User:Anoop.singh1980/sandbox

The Convener, Admission cell, Administrative Block, Bundelkhand University, Kanpur Road, Jhansi, Uttar Pradesh 284128
1.0	PURPOSE To lay down the procedure for documentation and data control Recognizing the legal status of USP and NF standards, it is essential, therefore, that proposal for adoption of new or revised compendial analytical methods be supported by sufficient laboratory data to document their validity.

2.0	SCOPE

3.0	RESPONSIBLITY Officer/ Executive / Manager – Quality control and Quality Assurance.

4.0	PROCEDURE 4.1	VALIDATION 4.1.1	Validation of an analytical method is the process by which it is established, by laboratory studies, that the performance characteristics of the method meet the requirements for the intended analytical applications. Typical analytical performance characteristics that should be considered in the validation of the types of methods described in this document are listed in Table 1. Table 1. Typical Analytical Characteristics Used in Method Validation Accuracy Precision Specificity Detection Limit Quantitation Limit Linearity Range In the case of compendial methods, revalidation may be necessary in the following cases: A submission to the USP of a revised analytical method; or the use of an established general method with a new product or raw material (see below under Data Elements Required for Assay Validation). The ICH documents give guidance on the necessity for revalidation in the following circumstances: Changes in the synthesis of the drug substance; changes in the composition of the drug product; and changes in the analytical procedure.

4.2	ANALYTICAL PERFORMANCE CHARACTERISTICS 4.2.1	Accuracy Definition: The accuracy of an analytical method is the closeness of test results obtained by that method to the true value. The accuracy of an analytical method should be established across its range. Determination: In the case of the assay of a drug substance, accuracy may be determined by application of the analytical method to an analyte of known purity (e.g., a Reference Standard or Working standard) or by comparison of the results of the method with those of a second, well-characterized method, the accuracy of which has been stated or defined. In the case of the assay of a drug in a formulated product, accuracy may be determined by application of the analytical method to synthetic mixtures of the drug product components to which known amounts of analyte have been added within the range of the method. If it is not possible to obtained sample of all during product components, it may be acceptable either to add known quantities of the analyte to the drug product (i.e. “to spike”) or to compare results with those of a second, well-characterized method, the accuracy of which has been stated or defined. In the case of quantitative analysis of impurities, accuracy should be assessed on samples (of drug substance or drug product) spiked with known amounts of impurities. Where it is not possible to obtain samples of certain impurities or degradation products, results should be compared with those obtained by an independent method. In the absence of other information, it may be necessary to calculate the amount of an impurity based on comparison of its response to that of the drug substance; the ratio of the responses of equal amounts of the impurity and the drug substance (response factor) should be used if known. Accuracy is calculated as the percentage of recovery by the assay of the known added amount of analyte in the sample, or as the difference between the mean and the accepted true value, together with confidence intervals. The ICH documents recommend that accuracy should be assessed using a minimum of nine determinations over a minimum of three concentration levels, covering the specified range (i.e. three concentrations and three replicates of each concentration). 4.2.2	Precision Definition: The precision of an analytical method is the degree of agreement among individual test results when the method is applied repeatedly to multiple samplings of a homogeneous sample. The precision of an analytical method is usually expressed as the standard deviation or relative standard deviation (coefficient of variation) of a series of measurements. Precision may be measure of either the degree of reproducibility or of repeatability of the analytical method under normal operating conditions. In this context, reproducibility refers to the use of the analytical procedure in different laboratories, as in a collaborative study. Intermediate precision expresses within laboratory variation, as on different days, or with different analysts or equipment within the same laboratory. Repeatability refers to the use of the analytical procedure within a laboratory over a short period of time using the same analyst with the same equipment. For most purposes, repeatability is the criterion of concern in USP analytical procedures; repeatability is the criterion of concern in USP analytical procedures, although reproducibility between laboratories or intermediate precision may well be considered during the standardization of a procedure before it is submitted to the Pharmacopoeia. Determination: The precision of an analytical method is determined by assaying a sufficient number of aliquots of a homogeneous sample to be able to calculate statistically valid estimates of standard deviation or relative standard deviation (coefficient of variation). Assays in this context are independent analyses of samples that have been carried through the complete analytical procedure from sample preparation to final test result. The ICH documents recommend that repeatability should be assessed using a minimum of nine determinations covering the specified range for the procedure (i.e., three concentrations and three replicates of each concentration or using a minimum of six determinations at 100 % of the test concentration). 4.2.3	Specificity Definition: The ICH documents define specificity as the ability to assess unequivocally the analyte in the presence of components that may be expected to be present, such as impurities, degradation products, and matrix components. Lack of specificity of an individual analytical procedure may be compensated by other supporting analytical procedures. [Note: - Other reputable international authorities (IUPAC, AOAC) have preferred the term “selectivity”, reserving “specificity” for those procedures that are completely selective.] For the test or assay methods below, the above definition has the following implications:

IDENTIFICATION TESTS: Ensure the identity of the analyte.

PURITY TESTS: Ensure that all the analytical procedures performed allow an accurate statement of the content of impurities of an analyte (e.g., related substances test, heavy metals limit, and organic volatile impurity limit).

4.2.4	ASSAYS: Provide an exact result, which allows an accurate statement on the content or potency of the analyte in a sample. Determination: In the case of qualitative analysis (identification tests), the ability to select between compounds, of closely related structure that are likely to be present should be demonstrated. This should be confirmed by obtaining positive results (perhaps by comparison to a known reference material) from samples containing the analyte, coupled with negative results from samples that do not contain the analyte, coupled with negative results from samples that do not contain the analyte and by confirming that a positive response is not obtained from materials structurally similar to or closely related to the analyte.

In the case of analytical procedure for impurities, specificity may be established by spiking the drug substance or product with appropriate levels of impurities and demonstrating that these impurities are determined with appropriate accuracy and precision. In the case of assay demonstration of specificity requires that it can be shown that the procedure is unaffected by the presence of impurities or excipients. In practice, this can be done by spiking the drug substance or product with appropriate levels of impurities or excipients and demonstrating that assay result is unaffected by the presence of these extraneous materials. If impurity or degradation product standards are unavailable, specificity may be demonstrated by comparing the test results of samples containing impurities or degradation products to a second well-characterized procedure (e.g., a pharmacopeial or other validated procedure). These comparisons should include samples stored under relevant stress conditions (e.g., light, heat, humidity, acid/base hydrolysis, and oxidation). In the case of the assay, the results should be compared; in the case of chromatographic impurity tests, the impurity profiles should be compared.

The ICH documents state that when chromatographic procedures are used, representative chromatograms should be presented to demonstrate the degree of selectivity, and peaks should be appropriately labeled. Peak purity tests (e.g., using diode array or mass spectrometry) may be useful to show that the analyte chromatographic peak is not attributable to more than one component.

4.2.5	Detection Limit Definition: The detection limit is a characteristic of limit tests. It is the lowest amount of analyte in a sample that can be detected, but not necessarily quantitated, under the stated experimental conditions. Thus, limit tests merely substantiate that the amount of analyte is above or below a certain level. The detection limit is usually expressed as the concentration of analyte (e.g., percentage, parts per billion) in the sample. Determination: For non-instrumental methods, the detection limit is generally determined by the analysis of samples with known concentrations of analyte and by establishing the minimum level at which the analyte can be reliably detected. For instrumental procedures, the same method may be used as for non-instrumental. In the case of methods submitted for consideration as official compendial methods, it is almost never necessary to determine the actual detection limit. Rather, the detection limit is shown to be sufficiently low by the analysis of samples with known concentrations of analyte above and below the required detection level. For example, if it is required to detect an impurity at the level of 0.1 %, it should be demonstrated that the procedure will reliably detect the impurity at that level. In the case of instrumental analytical procedures that exhibit background noise, the ICH documents describe a common approach, which is to compare, measured signals from samples with known low concentration of analyte with those of blank samples. The minimum concentration at which the analyte can reliably by detected is established. Typically acceptable signal-to-noise ratios are 2 : 1 or 3 : 1. Other approaches depend on the determination of the slope of the calibration curve and the standard deviation of the responses. Whatever method is used, the detection limit should be subsequently validated by the analysis of a suitable number of samples known to be near, or prepared at, the detection limit. 4.2.6	Quantitation limit Definition: The quantitation limit is a characteristic of quantitative assays for low levels of compounds in sample matrices, such as impurities in bulk drug substances and degradation products in finished pharmaceuticals. It is the lowest amount of analyte in a sample that can be determined with acceptable precision and accuracy under the stated experimental conditions. The quantitation limit is expressed as the concentration of analyte (e.g., percentage, parts per billion) in the sample. Determination: For non-instrumental methods, the quantitation limit is generally determined by the analysis of samples with known concentrations of analyte and by establishing the minimum level at which the analyte can be determined with acceptable accuracy and precision. For instrumental procedures, the same method may be used as for non-instrumental. In the case of methods submitted for consideration as official compendial methods, it is almost never necessary to determine the actual Quantitation limit. Rather, the quantitation limit is shown to be sufficiently low by the analysis of samples with known concentrations of analyte above and below the quantitation level. For example, if it is required to assay an analyte at the level of 0.1 mg per tablet, it should be demonstrated that the method would reliably quantitate the analyte at that level. In the case of instrumental analytical methods that exhibit background noise, the ICH documents describe a common approach, which is to compare measured signals from samples with known low concentrations of analyte with those of blank samples. The minimum concentration at which the analyte can reliably be quantified is established. A typically acceptable signal-to-noise ratio is 10:1. Other approaches depend on the determination of the slope of the calibration curve and the standard deviation of responses. Whatever method is used, the quantitation limit should be subsequently validated by the analysis of a suitable number of samples known to be near, or prepared at, the quantitation limit.

4.2.7	Linearity and range Definition of Linearity: The linearity of an analytical method is its ability to elicit test results that are directly, or by a well-defined mathematical transformation, proportional to the concentration of analyte in samples within a given range Definition of Range: The range of an analytical method is the interval between the upper and lower levels of analyte (including these levels) that have been demonstrated to be determined with a suitable level of precision, accuracy, and linearity using the method as written. The range is normally expressed in the same units as test results (e.g., percent, parts per million) obtained by the analytical method. Determination of Linearity and Range: Linearity should be established across the range of the analytical procedure. It should be established initially by visual examination of a plot of signals as a function of analyte concentration of content. If there appears to be linear relationship, test results should be established by appropriate statistical methods (e.g., by calculation of a regression line by the method of least squares). In some cases, to obtain linearity between the response of an analyte and its concentration, the test data may have to be subjected to a mathematical transformation. Data from the regression line itself may be helpful to provide mathematical estimates of the degree of linearity. The correlation coefficient, y-intercept, slope of the regression line, and residual sum of squares should be submitted. The range of the method is validated by verifying that the analytical method provides acceptable precision, accuracy, and linearity when applied to samples containing analyte at the extremes of the range as well as within the range. ICH recommends that, for the establishment of linearity, a minimum of five concentrations normally be used. It is also recommended that the following minimum specified ranges should be considered:

ASSAY OF A DRUG SUBSTANCE (or a finished product): from 50 % to 150 % of the test concentration.

DETERMINATION OF AN IMPURITY: from quantitation limit to 120 % of the specification.

FOR CONTENT UNIFORMITY: a minimum of 70 % to 130 % of the test concentration, unless a wider or more appropriate range, based on the nature of the dosage form (e.g., metered-dose inhalers) is justified.

FOR DISSOLUTION TESTING:  20 % over the specified range (e.g., if the specifications for a controlled – release product cover a region from 20 %, after 1 hour, and up to 90 %, after 24 hours, the validated range would be 0 % to 110 % of the label claim). 4.2.8	Ruggedness Definition: The ruggedness of an analytical method is the degree of reproducibility of test results obtained by the analysis of the same samples under a variety of conditions, such as different laboratories, different analysts, different instruments, different lots of reagents, different elapsed assay times, different assay temperatures, different days, etc. Ruggedness is normally expressed as the lack of influence on test results of operational and environmental variables of the analytical method. Ruggedness is a measure of reproducibility of test results under the variation in conditions normally expected from laboratory to laboratory and from analyst to analyst. Determination: The ruggedness of an analytical method is determined by analysis of aliquots from homogeneous lots in different laboratories, by different analysts, using operational and environmental conditions that may differ but are still within the specified parameters of the assay. The degree of reproducibility of test results is then determined as a function of the assay variables. This reproducibility may be compared to the precision of the assay under normal conditions to obtained a measure of the ruggedness of the analytical method

4.2.9	Robustness Definition: The robustness of an analytical method is a measure of its capacity to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during normal usage. System suitability If measurement are susceptible to variations in analytical conditions, these should be suitably controlled, on a precautionary statement should be included in the method. One consequence of the evaluation of robustness and ruggedness should be that a series of system suitability parameters is established to ensure that the validity of the analytical method is maintained whenever used. Typical variations are the stability of analytical solutions, different equipment, and different analysts. In the case of liquid chromatography, typical variations are the pH of the mobile phase, the mobile phase composition, different lots or suppliers of columns, the temperature, and the flow rate. In the case of gas chromatography, typical variations are different lots or suppliers of columns, the temperature and the flow rate.

System suitability tests are based on the concept that the equipment, electronics, analytical operations, and samples to be analyzed constitute an integral system that can be evaluated as such. System suitability test parameters to be established for a particular method depend on the type of method being evaluated. They are especially important in the case of chromatographic methods, and submissions to the USP should make note of the requirements under the system suitability section in the general test chapter Chromatography.

DATA ELEMENTS REQUIRED FOR ASSAY VALIDATION Compendial assay procedures vary from highly exacting analytical determinations to subjective evaluation of attributes. Considering this variety of assays, it is only logical that different test methods require different validation schemes. This chapter covers only the most common categories of assays for which validation data should be required. This categories are as follows : Category I – Analytical methods for quantitation of major components of bulk drug substances or active ingredients (including preservatives) in finished pharmaceutical products.

Category II – Analytical methods for determination of impurities in bulk drug substances or degradation compounds in finished pharmaceutical products. These methods include quantitative assays and limit tests.

Category III– Analytical methods for determination of performance characteristics (e.g. dissolution, drug release). Category IV – Identification tests. For each assay category, different analytical information is needed. Listed in Table 2 are data elements that are normally required for each of the categories of assays.

Table 2. Data Elements Required for Assay Validation

Assay Category II Analytical Performance Characteristics	Assay Category I	Quantitative	Limit Tests	Assay Category III	Assay Category IV Accuracy	Yes	Yes	*	*	No Precision	Yes	Yes	No	Yes	No Specificity	Yes	Yes	Yes	*	Yes Detection Limit	No	No	Yes	*	No Quantitation Limit	No	Yes	No	*	No Linearity	Yes	Yes	No	*	No Range	Yes	Yes	*	*	No


 * May be required, depending on the nature of the specific test.

Already established general assays and tests (e.g., titrimetiric method of water determination, bacterial endotoxins test) should be revalidated to verify their accuracy (and absence of possible interference) when used for a new product or raw material.

The validity of an analytical method can be verified only by laboratory studies. Therefore, documentation of the successful completion of such studies is a basic requirement for determining whether a method is suitable for its intended applications. Appropriate documentation should accompany any proposal for new or revised compendial analytical procedures.

EXPERIMENTAL PLAN The following are the experimental design and respective acceptance criteria for the method validation to be conducted at. Sample selection for method validation:

Incase of multiple strength formulation, which is scale up / scale down, the method validation in such cases shall be carried out on the highest strength. In case of multiple strength formulation which are look a like, the validation in such cases shall be carried out on the lowest strength (having highest placebo concentration present in it). The entire validation shall be carried out on a single batch sample.

Parameter	Experiment	Acceptance Criteria* System Precision	Five replicate injections of standard solution (wherever RSD is below 2.0 %). For HPLC: RSD should not be more than 1.0 / 2.0 / 5.0 / 10.0 %

For G.C: RSD should not be more than 5.0 / 10.0 / 15.0 %

For UV: RSD should not be more than 2.0 %

Six replicate injections of standard solution (wherever RSD is above 2.0 %). Measuring the absorbance of the standard solution five times. System suitability	Evaluate the system suitability parameters as mentioned in the individual test method. As per individual test procedure

Specificity a.	Interference from placebo and related impurities           	Observation of blank gradient run. No interference at the retention time of analyte peak.

Peak purity of analyte peak should be not less than 990/0.995

Placebo should not consume any titrant

Injection/scan of blank (diluent used in the method) Injection/scan of placebo solution prepared in triplicate (in case there are different strengths available and if they are not scale up –scale down then placebo solution for all the strengths need to be injected)


 * Choose the appropriate acceptance criteria for the individual validation study.

Injection/scan of sample solution (in case there are different strengths available and if they are no scale up – scale down then sample solution for all the strengths need to be injected) Injection of placebo solution spiked with analyte at a concentration as specified in the test method (in case there are different strengths available and if they are no scale up – scale down then placebo solution for all the strengths need to be Spiked). Incase of titrimetry, perform the titration in triplicate with placebo. Injection of all the individual known related substances Injection of placebo solution spiked with known related substances/at levels as per specs. Placebo absorbance should be not more than 2.0 % of the standard absorbance

Interference from capsule shell should not be more than 10% of the labeled content

Note: Correction factor shall be applied wherever appropriate if the interference from the capsule shell exceeds 2 %

% Interference from placebo to be determined in triplicate by weighing three samples each of the placebo blend (the coating blend, inks and sinker are also to be added wherever appropriate) and performing dissolution in the test medium at 37C, at 150 RPM for an hour using the method apparatus (in case there are different strengths available and if they are not scale up – scale down, then % interference for all the strengths need to be determined) Note: For Extended release products, a placebo version of the finished dosage form shall be used and the interference must be determined at each sampling point in the release profile. % Interference from capsule shell to be determined by performing dissolution on six empty capsule shells in the test medium at 37C, at 150 RPM for an hour using the method apparatus (in case there are different strengths available and if shells used are different, then % shell interference for all the strengths need to be determined)

b.	Interference From  degradants (Forced degradation studies)	Control sample (No treatment) Peak purity of analyte peak should be not less than 990/0.995. Treat with suitable concentration of HCl for acid hydrolysis. Treat with suitable concentration of NaOH for alkali degradation. Treat with suitable concentration of H2O2 for peroxide oxidation. Expose at suitable temperature in oven for minimum 5 days. Expose under UV light for minimum 5 days. Expose in Humidity chamber at 40C and 75 % RH for minimum 5 days. Placebo and API to be treated in the same way. Method precision	Six test preparations of a single batch sample to be analysed as per test method.

For Content uniformity: Ten individual test units of a single batch sample to be analysed as per test method.

Note-1: Incase of related substances, spike all the known impurities at about specification limit.

Note-2: Incase of residual solvents, spike the solvents at about specification limit, wherever applicable.

Note-3: Prepare standard solution in duplicate, wherever applicable.

For assay: RSD should not be more than 2.0 % For Content uniformity: RSD should not be more than 6.0 % For Preservative/antioxidant content: RSD should not be more than 10.0 % For Residual solvents: RSD should not be more than 15.0 % For related substances: Impurity level              RSD 0.05 % to 0.10 %         25.0 % 0.11 % to 0.50 %          15.0 % 0.51 % to 1.0 %           10.0 % more than 1.0 %           5.0 % General (Wherever applicable): The response ratio of two different standard solutions should be : between 0.98 and 1.02 (for HPLC/UV methods) Between 0.9 and 1.1 (for GC methods).

Stability of analytical solution For assay and related substances (by HPLC): Resolution or system suitability solution to be injected initially and on different days for at least 7 days, after storing them at room temperature (25oC) and refrigerator ( 2 to 8C) For assay, dissolution & preservative / antioxidant content: The response of standard and sample solution should not differ by more than 2.0 % / 5.0 % from the initial response. For related substances: The individual impurity should not differ by more than 0.05 and total impurities by more than 0.1 from the initial value. For assay and related substances: System suitability requirements should be met. The difference in response of each component in the resolution solution should not differ by more than 20 % of initial response. For assay/ dissolution / preservative content or anti oxidant estimation by HPLC: Standard solution and Sample solution to be injected initially and at different time intervals for at least 24 hours at room temperature (25o C)

Note: For API validations perform solution stability for at least 12 hours. For related substances: Sample solution spiked with all known related substances at about specification limits to be injected initially and at different time intervals for at least 24 hours at room temperature (25oC). For dissolution/assay by UV: The deviation in test result should not be more than  2 % of the initial value.

For dissolution/ Assay by UV: Standard solution stability: Absorbance of standard solution to be measured initially and at different time intervals for at least 24 hours by storing it in a glass volumetric flask at room temperature, using a freshly prepared standard solution at each time interval for comparison. Sample solution stability: Absorbance of sample solution to be measured initially and at different time intervals for at least 24 hours by storing it in a glass tube wrapped in Para film or in a closed vial at room temperature, using a freshly prepared standard solution at each time interval for comparison.

Note: If the standard solution or sample solution is not stable for less than 8 hours then establish solution stability under refrigerator condition (i.e., 2 C to    8 C) Limit of detection (LOD) and Limit of Quantification (LOQ)	Determination of LOD/LOQ based on Residual standard deviation and slope of the linearity data and showing precision at that level. (as per ICH Q2B)

3.3              LOD =    S

10                          LOQ =    S - Residual standard deviations of regression line S - Slope of regression line

Note-1: Determine LOD/LOQ of known related impurities using the respective related impurities.

Note-2: Determine LOD/LOQ of unknown impurity using the analyte standard. LOD is the concentration for which detector shows a positive response.

LOQ is the concentration for which the RSD of six replicate injections is less than 10.0* / 25.0** %.

Linearity	For assay : Linearity to be performed in the range of about 50 – 150% of standard concentration. (Minimum 5 points) For assay/dissolution/preservative Or antioxidant content: Correlation coefficient should be not less than 0.995. Y-intercept should be  2 % of 100 % linearity level response. For related substances/residual solvents: Correlation coefficient should be not less than 0.980. For dissolution and preservative content: Linearity to be performed in the range of about 20 – 160% of standard concentration. (Minimum 5 points)
 * For HPLC methods
 * For GC methods

For control release formulation: Linearity to be performed in the concentration range –20% absolute of the lowest specification & +20 % of the highest specification (control release) For related substances/residual solvents: Linearity to be performed in the range of about LOQ to 150% of shelf life specification limit. (Minimum 5 points) Note-1: Incase of HPLC analysis, Plot a graph of concentration Vs peak response. Note-2: Incase of titrimetry analysis, plot a graph of mg of analyte added Vs volume of titrant consumed (ml) Accuracy	For assay /preservative/antioxidant content /Content uniformity: Prepare accuracy samples in the range 50% to 150% (50 %, 100 % and 150 %) of sample concentration in triplicate by spiking the analyte with placebo. For related substances/residual solvents: Inject as such sample preparation in triplicate and calculate the mean of each individual impurity. Prepare accuracy samples in the range LOQ to 150% (LOQ, 50 %, 100 % and 150 %) of shelf life limit specification in triplicate by spiking the impurity/solvent with sample/placebo.

For dissolution: Prepare accuracy samples in the range 25% to 150% (25 %, 100 % and 150 %) of sample concentration in triplicate by spiking the analyte with placebo inside a suitable volumetric flask and add the quantity of dissolution medium mentioned in the test method, which was preheated to 37  0.5 C. Sonicate the recovery (accuracy) samples for specified time.

Note 1: The amount of placebo blend weighed for each level is to be the same as the total excipients weight of each dosage form for the dosage strength being validated. Note 2: Prepare standard solution in duplicate, wherever applicable. Test                       % Recovery       RSD

Assay                    98.0 to 102.0     2.0%

Dissolution /          95.0 to 105.0    5.0% Content  uniformity

Preservative           90.0 to 110.0    5.0% Content/anti Oxidant Related substances/residual solvent Impurity level           % Recovery 0.05 % to 0.10 %       50.0 to 150.0 % 0.11 % to 0.50 %       70.0 to 130.0 % 0.51 % to 1.0 %         80.0 to 120.0 % more than 1.0 %        90.0 to 110.0 %

General (Wherever applicable): The response ratio of two different standard solutions should be : between 0.98 and 1.02 (for HPLC/UV methods) Between 0.9 and 1.1 (for GC methods).

Ruggedness	For assay/ related substances / antioxidant / preservative content / residual solvents: Matrix design for HPLC/GC Methodology:

Day	Instrument	Analyst	Column 1	1	1	1 2	1	2	1 3	2	1	2

•	Method precision data shall be referred for Day-1 Analysis.

For related substances/residual solvents: Prepare and make six replicate injections of impurity solutions at LOQ level and determine the RSD, to be established on two different instruments. For assay/content uniformity/ preservative/antioxidant content: The RSD of six / ten test results obtained under normal and changed condition should be not more than the limit specified under method precision. The difference in test result between normal condition and changed condition should be not more than 3.0 % / 5.0 % / 10.0 %.

For related substances/residual solvents: The difference in individual impurity between normal condition and changed condition should be not more than 0.1 and that of total impurities should be not more than 0.2. RSD of six replicate injections of impurity / solvent solution at LOQ level should be not more than 10.0 %* /     25.0 %**.


 * For HPLC methods
 * For GC methods

For dissolution: For immediate release product: Dissolution profile to be performed separately on a single batch (preferably on a batch having tight content uniformity) at a release time point and  15 min of release time point on 6 units, on two different instruments, on two different days and by two different analysts.

For control release product: Drug release profile to be performed separately on a single batch (preferably on a batch having tight content uniformity) at specified time points on 6 units, on two different instruments, on two different days and by two different analysts.

Note 1: Intermediate precision to be performed on highest and lowest strength.

Note-2: For ruggedness experiment preferably use an instrument of different manufacturer (make)

Note 3: Prepare standard solution in duplicate wherever applicable. For dissolution: The difference in the mean dissolution results from the first analyst to second analyst does not exceed an absolute 10 % at time points with less than 85 % drug release and does not exceed an absolute 5 % for time points with above 85 % drug release.

For Residual solvents: The RSD of six test results obtained under normal and changed condition should be not more than the limit specified under method precision.

The difference in content of solvent between normal and changed condition shall be: 1.	NMT 20 % of mean value of normal condition. 2.	NMT 50 % of mean value of normal condition.

General (Wherever applicable): The response ratio of two different standard solutions should be : between 0.98 and 1.02 (for HPLC/UV methods) Between 0.9 and 1.1 (for GC methods).

Robustness	For assay/related substances/residual solvents and dissolution by HPLC/GC: Perform system suitability under normal and each of the changed conditions mentioned below For related substances/residual solvents: System suitability parameters specified in the test method should pass.

For assay and dissolution by HPLC : System suitability parameters specified in the test method should pass.

The difference in assay result between the normal and changed condition Should be not more than 3 %. Sample solution spiked with all the known related substances to be injected under each of the following variable to monitor the RRTs. By changing the column oven temperature by + 5o C. (in case when the column oven temperature is not specified in the method then robustness to be shown at 35 oC) By changing organic phase ratio of the mobile phase by + 2 % absolute. By changing pH of buffer/mobile phase by  +   0.2 By changing the flow rate by    10 %

NOTE: Incase of assay, if the system suitability fails under a particular changed condition, then prepare triplicate sample as per test method and determine the mean assay. Compare the average assay against the assay value obtained in precision. For dissolution: 1.	Dissolution to be performed using 90 %, 100 % and 110 % of the medium concentration (e.g., surfactant, w/v %), wherever appropriate.

2.	For buffered media dissolution to be performed with method pH, and by altering the pH  0.5 unit.

3.	Dissolution to be performed by altering the RPM by  4 % of method RPM.

4.	Dissolution results to be determined by altering the wavelength by  5nm of method wavelength.

Note: Perform the robustness study at single point (release time point).

For dissolution: The absolute difference in the average dissolution result between normal condition and changed condition should be not more than 5 %.

Partial method validation Partial method validation shall be performed under the following circumstances. If a Pharmacopoeial or a DMF method is adopted for testing the API, then a partial validation (mini validation) shall be conducted in which the following performance characteristics need to be established.

Performance characteristics	Assay	Related Substances	Residual Solvent System precision	Yes	Yes	Yes System suitability	Yes	Yes	Yes Method precision	Yes	Yes	Yes Solution stability	Yes	Yes	No LOD / LOQ	No	Yes	Yes

In case of minor changes in quantitative composition of validated formulation after the completion of method validation, a partial validation (supplement validation) shall be carried out on the revised formulation, in which following performance characteristics needs to be established for the revised formulation based on nature of changes (above SUPAC LEVEL 1).

Note 1: Wherever changes are made in core formulation, supplement validation shall be carried out for assay and related substances test methods.

Note 2: Wherever changes are made in functional coating composition, supplement method validation shall be carried out for assay, dissolution and related substances.

Performance characteristics to be considered for supplement method validation.

1.	System precision 2.	System suitability 3.	Method precision 4.	Solution stability 5.	Accuracy Intermediate precision (incase of dissolution and drug release)

Re-Validation: Whenever critical changes have been made in the validated test method, re-validation should be done for the method that has undergone a critical change. Wherever multiple sources of API are employed, a partial method validation shall be carried out for the formulation formulated with alternate source API provided the test method remains identical as that of the formulation with primary source API.

The partial validation shall be conducted for assay and related substances test method in which the following performance characteristics shall be evaluated.

1.	System Precision. 2.	System suitability 3.	Method Precision 4.	Solution stability Ruggedness (for analyst to analyst variability)

Before initiating any method validation, a specific method validation protocol shall be prepared based on this SOP and it shall be approved by Quality Assurance – Head. The analysis details and results shall be documented in method validation laboratory note book (black LNB). After execution of method validation the data shall be reviewed for compliance and a method validation report shall be prepared which shall be certified by Quality Assurance – Head.

5.0	DISTRIBUTION Master copy                               :  QA department Control copy no.                        :  Quality control and Quality assurance Control copy no.                        :  Display copy if required 6.0	ANNEXURES (Attached with SOP) Nil

7.0	REFERENCE USP-32

8.0	ABBREVIATIONS

SOP	Standard operating procedure CC #	Change Control Number G	General QA 	Quality assurance QC	Quality control Form #	Form Number SOP #	SOP Number NA	Not Applicable IUPAC	International union of pure and applied chemistry LOD	Limit of detection LOQ	Limit of Quantitation RSD	Relative standard deviation SD	Standard deviation

9.0	HISTORICAL INFORMATION

Sr. No. Revision no. Reason(s) For The Revision Effective date	Change Control No./ date	Changed By 1.	00	New SOP	10/12/2007	N.A.	- 2.	01	Annual Review	06/12/2008	N.A.	Shalini 3.	02	Annual Review	03/12/2010	N.A.	Priyanka 4.					5.					6.					7.					8.					9.					10.					New SOP #: