Bio-Equivalence : Module 8


All analytical test methods used to determine the active compound and/or its biotransformation product in the biological fluid must be well characterized, fully validated and documented. The objective of the validation is to demonstrate that a particular method used for quantitative measurement of analytes in a given biological matrix, such as blood, plasma, serum or urine, is reliable and reproducible for the intended use. Applicable principles of GLP should be followed in the conduct of chemical analysis. Bioanalytical methods should meet the requirements of specificity, sensitivity, accuracy, precision and reproducibility. Knowledge of the stability of the API and/or its biotransformation product in the sample material is a prerequisite for obtaining reliable results.

Some of the important recommendations are:

• Validation comprises pre-study and within-study phases. During the prestudy phase stability of the stock solution and spiked samples in the biological matrix, specificity, sensitivity, accuracy, precision and reproducibility should be provided. Within-study validation proves the stability of samples collected during a clinical trial under storage conditions and confirms the accuracy and precision of the determinations.

• Validation must cover the intended use of the assay

• The calibration range must be appropriate to the study samples. A calibration curve should be prepared in the same biological matrix as will be used for the samples in the intended study by spiking the matrix with known concentrations of the analyte. A calibration curve should consist of a blank sample, a zero sample, and 6–8 non-zero samples covering the expected range. Concentrations of standards should be chosen on the basis of the concentration range expected in a particular study.

• Spiked quality control samples at a minimum of three different concentrations in duplicate should be used for accepting or rejecting the analytical run.

All the samples from one subject (all periods) should be analysed in the same analytical run, if possible. Validation procedures, methodology and acceptance criteria should be specified in the analytical protocol, and/or the SOP. All experiments used to support claims or draw conclusions about the validity of the method should be described in a report (method validation report). Any modification of the method during the analysis of study samples will require adequate revalidation. The results of study sample determination should be given in the analytical report together with calibration and quality control sample results, repeat analyses (if any), and a representative number of sample chromatograms.

For more detail on Bioanalytical method validation please refer following web-link

FDA Guideline on Bioanalytical method validation

European Guideline Bioanalytical method validation

Dr. Manoj Karwa


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