Bioanalytical Method Validation

Bioanalytical method validation parameters

Hi,

     Friends here i try to explain the bioanalytical method validation parameters as per the guideline for easy understanding.

Selectivity

Selectivity has the ability to differntiate the bioanalytical method and measure the analyte in the presence of potential Interfering substance in the biological matrix.Perform selectivity exercise with 6 indivial sources/lots of biological matrix in plain, haemolyed and lipemic.

Selectivity perform for both analyte and internal standard.

Selectivity exercise perform using blank samples (matrix samples processed without addition of an analyte or IS).The results of Selectivity Should be,no significant response contribute to interfering components is observed at the retention time(s) of the analyte or the IS in the blank samples. If Responses detected and attributable to interfering components should not be more than 20% of the analyte response at the LLOQ sample and not more than 5% of the IS response in the LLOQ sample for each matrix.

Specificity

Specificity is the ability of a bioanalytical method to detect and differentiate the analyte from other substances, including its related substances (e.g., substances that are structurally similar to the analyte, metabolites, isomer, impurities, degradation products formed during sample preparation, or concomitant medications that are expected to be used in the treatment of patients with the intended indication.

Note:

concomitant medications are like paracetamol, caffine, cetrizine, diclofenac, Ibuprofen etc.If concomitant medications drugs are used during study then selectivity for that particular drugs has to performed.

If concomitant medications are given during study then impact of such substances should be evaluated during method validation or alternatively in the pre-dose study samples.

In the case of LC-MS based methods, to assess the impact of such substances, the evaluation may include comparing the molecular weight of a potential interfering related substance with the analyte and chromatographic separation of the related substance from the analyte. Responses detected and attributable to interfering components should not be more than 20% of the analyte response at the LLOQ and not more than 5% of the IS response in the LLOQ sample.The possibility of back-conversion of a metabolite into the parent analyte during the successive steps of the analysis (including extraction procedures or in the MS source) should also be evaluated when relevant (i.e., potentially unstable metabolites such as ester analytes to ester/acidic metabolites, unstable N-oxides or glucuronide metabolites, lactone-ring structures).It is acknowledged that this evaluation will not be possible in the early stages of drug development of a new chemical entity when the metabolism is not yet evaluated.

Matrix effect

A matrix effect is defined as an variations of the analyte response due to interfering and often unidentified component(s) in the sample matrix. During method validation it is necessary to evaluate the matrix effect between different independent sources/lots.

The matrix effect should be evaluated by analysing at least 3 replicates of low and high QCs, each prepared using matrix from at least 6 different sources/lots. The accuracy should be within ±15% of the nominal concentration and the precision (%CV) should not be greater than 15% in all individual matrix sources/lots.Matrix effect has to performed in plain, haemolyed and lipemic matrix.

Reference:Bioaanlytical method validation-FDA

Regards,

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