11 THE EXP ANDING ROLE OF HPLC IN DRUG DISCOVERY Daniel B. Kassel 11.1 INTRODUCTION Great efficiencies have been achieved in the drug discovery process as a result of technological advances in target identification, high-throughput screening, high-throughput organic synthesis, just-in-time in vitro ADME (absorption, distribution, metabolism, and excretion), and early pharmacokinetic screening of drug leads. These advances, spanning target selection all the way through to clinical candidate selection, have placed greater and greater demands on the analytical community to develop robust high-throughput methods. This review highlights the various roles of high-performance liquid chromatogra- phy/mass spectrometry (HPLC/MS) in drug discovery and how the field has evolved over the past several years since the introduction of myriad high- throughput drug discovery technologies. Included are significant develop- ments in HPLC/MS to support target selection (proteomics), biological screening and assay development, high-throughput compound analysis and characterization, UV- and mass-directed fractionation for unattended, automated compound purification, and high-throughput in vitro ADME screening. Focus within the pharmaceutical industry has been to increase the likeli- hood of successfully developing clinical candidates by optimizing the compo- nents of the discovery process (i.e., spanning target identification → chemical 535 HPLC for Pharmaceutical Scientists, Edited by Yuri Kazakevich and Rosario LoBrutto Copyright © 2007 by John Wiley & Sons, Inc. design → synthesis → compound analysis and purification → registration → biological and ADME screening. By optimizing each step in the iterative dis- covery process, it is expected that the compound attrition rate will be reduced dramatically as compounds advance into preclinical development. Both HPLC and LC/MS enjoy important roles throughout the discovery process, as will be highlighted in detail in this review. Once considered primarily an enabling tool for medicinal chemists, HPLC and LC/MS are now key technologies incorpo- rated at just about every stage of the drug discovery process. Drug discovery programs typically initiate, as follows. Assuming that the relevant therapeutic area (e.g.,oncology, metabolic diseases,inflammation, pain,CNS,etc.) has been selected, the next step is to identify a biological target relevant to the disease. As will be discussed shortly, numerous technological advances in the field of analytical chemistry (e.g., nanocolumn HPLC/MS/MS) that have greatly facilitated protein/target identification have been made since the human genome initiative was launched. Following on the heels of target selection is the requirement to establish tools for “just-in-time” high-throughput screen- ing of compound repositories (so-called corporate collections) and synthetic libraries as a means for identifying initial hits/actives. In combination with structure–activity relationship (SAR) data generated from these high- throughput screens, chemists incorporate knowledge of protein three- dimensional structures and utilize computational tools (i.e., in silico methods that measure diversity and “drug-likeness” as well two-dimensional and three- dimensional pharmacophore models [descriptors] that predict biological activ- ity) to support iterative compound design, synthesis, and biological testing. Once the hits or actives have been identified, the process of hit refinement and lead optimization is initiated.At this stage, a chemistry team is established and both parallel synthesis and more traditional medicinal chemistry strategies are incorporated to rapidly converge on qualified leads (so-called hit-to-lead stage). HPLC and LC/MS play an extremely important role in the hit-to-lead stage of discovery, providing key enabling analysis and purification capabili- ties to the medicinal chemist. Furthermore, activities that were traditionally relegated to drug metabolism and pharmacokinetics departments within development organizations are now integrated into early discovery so as to provide early measurements and predictions of in vivo properties. Again, LC/MS has played an extremely important role in enhancing the drug devel- opability of these hits and leads. All of these advances have helped to stream- line the discovery phase of pharmaceutical drug discovery and development and are presented within. 11.2 APPLICATIONS OF HPLC/MS FOR PROTEIN IDENTIFICATION AND CHARACTERIZATION The human genome initiative that took a stronghold on biotechnology com- panies in the early 1990s through the first few years of the twenty-first century 536 THE EXPANDING ROLE OF HPLC IN DRUG DISCOVERY spawned a completely new field that had analytical chemistry as its corner- stone . Specifically, high-resolution capillary and nano-column HPLC coupled with tandem mass spectrometry became one of the tools of choice for char- acterizing proteins and identifying potential therapeutic protein targets. Although capillary HPLC/MS/MS was applied as early as 1989–1991 to the characterization of proteins and for identifying sites of post-translational mod- ification [1–4], the field took off in earnest following the genomics boom and became known as proteomics, coined by Wilkins et al. [5]. In essence, the mandate of the proteomics field since its inception has been to identify dif- ferences at the protein level, in cells, tissues, plasma, and so on, between a disease state and control (“normal”). The basic premise is that proteins will be either up- or down-regulated (i.e., over- or underexpressed) in the disease state relative to “normal” state, and these differences can be identified and quantified by mass spectrometry.There have been several analytical advances made in the field of proteomics since its inception, far too numerous to capture in this review. One noteworthy advance in proteomics is the technique of multidimensional protein identification technology (MUDPIT), developed by Yates and co-workers, which has been used widely in place of the more labo- rious, less automated method of 2D-polyacrylamide electrophoresis [6]. MUDPIT is a column chromatography method whereby ion-exchange chro- matography is used in the first dimension of chromatography to simplify the complexity of the complex mixture of peptides by separating them based on charge followed by reversed-phase HPLC for the higher-resolution separation based on molecular weight and hydrophobicity. An equally important devel- opment in the field of proteomics has been isotope-coded affinity tags (ICAT) technology, a method whereby isotopic labeling of peptides containing cys- teine residues is performed so as to facilitate peptide quantitation and identi- fication of putative biological targets [7]. The reader is directed to the following review in the field of proteomics for more information [8]. A wealth of preclinically validated targets has emerged as a result of mouse genetics [9] and siRNA technology [10]. For both techniques, a single gene knockout is performed, and the effect of the deletion is monitored/evaluated. Proteomics, on the other hand, generally takes a shotgun approach to identi- fying the targets that are relevant and specific to the disease. Unfortunately, because many diseases are polygenic in origin and because protein pathways are extremely complex (e.g., intracellular protein signaling pathways [11]), proteomics has been best at identifying a short list of “candidate” protein targets rather than a single protein target completely unique to the disease. The challenge has been to sift through all the proteins that have been identi- fied as altered in a disease state relative to healthy state, and this has proved extremely challenging. The focus of proteomics has turned to identifying potential biomarkers of disease. A biomarker, by definition, is (a) a molecular indicator for a specific biological property or (b) a feature or facet that can be used to measure the progress of disease or the effects of treatment. As an example, a biomarker APPLICATIONS OF HPLC/MS 537 for Type II diabetes is higher fasting blood glucose levels relative to age- matched controls . Another, more definitive biomarker of type II diabetes is elevated HbA1c levels. For many diseases, however, the relevant biomarkers are less well understood. This is especially true in the fields of oncology and inflammation research. Biomarker research is a particularly intense area of focus for many pharmaceutical companies, with new departments being formed for the purpose of identifying both preclinical and clinical biomarkers to facilitate their drug discovery and development programs. Like the field of proteomics, the field of biomarker research is far too vast to warrant its review here. A very nice review article by the late Wayne Colburn, a pioneer in dia- betes biomarker research, describes this maturing field [12]. 11.3 APPLICATIONS OF HPLC/MS/MS IN SUPPORT OF PROTEIN CHEMISTRY Independent of the tool used to identify the protein target,whether it be mouse genetics, siRNA technology, or proteomics, once a protein has been identified as a suitable target for drug discovery,the next step in the drug discovery process is to express and purify the protein (carried out combining molecular biology and protein chemistry techniques) in sufficient quantities so as to support bio- logical screening, X-ray crystallography, and any other drug discovery studies requiring purified protein material.The traditional method for assessing protein expression and purification has been to use 1D-polyacrylamide gel elec- trophoresis. 1D-PAGE is capable of separating proteins based on molecular weight and charge (pI).However,the technique is unable to provide more than a crude assessment of protein molecular weight.Recently,open-access or walk- up LC/MS has been incorporated into protein chemistry and molecular biology labs and has greatly facilitated confirmation of protein expression [13–15]. Generic gradient LC-MS methods are used to trap and elute expressed, purified proteins by RP-HPLC/ESI/MS. Open-access protein QC is a bit more challenging than its small-molecule counterpart in that not all proteins “fly” by electrospray ionization, identifying a “universal” HPLC method for their sep- aration can be challenging, and instrument calibration and mass accuracy are of paramount importance. We developed a fast, 5-minute protein QC method using a Poroshell 1-mm-i.d. column and found the method to be satisfactory for the vast majority of protein separations and analyses performed in our labora- tory. To achieve adequate mass accuracy for protein molecular weight deter- minations,an external calibration with myoglobin is performed at the beginning and end of each overnight queue of protein samples so as to ensure that the instrument calibration is maintained over the course of the batch analysis.Mol- ecular weights of deconvoluted protein spectra are then compared to the pre- dicted protein molecular weight,and the results are captured graphically (in the form of a microtiter plate view) as well as in tabular format, amenable to data- base uploading, as shown in Figure 11-1. 538 THE EXPANDING ROLE OF HPLC IN DRUG DISCOVERY 11.4 APPLICATIONS OF HPLC/MS/MS IN SUPPORT OF ASSAY DEVELOPMENT AND SCREENING The overwhelming majority of biological assays have been developed in microtiter plate format (typically 96-well, 384-well, 1536-well) and with paral- lel detection methods such as fluorescence polarization. The vast majority of druggable targets, including enzymes, ligand gated ion channels, and G- protein-coupled receptors, are all amenable to screening in high-throughput microtiter plate format. In general, serial-based chromatographic methods, such as HPLC and HPLC/MS, are unable to compete with the high-throughput screening tech- nologies. However, a small number of targets, such as those involved in medi- ating protein–protein interactions, are not well-suited to HTS methodologies. For this class of targets, HPLC coupled with mass spectrometry has proved to be a very reliable, albeit lower throughput, alternative. The technique that has been used most widely for directly assessing protein–small molecule and protein–protein interactions is affinity chromatography–mass spectrometry. Kassel et al. [16] presented one of the first papers coupling affinity chro- matography with mass spectrometry. In their work, a two-dimensional LC/LC/MS method was developed to assess protein–ligand binding. Affinity chromatography was used in the first dimension of separation, followed by reversed-phase chromatography coupled with mass spectrometry for the identification of binders. Kaur et al. [17] showed the power of size exclusion APPLICATIONS OF HPLC/MS/MS IN SUPPORT 539 Figure 11-1. Automated protein AnalysisOpenLynx LC/MS for protein molecular weight confirmation. chromatography (SEC) coupled with reversed-phase HPLC/MS for identify- ing ligands for a receptor derived from a 576-component combinatorial library . Today, size-exclusion columns are available in microtiter plate format, permitting higher-throughput characterization of protein–protein and protein–ligand interactions. Berman et al. [18] pioneered one of the earliest applications of HPLC in support of assay development.They showed the power of HPLC for the deter- mining preferred substrates of the enzyme collagenase, a metalloprotease. Complex mixtures (pools of 100 components each) of probe substrates for collagenase were prepared by combinatorial methods. Each of the pooled libraries was incubated with enzyme. Substrate disappearance (turnover) and product appearance profiles were monitored by HPLC and the optimal sub- strate(s) identified. Recently, Lambert et al. [19] published a two-dimensional LC/LC/MS method for the identification and optimization of substrates for TNF convertase. Scientists at Nanostream, Inc., a company dedicated to high- throughput HPLC, introduced a parallel capillary LC/fluorescence method to support screening for kinase inhibitors. Their method complements the more traditional (and higher-throughput) fluorescence-based screening approach but offers the advantage of chromatographic separation of phosphorylated and unphosphorylated products, thereby reducing background interference. Another emerging role of HPLC/MS is in support of cell-based assays for which no direct measures of drug effect are possible and require indirect methods for detection. A recent publication by Clark et al. highlights the power of LC-MS for screening inhibitors of HMG-CoA reductase (a rate- determining enzyme in the cholesterol biosynthesis) [20]. In addition, Thibodeaux et al. [21] and Xu et al. [22] reported on methods for directly assessing the cell-based activity of inhibitors of the metabolic disease target, 11β-hydroxysteroid dehydrogenase-1 (11β-HSD-1). LC-MS was used to measure the effect of 11β-HSD-1 inhibitors on the intracelleular conversion of cortisol and cortisone using LC/MS/MS. 11.5 SOURCES OF COMPOUNDS FOR BIOLOGICAL SCREENING Once the assay and assay format have been decided upon, the next step in the discovery process is to initiate compound screening for the purpose of identi- fying hits or lead compounds. The fundamental requirement is that the assay results identify a collection of actives or “hits.” The definition of “hit” varies between organizations, but most accept the definition that the compound shows a confirmed structure, shows a confirmed dose response, exhibits an IC50 ≤ 10µM potency, and is a member of a chemotype that is amenable to analoging and fast follow-on synthesis. What is the source of these initial actives or hits? There is a wide array of compound sources. Generally, pharmaceutical and biotechnology organiza- tions initiate screening by accessing their internal compound repositories (so- 540 THE EXPANDING ROLE OF HPLC IN DRUG DISCOVERY called corporate collections or compound archives). Often, the corporate col- lections are not particularly diverse but are biased to the therapeutic focus(es) of the organization. Consequently, the screening libraries are often augmented by addition of commercially available screening libraries that are (a) gene- family focused (e.g., GPCR-targeted libraries, kinase-targeted libraries, etc.) and/or (b) general diversity sets. Further augmentation of the initial screening activities is to include custom synthesis compound libraries (typically pro- duced by automated high-throughput organic synthesis (HTOS) methodolo- gies, such as those described by Nikolaou et al. [23]. One of the challenges with compound collections is that they are historical by nature. For large Pharma, it is not uncommon for corporate collections to include compounds that were synthesized more than 25 years ago. At the time of synthesis, it can be presumed that the compounds met the purity criteria for compound registration. However, it can also be presumed that a high like- lihood exists that the compounds have degraded over extended storage time. Another reason for poorer quality of compound collections is attributable to the fact that most compounds are stored as DMSO stock solutions as opposed to storage as solid materials.Storage of compounds in DMSO is done primarily for the reason that (a) DMSO is considered a “universal” solvent and (b) solu- tions are much easier to handle in plate-based high-throughput biological screening systems. However, the drawback to DMSO is that it is a very hydro- scopic solvent and unless the compounds are stored under inert conditions, they are prone to hydrolysis. Kozikowski et al. [24] evaluated the effect of freeze/thaw cycles on stability of compounds stored in DMSO. Until very recently, with the introduction of high-throughput analytical technology, these compound sources were far too large to merit re-analysis and/or re-purification and hence were screened “as is.”The result was (and has been observed frequently) that hits could not be reconfirmed during follow- on bioassay screening, and subsequent evaluation of the compounds by tech- niques such as HPLC/MS and NMR showed that the expected compound was not pure and, in some cases, was completely absent! The adage “garbage in, garbage out” became a mantra of many high-throughput screening laborato- ries and forced companies to take a much more serious look at the quality of their compound collections. Morand et al. [25] from Proctor and Gamble set out to fully assess the quality of their >500,000 compound corporate collec- tion.They achieved this goal through incorporation of a massively parallel flow injection–mass spectrometry system, capable of analyzing a plate of samples in less than 2 minutes.The throughput of their technique was one to two orders of magnitude faster than typical flow injection–mass spectrometry systems used for reaction monitoring [26]. In addition to quality control over compound collections, the issue of purity of synthetic libraries derived using combinatorial chemistry quickly came under the microscope. In the early to mid-1990s, “combichem” became a household word throughout the pharmaceutical industry and was believed to be a key technology that would revolutionize drug discovery. The basis of SOURCES OF COMPOUNDS FOR BIOLOGICAL SCREENING 541 combinatorial chemistry was the ability to perform split-mix synthesis on solid support and to take advantage of the combinatorial nature of the process to generate vast arrays of compounds . Combinatorial libraries were purported to be pure, owing to the fact that they were synthesized on solid support and amenable to extensive washing to remove excess reagents and, therefore, directly amenable to high-throughput screening. However, these combinator- ial libraries synthesized on solid support suffered from the same problems that have long plague solution-phase synthesis—that is, the generation of unex- pected and unwanted by-products. Due to the shear size of these compound libraries and the relatively small amounts available following resin cleavage, it was not possible to either characterize or purify the expected products. Con- ventional split-mix combinatorial methods, though still popular with some bench chemists, have been replaced largely by the technique of directed par- allel solution and parallel solid-phase organic synthesis. 11.6 HPLC/MS ANALYSIS TO SUPPORT COMPOUND CHARACTERIZATION Combinatorial chemistry paved the way for high-throughput, parallel organic synthesis techniques, now mainstream in the pharmaceutical and biotechnol- ogy industries for lead generation activities. The ability to synthesize com- pound libraries rapidly using automated solution-phase and solid-phase parallel synthesis has led to a dramatic increase in the number of compounds now available for high-throughput screening. The unprecedented rate by which compound libraries are now being generated has forced the analytical community to implement high-throughput methods for their analysis and characterization. As early as 1994, groups adopted high-speed, spatially addressable auto- mated parallel solid-phase and solution-phase synthesis of discretes [27–31]. Both solution-phase and solid-phase parallel synthesis permits the production of large numbers as well as large quantities of these discrete compounds, eliminating the need for extensive decoding of mixtures and re-synthesis following identification of “active” compounds in high-throughput screening of combinatorial libraries. Importantly, parallel synthesis is performed readily in microtiter plate format amenable to direct biological screening, as was touched upon earlier. The relative ease of automation of parallel synthesis led to a tremendous in flux of compounds for lead discovery and lead optimization. Almost all of the analytical characterization tools (e.g., HPLC, NMR,FTIR, and LC/MS) are serial-based techniques, and parallel synthesis is inherently parallel. Consequently, this led rapidly to a new bottleneck in the discovery process (i.e., the analysis and purification of compound libraries). Parallel syn- thesis suffers from some of the same shortcomings of split and mix synthesis (e.g., the expected compound may not be pure, or even synthesized in suffi- 542 THE EXPANDING ROLE OF HPLC IN DRUG DISCOVERY cient quantities).The analytical community was faced with the decision of how to analyze these parallel synthesis libraries . The traditional method for assessing compound purity has been to perform the following: Purify the desired product to homogeneity by crystallization or column chromatography (e.g., RP- or NP-HPLC),acquire a 1D-NMR and 2D- NMR spectrum on the isolated product, obtain confirmatory molecular weight information by mass spectrometry, perform a C, H, and N combustion analy- sis, generate an exact mass measurement (to within 5ppm of the expected mass) by high-resolution mass spectrometry, and determine the amount of iso- lated product by weight—all prior to compound submission and biological screening. In the era of high-throughput compound library synthesis, however, this extensive characterization is simply not possible. Therefore, groups have focused principally on a limited number of analytical measurements for compound identity and purity—in particular, LC/MS analysis incorporating orthogonal detection methods, such as UV and evaporative light scattering detection (ELSD) and flow-probe 1D-NMR [32]. The most commonly employed technique for characterizing compound libraries is to incorporate LC/MS with electrospray or atmospheric pressure chemical ionization with UV and ELSD and, more recently, photoionization [33]. LC/MS emerged as the method of choice for the quality control assessment to support parallel synthesis because the technique, unlike flow injection mass spectrometry, provides the added measure of purity (and quantity) of the com- pound under investigation. In addition, “universal-like” HPLC gradients (e.g., 10% to 90% acetonitrile in water in 5 minutes) have been found to satisfy the separation requirements for the vast majority of combinatorial and parallel synthesis libraries. Fast HPLC/MS has been found to serve as good surrogate to conventional HPLC for assessing library quantity and purity [34–37]. Fast HPLC/MS is simple in concept. It involves the use of short columns (typically 4.6mm i.d. × 30mm in length) operated at elevated flow rates (typically 3– 5mL/min). Typically, short columns are used for compound analysis because they allow for fast separations to be carried out at ultrahigh flow rates. Also, these columns tend to be more robust than narrow bore columns (1-mm and 2-mm i.d.) (i.e., less clogging is experienced and longer lifetimes are observed when these columns are subjected to unfiltered chemical libraries).A typical LC/MS analysis consists of injection a small aliquot (10–30µL) of the reaction mixture (total concentration of 0.1–1.0mg/mL) and performing the separation using a “universal” gradient of 10–90% Buffer B in 2–5 minutes. Buffer A is typically H 2 O containing 0.05% trifluoroacetic acid (or formic acid), and Buffer B is typically acetonitrile containing 0.035% trifluoroacetic (or formic acid). HPLC columns are operated typically at flow rates of 3–5mL/min (depending on their dimensions), and the cycle time between injections is 3–5 minutes. An example of a fast LC/MS analysis of a combinatorial library compo- nent is shown in Figure 11-2. Fast LC/MS run times incorporating these short columns is typically between 3 and 5 minutes including re-equilibration. HPLC/MS ANALYSIS TO SUPPORT COMPOUND CHARACTERIZATION 543 Recent reports by Kyranos et al. [38] suggest that “pseudo-chromatography” (in essence , step elution chromatography) provides a more rapid and reliable assessment of the quality of library synthesis than methods such as flow injec- tion mass spectrometry. 11.6.1 Purity Assessment of Compound Libraries The issue of compound purity has received a great deal of attention over the last several years as more and more chemists have adopted high-throughput organic synthetic protocols but are unwilling to compromise the quality of the molecules submitted for biological evaluation. The general consensus target purity of a compound library compound before it is to be archived or screened for biological activity is between 90% and 95% pure. This purity criterion is more stringent than in the past, where 85–90% (based on UV detection) was considered acceptable. This may be attributed primarily to a shift toward smaller, focused (or biased) libraries than larger, diverse collections of com- pounds. The majority of mass spectrometry manufacturers now offer software packages that aid in the automatic determination of purity. UV chromatograms are typically used, rather than the total ion current chromatogram, to assess purity. This is because the total ion current chro- matogram is a measure of a compound’s “ionizability,” which is well known to vary dramatically from one compound to the next. Orthogonal detection methods, such as chemiluminescence nitrogen detection (CLND) [39] and ELSD [40, 41], have been proposed to be more universal detection methods than UV and hence are being used with increasing frequency to assess reac- tion yields and purity. CLND, as indicated from its name, measures the amount of nitrogen in a sample. In this method, a compound is transferred to a high- 544 THE EXPANDING ROLE OF HPLC IN DRUG DISCOVERY Figure 11-2. (A) A 4-minute HPLC/MS separation of a solution-phase parallel synthesis library. The gradient profile for fast HPLC/MS was 10–90% acetonitrile in H 2 O in 4 minutes with a 1-minute equilibration time. (B) A 1-minute, total cycle time chromatographic separation of the same crude product. (Reprinted from reference 42, with permission.) [...]... highly selective detector for massdirected fractionation and isolation This technique provides a means for reducing dramatically the number of HPLC fractions collected per sample and virtually eliminates the need for post-purification analysis to determine the mass of the UV-fractionated compound Preparative LC/MS is now widely incorporated in the pharmaceutical industry Systems for preparative LC/MS are... assay only high-quality compounds Therefore, great effort has been devoted to the development of automated purification technology designed to keep pace with the output of high-throughput combinatorial/parallel synthesis Automated methods are now available to the chemist to perform highthroughput purification Although HPLC has long been a method available to the chemist for product purification, only recently... (e.g., UV thresholds for initiating and terminating fraction collection) can be used to reduce the total number of fractions, all, to some extent, will contain impurities The exact number of chromatographic peaks for a given sample will be hard to predict, and therefore the footprint for fraction collection will be difficult to predict Experience has shown that it is not uncommon for 5–10 fractions to... of the first scientists to implement a fully automated preparative LC/MS system for combinatorial library purification His approach was to perform a scouting analytical run prior to purification so as to optimize chromatographic method and fraction collection parameters Fraction location and molecular weight information were captured through a custom LIMS system The added mass spectrometric information... ROLE OF HPLC IN DRUG DISCOVERY review focuses exclusively on HPLC- and HPLC/ MS-based purification methods 11.7.1 UV-Directed Purification Both activity and inactivity data are being used increasingly to generate SAR and direct subsequent synthetic efforts Consequently, organizations have recognized the importance of confirming the purity of compounds prior to screening, and not only those compounds for which... of 10 mM in DMSO Many in vitro ADME screens may be performed in micro titer plate format, and it is at the time of biological screening that a number of daughter plates may also be generated for highthroughput ADME, as shown in Figure 11-12 In addition to the metabolic stability assays, plasma protein binding can be performed in microtiter plate format using both the ultrafiltration method [80] and equilibrium... analysis strategy 562 THE EXPANDING ROLE OF HPLC IN DRUG DISCOVERY Figure 11-13 Time-course human liver microsomal incubation profiles for a number of positive and negative controls, as well as project compounds Metabolic stability profiles are represented both in tabular format and graphically above is to extract the most appropriate information required for decision-making in as streamlined a manner... compounds are color-coded for easy visualization: green, >80%; orange, 80–40%; red, . identification → chemical 535 HPLC for Pharmaceutical Scientists, Edited by Yuri Kazakevich and Rosario LoBrutto Copyright © 2007 by John Wiley & Sons,. research is a particularly intense area of focus for many pharmaceutical companies, with new departments being formed for the purpose of identifying both preclinical