Application Of Validated Method To Routine Drug Analysis


Assays of all samples of an analyte in a biological matrix should be completed within the time period for which stability data are available. In general, biological samples can be analyzed with a single determination without duplicate or replicate analysis if the assay method has acceptable variability as defined by validation data. This is true for procedures where precision and accuracy variabilities routinely fall within acceptable tolerance limits. For a difficult procedure with a labile analyte where high precision and accuracy specifications may be difficult to achieve, duplicate or even triplicate analyses can be performed for a better estimate of analyte.

A calibration curve should be generated for each analyte to assay samples in each analytical run and should be used to calculate the concentration of the analyte in the unknown samples in the run. The spiked samples can contain more than one analyte. An analytical run can consist of QC samples, calibration standards, and either (1) all the processed samples to be analyzed as one batch or (2) a batch composed of processed unknown samples of one or more volunteers in a study. The calibration (standard) curve should cover the expected unknown sample concentration range in addition to a calibrator sample at LLOQ. Estimation of concentration in unknown samples by extrapolation of standard curves below LLOQ or above the highest standard is not recommended. Instead, the standard curve should be redefined or samples with higher concentration should be diluted and reassayed. It is preferable to analyze all study samples from a subject in a single run.

Once the analytical method has been validated for routine use, its accuracy and precision should be monitored regularly to ensure that the method continues to perform satisfactorily. To achieve this objective, a number of QC samples prepared separately should be analyzed with processed test samples at intervals based on the total number of samples. The QC samples in duplicate at three concentrations (one near the LLOQ (i.e., _3 x LLOQ), one in midrange, and one close to the high end of the range) should be incorporated in each assay run. The number of QC samples (in multiples of three) will depend on the total number of samples in the run. The results of the QC samples provide the basis of accepting or rejecting the run. At least four of every six QC samples should be within _15% of their respective nominal value. Two of the six QC samples may be outside the _15% of their respective nominal value, but not both at the same concentration.

The following recommendations should be noted in applying a bioanalytical method to routine drug analysis:

    · A matrix-based standard curve should consist of a minimum of six standard points, excluding blanks (either single or replicate), covering the entire range.

    · Response Function: Typically, the same curve fitting, weighting, and goodness of fit determined during prestudy validation should be used for the standard curve within the study. Response function is determined by appropriate statistical tests based on the actual standard points during each run in the validation. Changes in the response function relationship between prestudy validation and routine run validation indicate potential problems.

    · The QC samples should be used to accept or reject the run. These QC samples are matrix spiked with analyte.

    · System suitability: Based on the analyte and technique, a specific SOP (or sample) should be identified to ensure optimum operation of the system used.

    · Any required sample dilutions should use like matrix (e.g., human to human) obviating the need to incorporate actual within-study dilution matrix QC samples.

    · Repeat Analysis: It is important to establish an SOP or guideline for repeat analysis and acceptance criteria. This SOP or guideline should explain the reasons for repeating sample analysis. Reasons for repeat analyses could include repeat analysis of clinical or preclinical samples for regulatory purposes, inconsistent replicate analysis, samples outside of the assay range, sample processing errors, equipment failure, poor chromatography, and inconsistent pharmacokinetic data. Reassays should be done in triplicate if sample volume allows. The rationale for the repeat analysis and the reporting of the repeat analysis should be clearly documented.

    · Sample Data Reintegration: An SOP or guideline for sample data reintegration should be established. This SOP or guideline should explain the reasons for reintegration and how the reintegration is to be performed. The rationale for the reintegration should be clearly described and documented. Original and reintegration data should be reported.