Overview: Handbook of Pharmaceutical Analysis by HPLC

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OVERVIEW: HANDBOOK OF PHARMACEUTICALANALYSIS BY HPLC SATINDER A H U J A

Ahuja Consulting,Calabash,NC

I. II. III. IV. V. VI. VII. VIII. IX. X. XI. XII. XIII. XIV. XV. XVI. XVII. XVIII. XIX. XX. XXI. XXII.

INTRODUCTION KEY CONCEPTS IN HPLC HPLC INSTRUMENTATION IN PHARMACEUTICAL ANALYSIS HPLC COLUMNS IN PHARMACEUTICAL ANALYSIS SAMPLE PREPARATION METHOD DEVELOPMENT METHOD VALIDATION ION CHROMATOGRAPHY HOW TO BE MORE SUCCESSFULWITH HPLC ANALYSIS REGULATORY PRACTICES HPLC SYSTEM CALIBRATION SYSTEMVALIDATION ASSAYAND STABILITY TESTING IMPURITY EVALUATIONS DISSOLUTION TESTING CLEANING VALIDATION HIGH-THROUGHPUT SCREENING CHIRAL SEPARATIONS LC-MS LC-NMR CHROMATOGRAPHY DATA PROCESSING NEW DEVELOPMENTS IN HPLC REFERENCES

I. INTRODUCTION A variety of methods are available for analyzing pharmaceutical compounds; however, high-pressure liquid chromatography is currently the method of choice for the analysis of these compounds. High-pressure 9 2005 ElsevierInc. All rights reserved. Handbook of PharmaceuticalAnalysisby HPLC, S. Ahulaand M.W.Dong,editors.

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liquid chromatography is at times called high-performance liquid chromatography because it offers high performance over ambient pressure or low-pressure liquid chromatography. The use of the term high performance can be debated; however, its use in the context described above is acceptable. The terms high-pressure or high-performance liquid chromatography have been used interchangeably in this book. Coincidentally, they both have the same abbreviation, i.e., HPLC. More commonly, this technique is simply called liquid chromatography or LC. HPLC is used in the pharmaceutical industry for a wide variety of samples. It is the method of choice for checking the purity of new drug candidates, monitoring changes or the scale up of synthetic procedures, in-process testing for developing new formulations, and quality control/assurance of final drug products. A large number of books have been published on HPLC and are referenced throughout this text; a select list of books that may be found useful by the interested reader is provided at the end of this chapter (1-15). This handbook provides busy pharmaceutical scientists with a complete yet concise reference guide for utilizing the versatility of HPLC in new drug development and quality control. It can be broadly divided into six major sections: 1. 2. 3. 4. 5. 6.

Overview, theory, and instrumentation (Chapters 1-3) HPLC methods and practices (Chapters 4-9) Regulatory aspects (Chapters 10-12) Applications (Chapters 13-18) Post-chromatographic techniques (Chapters 19-21) New developments in HPLC (Chapter 22)

All of the chapters have been written by selected experts in their respective fields, and will undoubtedly provide the readers with an indepth understanding of HPLC theory, hardware, methodologies, regulations, and applications. Pharmaceuticals, for the purpose of this book, means chemical compounds that are used in pharmaceutical production. This can comprise the active ingredient, which is also called active pharmaceutical ingredient (API) or drug substance or drug product; and the inert pharmaceutical ingredients (excipients) that are used to formulate a drug product in the form of tablets, capsules, ointments, creams, lotions, parenterals, inhalers, and a variety of drug delivery systems. The primary object of this book is to provide the HPLC practitioner with a handy guide to the use of HPLC for analyzing pharmaceutical compounds of interest. This means familiarizing the practitioner with the theory, instrumentation, regulations, and numerous applications of HPLC. This handbook provides practical guidelines using case studies on sample preparation, column or instrument selection, and summaries of "best practices" in method development and validation, as well as "tricks

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of the trade" in HPLC operation. It describes the essence of major pharmaceutical applications of HPLC (assays, stability testing, impurity testing, dissolution testing, cleaning validation, high-throughput screening, and chiral separations) in addition to nuances in interpreting International Conference on Harmonisation (ICH) guidelines and instrument qualification. This book also highlights the latest developments in hyphenated techniques, such as LC-NMR or LC-MS, and in data handling. Finally, it discusses new and upcoming developments in HPLC. The contents of this book have been highlighted below to provide the reader with an overview of pharmaceutical analysis by HPLC.

II. KEY CONCEPTS IN HPLC

A concise view of HPLC concepts essential to the performance of successful pharmaceutical analysis is provided in Chapter 2 for the benefit of laboratory analysts and pharmaceutical professionals. The goal is to present the basic theory and terminology to the beginner, and, at the same time, delineate the more advanced concepts in gradient elution and high-pH separations for the more experienced practitioners. The important concepts in HPLC that are covered include retention, selectivity, efficiency, and resolution. This chapter discusses their relationship to key column and mobile phase parameters such as particle size, column length and diameter, mobile phase strength, pH, and flow rate. The impact of various other concepts important in chromatographic analysis such as peak capacity, gradient time, void volume, and limit of quantification are also discussed.

III. HPLC INSTRUMENTATION IN PHARMACEUTICAL ANALYSIS

An overview of HPLC instrumentation, operating principles, and recent advances or trends that are pertinent to pharmaceutical analysis is provided in Chapter 3 for the novice and the more experienced analyst. Modern liquid chromatographs have excellent performance and reliability because of the decades of refinements driven by technical advances and competition between manufacturers in a two billion-dollar-plus equipment market. References to HPLC textbooks, reference books, review articles, and training software have been provided in this chapter. Rather than summarizing the current literature, the goal is to provide the reader with a concise overview of HPLC instrumentation, operating principles, and recent advances or trends that lead to better analytical performance. Two often-neglected system parametersmdwell volume and instrumental bandwidth--are discussed in more detail because of their impact on fast LC and small-bore LC applications.

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IV. HPLC COLUMNS IN PHARMACEUTICALANALYSIS An accurate choice of column configuration and column chemistry is very important for carrying out pharmaceutical analysis by HPLC successfully (see Chapter 4). In the section on column configuration, it is emphasized that column performance should be seen from the standpoint of the ratio of column length to particle size. The overall performance characteristics of a column remain constant when this ratio is kept constant: the same maximum resolving power and the same shortest analysis time can be achieved. Other subjects discussed are elevated temperature, column diameter, high-speed analysis, and monolithic columns. In the section on column chemistry, the silica-based stationary phases are described in great detail, including new developments such as waterwettable stationary phases and packings with embedded polar groups. An overview of commonly applied surface chemistries of packings used in reversed-phase applications is included. A separate discussion is dedicated to hybrid packings and zirconia-based packings. Because the long-term reproducibility of an assay is of utmost importance in the pharmaceutical industry, the brief review of the reproducibility of stationary phases should be of great interest to the readers. As many modern chemical entities are polar compounds, hydrophilic interaction chromatography may be a suitable technique for these compounds. Finally, a synopsis of what is currently understood about column selectivity in reversed-phase HPLC is provided.

V. SAMPLE PREPARATION Sample preparation (SP) is generally not given adequate attention in discussions of pharmaceutical analysis even though its proper execution is of paramount importance in achieving fast and accurate quantification (see Chapter 5). Non-robust SP procedures, poor techniques, or incomplete extraction are the major causes of out-of-trend and out-ofspecification results. The common SP techniques have been reviewed with a strong focus on tablets or capsules, as they are the primary products of the pharmaceutical industry. Detailed descriptions of SP methods for assays and impurity testing are provided with selected case studies of single- and multi-component products. Sample preparation refers to a family of solid/liquid handling techniques to extract or to enrich analytes from sample matrices into the final analyte solution. While SP techniques are well documented, few references address the specific requirements for drug product preparations, which tend to use the simple "dilute and shoot" approach. More elaborate SP is often needed for complex sample matrices (e.g., lotions and creams). Many newer SP technologies such as solid-phase extraction

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(SPE), supercritical fluid extraction (SFE), pressurized fluid extraction or accelerated solvent extraction (ASE), and robotics are topics of numerous research papers, symposia and commercial promotion, and are adequately referenced in this chapter. Both novices and experienced analysts can learn the usefulness of sample preparation techniques for the solid dosage forms. The "best practices" are described in the dilute and shoot approach, and the "tricks of the trade" in grinding, mixing, sonication, dilution, and filtration of drug products. Selected case studies of sample preparation for assays and impurity testing are used to illustrate the strategies, trade-offs, and potential pitfalls encountered during method development.

VI. METHOD DEVELOPMENT

The method development of pharmaceuticals by HPLC begins with the generation of an orthogonal array of methods suitable for the separation of early drug substance lots, synthetic intermediates and starting materials, excipients (where known), and products from forced decomposition studies (see Chapter 6). From the array, two orthogonal methods are selected to support subsequent stages of pharmaceutical development. One of these methods is optimized to separate all known related substances and excipients, using chromatographic optimization software such as DryLab. The other method is used to analyze pivotal lots of drug substance and drug product formulations to assure that the primary method continues to be viable for the separation of all relevant components. Following finalization of drug substance synthetic routes and drug product formulation, the focus shifts to the development of robust and transferable methods for post-approval support at quality control units. It is important to remember during the final stage of method development that achievement of separation conditions is only one of the necessary parameters for successful method implementation. Extensive studies to measure robustness and quantitative method performance are conducted to assure that the method performs as intended in quality control laboratories. It should be emphasized that successful method development requires extensive cooperation between the development laboratory and the receiving quality control laboratories.

VII. METHOD VALIDATION

Method validation is discussed in Chapter 7 as it relates to the HPLC methods of analysis. Validation is a process required by law, and the concept is described by regulatory agencies in the guidance documents. The analyst performing method validation is responsible for interpreting the

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guidance into acceptable practices. Establishing a clear, well-planned validation program that incorporates the concepts discussed here is critical to success. Method validation is not a one-time process that can be ignored once completed; it is a constant, evolving process. A well-executed and documented validation serves as evidence to regulatory agencies that the method in question is compliant and performs as intended. Periodic reviews should occur to evaluate validation data for compliance with the current regulatory requirements. To be successful, applicants need to remain up to date with the current thinking of regulatory agencies and anticipate the changes to occur in the regulations. Validation programs that are able to incorporate these changes quickly and efficiently save both time and money for the applicant and help streamline the process to an approvable application.

VIII. ION CHROMATOGRAPHY

Ion chromatography has become an essential tool for the pharmaceutical analytical chemist. The high sensitivity of the technique, coupled with the wide dynamic operating range made possible with modern highcapacity stationary phases, makes it ideal for the analysis of ions in pharmaceutical applications (see Chapter 8). The combination of gradients and suppressed conductivity detection provides a powerful screening tool for the analysis of ions in drug substances and in pharmaceutical formulations, thus offering the basis for analysis of counter ions, additives, and manufacturing by-products. Ion chromatography has been expanded on a number of fronts, now covering an extremely wide variety of analytes, and has seen significant improvements in stationary phase design, chromatographic performance, detection sensitivity, suppressor design, and electrolytic generation of eluents. With all of these improvements, ion chromatography is much more than simply ion-exchange chromatography by another name. Ion chromatography is used in HPLC for separation of inorganic anions, small organic acids, and simple amines. This chapter reviews the underlying principles of ion chromatography and its application in pharmaceutical analysis. It provides an overview of eluent systems, applications of gradients, electrolytic eluent generation, suppressors, and stationary phases. Applications of ion chromatography to the confirmation of counter ions, active ingredient analysis, competitive analysis and development work are discussed.

IX. HOW TO BE MORE SUCCESSFUL WITH HPLC ANALYSIS

Chapter 9, by summarizing a series of standard operating procedures representing the best practices of experienced HPLC analysts, describes

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how we can be more successful in HPLC operation. It offers brief guidelines on maintenance and troubleshooting and the means for enhancing HPLC assay precision. Many years of hands-on experience is required to be truly proficient in HPLC operation. Most analysts agree that the best way to learn HPLC is to be an apprentice to an expert chromatographer. Barring that, it may be desirable to use various reference books, user's manuals, training videos, hands-on training courses, and computer-based training software. This chapter focuses on the practical aspects of HPLC including the "best practices" or operating guides for mobile phase preparation and HPLC system operation. Guidelines on maintenance and troubleshooting are highlighted as well as ways to improve HPLC assay precision. The goal is to offer the analyst a concise overview and a guide on how to be more successful in HPLC analysis.

X. REGULATORYPRACTICES To guarantee the quality, clinical performance, efficacy, and safety of a pharmaceutical product, specifications are fixed and approved by the competent regulatory authorities of each country in which the drug is marketed. Analytical testing ensures that these specifications are met by confirming the identity, purity, and potency of drug substances and drug products prior to their release for commercial use (see Chapter 10). To assure reliable bioavailability, drug products' performance testing is needed. This performance testing comes in the form of either dissolution or disintegration tests. HPLC has become the most important separation technique for analyzing drug substance and drug product. In today's laboratory, testing is for the most part conducted by HPLC, and most wet chemistry methods have become obsolete. For drug substances, HPLC testing is focused on assay and impurity testing. For drug products, HPLC is also used for dissolution testing (an indicator of bioavailability) and content uniformity {used to ensure that the content of the drug substance is uniform throughout a batch). The technology surrounding HPLC instruments themselves and the software used to run them have advanced to such a degree that in many labs analyses are run overnight and in some cases 24 hours a day unattended. This chapter reviews the regulatory guidelines and requirements for the successful use of HPLC in drug substance and drug product testing in development and routine quality control.

Xl. HPLC SYSTEM CALIBRATION Calibration, in current Good Manufacturing Practices (GMP) terminology, refers to instrument "qualification" or performance verification

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of HPLC. In most pharmaceutical laboratories, calibration is performed every 6-12 months. If the results are acceptable according to some predetermined criteria, a sticker is placed on the instrument to indicate its readiness for GMP work. This periodic calibration, coupled with initial system qualification (Installation Qualification and Operation Qualification or IQ/OQ) and daily system suitability testing, is part of an overall system validation program to ensure data validity and regulatory compliance (see Chapter 11). A company-wide system calibration program can also facilitate method transfer by minimizing system-to-system variability among different testing sites. This chapter reviews the principles and strategies used for HPLC system calibration that includes the pump, the detector, the autosampler, and the column oven. A case study is used to illustrate the development of the calibration procedures for all system modules and the rationale of setting up acceptance criteria that balance productivity and compliance.

Xll. SYSTEM VALIDATION Chapter 12 defines the terms, responsibilities, requirements, and recommended procedures involved in pre-installation: Installation Qualification (IQ), Operational Qualification (OQ) and Performance Qualification (PQ); all of these are part of a typical HPLC system validation process. As the Food and Drug Administration (FDA) does not publish a definitive reference or "cookbook" for these procedures, the suggestions listed are only recommendations. The information in this chapter should be viewed as guidelines and not as inflexible mandates. The important thing is to take reasonable, verifiable action, armed with well-thought-out SOPs, protocols, security, and documentation procedures to verify that an HPLC system used in the development or production of pharmaceutical products is performing both in accordance with the Functional Specifications set forth by the vendor and the User Requirement Specification (URS) set forth by the user. Remember that the only thing worse than having no validation protocol is to have one that is poorly designed and/or one that is not followed. There are various stages to the validation process and all have to be thought out as part of an overall time line. The validation process starts with preliminary planning, and selecting/training those qualified in defining and designing the various procedures, experiments, and pertinent documents. This leads to the drafting of specific SOPs and IQ/OQ/PQ protocols, then moves on to carrying out the pre-installation procedures, and finally culminates with implementing the IQ, OQ, and PQ procedures, in that order. One must also have plans/procedures for continued maintenance and re-calibration. All of this is ultimately the responsibility of the user, but especially for the IQ and OQ the vendor

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can be of invaluable help, both in supplying the needed documentation and in implementing the various tasks.

XlU. ASSAYAND STABILITYTESTING

The general concept of drug stability and the factors affecting drug substance and drug product stability are covered in Chapter 13. This chapter also discusses the guidelines from the ICH for stability testing on drug substances and products and the requirements at different phases of the drug development process in the pharmaceutical industry. The role of HPLC in stability testing along with the chromatographic techniques and the procedures involved in developing a stability-indicating method are also described. Case studies of challenging HPLC method development on dual drug systems in solid dosage and surfactants with different molecular weights in liquid formulation are presented. Stability testing is an integral part of pharmaceutical product development and is ongoing throughout the entire drug development process. Product integrity and shelf life are based on stability testing. The pharmaceutical industry is challenged by the frequently changing regulatory requirements. The need for improvement in analytical techniques poses challenges and opportunities in pharmaceutical stability testing. A wealth of specific information is available in the FDA stability guidance and in ICH stability guidelines. HPLC and other analytical techniques play important roles in stability testing. The precision, ease of use, and ruggedness of HPLC methods are preferred by far over other separation and quantification techniques. A continuous effort to learn updated technology and to remain informed of constantly changing regulations is the only way to succeed in the pharmaceutical industry.

XIV. IMPURITY EVALUATIONS

Impurities in drug substances and drug products continue to be the source of great concern, discussion, debate, and research (see Chapter 14). These concerns and debates typically center on the potential safety risks associated with impurities resulting from contamination. However, most of the work being performed in the pharmaceutical industry with respect to impurities is focused on the isolation, identification, and quantification of impurities that could potentially be found as part of the manufacturing process or through chemical decomposition. On the surface it appears that there is a divergence between the perceived need for public safety and the industry focus. However, it should be appreciated that a thorough understanding of all known potential impurities (also known as the impurity profile} helps find unknown impurities and contaminants.

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The most effective means to increasing public safety is to create and maintain a public specification, e.g., those given in the United States Pharmacopoeia (USP). In theory, a public specification defines the boundaries of safety and efficacy of a given drug substance and product. The boundaries are generally set by the manufacturer and approved by the regulatory agency (e.g., the United States Food and Drug Administration or the Ministry of Health in Japan). These boundaries are created by evaluating critical parameters of a drug substance/product, with clinical data collected on human subjects. The first lot of material that is tested in the clinic is often called the pivotal lot. Once the pivotal lot is manufactured, it is evaluated by a battery of tests to ensure that the material is well characterized. Through this testing, a manufacturer may qualify impurities as nontoxic, and can create goalposts to evaluate additional lots, and include this information in the submission to the appropriate regulatory agency. The tests and procedures used to evaluate and characterize the pivotal lot and the acceptance criteria created to define the goalposts are the basis of a public specification. A public specification such as those found in the USP or the British Pharmacopoeia (BP) provides a benchmark of expected impurities, both toxic and nontoxic, that are typically found in the manufacture of a drug substance. With this information and a standardized approach, an analyst can ensure that additional lots of a material are equivalent to the pivotal lot. More important in terms of public safety, these public specifications aid the industry, government, and the affected public look for new impurities and contaminants. Therefore, it is vital that the pharmaceutical industry regularly update these publications for the continued safety of the patient. Impurity testing is pivotal in pharmaceutical development for establishing drug safety and quality. In this chapter, an overview of impurity evaluations of drug substances and products by HPLC is presented from both the laboratory and regulatory standpoints. Concepts from the development of impurity profiles to the final establishment of public specifications are described. Useful strategies in the identification and quantification of impurities and degradation products are summarized with practical examples to illustrate impurity method development.

XV. DISSOLUTIONTESTING HPLC instrumentation and column technologies have progressed to the point that pharmaceutical researchers have tremendous flexibility in selecting an optimal method for any dissolution testing method during drug development and routine quality control tests (see Chapter 15). As formulation development tends toward increasing complexity, HPLC methodology will be embraced even more in dissolution testing, as it can help solve unique analytical challenges posed either by the media or by

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dosage form characteristics. Equipped with a variety of detection methods, HPLC can be used to analyze dissolution samples that otherwise cannot be analyzed by UV methods either because of significant interferences or poor UV absorbance. Automation of dissolution systems with on-line HPLC analysis has been accomplished by the development of fast chromatography, which is being increasingly used in the pharmaceutical industry to improve throughput and productivity.

XVI. CLEANINGVALIDATION The HPLC method used for validation of cleaning of the manufacturing equipment has to be linear; this usually requires additional evaluations to extend the lower end of the linear range used for the potency assay (see Chapter 16). In many laboratories, analysts realize that they will have to evaluate cleaning samples and push the linear range as low as possible in the initial HPLC method development for the API potency assay. Many times during the development cycle, toxicology or clinical data indicate that the cleaning limit must be lowered. It is important for the analyst to know the limits of existing methodology to ascertain the possibility of accommodating lower cleaning limits. Poor chromatographic response by many of today's molecules above 220-240 nm means that many cleaning assays are forced into the lower end of the UV range. This presents additional challenges for the analyst in both linearity and sample analysis because of solvent and impurity interferences at the low wavelengths.

XVII. HIGH-THROUGHPUT SCREENING There is a significant increase in demand for new drug candidates that exhibit improved efficacy, selectivity, and safety. In response to this demand, drug discovery strategy has dramatically changed during the last two decades. Automated and parallel organic synthesis can generate a large number of compounds that can be screened for biological activity and potency against the appropriate targets. These high-throughput screens (HTS) usually identify many hits with potent in vitro pharmacological activity (see Chapter 17). The need for a high-throughput ADME (absorption, distribution, metabolism, and elimination) screen is driven by the necessity for not only acceleration of the discovery process identifying new leads rapidly, but also for the improvement of the success rate of clinical development. Therefore, ADME screens for desirable drug-like properties have been developed to help identify promising new chemical entities (NCEs) among libraries in order to optimize the ADME properties and minimize the potential of toxic liabilities. These ADME analyses generate a significant amount of data that can be utilized to

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categorize and rank NCEs according to their aqueous solubility, chemical stability, intestinal permeability, hepatic metabolism, etc. The results from ADME screens can be utilized by medicinal chemists to design and synthesize NCEs with more favorable ADME properties in the lead optimization phase. This chapter reviews some of the applications of high-throughput liquid chromatography-mass spectrometry (LC/MS) techniques in ADME screens in the lead optimization phase of drug discovery. Several routinely used techniques that are utilized in performing these ADME assays are described, with a strong focus on either the instrumental methods or experimental designs that significantly improve the throughput. Selected case studies on both instrumental methods and experimental setups are discussed to illustrate the strategies, trade-offs, and any potential pitfalls of these various techniques. The preparation of biological samples is also presented in great detail for the LC/MS quantitative bioanalysis. HPLC coupled with mass spectrometry is widely used in drug discovery and development. Mass spectrometric applications are being used in qualitative analysis, quantitative analysis, and preparative HPLC analysis where the mass spectrometer serves as a detector to trigger fraction collection. This chapter presents applications of LC/MS tandem mass spectrometry (LC/MS and LC/MS/MS) in in vitro ADME screens and bioanalysis of in vivo biological samples for evaluation of hits as well as the lead optimization phase in drug discovery. An effort has been made to report the most recent advances in LC/MS software and hardware, analytical instrumentation, and automated sample handling and processing techniques where a significant increase in analysis throughput has been reported.

XVlll. CHIRAL SEPARATIONS Chiral separation of drug molecules and of their precursors, in the case of synthesis of enantiomerically pure drugs, is one of the important application areas of HPLC in pharmaceutical analysis. Besides HPLC, capillary electrophoresis (CE) is another technique of choice for chiral separations. Chapter 18 provides an overview of the different modes (e.g., direct and indirect ones) of obtaining a chiral separation in HPLC and CE. The direct approaches, i.e., those where the compound of interest is not derivatized prior to separation, are discussed in more detail since they are currently the most frequently used techniques. These approaches require the use of the so-called chiral selectors to enable enantioselective recognition and enantiomeric separation. Many different molecules have been used as chiral selectors, both in HPLC and CE. They can be classified into three different groups, based on their

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structure: (i) low-molecular-weight selectors, (ii) macrocyclic selectors, and (iii) macromolecular selectors. Finally, some practical guidelines about screening conditions to test the enantioselectivity of a given compound on a limited number of chromatographic systems, and about method optimization when an initial separation is already obtained, are given briefly. The possibilities potentially applicable to induce the separation of enantiomers of interest are numerous. Not only can different techniques be applied, but also within each technique large numbers of chiral selectors might be used. This assures that almost any chiral separation can be achieved with at least one of the dozens of commercially available chiral selectors for the different techniques. Difficulties, however, originate from the selection of the appropriate chiral selector for a given separation. Since chiral recognition mechanisms are complex, the prediction of a suitable chiral selector is very difficult. Frequently, the identification of suitable selectors for a specific pair of enantiomers requires considerable experimentation and might therefore be highly demanding with respect to time, material, and labor. There is a clear need for fast method development strategies to avoid to some extent trial-and-error approaches, and recently some attempts in this context have been made. Among the different chiral stationary phases (CSPs) used in chiral HPLC, the polysaccharide CSPs have proved to be very useful and might be recommended as first-choice columns when one wants to develop chiral separations. These CSPs know many applications, both in normalphase and reversed-phase mode. The chiral selectors of these CSPs have a broad enantiorecognition range (80% of marketed drugs), which makes them excellent candidates for developing both screening strategies and individually optimized separations. Moreover, the number of different types of stationary phases can be limited to three or four (Chiralcel OD-H and OJ, Chiralpak AD, and occasionally, AS) since both reversedphase and normal-phase versions exist, while the latter can even be used for chiral SFC.

XIX. LC-MS

The power and versatility of LC/MS and LC/MS/MS are discussed in Chapter 19 on the basis of successful practices and how an individual investigator can follow these practices to obtain useful information during different stages of the drug development process. The primary focus is on various applications of LC and electrospray mass spectrometry for pharmaceutical analysis. The goal is to provide some ideas and experimental designs for the solution of qualitative problems including drug impurity profiling, HPLC method development, deficit in drug mass balance, drug discoloration, and drug counterfeiting. Many examples are

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presented to illustrate how LC/MS and LC/MS/MS can be used to provide answers to these real-world problems. LC/MS has emerged as one of the most important techniques in pharmaceutical analysis. It is used to ensure the quality and safety of pharmaceutical products. Mass spectrometry can detect molecules in the positive and negative ionization modes, in which molecules are transformed, primarily, into protonated or deprotonated pseudo-molecular ions. The obvious benefit of using LC/MS is the opportunity it affords for the rapid identification of unknown compounds by generating compound-specific information such as molecular weight and elemental composition. Other benefits can also be realized, such as increased gain in confidence and decreased time required for HPLC method development. Using a "generic HPLC/MS method," data can be collected in a consistent manner throughout the development life cycle of a pharmaceutical product. However, LC/MS is not a panacea. In mass spectrometry, molecules must exist as ionic species in order to be detected. The ionization efficiency may vary over many orders of magnitude, depending on the structure of the compounds studied. For non-polar and neutral drug compounds, an ESI or APCI interface may not be sensitive enough to allow meaningful structure determination. Although assignment of various signals in an ESI/MS mass spectrum is usually straightforward, artifacts can arise from electrochemical reactions during the electrospray ionization process. Thermally induced degradation is also a common phenomenon with both ESI and APCI interfaces because of the heat input that has been used to aid in the evaporation of solvents. The presence of artifacts will obviously complicate accurate determination of the molecular weight for trace-level drug impurities. A thorough knowledge of the sample relating to its formulation, packaging materials, synthetic route, chemical activity of drug compound, etc. is beneficial in the identification of unknown impurities.

XX. LC-NMR Hyphenated analytical techniques such as LC-MS, which combines liquid chromatography and mass spectrometry, are well-developed laboratory tools that are widely used in the pharmaceutical industry. For some compounds, mass spectrometry alone is insufficient for complete structural elucidation of unknown compounds; nuclear magnetic resonance spectroscopy (NMR) can help elucidate the structure of these compounds (see Chapter 20). Traditionally, NMR experiments are performed on more or less pure samples, in which the signals of a single component dominate. Therefore, the structural analysis of individual components of complex mixtures is normally time-consuming and less cost-effective. The

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combination of chromatographic separation techniques with NMR spectroscopy offers advantage for the on-line separation and structural elucidation of unknown compounds. Mixtures such as crude reaction mixtures in drug discovery can be analyzed without prior separation. Experiments combining an HPLC with NMR on the study of mixtures were introduced to the scientific community in the early 1980s. However, LC-NMR was not widely practical because of its low sensitivity, approximately six orders in magnitude inferior to that of MS. Another challenge is from the measurement of proton signals in mobile phase. Recent developments in higher magnetic field strength and electronics that have improved the sensitivity of probes, together with advanced solvent suppression techniques, have made LC-NMR measurement practical. During the last decade, LC-NMR has been fully commercialized, and its applications in both academia and industry have been growing rapidly. One example is LC-NMR-MS, which is a more powerful hyphenated technique combining LC-MS and LC-NMR to provide complementary information about structures simultaneously and offer a method for more accurate and rapid structural analysis. In this chapter, some routine subjects such as the theory and design of flow probes have been avoided, as there are many papers published in the 1990s that have covered these topics comprehensively. The emphasis is therefore on describing experimental design, practical applications, and recent developments in technology. With all the applications to date, LC-NMR spectroscopy is still a relatively insensitive technique because of the poor mass sensitivity of the NMR detection system. To this end, several other hyphenated NMR techniques have been developed to enhance the sensitivity of the technique. LC-SPE-NMR dramatically increases the sensitivity up to a factor of four by utilizing a solid-phase extraction (SPE) device after the LC column. Capillary LC-NMR also significantly lowers the detection limit to low nanogram range through the integration of capillary LC with NMR detection. Other breakthroughs, such as cryoLC-probe technology, combine the advantages of sample flow and enhanced sensitivity from a cryogenically cooled NMR probe. Most of the latest developments in LC-NMR technology have been covered in this chapter. In general, LC-NMR is an efficient analytical technique for the identification of components in pharmaceutical mixtures. It is especially efficient when it is combined with MS; the two on-line detectors are complementary in providing unequivocal structural identification for both expected components and unknown substances. However, the relatively low NMR sensitivity is still the major drawback that hampers the utility of LC-NMR technique. Hopefully, the newly developed cryogenic LC-NMR probes coupled with recent interface enhancements and higher magnetic field strengths will further enhance the utility of this technique in solving more challenging problems.

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XXI. CHROMATOGRAPHYDATA PROCESSING Chromatographic techniques represent one of the most significant sources of analytical data found in today's pharmaceutical laboratories. All of the chromatographic techniques produce data that must be acquired, interpreted, quantified, compared, reported, and finally archived (see Chapter 21). Whether the analysis is qualitative or quantitative in nature, the data must somehow be interpreted and reported so that meaningful decisions can be made. It may be as simple as a qualitative decision that indicates whether a reaction has reached completion successfully, or it may be a series of quantitative analyses that help determine if a batch or lot of product meets its specifications and may now be released. This chapter also examines the evolution and perhaps the revolution that has taken place within the chromatography data system (CDS) marketplace.

XXII. NEW DEVELOPMENTS IN HPLC A few glimpses have been provided into the new developments in HPLC that will impact on the future progress in this field (see Chapter 22). Many of these advances revolve around reducing not only HPLC cycle times by improving the columns and the instrumentation but also improving sample preparation techniques. Under Simplifying Sample Preparation, the latest trends in reducing sample preparation time, such as direct sample infusion/injection and on-line SPE, are discussed. In the section on Column Technologies, newer trends in stationary phases such as metal oxide-based phases and hybrids may offer novel selectivities as compared to C18-based phases. The section on Improvements in Detectors provides some observations on how new detectors are increasing the versatility of HPLC. In the last section, on Improvements in HPLC Throughput, monolithic columns, small particles packed in short columns, high-temperature LC, ultra high-pressure LC, and parallel injection techniques are covered. All of these approaches focus on developing both columns and instruments capable of achieving the types of separations that theory predicts. It is expected that new developments on lab-on-chip will eventually produce more functional systems for small molecules. All in all, the future of HPLC is exciting, and time will unravel newer advances that will make the work of the chromatographer even faster and more productive.

REFERENCES 1. Ahuja, S. and Alsante, K., Handbook of Isolation and Characterization of Impurities in Pharmaceuticals, Academic Press, New York, 2003.

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