Method validation in the bioanalytical laboratory.

Bioanalytical methods, based on a variety of physico-chemical and biological techniques such as chromatography, immunoassay and mass spectrometry, must be validated prior to and during use to engender confidence in the results generated. The fundamental criteria for assessing the reliability and overall performance of a bioanalytical method are: the evaluation of drug and analyte stability, selectivity, limits of quantification and detection, accuracy, precision, linearity and recovery. The extent to which a method is validated is dependent on its prospective use, the number of samples to be assayed and the use to which the data are put. Specific analytical techniques may require additional validation such as antibody-binding characteristics, peak purity determination, evaluation of matrix effects or structural confirmation of the analyte. Ideally each assay should be cross-validated with a method utilizing a highly specific detector such as a mass spectrometer. Once in use, the performance of the method should be monitored using quality control standards. If a method is set up in another laboratory, the performance of that assay should be monitored with quality control standards sent from the originating laboratory.

Validation of a radioimmunoassay for the determination of human plasma concentrations of lamotrigine.

A precise and sensitive radioimmunoassay to determine human plasma lamotrigine (430C78), Lamictal) is described. The method is a direct double antibody procedure employing a rabbit polyclonal first antibody raised to a BSA conjugate of lamotrigine, and an iodinated tyrosine methyl ester of lamotrigine as the tracer. Both reagents are added simultaneously to samples containing lamotrigine prior to an overnight incubation at 4 degrees C. The method has a sensitivity of 20 ng ml-1, when plasma samples are initially diluted 1:20 with phosphate buffer and sample volumes of 20 microliters are used. The intra-assay precision at 40, 80 and 160 ng ml-1 was 6.2, 2.1 and 4.8%, respectively, and the inter-assay precision at 500, 2000, 5000 and 10,000 ng ml-1 was 4.6, 5.7, 4.6 and 5.9%, respectively. The method was specific and showed reasonable correlation with an HPLC method [1].