9 METHOD VALIDATION Rosario LoBrutto and Tarun Patel 9.1 INTRODUCTION The method validation process is to confirm that the method is suited for its intended purpose. Although the requirements of validation have been clearly documented by regulatory authorities [ICH, USP, and FDA], the approach to validation is varied and open to interpretation. Validation requirements differ during the development process of pharmaceuticals. The method validation methodologies in this chapter will focus on the method requirements for preliminary and full validation for both drug substance and drug product. Preliminary method validation is generally performed in the earlier phases of development up to Phase IIa because at this time ICH Q2A and Q2B [1] are not yet binding. A more extensive validation (full validation) is performed for methods used in later stages of drug development (after Phase IIa) and for methods that will be used to evaluate marketed products. Specific require- ments or methodologies for validation depending on the life cycle of the potential drug candidate in each specific area in the drug development process will be addressed in the corresponding chapter. An analytical method is a laboratory procedure that measures an attribute of a raw material, drug substance, or a drug product. Analytical method vali- dation is the process of demonstrating that an analytical method is reliable and adequate for its intended purpose. Any method that is utilized to deter- mine results during drug substance and formulation development will have to be validated. Reliable data for release of clinical supplies, stability, and setting shelf life can only be generated with appropriate validated methods. 455 HPLC for Pharmaceutical Scientists, Edited by Yuri Kazakevich and Rosario LoBrutto Copyright © 2007 by John Wiley & Sons, Inc. Validation of high-performance liquid chromatography (HPLC) methods focus mainly on the following: • Identification tests • Quantitative measurements of the content of related substances* • Semiquantitative and limit tests for the control of related substances* • Quantitative tests for the assay of major components (e.g., drug substance and preservatives) in samples of drug substance or drug product (assay, content uniformity, dissolution rate, etc.) Moreover, HPLC methods that are described in pharmacopeias may not have to be validated but should be verified, if necessary. Well-characterized refer- ence materials with documented purity should be used throughout the vali- dation study, especially during full development. Validation experiments and analyses must be carried out on fully qualified and calibrated instrumentation, and some references have been published on this subject [2–6]. Analytical method validation is established through documented evidence demonstrating the accuracy, precision, linearity, selectivity, ruggedness, and/or robustness of that particular test method which will be utilized to generate test results for a drug substance or drug product. Different test methods require different validation parameters. All analytical procedures require some form of method validation, regardless of whether the test method is utilized for the testing of Good Laboratory Practice (GLP) toxicology, shelf-life determina- tion (stability indicating), in-process controls [7], clinical release, or release of products for open market [8]. As development of the project progresses and as more analytical and product-specific information is acquired, the analytical methods evolve and are gradually updated. The extent of validation increases and the documentation is completed. During the early development phase, depending on the analytical labora- tory, generic validation protocols may be used because project-specific pro- tocols are not required. Sometimes an internal Standard Operating Procedure (SOP) suffices and a generic validation protocol does not need to be used. Usually, for Phase I, validation experiments may be carried out concurrently with the analysis of the first batch of clinical supplies or the first delivery of drug substance to be used for clinical supplies. However, depending on the pharmaceutical organization method validation may need to be performed prior to the analysis of material that will be used for clinical supplies. For analytical method validation during full development (after final syn- thesis has been set for drug substance and after final market formulation has been set for drug product) corresponding to the definitive control procedure for new drug application (NDA), a specific validation protocol has to be written. Before start of the experimental work, the protocols must be written 456 METHOD VALIDATION *Related substances described in this chapter encompass degradation products, and synthetic by-products. by an analytical chemist and approved by a quality assurance department. Some of the items that are necessary to be specified in the validation proto- col are listed below: • The analytical method for a given product or drug substance • The test to be validated • The test parameters for each test, including type and number of solutions and number of injections • The acceptance criteria for each parameter based on an internal SOP (product or method-specific adaptations may be necessary and are accept- able, if justified) • List of batches of drug substance and/or drug products • For a drug product the grade/quality of the excipients used in the formulation • List of reference materials to be used in the validation experiments • Information on the instruments and apparatus to be used • Responsibilities [author, chemists, analytical research project leader, quality assurance (QA), etc.] Depending upon a company’s culture, a method validation protocol could be simple (listed items above) or exhaustive (in addition to the listed items above, each parameter to be validated is described in detail): How solutions are going to be made, the experimental design, how the calculations are going to be performed, any software to be utilized (e.g., Excel). If a full-length protocol is required within a particular company, then the writing of this protocol and approval of the protocol would need to be completed prior to the com- mencement of the validation work. Otherwise, there may be many deviations to the protocol which will be needed to be referenced to in the final method validation report. Some companies also have templates for the validation reports, thereby allowing for facile population of the results. Once populated, the file is reviewed to determine if all validation parameters and acceptance criteria were met. If they were not, then a deviation is added and the proper justification must be given. If it is deemed that the justification is not ap- propriate, then an action plan for the specific figure of merit in question is determined (i.e., repeat analysis, change of the analytical procedure, and revalidation). Also, if the analytical method has not yet been approved at the time of writing the validation protocol, it is recommended to attach a final draft of the method to the protocol. The final HPLC method must also be approved with the validation report submission. 9.2 VALIDATION REPORT A validation report is written during early and full development, and approval by QA is required. Existing method validation data from earlier stages of VALIDATION REPORT 457 development may be used for full development if the HPLC method has not changed. Minor changes such as change in equilibration time may be accept- able, and the preliminary validation performed for early phase may be used. These data can be referred to in the validation report, and reference to the original data must be given. The validation report should contain reference to the analytical methods (specific code number used as identifier within the pharmaceutical organiza- tion) and the corresponding drug substance or product name. Note that for early-phase method validation reports the results maybe filled in a predefined table and compared against the acceptance criteria. However, for late-phase validation, more explicit reports are generated explaining each and every experiment, with detailed steps of sample and standard preparation. The list of reference materials (reference standards with the appropriate certificate of analysis) as well as the list of calibrated and qualified instruments used in the validation experiments should be documented in the report. For drug substances the list of the batches of drug substances, notebook number/reference number for any individual impurities, or solutions or inter- mediates used should be listed. For drug products the list of the batches of drug substances, drug product, and the grade/quality of excipients should be listed. The test parameters and acceptance criteria should be listed together with the results for each test, and the results passed or failed should be indi- cated. The validation report should also contain whether the method valida- tion was successful and if any changes had to be applied to the analytical method, and then the final analytical method must be resubmitted for QA approval. 9.3 REVALIDATION After any major changes in the HPLC method (solution preparation, experi- mental conditions, etc.) or after change in route of synthesis of the drug sub- stance or drug product manufacture (change of process, change of equipment, change of analytical procedure), it must be assessed whether a new validation or a partial validation is required, addressing all the validation parameters that may be affected by the methodological change. If revalidation is not deemed necessary, then the reasons behind the decision must be documented in the revision history of the test method and the proper change control initiated. The revision of the test method and any documents that refer to the original method, such as the analytical specifications, will then be approved by QA. When revalidation is deemed necessary, the reason for change must be docu- mented and any new validation activities must be performed according to an approved, updated HPLC validation protocol. The results would then be doc- umented in an update of the validation report or a supplement to the original validation report. 458 METHOD VALIDATION 9.4 ASSIGNMENT OF VALIDATION PARAMETERS The type and degree of validation depends on the nature of the test. In par- ticular, methods described in pharmacopeias may not have to be validated but should be verified, if needed. Different test methods require different valida- tion parameters. As development of the project progresses and as more ana- lytical and product-specific information is acquired, the analytical methods evolve and are gradually updated. The extent of validation increases and the documentation is completed. Table 9-1 outlines the validation parameters that are usually required for the early development stage, and Table 9-2 outlines the validation parameters that are usually required for the full development stage. The proposed acceptance criteria in Table 9-3 should be included in the validation protocol, especially for the full development stage. There are numerous method validation examples in the literature [9–18]. Each company has their own approach and own set of acceptance criteria for different analytical assays, but these approaches must be within the confines of their line unit QA department and be in accordance with any regulatory provisions. In the next section a description for each of the parameters to be validated (figures of merit) are described in detail and examples are given for each. ASSIGNMENT OF VALIDATION PARAMETERS 459 TABLE 9-1. Early Development Type of Tests to Be Validated Weight Percent/Assay/Content Impurity Testing: Validation Parameters Identity Uniformity/Dissolution Quantitative Test a Specificity Yes Yes Yes Linearity No Yes b Yes b Accuracy No Yes c Yes d Precision (repeatability) No Yes Yes e Limit of detection No No Yes f Limit of quantitation No g No Yes d Stability of the solutions No Yes Yes a If impurities not available, with drug substance. b Four points may be adequate. c For drug product only (assay/CU/dissolution). d A spiking experiment carried out is adequate at this stage (only possible if impurity/impurities are available). e At least triplicate analysis. f Not required, but recommended. g For the identity test of a 0-mg formulation (placebo), it may be necessary to document the absence of drug substance, and an LOQ determination will then be required. 9.4.1 Accuracy The test for accuracy is intended to demonstrate the closeness of agreement between the value found and the value that is accepted either as a conven- tional true value or as an accepted reference value [19]. Therefore, accuracy can be defined as the agreement between the result obtained with method being validated and an accepted reference value. The accuracy can be inferred from precision, linearity, and specificity. The results for the method being validated can be compared to the results with those of a well-characterized, independent method. These results may be compared to an alternate reversed-phase HPLC method (phenyl versus C18 or separation run at dif- ferent pH using the same column) using the same detection scheme. In some, cases an orthogonal method is used to demonstrate accuracy. The methods should differ with respect to separation mode and therefore provide orthog- onal information concerning related substances and degradation products. For example, one method would use reversed-phase (RP) separation mode on a C18 column, and the second method would use a strong cation exchange (SCX) column [20]. The orthogonal methods may show different selectivities toward the degradation products, thereby demonstrating the orthogonal nature of the two separation techniques. The accuracy would be demonstrated 460 METHOD VALIDATION TABLE 9-2. Full Development Type of Tests to Be Validated Weight Percent/Assay/Content Impurity Testing: Validation Parameters Identity Uniformity/Dissolution Quantitative Test Specificity a Yes Yes Yes Linearity No Yes Yes Accuracy No Yes Yes Precision (repeatability) No Yes Yes Precision (intermediate No Yes Yes precision) b Precision (reproducibility) No cc Range No Yes Yes Limit of detection No No Yes e Limit of quantitation No d No Yes Stability of the solutions No Yes Yes Robustness f Yes Yes a Lack of specificity of one analytical procedure may be compensated for by other supporting analytical procedures. b In cases where reproducibility has been performed, intermediate precision not needed. c In exceptional cases. d For the identity test of a 0-mg formulation (placebo) it may be necessary to document the absence of drug substance and an LOQ determination will then be required. e Not required by ICH, but recommended. f May be required, depending on the nature of the test. ASSIGNMENT OF VALIDATION PARAMETERS 461 TABLE 9-3. Proposed Acceptance Criteria for Drug Product (DP) and Drug Substance (DS) Quality Characteristics Parameter to be Validated Acceptance Criteria Identity Selectivity/specificity All known peaks are separated. Major (API) peak is “pure” [Peak purity angle ≥ peak threshold angle]. {DS and DP} For the identity test of a 0-mg formulation (placebo), it may be necessary to document the absence of drug substance, and an LOQ determination will then be required. {only DP} Dissolution Accuracy (mean) (drug • Recovery 95–105% product) • S rel for recovery ≤2.5% Precision • Repeatability S rel ≤ 2.0%, n ≥ 6 {at Q time} • Intermediate precision Project specific. Linearity n ≥ 6 • Correlation coefficient r ≥ 0.990 • y-intercept (absolute value) ≤5% • Residual standard deviation ≤2.5% Stability of solutions • Sample ≤2.0% change over specified time • Reference standard ≤2.0% change over specified time Specificity • HPLC No interference from placebo solution at the retention time of API. Range (basket/paddle) IR: ±30% of specified range MR,SR: From 50% of Q-value to 130% of label claim. Content Precision As defined in assay uniformity Accuracy (CU) Stability of solutions Drug product Specificity Chromatographic peaks are separated. No indication of interference from placebo solution at the retention time of API. Linearity n ≥ 6 • Correlation coefficient r ≥ 0.990 • y-intercept ≤5.0% • Residual standard deviation ≤2.0% Range At least 70–130% of declared content Assay—drug Accuracy (mean)—DP product • Recovery 98.0–102.0% • S rel for recovery ≤2.0%, n ≥ 9 (at least three concentrations) 462 METHOD VALIDATION TABLE 9-3. Continued Quality Characteristics Parameter to be Validated Acceptance Criteria Weight Accuracy—DS percent— • Comparison of methods % difference of the mean of two drug (i.e., titration, DSC, PSA) methods ≤2.0% substance Precision • Repeatability S rel ≤ 2.0%, n ≥ 6, DP S rel ≤ 1.0%, n ≥ 6, DS • Intermediate precision S rel ≤ 2.0%, n ≥ 4 [when combined from two analysts] Linearity n ≥ 6 • Correlation coefficient r ≥ 0.998 • y-intercept ≤2.0% • Residual standard deviation ≤2.0% Stability of solutions • Sample ≤2.0% change over specified time (DP) • Reference standard ≤2.0% change over specified time (DP) • Sample ≤1.0% change over specified time (DS) • Reference standard ≤1.0% change over specified time (DS) Specificity • HPLC Chromatographic peaks are separated. No indication of interference from placebo solution at the retention time of API. No indication of another peak under the API peak. Range At least 80–120% of declared content (100% = concentration X of final sample stock solution) Ruggedness/robustness ≤1.0% difference for a defined range of intentionally altering sensitive parameters (pH of mobile phase, column, temperature, flow rate, wavelength, etc.) Drug product- Precision Related • Repeatability Level < 0.1%, S rel ≤ 30%, n ≥ 6 substances Level 0.1–<0.2%, S rel ≤ 20%, n ≥ 6 (degradation Level 0.2–<0.5%, S rel ≤ 10%, n ≥ 6 products) Level 0.5–<5%, S rel ≤ 5%, n ≥ 6 Level ≥ 5%, S rel ≤ 2.5%, n ≥ 6 Drug substance • Intermediate precision Level < 0.1%, S rel ≤ 40%, n ≥ 4 (synthetic by- [all replicates combined Level 0.1–<0.2%, S rel ≤ 30%, n ≥ 4 products and from two analysts] Level 0.2–<0.5%, S rel ≤ 15%, n ≥ 4 degradation Level 0.5–<5%, S rel ≤ 7.5%, n ≥ 4 products) Level ≥ 5%, S rel ≤ 4.0%, n ≥ 4 Specificity Known peaks are separated. • HPLC No indication of interference from placebo solution at the retention time of API. No indication of another peak under the API peak. ASSIGNMENT OF VALIDATION PARAMETERS 463 TABLE 9-3. Continued Quality Characteristics Parameter to be Validated Acceptance Criteria Linearity n ≥ 6 • Correlation coefficient r ≥ 0.990, DP and r ≥ 0.998, DS • y-intercept Level < 0.5%: ≤25% Level 0.5–<1%: ≤10% Level ≥1%: ≤5.0% • Residual standard deviation Level < 0.2%: ≤20% Level 0.2–<0.5%: ≤10% Level 0.5–<5%: ≤5.0% Level ≥ 5%: ≤2.5% Range LOQ to 120% of specification limit of largest impurity or related substance LOD Peak signal/noise ratio ≥3:1 LOQ Peak signal/noise ratio ≥10 : 1 and S rel ≤ 10%, n ≥ 5 Accuracy (mean) Level < 0.2%: 70–130% • Recovery Level 0.2–<0.5%: 80–120% Level 0.5–<5%: 90–110% Level ≥ 5%: 95–105% • S rel for recovery Level < 0.5%: ≤10%, Level 0.5–<5%: ≤5% Level ≥ 5%: ≤2.5% For all, n = 9 (at least three concentrations), a weighted average maybe used based on the level and the S rel . Stability of solutions [report two decimal places] • Reference standard Level < 5% of theoretical 100% Change ≤10% concentration over specified time Level ≥ 5% of theoretical 100% Change ≤2.0% concentration over specified time • Sample Related substances (impurities) Level < 0.5% Change ≤20% over specified time Level 0.5–<5% Change ≤10% over specified time Level ≥ 5% Change ≤5% over specified time No new peak ≥ reporting level Ruggedness/robustness Defined based on an experimental design and data (sensitive parameters and a range for each parameter in the final test method) if the overall purity in both of the methods would still be the same according to a predefined set of acceptance criteria. Different types of separation methods could also be used to show accuracy. For example, if normal-phase chromatography was used as the parent method, this could be compared to a separation obtained using supercritical chromatography. In another example, an electrophoretic method using capillary electrochromatography or capillary electrophoresis could be compared to an HPLC separation. Also, the HPLC weight percent (assay) method of the drug substance can be compared to nonchromatographic methods such as nuclear magnetic resonance (NMR) [21], Phase Solubility Analysis (PSA), and DSC [22] and to nonspecific titration and spectrophotometric assay methods that may have been used in early development before the qualification of a reference stan- dard. Potentiometric titration methods using nonaqueous or aqueous titrations are only amenable to ionizable compounds and are nonspecific because the impurities may contain the same ionizable functionality as the parent com- pound being titrated. Titration is a nonspecific method because synthetic by- products in drug substance may have a pK a similar to that of the main component (the endpoints for the by-products and the drug substance may overlap in this case) and results may be biased, leading to a higher weight percent of the material. However, these titration methods can be used in early development when a reference standard is not available. Also, the spectrometric-based assay methods such as ultraviolet (UV) may be nonspecific because most of the drug substance impurities contain a similar chromophore as the parent molecule. If UV is used, UV absorption is measured at one or more wavelengths and the absorbance value is recorded for a particular concentration. Sandor Gorog has critically evaluated the difference between specific and nonspecific assay methods in the European and US Pharmacopoeias [23]. The difference between the mean and the accepted true value with a defined confidence inter- val should be reported in the acceptance criteria. The accuracy can also be demonstrated by recovery of drug substance spiked into a placebo for a drug product. The accuracy can also be demon- strated by recovery of the impurity spiked to a drug substance or into a placebo with drug substance. The percentage recovery with the certain accep- tance criteria at each defined level is reported. Accuracy should be assessed using a minimum of nine determinations at a minimum of three concentration levels covering the specified range (e.g., 3 concentrations/3 replicate preparations of each in the total analytical proce- dure) within the ranges shown in Table 9-4. Accuracy is performed to determine recovery of an active or degradation products from a drug product or recovery of related substances from a drug substance. The experiment is designed to recover the total amount of active or degradation product from a drug product or a specific impurity or impuri- ties from a drug substance. For recovery of the active for assay and CU, a known amount of drug substance in solution is spiked into the placebo blend. The influence of sample preparation steps for tablets must be taken into con- 464 METHOD VALIDATION [...]... these percent difference values should show ≤10% difference to provide additional support for qualifying the proposed LOQ as the official LOQ for the method 9.4.3.1 Linearity Example (Assay and Content Uniformity) An example for linearity for Assay and Content Uniformity is given The target concentration is 1.0 mg/mL for this particular drug substance D Table 9-12 shows the table that could be included... ≤ 2.5% for 5.0, 7.0 and 9.0% Criteria set at %RSD ≤ 5% for 1.0, 2.0 and 3.0% Criteria set at %RSD ≤ 10% for 0.4% [31–33] The following acceptance criteria have been met as shown in Table 9-16 for the linearity part of this validation 9.4.3.2 Linearity Example (Related Substances, Low-Level Linearity) An example for linearity of related substances is given The target concentration is 1.0 mg/mL for compound... dosage form In contrast, if a stand-alone homogenizer was utilized for the same procedure for this dosage form, then the total sample extraction time was found to be about 5 minutes with intermittent stops required by the system software (e.g., TPWII takes about 2 to 5 seconds from end of one pulse to the start of another pulse) The homogenizer provides the energy needed to break the dosage form and... pulses at 8000 rpm for 10 seconds each Six pulses at 15,000 rpm for 15 seconds each Soak/settle time for 2 minutes (to allow all particles to settle to the bottom of the vessel, which allowed for a facile filtration step) 9.4.2 Precision Precision provides an indication of random errors and can be broken down into repeatability and intermediate precision This procedure should only be performed when the... the lower limit (LOQ to 1.0%) for the degradation products and (b) the higher limit (80–120%) for the assay of the active If the assay method is also used for content uniformity, the range 80–120% should be expanded accordingly to 70–130% As stated in the recovery section, if authentic degradation products or impurities are not available, then an API may be utilized to perform the linearity test at the... determined by HPLC analysis Then the percent recovery can be determined (actual overall/theoretical overall) × 100 9.4.1.1 Filter Check If for the drug product the sample preparation procedure (recovery procedure) requires filtering the sample solution prior to the solution being injected into an HPLC system, then a check for adsorption of the components onto the filter membrane must be performed The experiment... this linearity is for both assay and content uniformity TABLE 9-17 Linearity for Related Substances for Compound D Target % of Solutions 9.0 7.0 5.0 3.0 2.0 1.0 0.4 Target Concentration (mg/mL) Target Amount Injected (µg) Number of Injections 0.09 0.07 0.05 0.03 0.02 0.01 0.004 0.9 0.7 0.5 0.3 0.2 0.1 0.04 2 2 2 2 2 2 6 a Note that in this theoretical example the specification limit for compound D is... development gives a drug release profile for at least 8 hours This corresponds to the release rate of drug substance or the API from the dosage form within 8 hours at 37°C in the dissolution media (apparatus I as described in the United States Pharmacopoeia (USP) 〈711〉 at 100 rpm) For example, when a sample preparation procedure was developed for assay and degradation products for a modified release drug product,... industry and are accepted by health authorities as well To determine LOD and LOQ, progressive dilutions of the analyte are prepared and analyzed For HPLC, the normal range for LOD/LOQ is between 0.01% and 0.2% for non-peptide/protein-related products; however, for protein/peptides the LOD/LOQ range is typically between 0.1% and 0.5% From the injected series, a peak is selected whose height hs is about... filter (e.g., 2–6 mL) Therefore, the minimum volume that needs to be passed through the filter to get constant peak areas that are comparable to the centrifuged peak areas must be determined If those areas are within (2.0%), then the designated amount of volume that is needed to pass through the filter before the 469 ASSIGNMENT OF VALIDATION PARAMETERS solution can be collected for HPLC analysis must be noted . Reliable data for release of clinical supplies, stability, and setting shelf life can only be generated with appropriate validated methods. 455 HPLC for Pharmaceutical. solution can be collected for HPLC analysis must be noted. These types of filter experiments must be performed every time the drug product formula- tion has changed