Ministry of Health MS Brazilian Health Surveillance Agency - ANVISA

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1 RESOLUTION OF COLLEGIATE BOARD RDC # 166, OF JULY 24, 2017 (Published in Brazilian Official Gazette #141 on July 25, 2017) It provides for the validation of analytical methods and other provisions. The Collegiate Board of the Brazilian Health Surveillance Agency, in the use of the attribution conferred by art. 15, III and IV allied to art. 7, III and IV of Law No. 9,782 of January 26, 1999, and to art. 53, V, Paragraph 1 and Paragraph3 of the Internal Regulations approved in accordance with Annex I of the Resolution of the Collegiate Board of Directors - RDC No. 61 of February 3 rd, 2016, resolves to adopt the following Resolution of the Collegiate Board of Directors, as resolved at a meeting held On July 11 th, 2017, and I, the Chief Executive Officer, hereby determine its publication. CHAPTER I INITIAL PROVISIONS Section I Objective Art. 1 This Resolution establishes criteria for the validation of analytical methods. Sole paragraph. Failure to comply with any of the criteria set forth in this Resolution must be technically justified and subject to analysis by ANVISA. Section II Scope Art. 2 This Resolution applies to analytical methods used in pharmaceutical ingredients, drugs and biological products at all stages of production. Paragraph 1 - Validation parameters and their respective acceptance criteria shall be defined according to the characteristics of the analyte and the nature of the method. Paragraph 2 - Analytical methods applied to the products under investigation used in clinical trials shall have their suitability demonstrated in accordance with this Resolution, as applicable for each phase of clinical development. I - The use of an alternative approach must be technically justified based on recognized scientific references. Paragraph 3 - The use of alternative approaches for the validation of analytical methods applied to biological products, such as biological and immunological tests, shall be permitted.

2 Paragraph 4 - Microbiological methods for which a technical justification for the chosen approach based on the Brazilian Pharmacopoeia or other official compendia recognized by ANVISA are excluded from this Resolution. Section III Definitions Art. 3 For the purposes of this Resolution, the following definitions are adopted: I.- Sample: representative quantity of pharmaceutical ingredient, intermediate product or finished product, duly identified, within the established period of validity; II.- Analyte: substance or set of substances of interest to be identified or quantified; III.- Characterization of chemical substance: this is the set of tests which unequivocally guarantees the authenticity and quality of the substance as regards its identity, purity, content and strength and shall include data obtained from techniques applicable to the characterization of each substance, for example, thermogravimetry, melting point, differential scanning calorimetry, infrared spectroscopy, mass spectrometry, nuclear magnetic resonance, elemental (carbon / hydrogen / nitrogen) analysis, X-ray diffraction, optical rotation, chromatographic assays, among others; IV.- Analytical run: set of measurements performed on a group of samples within a predetermined time point under the same repeatability conditions, such as method, analyst, instrumentation, location and conditions of use; V.- Matrix effect: effect of matrix components on analytical response; VI.- Test: a technical operation consisting of the determination of one or more characteristics of a given ingredient or product according to a specified method; VII.- Limit test: tests to verify that the amount of analyte is above or below a preestablished level, without quantifying it accurately; VIII.- Response factor: ratio between analytical signal and analyte concentration; IX.- Relative response factor: ratio between two response factors, which is used as correction in the calculation of the concentration of a substance when it is measured by means of the analytical response of another; X.- Quality management: this is what determines the implementation of the "Quality Policy", that is, the overall intentions and guidelines regarding quality, formally expressed and authorized by the superior management of the company;

3 XI.- Impurities: any component present in the pharmaceutical ingredient or the finished product other than the active pharmaceutical ingredient or the excipient; XII.- Pharmaceutical ingredient: any substance being part of the formulation in a pharmaceutical form; XIII.- Active pharmaceutical ingredient (API): a pharmaceutical ingredient that, when administered to a patient, acts as an active component, and can exert pharmacological activity or direct effect in the diagnosis, cure, treatment or prevention of a disease or even affect the structure and functioning of the human organism; XIV.- Complex matrix: one containing an undefined number of unmonitored substances, which cannot be obtained without the presence of the analyte; XV.- Matrix: composition that mimics the sample without the presence of the analyte; XVI.- Investigational product: experimental drug, placebo, active comparator or any other product to be used in the clinical trial; XVII.- Chromatographic purity: absence of interference in the chromatographic signal of the analyte; XVIII.- Peak purity: spectral homogeneity of a chromatographic peak, indicative of its chromatographic purity, and the criteria for concluding whether there is spectral homogeneity and the parameters adopted for the purity calculation are defined as previously established for the software used or by means of scientifically based evaluation technique; XIX.- Validation report: document in which the validation procedures, records, results and evaluation are consolidated and summarized; XX.- Revalidation of analytical method: partial or complete re-validation of an analytical method to ensure that it continues to comply with established requirements; XXI.- Chemical Reference Substance (CRS): substance or mixture of high purity chemical or biological substances, which has been carefully characterized to ensure its identity, quality, content and potency, including a characterized chemical reference substance and a pharmacopoeial chemical reference substance; XXII.- Characterized Chemical Reference Substance (CCRS): Substance or mixture of chemical or biological substances in which identity, quality, purity, content and strength have been ensured by a characterization process; XXIII.- Pharmacopoeial Chemical Reference Substance (PCRS): Substance or mixture of chemical or biological substances established and distributed by official compendia recognized by ANVISA;

4 XXIV.- Working Chemical Reference Substance (WCRS): Substance or mixture of chemical or biological substances used in the laboratory routine, standardized from a pharmacopoeial reference chemical or, in the absence thereof, from a characterized chemical reference substance, traceable to the CRS used for its standardization; XXV.- Method transfer: a documented process that qualifies a laboratory (receiving unit) for the use of an analytical method from another laboratory (transfer unit), ensuring that the receiving unit is knowledgeable and capable of performing the analytical method according to intended purpose; XXVI.- Analytical validation: is the systematic evaluation of a method by means of experimental tests in order to confirm and provide objective evidence that the specific requirements for its intended use are met; XXVII.- Partial validation: demonstration, through some validation parameters, that the previously validated analytical method has the necessary characteristics to obtain results with the required quality, under the conditions in which it is practiced; and XXVIII.- System suitability: Procedure to be carried out prior to an analytical run to demonstrate that the system is fit for the intended use, and the parameters of this procedure must be defined during the development and validation of the method. CHAPTER II GENERAL PROVISIONS Art. 4 Validation shall demonstrate that the analytical method produces reliable results and is appropriate to the intended purpose, in a documented manner and through objective criteria. Art. 5 The use of analytical method not described in an official compendium recognized by ANVISA requires the conduction of an analytical validation, according to the parameters established in this resolution, taking into account the technical-operational conditions. Art. 6 The typical parameters to be considered for validation depend on the test to be carried out and are set out in Table 1 of Annex I. Art. 7 Compendial analytical methods must have their demonstrated suitability for the intended use, under the operational conditions of the laboratory, by means of the presentation of a partial validation study.

5 Sole paragraph. The provisions of the caput exclude basic general compendial methods such as ph measurement, drying loss, sulfated ash, moisture, disintegration, among others, and the analytical methods described in individual compendial monographs of non-active pharmaceutical ingredients. Art. 8 Partial validationvalidations shall evaluate, at least, the parameters of precision, accuracy and selectivity. Paragraph 1 - In case of analytical methods for quantification of impurities, partial validation shall include the quantification limit. Paragraph 2 - In case of a limit test, instead of the parameters of the caput, the selectivity and detection limit parameters must be evaluated. Art. 9 In case of transfer of method between laboratories, this will be considered as validated, provided that a partial validation study is carried out in the premises of the receiving laboratory. Paragraph 1 - Transfer of method between laboratories with the same quality management system can be carried out by means of a partial validation study, according to art. 8, or by reproducibility assessment. Paragraph 2 - Another approach may be accepted, based on justification and presentation of protocol and transfer report, based on risk analysis and considering previous experience, knowledge of the receiving unit, complexity of product and method and specifications, as well as other applicable relevant aspects. Paragraph 3 - If the transfer also uses comparative tests, the similarity in the results should be proven by means of statistical tool. Paragraph 4 - The method transfer documentation shall be presented containing the copy of the validation report of the transferred method as proof that it was originally validated in accordance with specific standards and regulations approved/endorsed by ANVISA. Paragraph 5 - In the case of transfer of methods already approved by ANVISA a copy of the approved validation report or an indication of the file number of the petition in which the final version of that report has been filed. Art. 10. A revalidation of analytical method may consider the following circumstances: I.- changes in the synthesis or obtainment of API; II.- changes in product composition; III.- changes in analytical method; and

6 IV.- other changes that could significantly impact the validated method. Sole paragraph. The validation parameters to be evaluated depend on the nature of the changes made. Art. 11 System verification shall be carried out at each analytical run. Art. 12 The validation and partial validation documents submitted shall describe the procedures, analytical parameters, acceptance criteria and results, with sufficient detail to enable their reproduction and, where applicable, their statistical evaluation. Art. 13 The validation report to be filed, according to registration and post-registration resolutions, shall contain the data and calculations obtained during the conduction of the analytical validation, as well as the statistical approach used for data evaluation. Paragraph 1 - The raw data related to the selectivity parameter should be part of the report mentioned in the caput. Paragraph 2 - The raw data related to other parameters must be available at the company for evaluation at ANVISA s request. CHAPTER III CHEMICAL REFERENCE SUBSTANCES Art. 14 In the validation of analytical methods Pharmacopoeial Chemical Reference Substance (PCRS) should be preferably used, officially designated by the Brazilian Pharmacopoeia, or by other compendia officially recognized by ANVISA. Paragraph 1. The use of Characterized Chemical Reference Substance (CCRS) will be admitted by submitting a conclusive characterization report for the batch under study, including the technical reasons for choosing the tests used and the relevant raw data. Paragraph 2 - ANVISA and members of the Brazilian Health Surveillance System may request samples from the CRS for the purpose of evaluating the characterization process in the hypotheses of the previous paragraph and, when it is necessary to carry out a fiscal analysis, a sample of CRS should be provided for the purposes of realization of the necessary tests. Art. 15 The characterization report, depending on the analyte, shall contain the data obtained from techniques applicable to the characterization of each chemical such as thermogravimetry, melting point, differential scanning calorimetry, infrared spectroscopy, mass spectrometry, nuclear magnetic resonance, elemental analysis (carbon/hydrogen/nitrogen), X- ray diffraction, optical rotation, chromatographic methods, among others.

7 Paragraph 1 - In addition to the characterization data, the following information should be included in the report: I.- number and shelf-life of the batch of the substance used in the characterization; II.- Brazilian non-proprietary name or international non-proprietary name; III.- CAS number; IV.- chemical name; V.- synonymy; VI.- molecular and structural formula; VII.- molecular weight; VIII.- physical form; IX.- physicochemical properties; X.- impurity profile; XI.- handling and conservation care, and XII.- Analytical report proving the identity, content and validity of CRS. Paragraph 2 - For biological products, the characterization of the reference material/standard shall be carried out using appropriate state of the art methods. Art. 16 For medical gases, analytical instrument verification and analytical determinations shall be conducted using traceable reference materials distributed by metrology institutes or by bodies recognized as producers of certified reference materials. Sole paragraph. In the absence of reference materials, internal standards produced according to guides and bibliographic records may be used. Article 17 For biological products, the terms material/standard substitutes the term chemical substance in the definitions of CRS, PCRS, CCRS and WCRS. Art. 18 The use of WCRS for the purpose of analytical method validation is not allowed.

8 CHAPTER IV ANALYTICAL VALIDATION PARAMETERS Section I Selectivity Art. 19 The selectivity of the analytical method should be demonstrated by its ability to identify or quantify the analyte of interest unambiguously in the presence of components that may be present in the sample such as impurities, diluents and matrix components. Sole paragraph. In the case of chromatographic methods, the chromatographic purity of the analyte signal must be demonstrated, except for biological products. Art. 20 In identification methods, the ability to obtain a positive result for the sample containing the analyte and a negative result for other substances in the sample. Paragraph 1 - CRS shall be used in comparison with the response obtained for the analyte under Chapter III. Paragraph 2 - In order to demonstrate the selectivity of the identification methods, the tests shall be applied to substances structurally similar to the analyte, with the acceptance criterion being negative. Paragraph 3 - For active pharmaceutical ingredients derived from plants and medicinal products containing them, the ability of the method of distinguishing material of interest from other similar plant species, particularly those which may be present as adulterants or substituents. Paragraph 4 - To achieve the required level of selectivity, a combination of two or more analytical methods of identification may be required. Art. 21 For quantitative methods and limiting tests, the selectivity shall be demonstrated by verifying that the analytical response is exclusively due to the analyte, without interference from the diluent, matrix, impurities or degradation products. Paragraph 1 - To demonstrate the absence of interference degradation products, it is necessary to expose the sample to degradation conditions in a wide ph range, oxidation, heat and light. Paragraph 2 - The following cases shall be exempted from the demonstration described in Paragraph 1:

9 I.- products for which it has already been shown to comply with the resolution that establishes parameters for the notification, identification and qualification of degradation products in drugs. II.- performance methods; III.- non-chromatographic methods. Paragraph 3 - The use of a method with technical limitation for selectivity, under the caput, is only accepted by technical justification and joint application of another complementary method. Art. 22 For medicinal gases, selectivity should be demonstrated by comparing the result of reading the sample with the reading response of CRS under Chapter III. Sole paragraph. The maximum amount of possible interference should be justified. Section II Linearity Art. 23 The linearity of a method must be demonstrated by its ability to obtain analytical responses directly proportional to the concentration of an analyte in a sample. Art. 24 A linear relationship must be evaluated throughout the established range for the method. Art. 25 For the establishment of linearity, at least 5 (five) different concentrations of CRS must be used for solutions prepared in at least triplicate. Sole paragraph. The solutions used for linearity evaluation must be prepared independently, and diluted solutions of the same CRS stock solution may be used. Art. 26 All calculations for the assessment of linearity shall be performed from actual concentration data and individual analytical responses. Art. 27 For the assessment of linearity, the following data shall be presented: I.- graphical representation of the responses as a function of analyte concentration; II.- graph of dispersion of the residues, accompanied by its statistical evaluation; III.- equation of the regression line of y in x, estimated by least squares method;

10 IV.- evaluation of the linear association between the variables by means of the coefficients of correlation (r) and determination (r²); V.- significance evaluation of the angular coefficient. Paragraph 1 - Homoscedasticity of the data shall be investigated for use of the appropriate model. Paragraph 2 - Statistical tests should be used a significance level of 5% (five percent). Paragraph 3 - The correlation coefficient should be above Paragraph 4 - The angular coefficient shall be significantly different from zero. Section III Matrix Effect Art. 28 The provisions of this section apply to complex matrices. Art. 29 The matrix effect must be determined by comparing the angular coefficients of the calibration curves constructed with the CRS of the analyte in solvent and with the sample fortified with the CRS of the analyte. Sole paragraph. The curves shall be established in the same way as in the linearity for the same concentration levels, using at least 5 (five) different concentrations in at least triplicate. Art. 30 The parallelism of the lines is indicative of interference absence of the matrix constituents and its demonstration must be carried out by means of adequate statistical evaluation. Sole paragraph. The level of significance of 5% (five percent) should be adopted in the hypothesis test. Section IV Working range Art. 31 The working range must be established from the linearity studies, together with the results of precision and accuracy, being dependent on the intended application. Art. 32 The following working ranges should be considered: I.- for content: from 80% (eighty percent) to 120% (one hundred and twenty percent);

11 II.- for uniformity of content: from 70% (seventy percent) to 130% (center and thirty percent); III.- for dissolution test: from -20% (minus twenty percent) of the lowest expected concentration to + 20% (plus twenty percent) of the highest concentration expected from the dissolution profile; and IV.- for determination of impurities: from the limit of quantification up to 120% (one hundred and twenty percent) of the concentration at the limit of the specification of each individual impurity; V.- for simultaneous determination of content and impurities by the area normalization procedure: from the limit of quantification (LQ) up to 120% (one hundred and twenty percent) of the expected concentration of the active substance. Paragraph 1 - Larger working ranges than those defined in the caput may be used if technically justified. Paragraph 2 - For medicinal gases, alternate working ranges shall be accepted provided that the approach for the choice of range is justified. Section V Precision Art. 33 The precision shall evaluate the proximity between the results obtained by means of tests with samples prepared as described in the analytical method to be validated. Art. 34 Precision shall be expressed by means of repeatability, intermediate precision or reproducibility. Art. 35 Precision must be demonstrated by scattering the results, calculating the relative standard deviation (RSD) of the series of measurements according to the formula "RSD = (SD / DAC) X100", where SD is the standard deviation and DAC, determined average concentration. Art. 36 Samples for precision evaluation shall be prepared independently from the beginning of the procedure described in method. Sole paragraph. In the case of solid and semi-solid samples, diluted solutions of the same stock solution are not acceptable. Art. 37 Where the precision assessment involves contamination of the matrix with a very low-quantity substance which makes direct weighing impossible, a concentrated solution of the substance may be used following the procedure described in the analytical method for extraction and dilution of the sample.

12 Paragraph 1 - In the case of known impurities absent or present in concentrations below the specification limit in the sample, the sample shall be fortified with known concentrations of the impurities standard. Paragraph 2 - In the case of unknown impurities, the sample shall be evaluated using the response of the active added to the matrix in the concentration corresponding to the limit of the specification established for the impurity, provided that the same response factor for impurity and for the active. Art. 38 The determination of repeatability shall comply with the following criteria: I - evaluate the samples under the same operating conditions, same analyst and same instrumentation, in a single analytical run. II - using at least 9 (nine) determinations, taking into account the linear interval of the analytical method, i.e. 3 (three) concentrations: low, medium and high, with 3 replicates at each level or 6 (six) replicates at 100% (one hundred percent) of the test concentration individually prepared. Art. 39 The acceptance criteria shall be defined and justified in accordance with the following aspects: I - purpose of the method; II - intrinsic variability of the method; III work concentration; and IV sample analyte concentration in the sample. Art. 40 The determination of intermediate precision shall meet the following criteria: I.- express the proximity between the results obtained from the analysis of the same sample, in the same laboratory, on at least two different days, performed by different operators; and II. - contemplating the same concentrations and the same number of determinations described in the repeatability evaluation. Art. 41 Reproducibility should be obtained by means of the proximity of the results obtained in different laboratories. Paragraph 1 - Reproducibility is applicable in collaborative studies or in the standardization of analytical methods for inclusion in official compendia, by appropriate statistical tests. Paragraph 2 - The acceptance criteria for the relative standard deviation must be justified as recommended in art. 39.

13 Section VI Accuracy Art. 42 The accuracy of an analytical method must be obtained by the degree of agreement between the individual results of the method under study in relation to a value accepted as true. Art. 43 The accuracy must be verified from at least 9 (nine) determinations, considering the linear interval of the analytical method, i.e. 3 (three) concentrations: low, medium and high, with 3 (three) replicates at each level. Art. 44 Samples for evaluation of accuracy should be prepared independently and diluted solutions of the same CRS stock solution may be used. Art. 45 For the determination of accuracy, the most appropriate approach must be used, according to the analytical method under study: I.- for API: a) apply the proposed method using a substance with known purity (CRS); b) compare the results with those resulting from a second validated method, whose accuracy has been established; or c) in the case of complex matrix analyte, perform analysis by the CRS addition method in which known quantities of CRS are added to the sample. II.- for finished product: a) apply the proposed method in the analysis of a sample, in which known quantity of CRS was added to the matrix; b) in the unavailability of samples of all components of the drug, analysis may be carried out by the CRS addition method in which known amounts of CRS are added to the solution of the finished product; or c) compare the results obtained with those resulting from a second validated method. III.- for impurities: a) apply the standard addition method in which known amounts of impurities or degradation products are added to the sample; b) in the unavailability of samples of certain impurities or degradation products, a comparison of the results obtained with a second validated method and the use of the response factor relative to the API;

14 c) for unknown impurities, accuracy shall be assessed by comparing the API s CRS or known impurity response, according to the proposed method, in a concentration range that contemplates the working range of the method, provided that the same response factor is considered. Sole paragraph. In all cases, the method of calculating analyte concentrations shall be the same as that described in the concerned analytical method. Art. 46 The accuracy shall be expressed by the percentage recovery ratio of the known analyte added to the sample or by the ratio between the experimentally determined mean concentration and the corresponding theoretical concentration given by formula 1 of Annex II. Sole paragraph. When accuracy is determined from a previously validated method, the concentration of analyte determined by it should be considered, replacing the term "theoretical concentration". Art. 47 The standard relative deviation (SRD) should be calculated for each concentration. Art. 48 The acceptance criteria for percentages of recovery and relative standard deviation obtained must be justified according to the criteria recommended in art. 39. Section VII Detection Limit Art. 49 Detection limit shall be demonstrated by obtaining the smallest amount of analyte presented in a sample that can be detected but not necessarily quantified under the established experimental conditions. Art. 50 The determination of the detection limit can be carried out by means of a visual method, the signal-to-noise ratio, based on the determination of the blank or parameters of the calibration curve, considering the particularities of the analytical method used. Art. 51 For visual methods, the detection limit is determined by the lower concentration for which the visual effect expected. Art. 52 For instrumental methods, the limit of detection can be determined by the signal-tonoise ratio. Paragraph 1 - The method used to determine the signal-to-noise ratio shall be described and justified. Paragraph 2 - The signal-to-noise ratio must be greater than or equal to 2:1.

15 Art. 53 For the determination based on analytical curve parameters, the detection limit may be calculated by formula 2 of Annex II. Art. 54 In cases where an estimated value for the detection limit is obtained by calculation or extrapolation, this estimate must be confirmed according to art. 52. Section VIII Quantification Limit Art. 55 The quantification limit is the smallest amount of analyte in a sample that can be determined with acceptable precision and accuracy under established experimental conditions. Art. 56 The quantification limit shall be consistent with the impurity specification limit. Sole paragraph. For products suitable for the resolution setting out parameters for the notification, identification and qualification of degradation products in medicinal products, the quantification limit must be less than or equal to the notification limit. Art. 57 For the determination of this parameter the same procedure described in art. 53, and the signal-to-noise ratio must be at least 10:1. Art. 58 For the determination based on analytical curve parameters, the quantification limit may be calculated by formula 3 of Annex II. Art. 59 In cases where an estimated value for the quantification limit is obtained by calculation or extrapolation, this estimate must be confirmed according to art. 57. Art. 60 Precision and accuracy at the concentrations corresponding to the quantification limit must be tested. Section IX Robustness Art. 61 Robustness is a parameter typically carried out in the development of the analytical method that indicates its ability to withstand small and deliberate variations of analytical conditions. Sole paragraph. If the method is susceptible to variations in analytical conditions, these should be controlled by precautions described in the method.

16 Art. 62 In the case of quantitative methods, the impact of proposed variations on the results obtained shall be evaluated using the same criteria used for accuracy. Art. 63 In the case of qualitative methods, it should be checked whether the proposed variations interfere in the analytical response. Art. 64 Compliance to the system verification characteristics shall be demonstrated. Art. 65 The assessment of the parameters described in Table 1 of Annex III shall be included in the validation report. Paragraph 1 - Parameters that are considered relevant to the result, according to the characteristics of the method, should be additionally evaluated. Paragraph 2 - The absence of the evaluation of any of the variations shall be justified. CHAPTER V TRANSITIONAL PROVISIONS Art. 66 Analytical method validations will be accepted in compliance with Resolution RE No. 899/2003, since it has been finalized prior to the validity of this resolution and the petitions containing them have been filed within 550 (five hundred and fifty) calendar days after the validity of this resolution. Paragraph 1 - In case of the need to execute and re-present one or more validation parameters, as long as a new validation is not required, the company may follow Resolution RE No. 899, dated May 29 th, Paragraph 2 - In case of need to execute and submit a new validation, the company must follow this resolution. Paragraph 3 - After the deadline established in the caput for investigational products whose validation of the analytical method used in clinical development has been initiated before the validity of this regulation, the analytical validations carried out according to Resolution RE No. 899 dated May 29 th, 2003 will be accepted at the time of registration. CHAPTER VI FINAL PROVISIONS Art. 67 Additional documentation and tests may be requested at any time by ANVISA.

17 Art. 68 All relevant data obtained during the conduction of the analytical validation, as well as the formulas used for calculation, must be filed together with the petition of interest for the evaluation of ANVISA. Art. 69 Failure to comply with the provisions contained in this resolution constitutes a sanitary infraction, pursuant to Law No , dated August 20 th, 1977, without prejudice to the applicable civil and criminal liability. Art. 70 Resolution RE No. 899, dated May 29 th, 2003, Subsection XXXI of art. 1, sole paragraph of art. 11 and Annex I of Resolution RDC No. 31 dated August 11 th, 2010 are revoked. Art. 71 This Resolution shall enter into force within a period of 180 (one hundred and eighty) calendar days from the date of its publication. JARBAS BARBOSA DA SILVA JR.

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19 ANNEX I Table 1. Parameters to be considered in analytical validation. Evaluated Parameter Identification Quantitative Impurity Test Limit Test Assay - dissolution (quantification) - content uniformity - strength Accuracy No Yes No Yes Repeatability precision No Yes No Yes Intermediate Precision No Yes (1) No Yes (1) Selectivity (2) Yes Yes Yes Yes Detection Limit No No (3) Yes No Quantification Limit No Yes No No (3) Linearity No Yes No Yes Interval No Yes No Yes

20 (1) In cases where reproducibility has been conducted, it is not necessary to conduct intermediate precision. (2) In the case of identification tests, it may be necessary to combine two or more analytical procedures to achieve the required level of discrimination. (3) May be necessary in some cases. Formula 1. Accuracy calculation. ANNEX II Experimental mean concentration Recovery = x 100 Theoretical concentration CA (added sample) - CA (sample) Recovery = x 100 CTA Where: CA is the experimental concentration of the analyte and CTA is the theoretical concentration of the added analyte or LD = 3.3. σ IC Where: IC is the slope of the calibration curve, σ is the standard deviation and can be obtained in 3 ways: I - from the standard deviation of the Y-axis intercept of at least 3 calibration curves constructed containing analyte concentrations close to the assumed detection limit;

21 II - from the residual standard deviation of the regression line; III - from the estimation of noise coming from the analysis of an appropriate number of blank samples. Formula 3. Calculation of quantification limit. LQ = 10. σ IC Where: IC is the slope of the calibration curve,curve; σ is the standard deviation and can be obtained in 3 ways: I - from the standard deviation of the Y-axis intercept of at least 3 calibration curves constructed containing analyte concentrations close to the assumed detection limit; II - from the residual standard deviation of the regression line; III - from the estimation of noise coming from the analysis of an appropriate number of blank samples. ANNEX III Table 1. Conditions for the evaluation of the method robustness Stability of analytical solutions Sample preparation Spectrophotometry Extraction time Filter compatibility ph variation of the solution Different solvent batches or manufacturers ph variation of the mobile phase Liquid Chromatography Different column batches or manufacturers Temperature Mobile phase flow

22 Different column batches or manufacturers Gas Chromatography Other analytical techniques Temperature Carrier gas velocity The variations to be tested should be evaluated critically and their results should be presented