EQUIPMENT and METHOD VALIDATION

“Methods validation is the process of demonstrating that analytical procedures are suitable for their intended use”

Parameters of Method Validation Linearity Accuracy (also termed trueness) Precision (reproducibility) Robustness Specificity (selectivity) Limit of detection Limit of quantitation

Specificity Ability to assess the analyte in the presence of components which may be expected to be present . To determine the specificity of the methods, monitor by analyzing the placebo, standard solution and market preparation containing drugs. No peak should be detected close to the retention time/λmax/IR, NMR, MS absorbance of the drug proving the high degree of specificity of the method.

Linearity Ability to obtain test results which are directly proportional to the concentration of analyte in the sample Aspects: To evaluate linearity by visual inspection of the plot and by statistical techniques To calculate correlation coefficient, y-intercept, slope Linearity: Prepare a set of a minimum of 5 concentrations of analyte standard. Perform the determination and generate a standard curve. Acceptance Criteria: Linear Regression Coefficient of Determination (r²) = 0.995 to 1.

Accuracy/ Recovery The accuracy of an analytical method expresses the nearness between the expected value and the value found. There are guidelines as to how the accuracy should be evaluated. For eg- in case of HPLC technique- - Accuracy should be assessed by 9 determinations over a minimum of 3 concentration levels -To be reported as percent recovery (R%) [R% = % Recovery = (Recovered Conc./Injected Conc.) x 100] Acceptance Criteria: 97.0% - 103.0% recovery for raw APIs 95.0% - 105.0% for finished dosage forms.

Precision/ Reproducibility The precision of an analytical method is the closeness of agreement among individuals test result of the same homogeneous sample under prescribed conditions where the method is applied repeatedly. Expressed as RSD % There are guidelines as to how the accuracy should be evaluated. For eg- in case of HPLC technique- [RSD (%) = (Standard deviation/Mean) x 100 %], Std Deviation = [(Std. conc.- Recovered conc.)/Std. conc.] Acceptable Range: RSD ≤ 2 %.

Precision is usually investigated at three levels: repeatability, intermediate precision, and reproducibility. Repeatability (Precision). Repeatability is a measure of the precision under the same operating conditions over a short interval of time. It is sometimes referred to as intraday precision. A minimum of nine determinations covering the specified range for the procedure (e.g., three concentrations/three replicates as in the accuracy experiment) Intermediate Precision. Intermediate precision is defined as the variation within the same laboratory. It is sometimes referred to as interday precision. Typical parameters that are investigated include day-to-day variation, analyst variation, and equipment variation. Reproducibility. Reproducibility measures the precision between laboratories as in collaborative studies. This parameter should be considered in the standardization of an analytical procedure.

Robustness The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small but deliberate variations in the analytical procedure parameters. The robustness of the analytical procedure provides an indication of its reliability during normal use.

Limit of Detection (LOD): For chromatographic method, determine the minimum level at which a compound can be detected, using analyte solutions of decreasing concentration. LOD is generally defined as 3 times the noise level. Other scientifically-sound approaches may also be used. For other types of methods, estimate through visual evaluation the minimum level at which a compound can be detected, using analyte solutions of decreasing concentration. Limit of Quantitation (LOQ): For chromatographic method, determine the minimum level at which a compound can be quantitated, using analyte solutions of decreasing concentration. LOQ is generally defined as 10 times the noise level. For other types of methods, estimate through visual evaluation the minimum level at which a compound can be quantitated, using analyte solutions of decreasing concentration.

Typical variations affecting a method’s reproducibility are: · Differences in room temperature and humidity; · Operators with different experience and thoroughness; · Equipment with different characteristics for eg- different injection modes; · Variations in material and instrument conditions, for example different protocols of the mobile phases preparation; changes in composition, pH, flow rate of mobile phase in case of HPLC; · Equipments of different ages; · Columns from different suppliers or different batches; · Solvents, reagents and other material with different quality

Thank You