide data dependant MSn. The ability to maintain the fidelity of an externally calibrated accu-rate mass measurement across an HPLC peak, where the analyte concentrations are rapidly changing, is a significant ad-vance for this technology, as is the ability to perform data dependent MS/MS experiments on the chromatographic timescale. The MSn and accurate mass capabilities are routinely utilized to rapidly confirm the identification of expected me-tabolites or to elucidate the structures of unusual or unexpected metabolites. The combination of traditional high-flowchromatography and robust, externally calibrated accurate mass determination for both parent and product ions makes theLTQ-FTMS a very powerful analytical tool for the characterization of metabolites, identification of metabolic soft-spotsand for metabonomics studies.

Determination of the pharmacokinetics and metabolite identification have been an integral part of drug discov-ery and development to ensure that drugs have appropriate absorption, distribution, metabolism and excretion properties.Liquid chromatography interfaced with a mass spectrometer has greatly facilitated these studies. Nano-electrospray hasdistinct sensitivity advantages and the increased amount of time available to perform mass spectrometric experiments fa-cilitates structural characterization of metabolites. The recently developed silicon chip-based nano-electrospray devicesare more practical than pulled capillaries. The use of these devices for the determination of pharmacokinetics and metabo-lite identification will be described and particular attention will be paid to the distinct advantages and disadvantages thesedevices offer.