Nội dung

Chromatographic Analysis of Pharmaceuticals Second Edition, Revised and Expanded edited by John A. Adamovics Cytogen Corporation Princeton, New Jersey Marcel Dekker, Inc. New York-Basel «Hong Kong Preface ISBN: 0-8247-9776-0 The publisher offers discounts on this book when ordered in bulk quantities. For more information, write to Special Sales/Professional Marketing at the address below. This book is printed on acid-free paper. Copyright © 1997 by Marcel Dekker, Inc. All Rights Reserved. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher. Marcel Dekker, Inc. 270 Madison Avenue, New York, New York 10016 Current printing (last digit): 10 9 8 7 6 5 4 3 2 1 PRINTED IN THE UNITED STATES OF AMERICA The first edition of Chromatographic Analysis of Pharmaceuticals was published in 1990. The past years have allowed me to evaluate leads that I uncovered during the researching of the first edition, such as the first published example of the application of chromatography to pharmaceutical analysis of medicinal plants. This and other examples are found in a relatively rare book, Uber Kapillaranalyse und ihre Anwendung in Pharmazeutichen Laboratorium (Leipzig, 1992), by H. Platz. Capillary analysis, the chromatographic technique used, was developed by Friedlieb Runge in the mid-1850s and was later refined by Friedrich Goppelsroeder. The principle of the analysis was that substances were absorbed on filter paper directly from the solutions in which they were dissolved; they then migrated to different points on the filter paper. Capillary analysis differed from paper chromatography in that no developing solvent was used. We find that, from these humble beginnings 150 years ago, the direct descendant of this technique, paper chromatography, is still widely used in evaluating radiopharmaceuticals. This second edition updates and expands on coverage of the topics in the first edition. It should appeal to chemists and biochemists in pharmaceutics and biotechnology responsible for analysis of pharmaceuticals. As m the first edition, this book focuses on analysis of bulk and formulated drug products, and not on analysis of drugs in biological fluids. in IV Preface The overall organization of the first edition — a series of chapters on regulatory considerations, sample treatment (manual/robotic), and chromatographic methods (TLC, GC, HPLC), followed by an applications section—has been maintained. To provide a more coherent structure to this edition, the robotics and sample treatment chapters have been consolidated, as have the chapters on gas chromatography and headspace analysis. This edition includes two new chapters, on capillary electrophoresis, and supercritical fluid chromatography. These new chapters discuss the hardware behind the technique, followed by their respective approaches to methods development along with numerous examples. All the chapters have been updated with relevant information on proteinaceous pharmaceuticals. The applications chapter has been updated to include chromatographic methods from the Chinese Pharmacopoeia and updates from U.S. Pharmacopeia 23 and from the British and European Pharmacopoeias. Methods developed by instrument and column manufacturers are also included in an extensive table, as are up-to-date references from the chromatographic literature. The suggestions of reviewers of the first edition have been incorporated into this edition whenever possible. This work could not have been completed in a timely manner without the cooperation of the contributors, to whom I am very grateful. John A. Adamovics Contents Preface Contributors 1. REGULATORY CONSIDERATIONS THE CHROMATOGRAPHER John A. Adamovics I. II. III. IV. V. VI. VII. Introduction Impurities Stability Method Validation System Suitability Testing Product Testing Conclusion References 2. SAMPLE PRETREATMENT John A. Adamovics I. Introduction II. Sampling III. Sample Preparation Techniques IV. Conclusions References Contents V/ PLANAR CHROMATOGRAPHY John A. Adamovics and James C. Eschbach I. Introduction II. Materials and Techniques III. Detection IV. Methods Development V. Conclusion References GAS John I. II. III. IV. V. 7. CHROMATOGRAPHY A. Adamovics and James C. Eschbach Introduction Stationary Phases Hardware Applications Conclusion References 57 57 58 66 68 72 72 79 79 79 84 105 119 120 HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY John A. Adamovics and David L. Farb I. Introduction II. Sorbents III. Instrumentation IV. Method Development V. Conclusion References 135 CAPILLARY ELECTROPHORESIS Shelley R. Rabel and John F. Stobaugh I. Introduction II. Capillary Electrophoresis Formats HI. Instrumentation IV. Methods Development V. Conclusion References 209 SUPERCRITICAL FLUID CHROMATOGRAPHY OF BULK AND FORMULATED PHARMACEUTICALS James T. Stewart and Nirdosh K. Jagota I. Introduction II. Hardware 135 135 140 157 184 184 209 210 221 227 231 231 239 239 240 VIl Contents III. IV. V. Application of SFC to Selected Bulk and Formulated Pharmaceuticals Conclusions References APPLICATIONS John A. Adamovics I. Introduction II. Abbreviations III. Table of Analysis References Index 244 268 269 273 273 274 275 424 509 X • Contributors James T. Stewart, Ph.D. Professor and Head, Department of Medicinal Chemistry, College of Pharmacy, The University of Georgia, Athens, Georgia John F. Stobaugh, Ph.D. Department of Pharmaceutical Chemistry, Uni­ versity of Kansas, Lawrence, Kansas 1 Regulatory Considerations for the Chromatographer JOHN A. ADAMOVICS New Jersey Cytogen Corporation, Princeton, I. INTRODUCTION Analysis of pharmaceutical preparations by a chromatographic method can be traced back to at least the 1920s [1]. By 1955, descending and ascending paper chromatography had been described in the United States Pharmaco­ peia (USP) for the identification of drug products [2]. Subsequent editions introduced gas chromatographic and high-performance liquid chromato­ graphic methods. At present, chromatographic methods have clearly be­ come the analytical methods of choice, with over 800 cited. The following section describes challenges presented to scientists in­ volved in the analysis of drug candidates and final products, including the current state of validating a chromatographic method. И. IMPURITIES In the search for new drug candidates, scientists use molecular modeling techniques to identify potentially new structural moieties and screen natural sources or large families of synthetically related compounds, along with modifying exisiting compounds. Once a potentially new drug has been iden1 2 Adamovics tified and is being scaled up from the bench to pilot plant manufacturing quantities, each batch is analyzed for identity, purity, potency, and safety. From these data, specifications are established along with a reference stan­ dard against which all future batches will be compared to ensure batch-tobatch uniformity. A good specification is one that provides for material balance. The sum of the assay results plus the limits tests should account for 100% of the drug within the limits of accuracy and precision for the tests. Limits should be set no higher than the level which can be justified by safety data and no lower than the level achievable by the manufacturing process and analytical variation. Acceptable limits are often set for individual impurities and for the total amount of drug-related impurities. Limits should be established for by-products of the synthesis arising from side reactions, impurities in starting materials, isomerization, enantiomeric impurities, degradation prod­ ucts, residual solvents, and inorganic impurities. Drugs derived from biotechnological processes must also be tested for the components with which the drug has come in contact, such as the culture media proteins (albumin, transferrin, and insulin) and other additives such as testosterone. This is in addition to all the various viral and other adventitious agents whose absence must be demonstrated [3]. A 0.1% threshold for identification and isolation of impurities from all new molecular entities is under consideration by the International Con­ ference on Harmonization as an international regulatory standard [4,5]. However, where there is evidence to suggest the presence or formation of toxic impurities, identification should be attempted. An example of this is the 1500 reports of Eosinophilia-Mylagia Syndrome and more than 30 deaths associated with one impurity present in L-tryptophan which were present at the 0.0089% level [6]. The process of qualifying an individual impurity or a given impurity profile at a specified level(s) is summarized in Table 1.1. Safety studies can be conducted on the drug containing the impurity or on the isolated impu­ rity. Several decision trees have been proposed describing threshold levels Table 1.1 Criteria That Can Be Used for Impurity Qualification Impurities already present during preclinical studies and clinical trials Structurally identical metabolites present in animal and/or human studies Scientific literature Evaluation for the need for safety studies of a "decision tree" Regulatory Considerations for the Chromatographer 3 for qualification and for the safety studies that should be performed [4]. For example, a 0.1% threshold would apply when the daily dose exceeds 10 mg, and a 0.5% threshold at a daily dose of less than 0.1 mg. Alternatively, when daily doses exceed 1000 mg per day, levels below 0.1% would not have to be qualified, and for daily doses less than 1000 mg, no impurities need to be qualified unless their intake exceeds 1 mg. The USP [7] provides extensive discussion on impurities in sections 1086 (Impurities in Offical Articles), 466 (Ordinary Impurities), and 467 (Organic Volative Impurities). A total impurity level of 2.0% has been adopted as a general limit for bulk pharmaceuticals [5]. There have been no levels established for the presence of enantiomers in a drug substance/ product. This is primarily because the enantiomers may have similiar phar­ macological and toxicological profiles, enantiomers may rapidly interconvert in vitro and/or in vivo, one enantiomer is shown to be pharmacologi­ cally inactive, synthesis or isloation of the perferred enantiomer is not practical, and individual enantiomers exhibit different pharmacologic pro­ files and the racemate produces a superior therapeutic effect relative to either enantiomer alone [8,9]. For biotechnologically derived products the acceptable levels of for­ eign proteins should be based on the sensitivity/selectivity of the test method, the dose to be given to a patient, the frequency of administration of the drug, the source, and the potential immunogenicity of protein con­ taminants [10]. Levels of specific foreign proteins range from 4 ppm to 1000 ppm. The third category of drugs are phytotherapeutical preparations; 80% of the world population use exclusively plants for the treatment of illnesses [11]. Chromatography is relied on to guarantee preparations contain thera­ peutically effective doses of active drug and maintain constant batch com­ position. A quantitative determination of active principles is performed when possible, using pure reference standards. In many phytotherapeutic preparations, the active constituents are not known, so marker substances or typical constituents of the extract are used for the quantitative determi­ nation [11]. The Applications chapter of this book (Chapter 8) contains numerous references to the use of chromatographic methods in the control of plant extracts. Ш. STABILITY The International Conference on Harmonization (ICH) has developed guidelines for stability testing of new drug substances and products [1214]. The guideline outlines the core stability data package required for Registration Applications. 4 A. Adamovics Batch Selection For both the drug substance (bulk drug) and drug product (dosage form) stability information from accelerated and long-term testing should be provided on at least three batches with a minimum of 12 months' duration at the time of submission. The batches of drug substance must be manufactured to a minimum of pilot scale which follows the same synthetic route and method of manufacturer that is to be used on a manufacturing scale. For the drug product, two of the three batches should be at least pilot scale. The third may be smaller. As with the drug substance batches, the processes should mimic the intended drug product manufacturing procedure and quality specifications. B. Regulatory Considerations for the Chromatographer C. 5 Biologies Degradation pathways for proteins can be separated into two distinct classes; chemical and physical. Chemical instability is any process which involves modification of the protein by bond formation or cleavage. Physical instability refers to changes in the protein structure through denaturation, adsorption to surfaces, aggregation, and precipitation [15]. Stability studies to support a requested shelf life and storage condition must be run under real-time, real-temperature conditions [16,17]. The prediction of shelf life by using stability studies obtained under stress conditions and Arrhenius plots is not meaningful unless it has been demonstrated that the chemical reaction accounting for the degradation process follows first-order reaction. Storage Conditions IV. The stability storage conditions developed by the ICH are based on the four geographic regions of the world defined by climatic zones I ("temperate") and II ("subtropical"). Zones III and IV are areas with hot/dry and hot/ humid climates, respectively. The stability storage conditions as listed in Table 1.2 are arrived at by running average temperatures through an Arrhenius equation and factoring in humidity and packaging. Long-term testing for both drug substance and product will normally be every 3 months, over the first year, every 6 months over the second year, and then annually. A significant change in stability for drug substance is when the substance no longer meets specifications. For the drug product, a significant change is when there is a 5% change in potency, exceeded pH limits, dissolution failure, or physical attribute failure. If there are significant changes for all three storage temperatures, the drug substance/product should be labeled "store below 25 0 C." For instances where there are no significant changes label storage as 15-30 0 C. There should be a direct link between the label statement and the stability characteristics. The use of terms such as ambient or room temperature are unacceptable [12-14]. Table 1.2 Filing Stability Requirements at Time of Submission • 12 months long-term data (25°C/60% RH) • 6 months accelerated data (40°C/75% RH) • If significant change, 6 months accelerated data (30°C/60% RH) METHODVALIDATION The ultimate objective of the method validation process is to produce the best analytical results possible. To obtain such results, all of the variables of the method should be considered, including sampling procedure, sample preparation steps, type of chromatographic sorbent, mobile phase, and detection. The extent of the validation rigor depends on the purpose of the method. The primary focus of this section will be the validation of chromatographic methods. The four most common types of analytical procedures are identification tests, including quantitative measurements for impurities, content, limit tests for the control of impurities, and quantitative measure of the active component or other selected components in the drug substance [18]. Table 1.3 describes the performance characteristics that should be evaluated for the common types of analytical procedures [18]. A. Specificity The specificity of an analytical method is its ability to measure accurately an analyte in the presence of interferences that are known to be present in the product: synthetic precursors, excipients, enantiomers, and known (or likely) degradants that may be present. For separation techniques, this means that there is resolution of > 1.5 between the analyte of interest and the interferents. The means of satisfying the criteria of specificity differs for each type of analytical procedure: For identification, in the development phases, it would be proof of structure, whereas in quality control, it is comparison to Adamovics 6 >> о C о с се S I I I + I I S I с U тз а IU си 13 у >. "её С < оэ ее H H •- u с IU се ^ з о тз а S .-S , с U ' ев а § " 1 тз .S е 73 ,_ Г З < ° О и С О и ев U се Ui 3 и CJ < » м g тз ~, <- а Xi £ -в '§ S и.Ё и2 .§ S U .2 .* <л а и а а -2 о ' 3 «" S" иа S OS £ С* о и с ее Q С IU а се IU с J iu C ^_ тз • - — D ТЗ ТЗ U U U UO с се ой IU а ее тз < с тз тз V V u •S?5 i=i 8<-* « о о Szz о Regulatory Considerations for the Chromatographer 7 a reference substance [17]; for a purity test, to ensure that all analytical procedures allow an accurate statement of the content of impurities of an analyte [18,19]; for assay measurements, to ensure that the signal measured comes only from the substance being analyzed [18,19]. One practical approach to testing the specificity of an analytical method is to compare the test results of samples containing impurities ver­ sus those not containing impurities. The bias of the test is the difference in results between the two types of samples [20]. The assumption to this approach is that all the interferents are known and available to the analyst for the spiking studies. A more universal approach to demonstrating specificity of chromato­ graphic methods has been outlined [21]. For peak responses in highperformance liquid chromatography (HPLC), gas chromatography (GC), capillary electrophoresis (CE), or supercritical fluid chromatography (SFC) or the spots (bands) in TLC or gel electrophoresis, the primary task is to demonstrate that they represent a single component. The peak homogeneity of HPLC and GC as well CE and SFC responses can be shown by using a mass spectrometer as a specific detector. The constancy of the mass spec­ trum of the eluting peak with time is a demonstration of homogeneity, albeit not easily quantified [22]. Multiple ultraviolet (UV) wavelength detection has become a popular approach to evaluating chromatographic peak homogeneity. In the simplest form, the ratio between two preselected wavelengths is measured, and for a homogenous peak, the ratio remains constant. A ratio plot of pure com­ pounds appears as a square wave, whereas an impurity distorts the square (Fig. 1.1). This technique is most useful when the spectral properties of the overlapping compounds are sufficiently different and total chromatographic overlap does not occur [23]. The ability to detect peak overlap can be enhanced by stressing (heat, light, pH, and humidity) the analyte of interest and evaluating the wavelength ratios. A degradation of 10-15% is consid­ ered adequate. The utility of this approach has been demonstrated for pipercuronium bromide [23]. Potentially, additional information about peak purity can be obtained by recording UV-vis data at the upslope, apex, PURE COMPOUND IMPURE SAMPLE -TL Ги Figure 1Д Ratio plots. 8 Adamovics and downslope of a chromatographic response using photodiode array detection [24-27]. An example of this approach has been published for a method used in assaying an analgesic [28]. Peak purity can be assessed with a higher degree of certainty only by additional analysis using a significantly different chromatographic mode. The collected sample should also be analyzed by techniques that can be sensitive to minor structural differences such as nuclear magnetic resonance (NMR) spectroscopy [29-31]. B. Linearity The evaluation of linearity can be best described as the characterization of the test method response curve. A plot of the test method response against analyte concentration is often expected to be linear over a specified range of concentrations. Some assays generate nonlinear curves. The function of the standard curve is to allow the prediction of a sample concentration interpolated from the standard data. This predictive feature does not require linearity of the assay response curve, but only that it be a reasonable description of the correlation between response and concentration. Attempting, a rigorous fit of a calculated curve fitting to the standard data may defeat the function because such rigorous curve fitting may emphasize the difference between the sample and the standard assay responses. The test method response curve is characterized by comparing the goodness of fit of calculated concentrations with the actual concentrations of the standards. For a linear response, this value would be the correlation coefficient derived from a linear regression using least squares. Nonlinear response curves require curve fitting calculations with the corresponding goodness-of-fit determinations [32]. Plotting the test results graphically as a function of analyte concentration on appropriate graph paper may be an acceptable alternative to the regression line calculation. Experimentally, linearity is determined by a series of injections of standards at six different concentrations that span 50-150% of the expected working range assay [20]. The AOAC recommends 25-200% of the nominal range of analyte [33] using standards and spiked placebo samples [34]. Response linearity for known impurities at 0.05-5.0% of the target analyte should also be evaluated [28]. A linear regression equation applied to the results should have an intercept not significantly different from zero; if it does, it should be demonstrated that there is no effect on the accuracy of the method [20]. The range of an analytical method is the interval between the upper Regulatory Considerations for the Chromatographer 9 and lower level of analyte in the sample, for which it has been demonstrated that the method has a suitable level of precision, accuracy, and linearity. C. Limit of Measurement There are two categories within the level of measurement, the first is the limit of detection (LOD). This is the point at which a measured value is larger than the uncertainty associated with it; for example, the amount of sample exhibiting a response three times the baseline noise [34]. The limit of detection is commonly used to substantiate that an analyte concentration is above or below a certain level, in other words, a limit test [30,35]. The second category is referred to as the limit of quantitation. This limit is the lowest concentration of analyte in a sample that can be determined with acceptable precision and accuracy; for example, the lowest amount of analyte for which duplicate injections resulted in a relative standard deviation (RSD) of <2% [34]. Limit of quantitation is commonly used for impurity and degradant assays of drug substances and products [35]. The limit of measurement for an analyte is not a unique constant because of day-to-day variation in detector response. Extensive discussions of these limits have been published [36,37]. D. Precision (Random Error) The precision of a test method expresses the closeness of agreement among a series of measurements obtained from multiple sampling of the same homogenous sample. The concept of precision is measured by standard deviations. It can be subdivided into either two or three categories. The European Community (EC) [19] divides precision into repeatability and reproducibility. Repeatability expresses precision under conditions where there is the same analyst, the same equipment, a short interval of time, and identical reagents. This is also termed intra-assay precision. Reproducibility expresses the precision when the laboratories differ, when there are reagents from different sources, different analysts, tested on different days, equipment from different manufacturers, and so on. The Food and Drug Administration (FDA) [18] uses a three-category definition of precision. The same definition is used by the EC and FDA for repeatability. The FDA differs from EC by the term "intermediate precision" (see Table 1.3) which is determined within laboratory variation: different days, different analysts, different equipment, and so forth. Reproducibility expresses the precision between laboratories (collaborative studies). Several organizations differ in their approaches to collaborative studies: the United States Pharmacopeia Adamovics 10 uses procedures validated by pubic comment and ruggedness testing rather than a collaborative study process [38], whereas the International Union of Pure and Applied Chemistry's and AOAC Offical Methods of Analysis have developed harmonized procedures for collaborative studies [39]. The reproducibility standard deviation is typically two to three times as large as that for repeatability. Precision decreases with a decrease in concentration. This dependence has been expressed as RSD = 2 (1 "°- 5explogC) , where RSD is expressed as a percentage and C is the concentration of the analyte [38]. For the concentration ranges typically found in pharmaceutical dosage forms (1-10" 3 ), the RSD under conditions of repeatability should be less than 1.0%, and less than 2.0% under conditions of reproducibility [21]. These are similiar to the 1.5 0Zo recommendation made for RSD of system repeatability after analyzing a standard solution six times [35]. For method repeatability, which includes sample pretreatment, six replicate assays are made with a representative sample. A RSD no greater than 2% should be obtained. E. Accuracy Accuracy is the closeness of agreement between what is accepted as a true value (house standard, international standard) and the value found (mean value) after several replicates. This also provides an indication of systematic error. Two of the most common methods of determining accuracy are by comparing the proposed test procedure to a second test procedure whose accuracy is known and the recovery of drug above and below the range of use. Average recovery of the drug should be 98-102% of the theoretical value. Recoveries can be determined by either external or internal standard methods. Quantification by external standard is the most straightforward approach because the peak response of the reference standard is compared to the peak response of the sample. The standard solution concentration should be close to that expected in the sample solution. Peak responses are measured as either peak height or area [41]. For the internal standard method, a substance is added at the earliest possible point in the analytical scheme. This compensates for sample losses during extraction, cleanup, and final chromatographic analysis. There are two variations in the use of the internal standard technique. One involves the determination of response factors which are the ratios of the analyte peak response to the internal standard peak response. The second is referred to as response ratios which are calculated by dividing the weight of the analyte by the corresponding peak response. An internal standard must be completely resolved from all other peak Regulatory Considerations for the Chromatographer 11 responses except where mass discrimination or isotopically labeled samples are used as the internal standard. The internal standard should elute near the solute to be quantified. The detector response should be similiar in area or height to the analyte of interest. The internal standard should be similiar in terms of chemical and physical properties to the analyte being measured. Substances that are commonly used as internal standards include analogs, homologs, isomers, enantiomers, and isotopically labeled analogs of the analyte. The internal standard should not be present or be a potential degradant of the sample. Finally, the internal standard should be present in reasonably high purity. Internal standards are often used in dissolution testing of oral dosage forms [42]. Internal standards should be avoided in stability-indicating assays due to the possible coelution with unknown degradation products. F. Ruggedness (Robustness) The ruggedness of an analytical method is the absence of undue adverse influence on its reliability of performance by minor changes in the laboratory environment [43]. This validation parameter is not recognized by all organizations with testing oversight, as this characteristic is implied by collaborative validation programs (see Section IV.D). The difference in chromatographic performance between columns of the same designation (i.e., C,g) is the most common source of chromatographic variability. To check the column-to-column ruggedness, the specificty (selectivity) of at least three columns from three different batches supplied by one column manufacturer should be checked [44]. A similarly designated column from another manufacturer should also be evaluated. Table 1.4 lists the specifications recommended to define a liquid chromatographic column [45,46]. Testing procedures have also evolved for the evaluation of gas chromatographic capillary columns [47]. Variability is also caused by the degradation of the chromatographic column. Besides the sorbent stability, consideration should also be given to the stability of the sample solution. The widespread use of automatic sample injectors makes it necessary to determine the length of time that a sample is stable. V. SYSTEM SUITABILITY TESTING After a method has been validated, an overall system suitability test should be routinely run to determine if the operating system is performing properly. An acceptable approach is to prepare a solution containing the analyte and a suitable test compound. If the method being used is to control the

This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.