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Guidance for Industry

Analytical Procedures and

Methods Validation

Chemistry, Manufacturing, and Controls Documentation

DRAFT GUIDANCE

This guidance document is being distributed for comment purposes only.

Comments and suggestions regarding this draft document should be submitted within 90 days of

publication in the Federal Register of the notice announcing the availability of the draft guidance. 

Submit comments to Dockets Management Branch (HFA-305), Food and Drug Administration, 5630

Fishers Lane, rm. 1061, Rockville, MD 20852. All comments should be identified with the docket

number listed in the notice of availability that publishes in the Federal Register. 

For questions on the contents of this draft document contact (CDER) Radhika Rajagopalan, 301-

827-5849 or (CBER) Alfred Del Grosso, 301-435-4988.

U.S. Department of Health and Human Services

Food and Drug Administration

Center for Drug Evaluation and Research (CDER)

Center for Biologics Evaluation and Research (CBER)

August 2000

CMC #
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Guidance for Industry

Analytical Procedures and

Methods Validation

Chemistry, Manufacturing, and Controls Documentation

Additional copies are available from:

Office of Training and Communications

Division of Communications Management

Drug Information Branch, HFD-210

Center for Drug Evaluation and Research (CDER)

5600 Fishers Lane

Rockville, Maryland 20857

(Tel) 301-827-4573

(Internet) http://www.fda.gov/cder/guidance/index.htm

or

Office of Communications

Training and Manufacturers Assistance, HFM-40

Center for Biologics Evaluation and Research (CBER)

1401 Rockville Pike

Rockville, Maryland 20852-1448

(Fax) 888-CBERFAX or 301-827-3844

(Voice Information) 800-835-4709 or 301-827-1800

(Internet) http://www.fda.gov/cber/guidelines.htm

U.S. Department of Health and Human Services

Food and Drug Administration

Center for Drug Evaluation and Research (CDER)

Center for Biologics Evaluation and Research (CBER)

August 2000

CMC #
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Table of Contents

I. INTRODUCTION...............................................................................................................................................................1

II. BACKGROUND.................................................................................................................................................................2

III. TYPES OF ANALYTICAL PROCEDURES .............................................................................................................3

A. REGULATORY ANALYTICAL PROCEDURE .................................................................................................................. 3

B. ALTERNATIVE ANALYTICAL PROCEDURE.................................................................................................................. 3

C. STABILITY-INDICATING ASSAY.................................................................................................................................... 4

IV. REFERENCE STANDARDS ............................................................................................................................................4

A. TYPES OF STANDARDS................................................................................................................................................... 4

B. CERTIFICATE OF ANALYSIS ......................................................................................................................................... 4

C. CHARACTERIZATION OF A REFERENCE STANDARD .................................................................................................. 4

V. METHODS VALIDATION FOR INDS ..........................................................................................................................6

VI. CONTENT AND FORMAT OF ANALYTICAL PROCEDURES FOR NDAS, ANDAS, BLAS, AND PLAS...6

A. PRINCIPLE....................................................................................................................................................................... 6

B. SAMPLING....................................................................................................................................................................... 7

C. EQUIPMENT AND EQUIPMENT PARAMETERS.............................................................................................................. 7

D. REAGENTS ...................................................................................................................................................................... 7

E. SYSTEM SUITABILITY TESTING .................................................................................................................................. 7

F. PREPARATION OF STANDARDS ..................................................................................................................................... 7

G. PREPARATION OF SAMPLES ......................................................................................................................................... 8

H. PROCEDURE .................................................................................................................................................................... 8

I. CALCULATIONS .............................................................................................................................................................. 8

J. REPORTING OF RESULTS.............................................................................................................................................. 8

VII. METHODS VALIDATION FOR NDAS, ANDAS, BLAS, AND PLAS ..............................................................9

A. NONCOMPENDIAL ANALYTICAL PROCEDURES.......................................................................................................... 9

1. Validation Characteristics....................................................................................................................................9

2. Other Methods Validation Information ............................................................................................................10

a. Robustness............................................................................................................................................................. 11

b. Stress Studies ......................................................................................................................................................... 11

c. Instrument Output/Raw Data ............................................................................................................................... 11

3. Recommended Validation Characteristics for Types of Tests.......................................................................13

B. COMPENDIAL ANALYTICAL PROCEDURES ............................................................................................................... 15

VIII. STATISTICAL ANALYSIS ......................................................................................................................................15

A. GENERAL....................................................................................................................................................................... 15

B. COMPARATIVE STUDIES ............................................................................................................................................. 16

C. STATISTICS................................................................................................................................................................... 16

IX. REVALIDATION.............................................................................................................................................................16

X. METHODS VALIDATION PACKAGE: CONTENTS AND PROCESSING........................................................17

A. METHODS VALIDATION PACKAGE............................................................................................................................ 17

B. SELECTION AND SHIPMENT OF SAMPLES................................................................................................................ 18

C. RESPONSIBILITIES OF THE VARIOUS PARTIES ....................................................................................................... 19
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XI. METHODOLOGY............................................................................................................................................................20

A. HIGH-PRESSURE LIQUID CHROMATOGRAPHY (HPLC)......................................................................................... 20

B. GAS CHROMATOGRAPHY (GC).................................................................................................................................. 22

C. SPECTROPHOTOMETRY, SPECTROSCOPY, SPECTROMETRY AND RELATED PHYSICAL METHODOLOGIES ... 23

D. CAPILLARY ELECTROPHORESIS (CE)....................................................................................................................... 23

E. OPTICAL ROTATION.................................................................................................................................................... 24

F. METHODOLOGIES RELATING TO PARTICLE SIZE ANALYSIS ............................................................................... 25

G. DISSOLUTION ............................................................................................................................................................... 26

H. OTHER INSTRUMENTATION........................................................................................................................................ 27

ATTACHMENT ANDA, ANDA, BLA, AND PLA SUBMISSION CONTENTS..............................................................28

ATTACHMENT BMETHODS VALIDATION PROBLEMS AND DELAY......................................................................29

REFERENCES .............................................................................................................................................................................30

GLOSSARY.................................................................................................................................................................................32
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Guidance for Industry11

2

3 Analytical Procedures and Methods Validation

4

5

6

7

8

9

10

11

12

13 If you plan to submit comments on this draft guidance, to expedite FDA review of your comments, please:

14

15 ! Clearly explain each issue/concern and, when appropriate, include a proposed revision and

16 the rationale and/or justification for the proposed change.

17 ! Identify specific comments by line numbers; use the pdf version of the document whenever

18 possible.

19 ! If possible, e-mail an electronic copy (Word or WordPerfect) of the comments you have

20 submitted to the docket to cunninghamp@cder.fda.gov.

21

22 I. INTRODUCTION

23

This guidance provides recommendations to applicants on submitting analytical procedures,224 validation

25 data, and samples to support the documentation of the identity, strength, quality, purity, and potency

of drug substances and drug products.326 This guidance is intended to assist applicants in assembling

27 information, submitting samples, and presenting data to support analytical methodologies. The

28 recommendations apply to drug substances and drug products covered in new drug applications

29 (NDAs), abbreviated new drug applications (ANDAs), biologics license applications (BLAs), product

license applications (PLAs), and supplements to these applications.430 The principles also apply to drug

31 substances and drug products covered in Type II drug master files (DMFs). If a different approach is

 

1

 This guidance has been prepared by the Analytical Methods Technical Committee of the Chemistry,

Manufacturing, and Controls Coordinating Committee (CMC CC) in the Center for Drug Evaluation and Research

(CDER) and the Center for Biologics Evaluation and Research (CBER) at the Food and Drug Administration (FDA).

2

Analytical procedure is interchangeable with method or test procedure.

3

 The terms drug substance and drug product, as used in this guidance, refer to human drugs and

biologics.

4

 Sponsors preparing investigational new drug applications (INDs) should also consider the

recommendations in this guidance. However, the amount and depth of the information that should be submitted to

support an IND depends in large part on the phase of the investigation and the specific testing proposed in

humans (see section V).

This draft guidance, when finalized, will represent the Food and Drug Administration=s current thinking

on this topic. It does not create or confer any rights for or on any person and does not operate to bind

FDA or the public. An alternative approach may be used if such approach satisfies the requirements of

the applicable statutes, regulations, or both.
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32 chosen, the applicant is encouraged to discuss the matter in advance with the center with product

33 jurisdiction to prevent the expenditure of resources on preparing a submission that may later be

34 determined to be unacceptable. 

35

36 The principles of methods validation described in this guidance apply to all types of analytical

37 procedures. However, the specific recommendations in this guidance may not be applicable to certain

38 unique analytical procedures for products such as biological, biotechnological, botanical, or

39 radiopharmaceutical drugs. For example, many bioassays are based on animal challenge models,

40 immunogenicity assessments, or other immunoassays that have unique features that should be

41 considered when submitting analytical procedure and methods validation information. Furthermore,

42 specific recommendations for biological and immunochemical tests that may be necessary for

43 characterization and quality control of many drug substances and drug products are beyond the scope

44 of this guidance document. Although this guidance does not specifically address the submission of

45 analytical procedures and validation data for raw materials, intermediates, excipients, container closure

46 components, and other materials used in the production of drug substances and drug products,

47 validated analytical procedures should be used to analyze these materials. For questions on

48 appropriate validation approaches for analytical procedures or submission of information not

49 addressed in this guidance, applicants should consult with the appropriate chemistry review staff at

50 FDA. 

51

52 This guidance, when finalized, will replace the FDA guidance for industry on Submitting Samples and

53 Analytical Data for Methods Validation (February 1987).

54

55

56 II. BACKGROUND

57

58 Each NDA and ANDA must include the analytical procedures necessary to ensure the identity,

59 strength, quality, purity, and potency of the drug substance and drug product, including bioavailability

60 of the drug product (21 CFR 314.50(d)(1) and 314.94(a)(9)(i)). Data must be available to establish

61 that the analytical procedures used in testing meet proper standards of accuracy and reliability (21

62 CFR 211.165(e) and 211.194(a)(2)).

63

64 Methods validation is the process of demonstrating that analytical procedures are suitable for their

65 intended use. The methods validation process for analytical procedures begins with the planned and

66 systematic collection by the applicant of the validation data to support the analytical procedures. The

67 review chemist evaluates the analytical procedures and validation data submitted in the NDA or

68 ANDA. On request from FDA, an NDA or ANDA applicant must submit samples of drug product,

69 drug substance, noncompendial reference standards, and blanks so that the applicant=s drug substance

70 and drug product analytical procedures can be evaluated by FDA laboratories (21 CFR 314.50(e)

71 and 314.94(a)(10)). The FDA laboratory analysis demonstrates that the analytical procedures are

72 reproducible by laboratory testing. The review chemists and laboratory analysts determine the

73 suitability of the analytical procedures for regulatory purposes. FDA investigators inspect the

74 analytical laboratory testing sites to ensure that the analytical procedures used for release and stability
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75 testing comply with current good manufacturing practices (CGMPs) (21 CFR part 211) or good

76 laboratory practices (GLPs) (21 CFR part 58), as appropriate.

77

78 Each BLA and PLA must include a full description of the manufacturing methods, including analytical

79 procedures, that demonstrate that the manufactured product meets prescribed standards of safety,

80 purity, and potency (21 CFR 601.2(a) and 601.2(c)(1)(iv)). Data must be available to establish that

81 the analytical procedures used in testing meet proper standards of accuracy and reliability (21 CFR

82 211.194(a)(2)). For BLAs, PLAs, and their supplements, the analytical procedures and their

83 validation are submitted as part of the license application or supplement and are evaluated by the

84 review committee. Representative samples of the product must be submitted and summaries of results

85 of tests performed on the lots represented by the submitted sample must be provided (21 CFR

86 601.2(a) and 601.2(c)(1)(vi)). The review committee chair may request analytical testing by CBER

87 laboratory analysts to evaluate the applicant=s analytical procedures and verify the test results.

88

89 All analytical procedures are of equal importance from a validation perspective. In general, validated

90 analytical procedures should be used, irrespective of whether they are for in-process, release,

91 acceptance, or stability testing. Each quantitative analytical procedure should be designed to minimize

92 assay variation.

93

94 Analytical procedures and validation data are submitted in the sections of the application on analytical

95 procedures and controls. Recommendations on information to be submitted are included in sections

96 III through IX and XI of this guidance. Information on submission of the methods validation

97 package to the NDA or ANDA and samples to the FDA laboratories is provided in section X.

98

99

100 III. TYPES OF ANALYTICAL PROCEDURES

101

102 A. Regulatory Analytical Procedure

103

104 A regulatory analytical procedure is the analytical procedure used to evaluate a defined

105 characteristic of the drug substance or drug product. The analytical procedures in the U.S.

106 Pharmacopeia/National Formulary (USP/NF) are those legally recognized under section

107 501(b) of the Food, Drug, and Cosmetic Act (the Act) as the regulatory analytical procedures

108 for compendial items. For purposes of determining compliance with the Act, the regulatory

109 analytical procedure is used.

110

111 B. Alternative Analytical Procedure

112

113 An alternative analytical procedure is an analytical procedure proposed by the applicant for

114 use instead of the regulatory analytical procedure. A validated alternative analytical procedure

115 should be submitted only if it is shown to perform equal to or better than the regulatory

116 analytical procedure. If an alternative analytical procedure is submitted, the applicant should

117 provide a rationale for its inclusion and identify its use (e.g., release, stability testing), validation
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118 data, and comparative data to the regulatory analytical procedure.

119

120 C. Stability-Indicating Assay

121

122 A stability-indicating assay is a validated quantitative analytical procedure that can detect

123 the changes with time in the pertinent properties of the drug substance and drug product. A

124 stability-indicating assay accurately measures the active ingredients, without interference from

125 degradation products, process impurities, excipients, or other potential impurities. If an

126 applicant submits a non-stability-indicating analytical procedure for release testing, then an

127 analytical procedure capable of qualitatively and quantitatively monitoring the impurities,

128 including degradation products, should complement it. Assay analytical procedures for

129 stability studies should be stability-indicating, unless scientifically justified.

130

131

132 IV. REFERENCE STANDARDS

133

134 A. Types of Standards

135

136 A reference standard (i.e., primary standard) may be obtained from the USP/NF or other

137 official sources (e.g., CBER, 21 CFR 610.20). If there are questions on whether a source of

138 a standard would be considered by FDA to be an official source, applicants should contact

139 the appropriate chemistry review staff. When there is no official source, a reference standard

140 should be of the highest possible purity and be fully characterized.

141

142 A working standard (i.e., in-house or secondary standard) is a standard that is qualified

143 against and used instead of the reference standard.

144

145 B. Certificate of Analysis

146

147 A certificate of analysis (COA) for reference standards from non-official sources should be

148 submitted in the section of the application on analytical procedures and controls. For

149 standards from official sources, the user should ensure the suitability of the reference standard.

150 The standard should be stored correctly and used within the established use interval.

151

152 C. Characterization of a Reference Standard

153

154 Reference standards from USP/NF and other official sources do not require further

155 characterization. A reference standard that is not obtained from an official source should be of

156 the highest purity that can be obtained by reasonable effort, and it should be thoroughly

157 characterized to ensure its identity, strength, quality, purity, and potency. The qualitative and

158 quantitative analytical procedures used to characterize a reference standard are expected to

159 be different from, and more extensive than, those used to control the identity, strength, quality,

160 purity, and potency of the drug substance or the drug product. Analytical procedures used to
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161 characterize a reference standard should not rely solely on comparison testing to a previously

162 designated reference standard.

163

164 Generally, this characterization information should include:

165

166 ! A brief description of the manufacture of the reference standard, if the manufacturing

167 process differs from that of the drug substance. Any additional purification

168 procedures used in the preparation of the reference standard should be described.

169

170 ! Legible reproductions of the relevant spectra, chromatograms, thin-layer

171 chromatogram (TLC) photographs or reproductions, and other appropriate

172 instrumental recordings.

173

174 ! Data establishing purity. The data should be obtained by using appropriate tests, such

175 as TLC, gas chromatography (GC), high-pressure liquid chromatography (HPLC),

176 phase solubility analysis, appropriate thermometric analytical procedures, and others

177 as necessary.

178

179 ! Appropriate chemical attribute information, such as structural formula, empirical

180 formula, and molecular weight. Information to substantiate the proof of structure

181 should include appropriate analytical tests, such as elemental analysis, infrared

182 spectrophotometry (IR), ultraviolet spectrophotometry (UV), nuclear magnetic

183 resonance spectroscopy (NMR), and mass spectrometry (MS), as well as applicable

184 functional group analysis. Detailed interpretation of the test data in support of the

185 claimed structure should be provided.

186

187 ! A physical description of the material, including its color and physical form.

188

189 ! Appropriate physical constants such as melting range, boiling range, refractive index,

190 dissociation constants (pK values), and optical rotation.

191

192 ! A detailed description of the analytical procedures used to characterize the reference

193 standard.

194

195 For biotechnological/biological product reference standards, the recommendations on

196 characterization information above may apply and should be considered. However, additional

197 and/or different tests would be important to assess physicochemical characteristics, structural

198 characteristics, biological activity, and/or immunochemical activity. Physicochemical

199 determinations may include isoform, electrophoretic, and liquid chromatographic patterns, as

200 well as spectroscopic profiles. Structural characterization may include a determination of

201 amino acid sequence, amino acid composition, peptide map, and carbohydrate structure. 

202 Biological and/or immunochemical activity should be assessed using the same analytical

203 procedures used to determine product potency. These can include animal-based, cell culture-
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204 based, biochemical, or ligand/receptor-binding assays. While these tests may be needed for

205 complete characterization of certain reference standards, specific recommendations for

206 validation of biological and immunochemical tests are not contained in this guidance document.

207

208

209 V. METHODS VALIDATION FOR INDs

210

211 For an investigational new drug, sufficient information is required in each phase of an investigation to

212 ensure proper identification, quality, purity, strength, and/or potency. The amount of information on

213 analytical procedures and methods validation necessary will vary with the phase of the investigation

214 (21 CFR 312.23(a)(7)).

215

216 For general guidance on analytical procedures and methods validation information to be submitted for

217 phase 1 studies, sponsors should refer to the FDA guidance for industry on Content and Format of

218 Investigational New Drug Applications (INDs) for Phase 1 Studies of Drugs, Including Well219 Characterized, Therapeutic, Biotechnology-Derived Products (November 1995). General

220 guidance regarding analytical procedures and methods validation information to be submitted for phase

221 2 or phase 3 studies will be provided in the FDA guidance for industry INDs for Phase 2 and 3

222 Studies of Drugs, Including Specified Therapeutic Biotechnology-Derived Products, Chemistry,

223 Manufacturing, and Controls Content and Format, when finalized (draft guidance published April

224 1999).

225

226 All analytical procedures should be fully developed and validation completed when the NDA, ANDA,

227 BLA, or PLA is submitted.

228

229

230 VI. CONTENT AND FORMAT OF ANALYTICAL PROCEDURES FOR NDAs,

231 ANDAs, BLAs, AND PLAs

232

233 Any analytical procedure submitted in an NDA, ANDA, BLA, or PLA should be described in

234 sufficient detail to allow a competent analyst to reproduce the necessary conditions and obtain results

235 comparable to the applicant=s. Aspects of the analytical procedure that require special attention

236 should be described. If the analytical procedure used is in the current revision of the USP/NF or other

237 FDA recognized standard references (e.g., AOAC International Book Of Methods) and the

238 referenced analytical procedure is not modified, a statement indicating the analytical procedure and

239 reference may be provided rather than a description of the method (21 CFR 211.194). A description

240 of analytical procedures from any other published sources should be provided, because the referenced

241 sources may not be readily accessible to the reviewer.

242

243 The following is a list of information that should typically be included in a description of an analytical

244 procedure.

245

246 A. Principle
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247

248 A statement of the principle of the analytical procedure should be included. For example,

249 separation is based on isocratic reversed phase HPLC with detection by UV.

250

251 B. Sampling

252

253 The number of samples (e.g., vials, tablets) selected, how they are used (i.e., as individual or

254 composite samples), and the number of replicate analyses per sample should be described.

255

256 C. Equipment and Equipment Parameters

257

258 A listing of all equipment (e.g., instrument type, detector, column type, dimensions) should be

259 included, as well as a list of equipment parameters (e.g., flow rate, temperatures, run time,

260 wavelength settings). A drawing representing the experimental configuration (e.g., illustrating

261 positions for a spray pattern analytical procedure) should be provided, when appropriate.

262 

263 D. Reagents

264

265 A list of reagents and their grades (e.g., USP/NF, American Chemical Society (ACS)

266 Analytical Reagent) should be included. If in-house or modified commercial reagents are

267 used, directions for their preparation should be included. Unstable or potentially hazardous

268 reagents should be identified, and storage conditions, directions for safe use, and usable shelf

269 life for these reagents should be specified.

270

271 E. System Suitability Testing

272

273 System suitability test parameters and acceptance criteria are based on the concept that the

274 equipment, electronics, analytical operations, and samples to be analyzed constitute an

275 integrated system. System suitability testing ensures that the system is working properly at the

276 time of analysis. Appropriate system suitability criteria should be defined and included in the

277 analytical procedure. 

278

279 All chromatographic analytical procedures should include system suitability testing and criteria.

280 Parameters typically used in system suitability evaluations are defined and discussed in the

281 CDER reviewer guidance on Validation of Chromatographic Methods (November 1994). 

282

283 System suitability testing is recommended as a component of any analytical procedure, not just

284 those that involve chromatographic techniques. Regardless of the type of analytical

285 procedure, testing should be used to confirm that the system will function correctly

286 independent of the environmental conditions. For example, titration analytical procedures

287 should always include the evaluation of a blank (commonly referred to as a blank titration).

288

289 F. Preparation of Standards
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290

291 Procedures for the preparation of all standard solutions (e.g., stock, working standard

292 solutions, internal standards) should be included.

293

294 G. Preparation of Samples

295

296 Sample preparation for individual tests should be clearly described. Specific details should be

297 provided for unusual sample preparations (e.g., solid-phase extraction, derivatization).

298

299 H. Procedure

300

301 A step-by-step description of the procedure should be provided. The description should

302 include, where appropriate, equilibration times, injection sampling sequence, and system

303 suitability or start-up parameters. Unusual hazards should be identified. 

304

305 I. Calculations

306

307 Representative calculations, with a tabulation defining all symbols and numerical factors, and

308 specific instructions for the calculation of degradation products and impurities should be

309 included. Any mathematical transformations or formulas used in data analysis should be

310 described in detail. These may include logarithmic transformations used to obtain a linear

311 relationship from exponential data, or the use of multiple order regression analyses.

312

313 J. Reporting of Results

314

315 1. General

316

317 The format used to report results (e.g., percent label claim, weight/weight,

318 weight/volume, parts per million (ppm)) including the specific number of significant

319 figures to be reported should be provided.

320

321 2. Impurities Analytical Procedures

322

323 The name and location/identifier (e.g., retention time (RT), relative retention time

324 (RRT)) of impurities and the type of impurity (e.g., process, degradant, excipient

325 degradant) should be included in the analytical procedures for impurities in the drug

326 substance and drug product. The detection limit (DL) or quantitation limit (QL)

327 should be stated, as appropriate. The DL or QL can be set using the drug substance's

328 detection response. 

329

330 Reporting of organic impurities should cover (1) specified identified impurities by

331 name, (2) specified unidentified impurities by location/identifier, (3) any unspecified

332 impurities, and (4) total impurities. The total organic impurities for the drug product or
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333 drug substance is the sum of all impurities equal to or greater than their individual QL. 

334 See recommendations regarding appropriate QLs in FDA impurities guidances (see

335 references). Inorganic impurities and residual solvents should also be addressed.

336

337 For the drug product, drug substance process impurities may be excluded from

338 reporting if an acceptable rationale is provided in the sections on analytical procedures

339 and controls. Drug product impurities from the drug product manufacturing process,

340 packaging, and labeling should be addressed.

341

342 The above reporting information may not be strictly applicable to all products (e.g.,

343 biological, biotechnological, botanical, radiopharmaceutical drugs), but any significant

344 process and product-related impurities should be determined and reported.

345

346

347 VII. METHODS VALIDATION FOR NDAs, ANDAs, BLAs, AND PLAs

348

349 A. Noncompendial Analytical Procedures

350

351 In an NDA, ANDA, BLA, or PLA, data must be submitted to establish that the analytical

352 procedures used in testing meet proper standards of accuracy and reliability (21 CFR

353 211.194(a)(2)). Methods validation is the process of demonstrating that analytical

354 procedures are suitable for their intended use. At the time of submission, the NDA, ANDA,

355 BLA, or PLA should contain methods validation information to support the adequacy of the

356 analytical procedures.

357

358 The International Conference on Harmonisation (ICH) guidance Q2A Text on Validation of

359 Analytical Procedures (March 1995) and Q2B Validation of Analytical Procedures:

360 Methodology (November 1996) provide recommendations on validation of analytical

361 procedures. Analytical procedures outside the scope of the ICH guidances should still be

362 validated. 

363

364 1. Validation Characteristics

365

366 Applicants should submit information on the validation characteristics of their

367 proposed analytical procedures (see ICH Q2A and ICH Q2B). Although not all of

368 the validation characteristics are needed for all types of tests (see section VII.A.3),

369 typical validation characteristics are:

370

371 ! Accuracy

372 ! Precision (repeatability and intermediate precision)

373 ! Specificity

374 ! Detection limit

375 ! Quantitation limit
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376 ! Linearity

377 ! Range

378 ! Robustness

379

380 2. Other Methods Validation Information

381

382 Methods validation information should also include:

383

384 ! Data to demonstrate the stability of all analytical sample preparations through

385 the time required to complete the analysis.

386

387 ! Legible reproductions of representative instrument output or recordings (e.g.,

388 chromatograms) and raw data output (e.g., integrated areas), as appropriate. 

389 Instrument output for placebo, standard, and sample should also be provided

390 (see section VII.A.2.c).

391

392 ! Representative calculations using submitted raw data, to show how the

393 impurities in drug substance are calculated.

394

395 ! Information from stress studies (see section VII.A.2.b).

396

397 ! Impurities labeled with their names and location identifiers (e.g., RRT for

398 chromatographic data) for the impurity analytical procedure.

399

400 ! For drug substances:

401

402 C A discussion of the possible formation and control of polymorphic and

403 enantiomeric substances.

404

405 C Identification and characterization of each organic impurity, as

406 appropriate. This information may not be needed for all products

407 (e.g., botanicals). Other impurities (e.g., inorganics, residual solvents)

408 should be addressed and quantitated.

409

410 Recommendations on submitting information on impurities is provided

411 in various FDA guidances such as the ICH guidance Q3A Impurities

412 in New Drug Substances (January 1996).

413

414 C A list of known impurities, with structure if available, including process

415 impurities, degradants, and possible isomers. 

416

417 ! For drug products:

418
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419 C A degradation pathway for the drug substance in the dosage form,

420 where possible.

421

422 C Data demonstrating recovery from the sample matrix as illustrated by

423 the accuracy studies.

424

425 C Data demonstrating that neither the freshly prepared nor the degraded

426 placebo interferes with the quantitation of the active ingredient.

427

428 ICH Q2A and Q2B address almost all of the validation parameters. Areas that should

429 be provided in more detail are described below.

430

431 a. Robustness

432

433 Robustness, a measure of the analytical procedure's capability to remain unaffected by

434 small but deliberate variations, is described in ICH Q2A and Q2B. Such testing

435 should be performed during development of the analytical procedure and the data

436 discussed and/or submitted. In cases where an effect is observed, representative

437 instrument output (e.g., chromatograms) should be submitted.

438

439 b. Stress Studies

440

441 Degradation information obtained from stress studies (e.g., products of acid and base

442 hydrolysis, thermal degradation, photolysis, oxidation) for the drug substance and for

443 the active ingredient in the drug product should be provided to demonstrate the

444 specificity of the assay and analytical procedures for impurities. The stress studies

445 should demonstrate that impurities and degradants from the active ingredient and drug

446 product excipients do not interfere with the quantitation of the active ingredient. Stress

447 studies are described in various FDA guidances relating to the stability of drug

448 products (see references).

449

450 The design of the stress studies and the results should be submitted to the stability

451 section of the application. Representative instrument output (e.g., chromatograms)

452 and/or other appropriate data (e.g., degradation information obtained from stress

453 studies) should be submitted in the sections on analytical procedures and controls.

454

455 c. Instrument Output/Raw Data

456

457 i. Organic Impurities

458

459 Representative data should be submitted to support an assessment of the

460 organic impurities. Representative data for residual solvents are generally not

461 needed. Instrument output and the raw numerical values (e.g., peak area)
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462 with appropriate identification and labeling (e.g., RT for chromatographic

463 peaks, chemical shift (δ) and coupling constant (J) for NMR) should be

464 provided. The impurity profile should be assessed at the quantitation limit and

465 the instrument output provided. Additional information should be provided to

466 confirm that the impurity profile is adequately characterized. For example, a

467 representative chromatogram using detection at a low wavelength, such as

468 205 nm, and double the proposed total run time could be submitted to

469 support the specificity of the analytical procedure. 

470

471 For quantitation purposes, the response factor of the drug substance may be

472 used for impurities without a reference standard. In cases where the response

473 factors are not close, this practice may still be acceptable, provided a

474 correction factor is applied or the impurities are, in fact, being overestimated. 

475 Acceptance criteria and analytical procedures used to estimate identified or

476 unidentified impurities often are based on analytical assumptions (e.g.,

477 equivalent detector response). Assumptions should be discussed and justified.

478

479 ii. Drug Substance

480

481 Data should be submitted showing the separation and detection of impurities

482 using spiked or stress samples. Complete impurity profiles as graphic output

483 (e.g., chromatograms) and raw data (e.g, integrated peak areas) of

484 representative batches should be submitted in the sections on analytical

485 procedures and controls for the drug substance. For ANDAs and related

486 submissions, appropriate information for the batches used in the biobatch or

487 submission batch should be provided. All responses (e.g., peaks) should be

488 labeled.

489

490 The analytical procedure used should be capable of differentiating changes, if

491 any, between past and present batches. The quantitation limit and the type of

492 organic impurity (e.g., degradant, process impurity) should be stated. The

493 analytical procedure number, batch number, manufacturing date and site, and

494 date of analysis should be provided.

495

496 iii. Drug Product

497

498 Information such as instrument output (e.g., chromatograms) and raw data

499 (e.g., integrated peak areas) from representative batches under long-term and

500 accelerated stability conditions, and stressed samples should be submitted in

501 the sections on analytical procedures and controls of the drug product. For

502 ANDAs and related submissions, appropriate information for the biobatch or

503 submission batch should be provided. References to the raw data (e.g.,

504 chromatograms) should be included in the stability section of the application.
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505

506 At a minimum, the submission should include instrument output and raw data

507 for release testing and at the latest available time point for the same batch. All

508 responses (e.g., peaks) should be labeled and identified. In addition, the

509 analytical procedure number, batch number of the drug product,

510 manufacturing date, date of analysis, source and batch number of drug

511 substance, manufacturing site, and container/closure information should be

512 provided. The analytical procedures used should be capable of differentiating

513 changes, if any, between past and present batches. The quantitation limit and

514 the type (e.g., degradant, leachables from packaging) should be reported. 

515 Multiple methodologies can be used.

516

517 If process impurities from the drug substance and excipients with their related

518 impurities are not reported in the impurities analytical procedure, the potential

519 locations/identifier (e.g., RT, RRT) of these compounds should be described

520 and listed in the analytical procedure.

521

522 3. Recommended Validation Characteristics for Types of Tests

523

524 Table 1 is a summary of the validation characteristics that should be addressed during

525 validation of different types of analytical procedures. The same methodology can be

526 used for several purposes. The validation information should support the intended

527 purpose of the test. For example, if Raman spectroscopy is the methodology selected

528 to quantitate polymorphic forms as impurities, or chiral HPLC for enantiomeric

529 impurities, the recommended validation characteristics in Table 1 under quantitative

530 testing for impurities would apply. However, if Raman spectroscopy or chiral

531 HPLC are used for the purpose of identification or as specific tests, the recommended

532 validation characteristics listed for those types of tests would apply.
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533

534 Table 1. Recommended Validation Characteristics of the Various Types of Tests.

535

Type of Tests /

Characteristics

Identification Testing for Impurities Assay

Dissolution

(Measurement

Only),

Content/Potency

Specific

Tests

Quantitative Limit

Accuracy - + - + +

4

Precision-Repeatability - + - + +

4

Precision-Intermediate

Precision

- +

1

- +

1

+

4

Specificity +

2

+ + +

5

+

4

Detection Limit - -

3

+ - -

Quantitation Limit - + - - -

Linearity - + - + -

Range - + - + -

Robustness - + -

3

+ +

4

536

537 NOTE:

538 - Signifies that this characteristic is not normally evaluated.

539 + Signifies that this characteristic is normally evaluated.

540 1 In cases where reproducibility has been performed, intermediate precision is not needed.

541 2 Lack of specificity for an analytical procedure may be compensated for by the addition of a second

542 analytical procedure.

543 3 May be needed in some cases.

544 4 May not be needed in some cases.

545 5 Lack of specificity for an assay for release may be compensated for by impurities testing.

546

547 a. Identification

548

549 Identification analytical procedures may include tests such as IR, differential scanning

550 calorimetry (DSC), X-ray diffraction (XRD), UV, and HPLC retention time. A

551 specific identification test should be included for the active ingredient whenever

552 possible. In cases where a nonspecific identification analytical procedure is proposed

553 for the active ingredient, two independent analytical procedures are generally

554 sufficient, if justified. For other identification tests (e.g., a chiral HPLC retention time

555 as confirmation for the presence of an enantiomer, chloride test for a counterion) a

556 single test is acceptable. This concept of the number of identification tests is
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557 applicable to both the drug substance and drug product.

558

559 b. Impurities

560

561 The validation characteristics under quantitative testing for impurities, as described

562 in Table 1, apply, regardless of which methodology is used to quantitate impurities. If

563 the same analytical procedure is proposed as a limit test, validation characteristics

564 under limit testing for impurities will apply.

565

566 c. Assay

567

568 Assay includes the content of the active ingredient, preservative (if used), and

569 measurement of content in dissolution and content uniformity samples.

570

571 d. Specific Tests

572

573 Specific tests to control the drug substance, excipient, or drug product can include

574 tests such as particle size analysis, droplet distribution, spray pattern, dissolution

575 (excludes measurement), optical rotation, and methodologies such as DSC, XRD, and

576 Raman spectroscopy. The validation characteristics may differ for the various

577 analytical procedures. For example, accuracy, repeatability, intermediate precision

578 and robustness should be evaluated for molecular size distribution gel permeation

579 chromatography (GPC).

580

581 B. Compendial Analytical Procedures

582

583 The suitability of a compendial analytical procedure must be verified under actual conditions of

584 use (21 CFR 211.194(a)(2)). Information to demonstrate that USP/NF analytical procedures

585 are suitable for the drug product or drug substance should be included in the submission. 

586 Information on the specificity, intermediate precision, and stability of the sample solution

587 should be included. Compendial assay analytical procedures may not be stability-indicating,

588 and this should be considered when developing the specification (see section III.C). For

589 compendial items, additional analytical procedures, such as impurities or osmolality, may be

590 requested to support the quality of the drug product or drug substance. These additional

591 analytical procedures should be validated (see section VII.A). 

592

593

594 VIII. STATISTICAL ANALYSIS

595

596 A. General

597

598 Methods validation includes an assessment of the adequacy of the analytical procedure. 

599 Statistical analysis (e.g., linear regression analysis, relative standard deviation) of methods
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600 validation data is often used to demonstrate the validity of the method. The statistical

601 procedures for the analysis of the validation data should be determined prior to the start of any

602 validation study. The procedure followed, including the amount of data to collect and the

603 criteria used in determining the acceptability of the analytical procedure, should be specified.

604

605 The raw methods validation data and statistical procedures used to analyze the raw data

606 should be provided and discussed in the sections on analytical procedures and controls. All

607 statistical procedures used in the analysis of the data should be based on sound principles and

608 be suitable for evaluating the dataset.

609

610 B. Comparative Studies

611

612 Comparative studies are performed to evaluate intermediate precision (e.g., different

613 equipment, analysts, days). Comparative studies are also used to evaluate between

614 laboratory variability (i.e., reproducibility) when an analytical procedure is used in more than

615 one laboratory or to compare and evaluate the precision and accuracy of two analytical

616 procedures (e.g., regulatory analytical procedure and an alternative analytical procedure). 

617 When comparative studies are performed, homogeneous samples from the same batch should

618 be used, if feasible. Comparative results should be statistically analyzed and discussed and

619 any bias explained. 

620

621 C. Statistics

622

623 For information on statistical techniques used in making comparisons, as well as other general

624 information on the interpretation and treatment of analytical data, appropriate literature or texts

625 should be consulted (see references) .

626

627

628 IX. REVALIDATION

629

630 When sponsors make changes in the analytical procedure, drug substance (e.g., route of synthesis), or

631 drug product (e.g., composition), the changes may necessitate revalidation of the analytical

632 procedures. Revalidation should be performed to ensure that the analytical procedure maintains its

633 characteristics (e.g., specificity) and to demonstrate that the analytical procedure continues to ensure

634 the identity, strength, quality, purity, and potency of the drug substance and drug product, and the

635 bioavailability of the drug product. The degree of revalidation depends on the nature of the change. 

636 When a different regulatory analytical procedure is substituted (e.g., HPLC for titration), the new

637 procedure should be validated (see section VII).

638

639 If during each use an analytical procedure can meet the established system suitability requirements only

640 with repeated adjustments to the operating conditions stated in the analytical procedure, the analytical

641 procedure should be reevaluated, amended, and revalidated, as appropriate.

642
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643 FDA intends to provide guidance in the future on postapproval changes in analytical procedures.

644

645

646 X. METHODS VALIDATION PACKAGE: CONTENTS AND PROCESSING

647

648 Part of the methods validation process may include FDA laboratory analysis to demonstrate that an

649 analytical procedure is reproducible by laboratory testing. A methods validation package (see X.A)

650 and samples (see X.B) will be needed for this process.

651

652 A. Methods Validation Package

653

654 The methods validation package will usually include information copied from pertinent sections

655 of the application. To aid the review chemist, these copies should retain the original pagination

656 of the application sections.

657

658 For ANDA and NDA products, the archival copy and extra copies of the methods validation

659 packages should be submitted with the application. For ANDAs and related supplemental

660 applications, one archival copy and two extra copies of the methods validation package

661 should be submitted. For NDAs and related supplemental applications, one archival copy and

662 three extra copies should be submitted. For BLAs and PLAs, a separate methods validation

663 package need not be submitted. Information similar to that specified here should be included

664 in the BLA or PLA submission.

665

666 The methods validation package should include:

667

668 1. Tabular List of All Samples to Be Submitted

669

670 The list should include the lot number, identity (with chemical name and structure

671 where required for clarity), package type and size, date of manufacture, and quantity

672 of the samples.

673

674 2. Analytical Procedures

675

676 A detailed description of each of the analytical procedures listed in the specifications

677 should be submitted. The description should be sufficient to allow the FDA laboratory

678 analysts to perform the analytical procedure (see section VI).

679

680 3. Validation Data

681

682 Appropriate validation data to support the analytical procedures should be submitted.

683 Individual values as well as summary tables should be provided. Representative

684 instrument output and raw data and information regarding stress studies should be

685 included (see section VII).
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686

687 4. Results

688

689 The results obtained by the applicant for the submitted samples should be provided. 

690 Alternatively, COAs could be submitted. The dates of analysis should be stated.

691

692 5. Composition

693

694 The components and composition of the drug product should be provided.

695

696 6. Specifications

697

698 The specifications for the drug substance and the drug product should be included.

699

700 7. Material Safety Data Sheets

701

702 The applicant should include material safety data sheets (MSDSs) for all samples,

703 standards, and reagents (29 CFR 1910.1200(g)). As appropriate, MSDSs should be

704 provided for other materials used in the analytical procedures listed in the methods

705 validation package. In the case of toxic or hazardous materials, MSDSs should be

706 posted on the outside of the package to facilitate safe handling.

707

708 B. Selection and Shipment of Samples

709

710 On request from CDER, an NDA or ANDA applicant must submit samples of drug product,

711 drug substance, noncompendial reference standards, and blanks, so that the suitability of the

712 applicant=s drug substance and drug product analytical procedures can be evaluated by FDA

713 laboratories (21 CFR 314.50(e) and 314.94(a)(10)). For BLAs and PLAs, representative

714 samples of the product must be submitted, and summaries of the results of tests performed on

715 the lots represented by the submitted sample must be provided (21 CFR 601.2(a) and

716 601.2(c)(1)(vi)).

717

718 For CDER products, the number of sets of samples that should be submitted for methods

719 validation will be identified in the instructions forwarded to the applicant by the FDA

720 laboratory. In general, the quantity of samples in each set should be double the amount

721 needed to carry out the testing as performed by the applicant. Along with the drug substance

722 and the drug product samples, the applicant should submit internal standards, non-USP

723 reference standards, samples of impurities, degradation products, and unusual reagents. A set

724 of samples will be shipped to each assigned laboratory. 

725

726 For biological products, CBER should be consulted on the submission of samples and

727 supporting materials.

728
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729 Unless specified differently by the reviewer, samples from any batch, preferably samples from

730 an aged batch, may be selected for NDAs and NDA supplemental applications. The

731 submitted drug product samples should be from a batch made with the proposed market

732 formulation. For ANDAs and appropriate supplements, a sample of the finished product from

733 a batch being used to support approval of the submission should be used. If a sample is

734 selected from a batch not described in the application, an amendment containing a copy of the

735 batch record and certificate of analysis should be provided to the ANDA. For supplements

736 that do not require submission and review of an exhibit batch record and associated data, any

737 commercial batch may be submitted. For biological products, samples from several

738 consecutively manufactured batches should be submitted.

739

740 The drug product should be supplied in its original packaging. Bulk substances (e.g., drug

741 substances, impurities, excipients) should be stored in opaque nonreactive containers. To

742 prevent breakage during shipping, the samples should be adequately packaged in a sturdy

743 container. Samples shipped from outside the United States should contain the appropriate

744 customs forms to reduce delay in delivery.

745

746 If special storage precautions (e.g., freezing, use of an inert gas blanket) are required to

747 protect sample integrity, arrangements should be made in advance with the validating

748 laboratory for scheduled direct delivery. If a sample is toxic or potentially hazardous, the

749 container should be prominently labeled with an appropriate warning and precautionary

750 handling instructions.

751

752 C. Responsibilities of the Various Parties

753

754 1. Applicant

755

756 In the sections of the application on analytical procedures and controls, the applicant

757 should provide a name, address, telephone number, and facsimile number so that

758 samples can be requested. If this information is not provided, the contact person and

759 address listed in the NDA, ANDA, BLA, or PLA submission will be used. 

760

761 The methods validation packages should be compiled and submitted with the NDA or

762 ANDA submission. For BLAs and PLAs, a separate methods validation package

763 need not be submitted.

764

765 When an FDA laboratory contacts the applicant for samples, the applicant should

766 provide FDA laboratories with the samples within 10 working days. With the

767 exception of sample delivery arrangements, all communications concerning validation

768 at the FDA laboratories should be made through or with the knowledge of the review

769 chemist for CDER applications, or the BLA/PLA committee chair for CBER

770 applications.

771
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772 2. Review Chemist

773

774 The review chemist will review the application to determine that the analytical

775 procedures are adequate to ensure the identity, strength, quality, purity, and potency

776 of the drug substance and/or drug product. Any changes in the methods resulting from

777 the review of the application may require resubmission of the methods validation

778 package. The review chemist, in coordination with the appropriate FDA laboratories,

779 will decide which analytical procedures are to be validated. Comments from the FDA

780 laboratories, if any, will be forwarded by the review chemist to the applicant on

781 completion of the studies by the laboratories.

782

783 3. FDA Laboratory

784

785 An FDA laboratory will contact applicants with instructions on the submission of

786 samples and the addresses to which samples should be mailed. The laboratory will

787 test the samples according to the submitted analytical procedures to determine

788 whether the analytical procedures are acceptable for quality control and suitable for

789 regulatory purposes. Results and comments will be forwarded to the review chemist

790 on completion of the studies.

791

792 4. Investigator

793

794 The investigator inspects the analytical laboratory testing sites where the release and

795 stability testing are performed to ensure that the analytical procedures are performed

796 in compliance with CGMP/GLP.

797

798

799 XI. METHODOLOGY

800

801 Sections II through IX provide general information on the submission of analytical procedures and

802 methods validation information, including validation characteristics. Additional information on certain

803 methodologies is provided below.

804

805 A. High-Pressure Liquid Chromatography (HPLC)

806

807 The widespread use of HPLC analytical procedures and the multitude of commercial sources

808 of columns and packings frequently have created problems in assessing comparability. Many

809 of the following points may also apply to other chromatographic analytical procedures.

810

811 1. Column

812

813 The following characteristics are useful for defining a particular column and, if known,

814 should be included in the analytical procedure description. If method development has
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815 indicated that columns from only one commercial source are suitable, this information

816 should be included as part of the analytical procedure. If more than one column is

817 suitable, a listing of columns found to be equivalent should be included.

818

819 a. Column Parameters

820

821 ! Material: glass, stainless steel, plastic

822 ! Dimensions: length, inner diameter

823 ! Frit size

824 ! Filter type

825 ! Precolumn and/or guard column type, if used

826

827 b. Packing Material

828

829 ! Particle type: size, shape, pore diameter

830 ! Surface modification (e.g., bonded surface type, surface coverage, percent

831 carbon, additional silylation)

832 ! Recommended pH range for column use

833

834 2. System Suitability Testing

835

836 Each analytical procedure submitted should include an appropriate number of system

837 suitability tests defining the critical characteristics of that system. Criteria for all system

838 suitability testing should be provided. The system suitability tests listed below are

839 defined in CDER=s reviewer guidance on Validation of Chromatographic Methods

840 (November 1994).

841

842 ! Tailing factor

843 ! Relative retention

844 ! Resolution

845 ! Relative standard deviation (RSD)

846 ! Capacity factor 

847 ! Number of theoretical plates

848

849 The RSD is normally performed at the beginning of the run. However, for assays with

850 lengthy run times or as otherwise justified by the applicant, the reported average may

851 be taken from injections at the beginning and end of the run, or at the beginning,

852 middle, and end of the run. 

853

854 If an internal standard is used, the minimum acceptable resolution between the

855 internal standard and one or more active ingredients should be specified. If the

856 analytical procedure is used to control the level of impurities, the minimum resolution

857 between the active ingredient and the closest eluting impurity, or the two peaks
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858 eluting closest to each other, should be given.

859

860 3. Operating Parameters

861

862 The sequence of injection of blanks, system suitability standards, other standards,

863 and samples should be defined. Flow rates, temperatures, and gradients should be

864 described.

865

866 Complete details should be provided for the preparation of the mobile phase,

867 including the order of addition of the reagents and the methods of degassing and

868 filtration. The effect of adjustments in mobile phase composition on retention times

869 should be included in the analytical procedure. The rationale for the use of

870 precolumns and/or guard columns should be provided and justified. Any special

871 requirements, such as the use of inert tubing or injection valves, should be specified.

872

873 B. Gas Chromatography (GC)

874

875 At a minimum, the following parameters should be included in the description of a GC

876 procedure. Additional parameters should be specified if required by the analytical procedure.

877 If method development has indicated that columns from only one commercial source are

878 suitable, this information should be included as part of the analytical procedure. If more than

879 one column is suitable, a listing of columns found to be equivalent should be included.

880

881 1. Column

882

883 ! Column dimensions: length, internal diameter, external diameter

884 ! Stationary phase

885 ! Column material (e.g., silica, glass, stainless steel)

886 ! Column conditioning procedure

887

888 2. Operating Parameters

889

890 ! Gases: purity, flow rate, pressure

891 ! Temperatures: column, injector, detector (including temperature program, if

892 used)

893 ! Injection (e.g., split, splitless, on-column)

894 ! Detector

895 ! Typical retention time and total run time

896

897 3. System Suitability Testing

898

899 Appropriate system suitability criteria should be defined and included in all analytical

900 procedures.
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901

902 If an internal standard is used, the minimum acceptable resolution between the internal

903 standard and one or more active ingredient should be specified. If the analytical

904 procedure is used to control the level of impurities, the minimum resolution between

905 the active ingredient and the closest eluting impurity, or the two peaks eluting closest

906 to each other, should be given.

907

908 The RSD is normally performed at the beginning of the run. However, for assays with

909 lengthy run times or as otherwise justified by the applicant, the reported average may

910 be taken from injections at the beginning and end of the run, or beginning, middle, and

911 end of the run.

912

913 C. Spectrophotometry, Spectroscopy, Spectrometry and Related Physical

914 Methodologies

915

916 These analytical procedures include, but are not limited to, IR spectrophotometry, near IR

917 spectrophotometry (NIR), UV/visible spectrophotometry (UV/Vis), atomic emission and

918 atomic absorption, NMR, Raman spectroscopy, MS, and XRD. 

919

920 Spectrometric analytical procedures may not be stability-indicating. The bias of the analytical

921 procedure should be evaluated by comparing it with a chromatographic procedure, where

922 appropriate. When manually operated equipment is used, the description of the analytical

923 procedure should include an acceptance criterion for the amount of time that may elapse

924 between sampling and reading. Appropriate system suitability and/or calibration testing is

925 recommended. Validation criteria should include specificity (demonstrating no interference of

926 placebo), linearity, repeatability, intermediate precision, and robustness. 

927

928 D. Capillary Electrophoresis (CE)

929

930 At a minimum, the parameters listed below should be specified for a capillary electrophoretic

931 analytical procedure. Additional parameters may be included as required by the procedure. 

932 If method development has indicated that capillaries from only one commercial source are

933 suitable, this information should be included as part of the analytical procedure. If more than

934 one capillary is suitable, a listing of capillaries found to be equivalent should be included.

935

936 1. Capillary

937

938 ! Capillary dimensions: length, length to detector, internal diameter, external

939 diameter

940 ! Capillary material

941 ! Capillary internal coating (if any)

942

943 2. Operating Parameters
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944

945 ! Capillary preparation procedure: procedure to be followed before the first

946 use, before the first run of the day, before each run (e.g., flush with 100

947 millimolar sodium hydroxide, flush with running buffer)

948 ! Running buffer: composition, including a detailed preparation procedure with

949 the order of addition of the components

950 ! Injection: mode (e.g., electrokinetic, hydrodynamic), parameters (e.g., 

951 voltage, pressure, time)

952 ! Detector

953 ! Typical migration time and total run time

954 ! Model of CE equipment used

955 ! Voltage (if constant voltage)

956 ! Current (if constant current)

957 ! Polarity (e.g., polarity of electrode by detector)

958

959 3. System Suitability Testing

960

961 Each analytical procedure should include the appropriate system suitability tests

962 defining the critical characteristics of that system. Other parameters may be included

963 at the discretion of the applicant.

964

965 If an internal standard is used, the minimum acceptable resolution between the internal

966 standard and one or more active ingredient should be specified. If the analytical

967 procedure is used to control the level of impurities, the minimum resolution between

968 the active ingredient and the closest eluting impurity, or the two peaks eluting closest

969 to each other, should be given.

970

971 E. Optical Rotation

972

973 Optical rotation is used for the measurement of stereochemical purity. Visual polarimeters rely

974 on a monochromatic source, which traditionally was sodium D, but has expanded to virtually

975 any wavelength. 

976

977 If measurements are to be made at a wavelength other than sodium D, an explanation for

978 selecting the wavelength should be given, along with a comparison of the specific rotation at

979 sodium D and the wavelength to be used. Circular dichroism (CD) spectra may suffice for this

980 purpose. In addition to the provisions of USP <781>, procedures for measurement of

981 specific rotation should include the solvent, concentration, and, for aqueous solutions, the pH

982 to which the solution should be adjusted. The conditions and equipment should be shown to

983 be suitable to confirm the stereochemical identity of a racemate or an enantiomer.

984

985 The enantiomeric purity can be expressed as enantiomeric excess (e.e.), using the following

986 formula as an example:
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987

988 e.e. = 100% * {{M} - [m]}/{[M] + [m]}

989

990 where [M] and [m] are the concentrations of the major and minor enantiomers, respectively. 

991 This yields values of zero for a racemate and 100 percent for a pure enantiomer. An

992 intermediate concentration gives intermediate values; for example, 97:3 would give an e.e. of

993 94 percent.

994

995 Appropriate system suitability and/or calibration testing is recommended. Validation criteria

996 should include specificity, and intermediate precision.

997

998 F. Methodologies Relating to Particle Size Analysis

999

1000 Particle size analysis is an important element for quality control and regulatory evaluation of

1001 certain drug substances and drug products. The normal concepts of validation may differ for

1002 particle size methodologies as compared to other analytical methodologies such as HPLC. 

1003 However, a standard mixture may be used for calibration.

1004

1005 Particle size evaluation can include characteristics of size, morphology, surface, and population

1006 of particles. The following parameters are useful for describing particle size analysis for

1007 characterization of drug substances and drug products.

1008

1009 1. Particle Size Methods

1010

1011 Types of particle size methods include, but are not limited to:

1012

1013 a. Nonfractionation methods that evaluate an entire population of particles

1014

1015 ! Microscopy (optical, electron)

1016 ! Light scattering (dynamic, photon correlation, laser diffraction)

1017 ! Electrozone sensing

1018 ! Photozone sensing

1019

1020 b. Fractionation methods that use physical techniques to separate particles on the

1021 basis of size

1022

1023 ! Sieving

1024 ! Cascade impactor

1025 ! Sedimentation

1026 ! Size exclusion chromatography

1027

1028 2. Calibration and Validation Characteristics

1029
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1030 To ensure proper instrument operation, the system should be calibrated according to

1031 the manufacturer's and/or the laboratory's specification, as appropriate.

1032

1033 The methods validation usually involves evaluation of intermediate precision and

1034 robustness. Assurance should be provided that the data generated are reproducible

1035 and control the product's quality. See additional information in sections V and VII.

1036

1037 G. Dissolution

1038

1039 The equipment used for dissolution is covered by USP <711> or USP <724>. The

1040 dissolution procedure description and validation should include the following.

1041

1042 1. Dissolution Medium

1043

1044 A brief discussion of the reasons for selecting the medium.

1045

1046 2. Procedure

1047

1048 A dissolution test consists of a dissolution procedure and method of analysis

1049 (automated on-line analysis or manual sampling followed by HPLC analysis). The

1050 written procedure should cover the following items:

1051

1052 ! Apparatus

1053 ! Preparation of standard

1054 ! Preparation of sample

1055 ! Method of analysis (e.g., UV, HPLC)

1056 ! Sampling procedure (e.g., intervals, filtration, handling of samples, dilutions)

1057

1058 ! Calculations

1059 ! Acceptance criteria

1060

1061 Regardless of the method of analysis, system suitability criteria should be described. 

1062 Blank and standard solution spectra or chromatograms should be included.

1063

1064 3. Validation Characteristics

1065

1066 Both the dissolution procedure and the method of analysis should be validated. 

1067

1068 The time needed for the completion of the sample analysis should be stated in the

1069 procedure. Data should be submitted to support the stability of the dissolution sample

1070 during the procedure. If filters are used on-line or during sample preparation,

1071 appropriate recovery studies should be performed and documented and any bias

1072 should be addressed.
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1073 H. Other Instrumentation

1074

1075 1. Noncommercial Instrumentation

1076

1077 FDA encourages the development and use of the most appropriate instrumentation. 

1078 However, the use of rare or exotic systems not only places an undue burden on the

1079 regulatory laboratory, but also may delay the validation process.

1080

1081 When noncommercial instrumentation is used, the instrumentation should be capable

1082 of being constructed from commercially available components at a reasonable cost, if

1083 possible. For unique methodologies or instrumentation requiring contract fabrication,

1084 the applicant's cooperation with the FDA laboratories in helping facilitate duplication

1085 of the analytical procedure is important. In addition to design and equipment

1086 specifications, complete performance assessment procedures should be provided. 

1087 Such systems may be found suitable for regulatory use.

1088

1089 2. Automated Analytical Procedures

1090

1091 The use of automated analytical procedures, although desirable for control testing,

1092 may lead to delay in regulatory methods validation because FDA laboratories have to

1093 assemble and validate the system before running samples. To avoid this delay,

1094 applicants should demonstrate the equivalence of a manual procedure to the

1095 automated procedure based on the same principle whenever possible.
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1096 ATTACHMENT A

1097 NDA, ANDA, BLA, AND PLA SUBMISSION CONTENTS

1098

1099 The information relating to analytical procedures and methods validation that should be submitted in

1100 NDAs, ANDAs, BLAs, and PLAs is identified below with a cross-reference to the section of this

1101 guidance that provides recommendations and/or discussion on the topics.

1102

1103 Information that should be included in the analytical procedures and controls sections

1104

1105 ! Reference standard information Section IV

1106 • Analytical procedures Section III, VI

1107 • Validation data Section VII

1108 • Stress studies Section VII.A.2.c

1109 • Instrument output/raw data for impurities Section VII.A.2.b

1110 • Statistical analysis Section VIII

1111 • Revalidation, as needed Section IX

1112

Information that should be included in the methods validation package5 1113

1114

1115 • Contents of the MV Package Section XI

1116 • Representative instrument output/data for stress studies Section VII.A.2.c

1117 ! Representative instrument output and raw data for initial

1118 and oldest sample of a batch Section VII.A.2.b

1119

1120 Information that should be included in the stability section

1121

1122 ! Stress study designs and results Section VII.A.2.b

1123 ! Reference (volume and page number of submission)

1124 to instrument output and raw data submitted to the section

1125 dedicated to analytical procedures and controls Section VII.A 2.c

 5

 For BLAs and PLAs, a separate methods validation package need not be submitted. Information similar to

what is listed here should be included in the BLA or PLA submission.
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1126 ATTACHMENT B

1127

1128 METHODS VALIDATION PROBLEMS AND DELAY

1129

1130

1131 Listed below are examples of common problems that can delay successful validation.

1132

1133 ! Failure to provide a sample of a critical impurity, degradation product, internal standard, or

1134 novel reagent

1135

1136 ! Failure to submit well-characterized reference standards for noncompendial drugs

1137

1138 ! Failure to provide sufficient detail or use of unacceptable analytical procedures. For example:

1139

1140 C Use of arbitrary arithmetic corrections

1141 C Failure to provide system suitability tests

1142 C Differing content uniformity and assay analytical procedures without showing

1143 equivalence factors for defining corrections as required by the current USP chapter

1144 <905> - Uniformity of Dosage Units

1145

1146 ! Failure to submit complete or legible data. For example:

1147

1148 C Failure to label instrument output to indicate sample identity

1149 C Failure to label the axes

1150

1151 ! Inappropriate shipping procedures. For example:

1152

1153 C Failure to properly label samples

1154 C Failure to package samples in accordance with product storage conditions

1155 C Inadequate shipping forms (e.g., missing customs form for samples from outside the

1156 United States)

1157

1158 ! Failure to describe proper storage conditions on shipping containers
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1159 REFERENCES

1160

FDA Documents61161

1162

1163 Guidance for Industry: ANDAs: Impurities in Drug Products (Draft, December 1998).

1164

1165 Guidance for Industry: ANDAs: Impurities in Drug Substances (February 2000).

1166

1167 Guidance for Industry: CMC Content and Format of INDs for Phase 2 and 3 Studies of Drugs,

1168 Including Specified Therapeutic Biotechnology-Derived Products (Draft, December 1997).

1169

1170 Guidance for Industry: Content and Format of Investigational New Drug Applications (INDs)

1171 for Phase 1 Studies of Drugs, Including Well-Characterized, Therapeutic, Biotechnology1172 derived Products (February 1995).

1173

1174 Guidance for Industry: Investigating Out of Specification (OOS) Test Results for Pharmaceutical

1175 Production (Draft, September 1998).

1176

1177 Guidance for Industry: Stability Testing of Drug Substances and Drug Products (Draft, June

1178 1998).

1179

1180 Guidance for Industry: Submission of Chemistry, Manufacturing, and Controls Information for

1181 Synthetic Peptide Substances (November 1994).

1182

1183 Guidance for Industry: Submitting Documentation for the Stability of Human Drugs and

1184 Biologics (February 1987).

1185

1186 Reviewer Guidance: Validation of Chromatographic Methods (November 1994).

1187

1188 FDA CDER MAPP 5221.1 Requesting Methods Validation for ANDAs (November 1998).

1189

1190 International Conference on Harmonization Guidances

1191

1192 ICH Q1A: Stability Testing of New Drug Substances and Products (November 1994)

1193

1194 ICH Q1B: Photostability Testing of New Drug Substances and Products (November 1996)

1195

1196 ICH Q1C: Stability Testing for New Dosage Forms (May 1997)

1197

 

6

 Draft guidances have been included for completeness only. As draft documents, they are not intended to

be implemented until published in final form.
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1198 ICH Q2A: Text on Validation of Analytical Procedures (March 1995)

1199

1200 ICH Q2B: Validation of Analytical Procedures: Methodology (May 1997)

1201

1202 ICH Q3A: Impurities in New Drug Substances (January 1996)

1203

1204 ICH Q3B: Impurities in New Drug Products (May 1997)

1205

1206 ICH Q3C: Impurities: Residual Solvents (December 1997)

1207

1208 ICH Q5C: Quality of Biotechnological Products: Stability Testing of

1209 Biotechnological/Biological Products (July 1996)

1210

1211 ICH Q6A: Specifications: Test Procedures and Acceptance Criteria for New Drug Substances

1212 and New Drug Products: Chemical Substances (Draft (Step 2) November 1997)

1213

1214 ICH Q6B: Specifications: Test Procedures and Acceptance Criteria for

1215 Biotechnological/Biological Products (March 1999)

1216

1217 U.S. Pharmacopeia/National Formulary

1218

1219 Chapter <621> Chromatography; US Pharmacopeia 23, United States Pharmacopeial Convention,

1220 Inc., Rockville MD: 1994

1221

1222 Chapter <781> Optical Rotation, US Pharmacopea 23, United States Pharmacopeial Convention,

1223 Inc., Rockville, MD: 1994

1224

1225 Chapter <1225> Validation of Compendial Methods; US Pharmacopeia 23, United States

1226 Pharmacopeial Convention, Inc., Rockville MD: 1994

1227

1228 Interpretation and Treatment of Analytical Data; USP Pharmacopeial Forum, United States

1229 Pharmacopeial Convention, Inc., Rockville MD: 1994, Volume 24, Number 5, pp. 7051 - 7056

1230

1231 Other

1232

1233 Miller, J.C., J.N. Miller, and E. Horwood, Statistics for Analytical Chemistry, 3rd edition, Prentice

1234 Hall, 1993.

1235

1236 Saunders, B.D., and R.G. Trapp, Basic and Clinical Biostatistics, 2nd edition, Appleton and Lange,

1237 1994.
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1238 GLOSSARY

1239

1240

1241 Acceptance Criteria: Numerical limits, ranges, or other suitable measures for acceptance of the

1242 results of analytical procedures.

1243

1244 Active moiety: The molecule or ion, excluding those appended portions of the molecule that cause

1245 the drug to be an ester, salt (including a salt with hydrogen or coordination bonds), or other

1246 noncovalent derivative (such as a complex, chelate, or clathrate) of the molecule, responsible for the

1247 physiological or pharmacological action of the drug substance (21 CFR 314.108(a)). The active

1248 moiety is the entire molecule or ion, not the active site.

1249

1250 Detection Limit: The detection limit of an individual analytical procedure is the lowest amount of

1251 analyte in a sample that can be detected, but not necessarily quantitated as an exact value.

1252

1253 Drug Product: A finished dosage form, for example, a tablet, capsule, or solution that contains a

1254 drug substance, generally, but not necessarily, in association with one or more other ingredients (21

1255 CFR 314.3(b)).

1256

1257 Drug Substance/Active Ingredient: An active ingredient that is intended to furnish pharmacological

1258 activity or other direct effect in the diagnosis, cure, mitigation, treatment, or prevention of disease or to

1259 affect the structure or any function of the human body. The active ingredient does not include

1260 intermediates used in the synthesis of such ingredient. The term includes those components that may

1261 undergo chemical change in the manufacture of the drug product and be present in the drug product in

1262 a modified form intended to furnish the specified activity or effect (21 CFR 210.3(b)(7) and

1263 314.3(b)).

1264

1265 Placebo (or Blank): A dosage form that is identical to the drug product except that the drug

1266 substance is absent or replaced by an inert ingredient or a mixture of the drug product excipients

1267 quantitatively equivalent to those found in the drug product dosage form.

1268

1269 Quantitation Limit: The quantitation limit of an individual analytical procedure is the lowest amount

1270 of analyte in a sample that can be quantitatively determined with suitable precision and accuracy. The

1271 quantitation limit is a parameter of quantitative assays for low levels of compounds in sample matrices,

1272 and is used particularly for the determination of impurities and/or degradation products.

1273

1274 Reagent: For analytical procedures, any substance used in a reaction for the purpose of detecting,

1275 measuring, examining, or analyzing other substances.

1276

1277 Specification: The quality standards (i.e., tests, analytical procedures, and acceptance criteria)

1278 provided in an approved application to confirm the quality of the drug substances, drug products,

1279 intermediates, raw materials, reagents, and other components including container closure systems, and

1280 in-process materials.
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1281

1282 Spiking: The addition of a small known amount of a known compound to a standard, sample, or

1283 placebo, typically for the purpose of confirming the performance of an analytical procedure or the

1284 calibration of an instrument.

1285

1286 Stability-Indicating Assay: A validated quantitative analytical procedure that can detect the

1287 changes with time in the pertinent properties (e.g., active ingredient, preservative level) of the drug

1288 substance and drug product. A stability-indicating assay accurately measures the active ingredients

1289 without interference from degradation products, process impurities, excipients, or other potential

1290 impurities.

1291

1292 Working Standard: A standard that is qualified against and used instead of the reference standard

1293 (also known as in-house or secondary standard).

1294

1295

1296

1297