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Evaluation and Application of Best Practice in Analytical

Method Validation

Article  in  Journal of Liquid Chromatography & Related Technologies · February 2007

DOI: 10.1080/10826070601084753

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Evaluation and Application of Best Practice in Analytical Method

Validation
G. A. Shabir, T. K. Bradshaw, and S. A. Arain
LJLC208418 LJLC_030_003 Techset Composition Ltd, Salisbury, U.K. 11/10/2006

Journal of Liquid Chromatography & Related Technologiesw, 30: 1–23, 2007

Copyright # Taylor & Francis Group, LLC
1 ISSN 1082-6076 print/1520-572X online
2 DOI: 10.1080/10826070601084753
3
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8
Evaluation and Application of Best Practice
9
10 in Analytical Method Validation
11
12
13 Ghulam A. Shabir
14 Abbott Diabetes Care, Abbott Laboratories, Witney, Oxfordshire, U.K.
15
16 Tony K. Bradshaw
17 School of Life Sciences, Oxford Brookes University, Oxford, U.K.
18
19 Shafique A. Arain
20 Department of Pure and Applied Chemistry, University of Strathclyde,
Glasgow, U.K.
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22
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Abstract: Method validation is an important part of analytical chemistry to confirm
25
that the method employed for a specific test is suitable for its intended use. As such,
26
it is an essential requirement for any package of information submitted to regulatory
27 agencies in support of new product marketing or clinical trial applications. Currently,
28 there is no single source or final guideline on analytical method validation that helps
29 analysts to perform validation in a systematic manner. Therefore, industry depends
30 on the analyst’s knowledge and experience to develop simple and efficient
31 methods of analysis. The intention of this paper is to review regulatory requirements
32 and role of the pharmacopeias and to study how analytical method development and
33 validation are typically carried out at present, and to formulate this into a simple step
34 by step approach. Such a template was not only used as the foundation of this
research programme, but could also serve as a simple systematic guide for other
35
practitioners and those new to the field. Furthermore, it was recognized that
36
this protocol should satisfy the requirements of the most strategically important
37
regulatory bodies.
38
39 Keywords: Method validation, Pharmacopoeias role, Best practice, HPLC analysis,
40 Regulatory agencies
41
42
43 Address correspondence to Ghulam A. Shabir, Abbott Diabetes Care, Abbott
44 Laboratories, Witney, Oxfordshire OX29 0YL, U.K. E-mail: ghulam.shabir@abbott.
45 com

1
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2 G. A. Shabir, T. K. Bradshaw, and S. A. Arain

46 INTRODUCTION
47
48 Analytical method development and validation play a major role in the
49 discovery, development, and manufacture of pharmaceuticals. The official
50 test method that results from these processes are used by quality control lab-
51 oratories to ensure the identity, purity, potency, and performance of drug
52 product ‘quality’, essential for drug safety and efficacy. In the pharmaceutical
53 and biotechnology industries, a current major issue is the high cost of research
54 in introduction of new drugs. In essence, it takes several hundred million
55 dollars to discover, develop, and gain regulatory approval. One of the
56 reasons research and development (R&D) is so costly in pharmaceuticals is
57 that most new drug candidates fail to reach the market. Failure can result
58 from toxicity, carcinogenicity, manufacturing difficulties, inadequate
59 efficacy, and analytical problems. Therefore, there is a need for high through-
60 put in order to maximize patent lifetime and, consequently, generate the
61 profits to support the research and to increase the speed with which the
62 product can be delivered to the market. All the different stages of pharma-
63 ceutical R&D are underpinned by analysis so that high throughput is
64 actually dependent on effective and efficient analysis within which simple
65 effective method development and comprehensive analytical method vali-
66 dation is of fundamental importance. A wide variety of materials are used
67 in the pharmaceutical and diagnostic industries. All of these materials must
68 be analysed in some way or other and, just as importantly, the method of
69 analysis must be validated, i.e., it must be shown that the method is fit for
70 its intended purpose.
71 In the pharmaceutical industry, analytical method validation is very
72 much a major issue as analysis is used primarily to control drug quality.
73 This is important in its own right and, also, in that drug safety and
74 efficacy are dependent on it. Different chemical entities with varying
75 chemical and physical properties are used. These may include starting
76 materials, intermediates, final drug substances, and the final formulated
77 pharmaceutical products. The pharmaceutical analyst will be concerned
78 with applying analytical methods to the determination of stability/shelf
79 life, purity, side-product identity, dissolution, etc. Here, the analyst is
80 required to develop new methods of analysis appropriate to the information
81 required. In many cases, the analyte may be known but is present in a new
82 sample matrix, such that a new sample preparation method is needed. The
83 knowledge gained in the method development phase is important when it
84 comes to validating the method efficiently. Frequently, high performance
85 liquid chromatography (HPLC) is the analytical method of choice in
86 pharmaceutical analysis because of its specificity (i.e. all the components
87 of a sample are separated from one another before the measurement is
88 made, so that its results arise from the analyte and from nothing else).
89 Although HPLC is a relatively mature technique, the analyst is continually
90 required to innovate by adapting current methodology, or indeed,
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Analytical Method Validation 3

91 developing completely new protocols. For example, the coupling of HPLC

92 with another technique such as mass spectrometry (MS) can be especially
93 powerful.
94 In the diagnostic industry, the variety of materials is further expanded due
95 to the complexity of medical devices and their corresponding reagents. Such
96 materials may include polymers, surfactants, enzymes, cofactors, stabilizers,
97 etc. The diagnostic analyst is, therefore, required to apply other techniques
98 apart from HPLC in the analysis of key materials. An in depth knowledge
99 of the materials and their critical properties as applied to their use in the diag-
100 nostic device is necessary. Innovation is again needed if there is no directly
101 applicable methodology reported in the literature. Once an analytical
102 method is developed, validation is conducted in order to prove its use for
103 the intended application.
104 Validation is a critical step for any product release for marketing auth-
105 orization. The literature contains diverse approaches to performing method
106 validation.[1 – 8] Many analytical methods appearing in the literature have
107 not been through a thorough validation exercise and, thus, should be
108 treated with caution until full validation has been carried out.[9] Validation
109 of a new method is a costly and time consuming exercise. However, the
110 result of not carrying out method validation could result in litigation,
111 failure to get product approval, costly repeat analysis, and loss of
112 business and market share.[9] Validation is the proof needed to ensure
113 that an analytical method can produce results that are reliable, reproducible,
114 and are fit for the purpose intended. Choosing the validation criteria
115 depends on the method type. In general, method validation parameters
116 that should be studied are linearity, range, accuracy, precision (repeatability
117 and intermediate precision), specificity, limit of detection, and limit of
118 quantitation.
119 The International Conference on Harmonization (ICH) guidelines[10,11]
120 achieved a great deal in harmonizing the definitions of the required validation
121 characteristics and their basic requirements. However, they provide only a
122 basis for a general discussion of the validation parameters, their calculation
123 and interpretation. It is the responsibility of the analyst to identify parameters
124 that are relevant to the performance of the given analytical procedure, as well
125 as to design proper validation protocols including acceptance criteria and to
126 perform an appropriate evaluation.
127 Currently, there is no single source or final guideline on method vali-
128 dation that helps analysts to perform validation in a systematic manner.
129 Therefore, industry depends on the analyst’s knowledge and experience to
130 develop simple and efficient methods of analysis. The other major problem
131 pharmaceutical industries are facing in today’s world, is that different vali-
132 dation data requirements are required for regulatory submissions for product
133 approval depending upon the location of the regulatory body. For example,
134 the release of any pharmaceutical product in Europe, Japan, and USA
135 would require the use of ICH method validation criteria. However, the
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4 G. A. Shabir, T. K. Bradshaw, and S. A. Arain

136 release of the very same product, by the same industry, in any other part of the
137 world would force the use of their local regulatory guidelines. This inevitably
138 becomes a costly process due to issues of documentation and personnel
139 training, etc. Therefore, efforts are underway to streamline the method vali-
140 dation process through an idea commonly referred to as Harmonization by
141 ICH. As an example, Health Canada Drugs Directorate has already started
142 to align their method validation guidelines (Acceptable Methods) according
143 to ICH.
144 The outcome of ICH efforts has been accepted by most regulatory bodies
145 and pharmacopoeia, such as FDA[12,13] (the largest of the world’s drug regu-
146 latory agencies, FDA is responsible for the approval of all drug products used
147 in the USA) and USP.[14,15] Consequently, they have updated their general
148 chapters. The USP established in 1820, contains legally recognized standards
149 of identity, strength, quality, purity, packaging and labeling for drug sub-
150 stances, dosage forms, and other therapeutic products, including nutritional
151 and dietary supplements. USP also contains monographs, which are recog-
152 nized worldwide and may be enforceable by the US FDA and also by state
153 agencies in the US.
154 The ICH guidelines achieved a great deal in harmonizing the definitions
155 of the required validation characteristics and their basic requirements.
156 However, they provide only a basis for a general discussion of the validation
157 parameters, their calculation, and interpretation. However, this has not
158 removed the confusion in industries because ICH, as yet, has not explained
159 various other method types such as a response test (to detect a specific
160 substance in a sample as indicated by test signal response), concentration
161 test (for quantitation of a specific substance in a sample), physical test (for
162 determination of the physical characteristics of a product or material), and
163 cleaning test (for evaluating the cleanliness of equipment and areas used for
164 manufacturing). This impacts regulatory submissions.
165 The purpose of this review is (i) to critically evaluate current practices
166 in method development and analytical method validation, in order to
167 identify best practices, (ii) to apply best practices with some improvements,
168 in such a way as to ensure good quality and provide new knowledge on a
169 wide range of pharmaceutical substances, products, and compounds used in
170 pharmaceuticals, diagnostics, and, finally, (iii) to draw upon the outcomes
171 of the programme to be able to recommend the way forward with respect
172 to ensuring that the ever evolving approaches to analytical method develop-
173 ment and validation were enhanced, simple, systematic, efficient, and
174 effective, while still being compliant with the requirements of regulatory
175 agencies. Also, it is intended in this paper, to review and demonstrate
176 practical approaches to method validation in detail with reference to an
177 HPLC assay of 4-hydroxybenzoic acid ester (HBAE). HBAE alone or in
178 combination with other esters of p-hydroxybenzoic acid, or with other anti-
179 microbial agents, is used as a preservative in cosmetic and pharmaceutical
180 formulations.
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Analytical Method Validation 5

181 PHARMACOPOEIA ROLE AND GLOBAL HARMONISATION

182
183 Pharmacopoeial standards for medicinal products and auxiliary substances are
184 widely used for regulatory purposes in the fields of public health protection
185 and commerce. Small wonder, therefore, that, with the existence of some 37
186 pharmacopoeias worldwide, those engaged in the marketing of these sub-
187 stances on an international scale are often faced with the need to undertake
188 additional testing of their products beyond that which they may consider to
189 be necessary, in order to ensure compliance with the often divergent specifica-
190 tions of these different pharmacopoeias. Such additional testing is expensive
191 and difficult to justify in terms of patient protection. This problem has been
192 recognized by the three major pharmacopoeial authorities.
193 In June 1989, on the 25th anniversary of the European Pharmacopoeia
194 convention in Strasbourg and at the congress on the perspectives of inter-
195 national harmonization in Tokyo, multinational pharmaceutical companies
196 expressed their need for the harmonization of the pharmacopoeias of Japan,
197 Europe, and the US. The heads of these pharmacopoeias immediately
198 decided to organize regular contacts among themselves and a procedure for
199 rapprochement. In this way, the Pharmacopoeial Discussion Group (PDG)
200 was founded and meets twice a year. About 50 compendial monographs on
201 excipients and general methods of analysis proposed by national associations
202 of manufacturers of pharmaceutical products have been selected for conver-
203 gence and harmonization among the three pharmacopoeias. Proposals for har-
204 monized texts are regularly published in the forum of the three
205 pharmacopoeias for public enquiry (Pharmeuropa, US Pharmacopoeial
206 Forum and the Japanese Pharmacopoeial Forum).
207 As commented by Halperin,[16] harmonization at the world level rarely
208 means identical standards (unlike the results obtained in Europe), but rather
209 the elimination of elements of disharmony whenever possible and whenever
210 useful to international trade. Indeed, many parameters are involved and
211 there are many conflicts between monographs, methods of analysis, and
212 reagents. Furthermore, attaining identical standards is complicated by
213 expanding markets. Hence, the first stage of harmonization involves the elim-
214 ination of standards that are not scientifically justified, the revision of a
215 thorough evaluation of analytical test methods, obsolete specifications, and
216 the search for compatibility between the chosen standards. To be effective,
217 it requires much explanation and public relations between all the partners
218 concerned so that the constraints and limits of each are known.
219 The pharmacopoeias also participate in the work on the rapprochement of
220 licensing dossiers within the framework of ICH. ICH is a joint initiative
221 involving both regulatory bodies and the pharmaceutical research based
222 industry (Europe, Japan and the United States of America) as equal partners
223 in the scientific and technical discussions of the testing procedures that are
224 required to ensure and assess the safety, quality, and efficacy of medicines.
225 The pharmacopoeias have observer status in the quality working party and
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6 G. A. Shabir, T. K. Bradshaw, and S. A. Arain

226 biotech working party and, notably, have participated in the elaboration of
227 guidelines on analytical validation, impurities, residual solvents, and specifi-
228 cations. Where appropriate, they integrate the general principles of these
229 guidelines into their specifications.
230 Following the adoption of the last quality guidelines by ICH steering
231 committee in October 1989 on specifications, test procedures, and acceptance
232 criteria for new drug substances and new drug products, and chemical sub-
233 stances, it is intended that the regulators of the three regions (U.S. FDA,
234 EU/Europe and MHW Japan) will recognize as interchangeable, procedure
235 and acceptance criteria of any of the three pharmacopoeias where harmoniza-
236 tion of this procedure and criteria have been successfully completed. To
237 signify the harmonized status of these procedures, the three pharmacopoeias
238 have agreed to include a statement in their respective texts that indicates
239 that the procedures and acceptance criteria from all three pharmacopoeias
240 are considered equivalent and are, therefore, interchangeable. This
241 agreement takes effect as soon as the three pharmacopoeias publish the
242 common text that was agreed on.
243
244
245 CRITICAL EVALUATION OF CURRENT BEST PRACTICE IN
246 ANALYTICAL METHOD DEVELOPMENT AND VALIDATION
247
248 The first stage of the programme was to study how analytical method devel-
249 opment and validation is typically carried out at present, and to formulate
250 this into a simple step by step approach. Such a template[17] was not only
251 used as the foundation of this research programme but could also serve as a
252 simple systematic guide for other practitioners and those new to the field. Fur-
253 thermore, it was recognized that this protocol should satisfy the requirements
254 of the most strategically important regulatory bodies. These requirements
255 were critically evaluated, identifying the key similarities and, more impor-
256 tantly, differences between the validation requirements of the FDA, USP,
257 and ICH.[18] The aim of the field was to take forward to apply the identified
258 best practices to studies of a diverse range of pharmaceutical substances,
259 products, and compounds used in pharmaceuticals and diagnostics.[19 – 26]
260 Everyday, many analysts face the need and challenge to develop and
261 validate HPLC, LC-MS, and GC methods. Whereas individual’s approaches
262 may exhibit considerable diversity, a best practice method development and
263 validation follows the systematic approach (Figure 1). This is a highly suc-
264 cessful approach to a method development and validation process. Before
265 embarking on the development of a new method, always search the literature
266 to see if a suitable one already exists. If a suitable one is found, it will still be
267 necessary to perform some method optimisation and validation to prove that
268 the method can be successfully adapted for its intended use.
269 In the feasibility phase, the analyst will determine whether the assigned
270 task can be successfully accomplished by using available resources.
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Analytical Method Validation 7

271
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288
289 Figure 1. Systematic approach in analytical method development and validation.
290
291
292 Research is defined as the activity aimed at discovering new knowledge on the
293 compound in hopes that such information will be useful in developing a new
294 method. Development is the translation of research findings into a new
295 analytical method and the systematic use of knowledge or understanding
296 gained from research directed toward the analytical methods, including the
297 design and development of prototypes and processes. Robustness studies
298 must be considered in this phase. Robustness: Measure of a method’s
299 capacity to remain unaffected by small but deliberate variations in method par-
300 ameters. The development phase must also include system suitability testing
301 and stability of analytical solutions, as well as mobile phase.
302 In optimization study, a developed method can be further improved to
303 gain greater confidence on the generation of analytical data; the search for
304 the best solution among alternatives, or the extreme value of a variable. The
305 current developed approach emphasis the allocation of greater resources
306 during the development and optimization phases. This allows the analyst to
307 have more confidence on the quality of data generated and, therefore, con-
308 siderably reduces the resources that are required for the process of validation.
309 The purpose of the characterization study is to determine reliable method
310 performance limits from the analytical performance characteristics and set
311 acceptance criteria for the test method validation. As a best practice, the
312 characterization protocol needs to be written and approved before
313 execution. Prior to execution of the protocol, it is necessary that the analytical
314 system itself is adequately designed, maintained, calibrated, and qualified. In
315 all cases proper validation documentation should be archived to support the
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8 G. A. Shabir, T. K. Bradshaw, and S. A. Arain

316 qualification process. All personnel involved in the characterization protocol

317 activities must be trained prior to performing their function. On completion
318 of the characterization study, the results/data should be critically assessed
319 from a statistical point of view.
320 Validation is the last and critical step for the success of the whole method
321 development project. If the validation fails, it can be seen as a wasted resource
322 and inevitably can delay the product release date. Here, validation protocol
323 needs to be written and approved by an appropriate cross functional team.
324 Upon successfully completing the validation, the data and its acceptance
325 criteria should be statistically analyzed by appropriate experts in order to
326 test its validity.
327 Timely implementation/method transfer plays an important role in expedi-
328 ting drug candidates through development stages. Method transfer is not a trivial
329 task and requires careful planning and constant communication between the lab-
330 oratory personnel involved in the transfer. Method transfer could occur within
331 the same organization or between pharmaceutical companies and analytical
332 service providers. To have a successful transfer, the analytical method itself
333 must be robust and the equipment differences between the delivering and
334 receiving parties should be carefully evaluated. Unfortunately, very limited
335 information on method transfer can be found in the literature. Typically in
336 any organization, before the method transfer, scientists from both sites need
337 to go through the method details very carefully. As a best practice and successful
338 transfer of analytical method, prepare a method transfer validation protocol that
339 is agreed by the both sites, approved, and executed.
340
341
342 METHOD DEVELOPMENT AND MODERN ANALYTICAL
343 TECHNIQUES
344
345 Method development is not always a simple task since there are a substantial
346 number of parameters in HPLC, which may influence the final results that are
347 obtained. Specially, when the required method does not exist in the literature,
348 the analyst needs advanced knowledge and experience on both analytical
349 equipment and drug substance, or drug product, that need to be analyzed. In
350 this situation, applying a systematic approach, as discussed above, can
351 make a task simple, and reduces resources of the company.
352 Reversed-phase chromatography is probably the most commonly used
353 separation mechanism in liquid chromatography and consists of a non-polar
354 stationary phase (normally octadecyl, C18 or octyl C8 chains) bonded to a
355 solid support that is generally micro particulate silica gel (non-polar). The
356 mobile phase is polar and, therefore, the sample compounds are partitioned
357 between the mobile and the stationary phases. The separation is normally
358 performed using aqueous mobile phase containing different percentages of
359 organic modifiers (e.g., methanol, ethanol, acetonitrile, or THF) to increase
360 the selectivity between species. Solute retention is also influenced by eluent
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Analytical Method Validation 9

361 pH, which affects the dissociation level of the analyte and, therefore, its
362 partition between the mobile and stationary phases.
363 Mass spectrometry has progressed extremely rapidly during the last
364 decade: production, separation, and detection of ions, data acquisition, data
365 reduction, etc. This has led to the development of entirely new modern instru-
366 ments and applications.
367 The combination of chromatographic separations with mass spectro-
368 metric detection is considered an indispensable tool for problem solving in
369 analytical chemistry and, increasingly, for routine analytical methods. Mass
370 spectrometric detection brings an added level of information, complementary
371 to the chromatographic process that improves the certainty of identification
372 and the specificity of detection. Mass spectral information can generally be
373 obtained from a sample size typical of common analytical methods. In the
374 last 10 years, research efforts in the field of HPLC-MS have changed consider-
375 ably. HPLC-MS has rapidly matured to become a very powerful and useful
376 analytical tool that is widely applied in many areas of chemistry, pharma-
377 ceutical sciences, and biochemistry. Investigation into the coupling of
378 HPLC and MS began in the early 1970s. In the first 20 years, most of the
379 attention had to be given to solving interface problems and building new tech-
380 nology. However, most scientists with HPLC-MS today are only concerned
381 with application of the commercially available techniques in their field of
382 interest. Technological problems in interfacing appear to be solved, and
383 from the wide variety of interface developed over the years, basically only
384 two remain, i.e., electrospray ionization (ESI) and atmospheric-pressure
385 chemical ionization (APCI), which are both atmospheric-pressure ionization
386 (API) techniques. With ESI and APCI, HPLC-MS has been implemented in
387 analytical strategies in many application areas, e.g., environmental analysis,
388 drug development within the pharmaceutical industry, characterization of
389 natural products, and the characterization of biomolecules like peptides,
390 proteins, oligosaccharides, etc.
391 The selection of the appropriate HPLC conditions, whether reversed-
392 phase liquid chromatography, ion-pairing chromatography, capillary electro-
393 phoresis, or ion chromatography, and of the most sensitive ionization mode,
394 ESI or APCI, depends upon the polarity and acidity of the analyte. The ESI
395 is best applied to the highly polar nature of the analyte and APCI ionizes
396 most efficiently compounds with low to moderately high polarities and, in
397 this respect is complementary to electrospray, which gives the best sensitivity
398 for ionic compounds. Both interfaces, ESI and APCI, can be operated in
399 positive and negative ion mode. Often, an appropriate selection for a given
400 analyte can be made by considering that ESI transfers ions from solution
401 into the gas phase, whereas APCI ionizes in the gas phase. As a rule of
402 thumb, analytes occurring as ions in solution may be best analyzed by ESI,
403 while non-ionic analytes may be well suited for APCI.
404 As for the other detection principles discussed above, all of these contrib-
405 ute significantly to the present day success of hyphenation in HPLC. There is
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10 G. A. Shabir, T. K. Bradshaw, and S. A. Arain

406 no doubt that, also, today, HPLC-PDA UV plays an important role (detection
407 and peak-purity) in many research and development studies, and for a wide
408 variety of routine analyses.
409
410
411 EXPERIMENTAL
412
413 Chemicals and Reagents
414
415 4-hydroxybenzoic acid ester (Batch #1005425) was obtained from Lancaster
416 Synthesis (Morecambe, England). HPLC grade acetonitrile was obtained
417 from Merck (Darmstadt, Germany). Deionized distilled water was used
418 throughout the experimental study.
419
420
HPLC Instrumentation
421
422
HPLC analysis was performed using a Waters Alliance 2690 Separations
423
model with a 996 Waters PDA detector system (Waters, Elstree, UK). The
424
second HPLC system was used for intermediate precision studies and
425
consisted of Perkin Elmer (Norwalk, CT) equipped with a model series 200
426
UV Visible detector, series 200 LC pump, series 200 autosampler, and series
427
200 peltier LC column oven, using a Symmetry C18 column (3.9
150 mm,
428
5 mm) at ambient temperature. The mobile phase was acetonitrile/water
429
(65:35, v/v). The mobile phase was filtered through a 0.45 mm membrane
430
filter and degassed before use. The flow rate was set at 1.0 mL/min. UV
431
detection was performed at 254 nm and volume of sample injected was 20 mL.
432
433
434 Preparation of Standard and Sample Solutions
435
436 HBAE (100 mg) was accurately weighed and added to a 100 mL volumetric
437 flask before being dissolved in acetonitrile. A 2.0 mL aliquot of stock
438 solution was diluted to 100 mL in the mobile phase, yielding a final concen-
439 tration of 20 mg/mL. Standard solutions for the evaluation of HBAE
440 linearity were prepared over a concentration range of 5.0– 40 mg/mL, to 25,
441 50, 75, 100, 150, and 200% in the mobile phase.
442
443
444 RESULTS AND DISCUSSIONS
445
446 Method Validation
447
448 Prior to method validation in the pharmaceutical and diagnostic industries,
449 analytical equipment must be qualified (installation, operational and perform-
450 ance qualification), as well as software validated in compliance with the U.S.
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Analytical Method Validation 11

451 Code of Federal Regulations (FDA 21 CFR Part 11). Best practices in method
452 development and validation is equally important in the analysis of active and
453 inactive components in formulated products. In this study, a simple and robust
454 HPLC assay method for determining the content of HBAE was validated.
455
456
457
458 Linearity and Range
459
460 The linearity of the method should be tested in order to demonstrate a pro-
461 portional relationship of response versus analyte concentration over the
462 working range. The linearity range for evaluation depends on the intended
463 use of the analytical method. The ICH guidelines specified a minimum of
464 five concentration levels, along with certain minimum specified ranges. For
465 an assay, the minimum specified range is from 80– 120% of the target concen-
466 tration. For an impurity test, the minimum range is from the reporting level of
467 each impurity to 120% of the specification. Additional suggestions for the
468 appropriate range are available in other literatures.[27 – 32] Acceptability of
469 linearity data is often judged by examining the correlation coefficient and
470 y-intercept of the linear regression line for the response versus concentration
471 plot. The regression coefficient (r2) is .0.998 is generally considered as
472 evidence of acceptable fit of the data to the regression line. The y-intercept
473 should be less than a few percent of the response obtained for the analyte at
474 the target level. The percent relative standard deviation (RSD), intercept,
475 and slope should be calculated.
476 In the present study, linearity was studied in the concentration range
477 5.0–40 mg/mL (25–200% of nominal concentration, n ¼ 3) and
478 the following regression equation was found by plotting the peak area (y)
479 versus the HBAE concentration (x) expressed in mg/mL: y ¼
480 29935x þ 51338 (r2 ¼ 1.000). The correlation coefficient (r2) obtained for
481 the regression line demonstrates the excellent relationship between peak
482 area and concentration of HBAE (Table 1). The range is derived from
483 linearity studies and depends on the intended application of the test method.
484 It is established by confirming that the assay procedure provides an acceptable
485 degree of linearity, accuracy, and precision when applied to samples contain-
486 ing amounts of analyte within, or at the extremes, of the specified range of the
487 test method. The range is normally expressed in the same units as the test
488 results obtained by the method. In this study, the data obtained during the
489 linearity and accuracy studies was used to assess the range of the assay
490 method. The precision data for this assessment was the precision of the
491 three replicate samples analyzed at each level in the accuracy studies. The
492 valid analytical range of the method is that range of concentrations, which
493 pass the linearity and accuracy criteria, and yields an RSD of ,2%. The
494 linearity data described earlier demonstrates acceptable linearity for HBAE
495 over the range of 80 to 120% of the target concentration.
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12 G. A. Shabir, T. K. Bradshaw, and S. A. Arain

496 Table 1. Method validation results

497
Validation Acceptance
498
steps Parameter criteria Results
499
500 Repeatability Retention time (min) 2 0.09
501 (n ¼ 10) RSD (%)
502 Peak area RSD (%) 2 0.13
503 Peak height RSD (%) 2 0.16
504 Linearity Correlation coefficient (r2) .0.998 R 2 ¼ 1.000
505 (n ¼ 3)
506
Equation for regression line Y ¼ 29936x þ 51337
LOD s/n ratio s/n ¼ 3:1 (s/n 3.3), 2.5 hg ml
507
LOQ s/n ratio s/n ¼ 10:1 (s/n 10.2), 5.5 hg ml
508
509
510
511 Accuracy/Recovery Studies
512
513 The accuracy of an analytical method is the closeness of test results obtained
514 by that method to the true value. Accuracy is usually determined in one of four
515 ways. First, accuracy can be assessed by analyzing a sample of known concen-
516 tration (reference materials), and comparing the measured value to the true
517 value. The second approach is to compare test results from the new method
518 with results from an existing alternate well characterized procedure that is
519 known to be accurate. The third approach is based on the recovery of
520 known amounts of analytes. This is performed by spiking analytes in blank
521 matrices. For assay methods, spiked samples are prepared in triplicate at
522 three levels over a range of 50– 150% of the target concentration. The
523 percent recovery should then be calculated. The fourth approach is the
524 technique of standard additions, which can also be used to determine
525 recovery of spiked analytes. This approach is used if it is not possible to
526 prepare a blank sample matrix without the presence of the analyte. In this
527 respect, the mean recovery should be 100 + 2% at each concentration over
528 the range of 80– 120% of the target concentration. The ICH recommends col-
529 lecting data from a minimum of nine determinations over a minimum of three
530 concentration levels covering the specified range (e.g., three concentrations,
531 three replicates each).
532 In the present study, a number of different solutions were prepared with
533 known added amounts of HBAE and injected in triplicate. Percent recoveries
534 of response factor (area/concentration) were calculated. The results of
535 accuracy studies are shown in Table 2, and it is evident that the method is
536 accurate within the desired recovery range. The RSD values obtained for
537 the recovery of HBAE at 50, 75, 100, and 150% of target are 0.15, 0.19,
538 0.14, and 0.12%, respectively. Each value was the result of three individual
539 sample preparations and analyses. These data support a method range of 80
540 to 120% of the target concentration.
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Analytical Method Validation 13

541 Table 2. Recovery studies of HBAE from samples with known

542 concentration
543
Percent of Recovery (%) RSD
544
Sample nominal (n ¼ 3) (%)
545
546 1 50 99.66 0.15
547 2 75 99.79 0.19
548 3 100 99.88 0.14
549 4 150 99.86 0.12
550 Mean 99.80
551
552
553
Specificity
554
555
In order to design a chromatographic system for the analysis of an active
556
component of a pharmaceutical product, it is essential to have a good
557
knowledge of; (a) susceptibility of the drug to degradation and its degra-
558
dation pathway; (b) assay interference by possible degradants or synthesis
559
precursors; and (c) assay interference by chemicals employed in sample
560
preparation and excipients in the formulation. Degradation products may
561
be formed by acid/base hydrolysis, oxidation, Ultraviolet (UV) irradiation,
562
heat, light, etc.
563
In the present study, initially, a reference standard of HBAE was chro-
564
matographed. Figure 2 clearly demonstrates that HBAE is well separated
565
from any potential interference. Assay interference was investigated by
566
injecting a placebo. No interfering peaks (Figure 3) were observed. Forced
567
568
569
570
571
572
573
574
575
576
577
578
579
580
581
582
583
584
585 Figure 2. HPLC chromatogram of HBAE.
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14 G. A. Shabir, T. K. Bradshaw, and S. A. Arain

586
587
588
589
590
591
592
593
594
595
596
597
598
599
600
601
602 Figure 3. HPLC chromatogram of placebo.
603
604
605
degradation studies were also performed to evaluate the specificity of HBAE
606
under four stress conditions (heat, UV light, acid, base) (Table 3). Solutions Q1
607
of HBAE were exposed to 508C for 1 h, UV light using a Mineralight
608
UVGL-58 light for 24 h, acid (1 M HCl) for 24 h, and base (1 M NaOH)
609
for 4 h. A summary data of the stress results is presented in Table 4,
610
which showed no changes in retention times of HBAE and no degradation
611
peaks were detected. The peak at 1.66 min is identified as that due to
612
HBAE, since its UV spectrum matches that of a known sample of HBAE
613
as shown in Figure 4. Q1
614
615 Precision Studies
616
617 Precision is the measure of the degree of repeatability of an analytical
618 method under normal operation, and is normally expressed as the percent
619 relative standard deviation for a statistically significant number of
620
621
Table 3. Assay (%) of HBAE under stress conditions
622
623 Stress Sample RT (min) Assay (%)
624 conditions treatment (HBAE) (HBAE)
625
626 Reference Fresh solution 1.66 99.78
Acid 1 M HCl for 24 h 1.65 99.71
627
Base 1 M NaOH for 4 h 1.65 99.80
628
Heat 508C for 1 h 1.66 99.82
629
Light UV light for 24 h 1.65 99.79
630
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Analytical Method Validation 15

631 Table 4. Demonstration of the intermediate precision of the HPLC assay

632
HPLC1 HPLC2
633
634 S1 S2 S3 S1 S2 S3
635 Sample (50%) (100%) (150%) (50%) (100%) (150%)
636
637 Operator 1, day 1 99.84 99.80 99.77 99.75 99.82 99.82
638 Operator 1, day 2 99.77 99.75 99.75 99.81 99.79 99.77
639
Operator 2, day 1 99.70 99.76 99.76 99.76 99.76 99.70
Operator 2, day 2 99.54 99.62 99.59 99.78 99.81 99.80
640
Mean (HPLC1 & 2) 99.71 99.73 99.73 99.78 99.80 99.76
641
Mean (Operators) 99.80 99.79 99.78 99.70 99.73 99.71
642 RSD (criteria 2%) HPLC1 S1 þ HPLC2 S1 ¼ 0.05; HPLC1 S2 þ HPLC2
643 HPLC 1 & 2 S2 ¼ 0.06; HPLC1 S3 þ HPLC2 S3 ¼ 0.05
644 RSD (criteria 2%) HPLC1 S1 þ HPLC2 S1 ¼ 0.06; HPLC1 S2 þ HPLC2
645 operators S2 ¼ 0.04; HPLC1 S3 þ HPLC2 S3 ¼ 0.05
646
647
648 samples. Precision may be performed at three different levels: repeatability,
649 intermediate precision, and reproducibility. Repeatability (intra-day assay
650 precision) is the results of the method operating over a short time interval
651 under the same conditions (intra-assay precision). It should be determined
652 from a minimum of nine determinations covering the specified range of
653 the procedure (for example, three levels, three repetitions each), or from a
654 minimum of six determinations at 100% of the test or target concentration.
655 A precision criterion for an assay method is that the instrument precision
656 (RSD) will be 1%, and for the impurity assay, at the limit of quantitation,
657 the instrument precision (repeatability) will be 5%. Documentation in
658 support of precision studies should include the standard deviation, relative
659 standard deviation, coefficient of variation, and the confidence interval. In
660 this study, precision of the method was evaluated through the repeatability
661 of the method (intra-assay precision) by assaying ten replicate injections
662 of HBAE at the same concentration (20 mg/mL), during the same day,
663 under the same experimental conditions. The RSD values of the retention
664 time, area, and height of HBAE peak were found to be ,0.20% as shown
665 in Table 1.
666 Intermediate precision (inter-day variation) is the results from lab vari-
667 ations, due to random events, such as different days, analysts, equipment,
668 etc. In determining intermediate precision, experimental design should be
669 employed, so that the effects (if any) of the individual variables can be
670 monitored. Precision criteria for an assay method is that the intra-assay
671 precision will be 2%, and for an impurity assay at the limit of quantitation,
672 the instrument precision will be 5%, and the intra-assay precision will be
673 10%. In this study, intermediate precision (within-laboratory variation)
674 was demonstrated by two operators, using two HPLC systems, and evaluating
675 the relative percent purity data across the two HPLC systems at three
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16 G. A. Shabir, T. K. Bradshaw, and S. A. Arain

676
677
678
679
680
681
682
683
684
685
686
687
688
689
690
691
692
693
694
695
696
697
698
699
700 Figure 4. PDA UV match spectra of the middle of the peak corresponding to the RT
701 of the main component HBAE and a reference sample.
702
703
704 concentration levels (50%, 100%, 150%) that cover the HBAE assay
705 method range (5.0 –40 mg/mL). The mean and RSD across the HPLC
706 systems and analysts were calculated from the individual relative percent
707 purity mean values at 50, 100, and 150% of the test concentration.
708 The RSD values presented in Table 4 were less than 1% for both HPLC
709 systems and operators, and illustrated the good precision of the analytical
710 method.
711 Reproducibility is determined by testing homogeneous samples in
712 multiple laboratories, often as part of inter-laboratory crossover studies. An
713 example of reproducibility criteria for an assay method could be that the
714 assay results obtained in multiple laboratories will be statistically equivalent,
715 or the mean results will be within 2% of the value obtained by the primary
716 testing laboratory. For an impurity method, results obtained in multiple lab-
717 oratories will be statistically equivalent, or the mean results will be within
718 10% (relative) of the value obtained by the primary testing lab for impurities.
719 Reproducibility is not normally expected if intermediate precision is
720 performed.
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Analytical Method Validation 17

721 Limit of Detection and Quantitation

722
723 The limit of detection (LOD) and limit of quantitation (LOQ) tests for the
724 procedure are performed on samples containing very low concentrations of
725 analyte. LOD is defined as the lowest amount of analyte that can be
726 detected above baseline noise; typically, three times the noise level. LOQ is
727 defined as the lowest amount of analyte, which can be reproducibly quanti-
728 tated above the baseline noise, that gives s/n ¼ 10. In this study, LOD for a
729 20 mL injection of HBAE standard (s/n ¼ 3.3) was 2.5 hg/mL (Figure 5,
730 Table 1) and LOQ (s/n ¼ 10.2) was 5.5 hg/mL (Figure 6, Table 1) and
731 RSD ,2% (n ¼ 6).
732
733
CONCLUSIONS AND SUGGESTIONS FOR FURTHER WORK
734
735
The efficient analytical method validation is a critical element in the develop-
736
ment of pharmaceuticals. Indeed, the principle of the validation of these
737
methods is today widely spread in all the domains of activities where measure-
738
ments are made. Nevertheless, the simple question of acceptability, or not, of
739
an analytical method for a given application, remains incompletely deter-
740
mined in several cases, despite the various regulations relating to good
741
practices (GLP, GMP, . . .) and other documents of normative character
742
(ICH, USP, FDA, . . .). There are many official documents describing the
743
criteria of validation to be tested, but they do not propose any simple and sys-
744
tematic approach to experimental/validation activities and limit themselves
745
most often to the general concepts.
746
747
748
749
750
751
752
753
754
755
756
757
758
759
760
761
762
763
764 Figure 5. HPLC chromatogram for limit of detection of HBAE. Sample concen-
765 tration 2.5 hg/mL.
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18 G. A. Shabir, T. K. Bradshaw, and S. A. Arain

766
767
768
769
770
771
772
773
774
775
776
777
778
779
780
781
782 Figure 6. HPLC chromatogram for limit of quantitation of HBAE. Sample concen-
783 tration 5.5 hg/mL.
784
785
786 The concept of validation covered in the literature is mostly associated
787 with development and validation of chromatographic methods. The descrip-
788 tion of equipment qualification is also discussed in the literature to a lesser
789 extent.[33 – 35] However, the description of instrument qualification generally
790 does not include the need to validate the computer aspect of the instrument
791 (software and computer hardware), which should be considered an
792 important part of the qualification package prior to method validation.
793 Another one of the critical issues that have not been addressed by the
794 consensus reports is when method validation is necessary. In the current
795 highly cost conscious environment, the balance of costs and benefits is an
796 issue. The literature and regulatory agencies contain diverse approaches to
797 performing method validation as discussed above; there is a need for a
798 single guideline worldwide on performing method validation. ICH should
799 expand their effort on method validation globally for more input and set the
800 minimum standard “one world –one standard,” which ensures patient safety.
801 Alternative guidelines to ICH are not preferred; the world should stay with
802 ICH to achieve global harmonization. This is because it is easier to revise
803 existing guidelines that are already in operation. The guideline should cover
804 step by step approaches from drug development to marketing authorization.
805 The guideline should also cover all the prevalidation requirement activities,
806 such as analytical instrument qualification that is another aspect of method
807 validation. The benefits to the regulated industry of achieving the desired
808 state (globally harmonized) will ensure, better quality, less recalls, less sup-
809 plements, and facilitate new technology and continuous improvement.
810 Focus will be on critical quality attributes and controls and will reduce the
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Analytical Method Validation 19

811 regulatory burden of post approval changes. The benefits to the regulators are
812 that they will be receiving relevant information concerning product under-
813 standing, allow consideration of product design, critical process control, and
814 critical quality attributes, etc., for regulatory decision and, hence, reduce the
815 burden upon regulatory resources. USP and FDA have accepted ICH
816 documents and have updated general chapters, but old methods do not meet
817 the criteria (e.g., TLC) and are currently not being updated. A major
818 challenge to many pharmaceutical industries of today who still use old
819 validated methods is that they need to upgrade/revalidate in order to meet
820 current regulatory standards. The USP28-NF23 contains over 4000 mono-
821 graphs and over 180 general chapters. Approximately 200 drug substances,
822 excipients, and drug product monographs are needed. Approximately
823 800– 1200 current monographs need to be updated. USP works with the
824 European and the Japanese Pharmacopoeias to harmonize excipients, mono-
825 graphs, and general chapters. The goal is to achieve regulatory interchange
826 ability.
827
828
829 Current Status of Compendial Monographs/Specifications
830
831 Monograph Development
832
833 Anyone can be a sponsor of a monograph. The sponsor develops and submits
834 monographs to USP based on their company’s timeline. Most of company’s
835 timelines are approximately three years prior to patent expiry. The
836 monograph typically resembles the specification and methods, which are
837 filed and approved by the FDA. The USP submits the monograph to the appro-
838 priate expert committee for review.
839
840
841
Monograph Revision
842
843
Monographs in the USP are “live” documents and are constantly being
844
revised. Anyone can submit a revision to an official monograph. Limits are
845
continually being changed, tightened, and/or widened.
846
847
848 Challenges of the Current System
849
850 The USP expert committee may challenge the specifications and methods in
851 the monograph submission. Methods may be changed, this presents a
852 problem since limits and methods are linked. Despite the fact that the
853 methods and specifications are reviewed and approved by the FDA, the
854 expert committee may ask for additional information to justify the proposed
855 specifications and methods.
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20 G. A. Shabir, T. K. Bradshaw, and S. A. Arain

856 Consideration for the Future

857
858 So . . . where are we going? Should we start with a blank piece of paper and
859 develop a “desired state” for pharmacopoeias and the public standards?
860 Does it make sense to retrofit for compendial standards from 1820 to the
861 “21st century? The testing of compendial standards and the specification
862 process has remained nearly unchanged. Adoption for the future is a must
863 and reassessment, reevaluation, and evolution is necessary. It is critical
864 for the USP to be engaged with the changes and paradigm shift occurring
865 at FDA. Specifications must be based on FDA approved materials. The
866 purpose of a monograph needs to be reassessed. The role of the
867 monograph in release, stability, and marketing surveillance needs to be
868 reevaluated.
869
870
871 Evolution and Application of General Chapters
872
873 How will they be impacted by the changes and be applied (enforced) in the
874 future? How will general chapters be developed to adapt to new paradigm
875 and existing chapters be modified? Will there be dual standards in the USP
876 to accommodate the new approaches, and how will content uniformity be
877 addressed? Should it be addressed in the USP, or should the current chapter
878 remain and companies left with the option of different specifications/
879 methods based on agreements with the FDA? Chapters should not duplicate
880 efforts underway in other areas (e.g., American Society for Testing and
881 Materials (ASTM) International Standards).
882
883
884 Global Harmonization
885
886 The path that the USP takes must be carefully considered. Since the USP
887 has been engaged with the Pharmacopoeial Discussion Group (PDG) on
888 harmonization of general chapters and monographs, it is important to
889 move forward in collaboration with the other Pharmacopoeias and not be
890 in isolation. We must not “undo” the work that has been done by the
891 PDG. The USP should work with the PDG to ensure that the current har-
892 monized items are not negatively impacted and also work prospectively
893 to harmonize new concepts.
894 Also, input from the pharmaceutical industry and other users of their
895 volumes, is, therefore, essential in the provision of information as to what is
896 most needed in the prioritization and harmonization of the work programs.
897 In a continually changing environment, the PDG looks to industry to
898 produce suggestions as to what issues need to be addressed in the formulation
899 of its work programs. Industry professionals are, therefore, urged to take a
900 keen and active interest in the work of the PDG, to monitor its progress by
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Analytical Method Validation 21

901 reading the appropriate forums and to let it know where major problems are
902 occurring.
903
904
905
In Overall Conclusion
906
907
The status quo is no longer adequate, evolution is necessary. In order to
908
prepare for the future, we must now critically evaluate the role of monographs
909
and general chapters and consider what changes must occur. These must be
910
linked to the changes occurring at the FDA and industry. The USP must
911
engage the FDA, industry, and PDG in the evolution process.
912
913
914
915 REFERENCES
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Analytical Method Validation 23

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995
996
997 Received September 28, 2006
998 Accepted October 29, 2006
999 Manuscript 6957
1000
1001
1002
1003
1004
1005
1006
1007
1008
1009
1010
1011
1012
1013
1014
1015
1016
1017
1018
1019
1020
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