Analytical Method Validation BY Dr. Alka N Choudhary Division of Pharmaceutical Sciences S.G.R.R.I.T.S., Patel Nagar, Dehradun (UK)

WHAT IS VALIDATION? “Validation of an analytical procedure is the process by which it is established, by laboratory studies, that the performance characteristics of the procedure meet the requirements for its intended use.” WHY VALIDATE ANALYTICAL PROCEDURES? There are many reasons for the need to validate analytical procedures. Among them are regulatory requirements, good science, and quality control requirements. The Code of Federal Regulations (CFR) 311.165c explicitly states that “the accuracy, sensitivity, specificity, and reproducibility of test methods employed by the firm shall be established and documented”

CYCLE OF ANALYTICAL METHODS The analytical method validation activity is not a one - time study. This is illustrated and summarized in the life cycle of an analytical procedure in Figure 1.

Validation Parameter Typical validation characteristics which should be considered are:. Accuracy Precision Specificity Linearity Range Detection Limit Quantitation Limit Robustness Ruggedness Noise Trueness Sensitivity

ACCURACY “Accuracy is the closeness of an individual test result to the true value.” Measures exactness of the analytical method developed. Expressed as percent recovery by the assay of a known amount of analyte added. Determined by applying the method to samples + known amount of analyte added both above & below normal levels expected in the samples.

Calculated from The test results as the percentage of the analyte recovered by the assay. Dosage form assays commonly provide accuracy within 3-5% of the true value. The ICH recommend that accuracy should be assessed using a minimum of nine determinations over a minimum of three concentration levels, covering the specified range

PRECISION “Precision is a measure of the degree of reproducibility / repeatability of the analytical method under normal operating circumstances.” Repeatability involves analysis of replicates by the analyst using the same equipment, method and conducting the precision study over short period of time while reproducibility involves precision study at Different Occasions, Different Laboratories, Different Batch of Reagent, Different Analysts, Different Equipment.

Determination of Repeatability Procedure when repeated by same analyst under the same operating conditions (same reagents, equipment, settings and laboratory) over a short interval of time. (intra-day) Determination of reproducibility  Carried out by different analysts under different conditions (different reagents, equipment, laboratories or by carrying out the analysis at different times also provide valuable information) on separate, putatively identical samples taken from the same homogenous batch of material. (inter-day)

SELECTIVITY “A measure of the discriminating power of a given analytical procedure in differentiating between the analyte and other components in the test sample.” SPECIFICITY “Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present. Typically these might include impurities, degrades, matrix, etc. Lack of specificity of an individual procedure may be compensated by other supporting analytical procedure(s)”

SELECTIVITY & SPECIFICITY If an analytical procedure is able to separate and resolve the various components of a mixture and detect the analyte qualitatively If the method determines or measures quantitatively the component of interest in the sample matrix without separation Restricted to qualitative detection of the components of a sample Restricted to quantitative measurement of one or more analyte

Linearity “The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample” It may be demonstrated directly on the drug substance (by dilution of a standard stock solution) and/or separate weighing of synthetic mixtures of the drug product components, using the proposed procedure. Linearity should be evaluated by visual inspection of a plot of signals as a function of analyte concentration or content. If there is a linear relationship, test results should be evaluated by appropriate statistical methods, for example, by calculation of a regression line by the method of least squares.

The correlation coefficient, y-intercept, slope of the regression line and residual sum of squares should be submitted. A plot of the data should be included. In addition, an analysis of the deviation of the actual data points from the regression line may also be helpful for evaluating linearity. For the establishment of linearity, a minimum of 5 concentrations is recommended. Under normal circumstances, linearity is acceptable with a coefficient of determination (r 2 ) of >/=0.997 “The range of an analytical procedure is the interval between the upper and lower concentration (amounts) of analyte in the sample (including these concentrations) for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy and linearity.” The specified range is normally derived from linearity studies and depends on the intended application of the procedure. RANGE

LIMIT OF DETECTION “The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value. “ Several approaches for determining the detection limit are possible, depending on whether the procedure is a non-instrumental or instrumental. Approaches other than those listed below may be acceptable. Based on Visual Evaluation Based on Signal-to-Noise Based on the Standard Deviation of the Response and the Slope The detection limit (DL) may be expressed as: DL = 3.3 Sa / S Where Sa = the standard deviation of the response S = the slope of the calibration curve

Quantitation Limit The quantitation limit of an individual analytical procedure is the lowest concentration of analyte in a sample which can be quantitatively determined with suitable precision and accuracy.” Several approaches for determining the quantitation limit are possible, depending on whether the procedure is a non-instrumental or instrumental. Approaches other than those listed below may be acceptable Based on Visual Evaluation Based on Signal-to-Noise Based on the Standard Deviation of the Response and the Slope The quantitation limit (QL) may be expressed as: QL =10 Sa / S Where, Sa = the standard deviation of the response S = the slope of the calibration curve

ROBUSTNESS “Measure of its capacity to remain unaffected by small but deliberate variation in method parameters and provides an indication of its reliability during normal usage.” Determination requires that methods characteristic are assessed when one or more operating parameter varied. Examples of typical variations are: - stability of analytical solutions; - Extraction time. In the case of liquid chromatography, examples of typical variations are: - influence of variations of pH in a mobile phase; - influence of variations in mobile phase composition; - different columns (different lots and/or suppliers); - temperature; - Flow rate. In the case of gas-chromatography, examples of typical variations are:

RUGGEDNESS “Degree of reproducibility of test results obtained by the analysis of the same samples under a variety of normal test  conditions within the specified parameters of the assay.” Determination involves the degree of reproducibility of test result is determined as function of the assay variable. This reproducibility may be compared to the precision of the assay under normal condition to obtain a measure of the ruggedness of the analytical method. According to USP, ruggedness is determined by analysis of aliquots from homogeneous batches in different laboratories, by different analysts, using operational and environmental conditions prescribed for the assay. The degree of reproducibility is then evaluated by comparison of the results obtained under varied conditions with those under standard conditions.

NOISE “A phenomenon defined as fast changes in the intensity and frequency of a measured signal irrespective of the presence or absence of the analyte.” The speed of change is significantly different from the normally expected detector response. A measure of noise is the measured difference between the highest and lowest value of the measured signal with no analyte present, observed in a relatively short time-span, as compared to the time-span necessary for measurement of the analyte. TRUENESS The closeness of agreement between the average values obtained from a large series of test results and an accepted reference value. The measure of trueness is usually expressed in terms of bias.

SENSITIVITY The change of the measured signal as a result of one unit change in the content of the analyte. The change is calculated from the slope of the calibration line of the analyte

REFERENCE U.S.Pharmacopoeia 2007,validation of compendia procedure,chapter-1225.,pp 680-681 ICH Q2 (R1): Validation of Analytical Procedures: Text and Methodology, ICH Harmonized Tripartite Guideline, Current Step 4 version S.Iyer, Validation of analytical Procedure, Guidelines on CGMP and Quality of Pharmaceutical Products, D. K. Publications, chapter-8, pp145-150.