
	Problems posed by the validation of drug immunoassay

Immunoanalysis offers advantages of sensitivity and analytical simplicity which are particularly attractive for pharmacokinetic studies during clinical trials and therapeutic monitoring. This is particularly true for compounds whose low posology or whose intensity of first passage effect leads to circulating plasma levels below the nanogram per millilitre range.

The need to ensure the safety of a medication has im-posed on the pharmaceutical industry the development of rigorous controls for the manufacture of galenical forms as well as for development studies. For studies concerning pharmacokinetics or bioequivalence, the need to harmonise the validation criteria of the analytical methods has led to a group of experts, originating nota-bly from the Food and Drug Administration (USA), to propose certain recommendations (1). Further more the Committee for Pharmaceutical Products of the Euro-pean Union has established a basis for validation for assays in biological media (2). The required validation criteria principally concern the specificity, the accuracy, the precision, the linearity and measurement of the lim-its of detection and quantitation. The criteria, initially de-fined by reference to current classical physico-chemical methods (liquid or gas chromatography), require an ap-proach which is different from that of immunoanalysis (3). For this technique, the validation presents particular problems due, for example to the problem of specificity of the antibodies, to the method of preparation of the sample as well as to the modelisation of the calibration curve.

When applied to drugs, immunoanalysis profits from the experience in validation acquired by clinical biology, but shows specific restrictions: recognition of metabolites or isomers by antibodies, necessity of validating several biological matrices biopsied in different animal species in addition to man. The basic principle of an immunological assay is the interaction between the analyte (antigen or hapten) and a specific antibody and is subject to the law of mass action. When applied to a biological medium, this reaction may be modified by various factors (Figure 1), the existence of which and proof of its elimination will ensure specificity and which constitute the principal component of the validation. This approach will necessarily be supplemented by the respect of certain rules concerning Good Laboratory Practice, concerning measuring instruments (balances, pipettes, detectors) and the quality control of the physico-chemical purity of the reagents (purity of the standards, stability of the samples). Furthermore, the demonstration of the criteria of linearity, the exactitude and reproducibility will be carried out according to protocols which have already been accepted for the validation of physico-chemical methods. E.E.

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