Development and validation of assay methods for the quantitative determination of drugs and their metabolites in biological specimens (piroxicam and nabumetone)

English: The development and validation of bioanalytical assay methods suitable for the quantitation of drugs in biological matrices is discussed, as well as general principles applicable to this particular aspect of drug development. Relevant literature is consulted, with a view to exemplifying what constitutes good assay method development strategy, as well as to reflect current international policy in this field, with particular reference to bioequivalence studies. Comparisons are made between international practices and those in place at FARMOVSPAREXEL Bioanalytical Services Division®. Attention is given inter alia to detector selection, chromatographic optimisation, extraction procedures and method validation, with reference to new assay methods for two drugs in particular that have been developed and validated according internationally acceptable standards. In the first instance, a highperformance liquid chromatographic method with ultraviolet detection was developed for the determination of 6-methoxy-2-naphthylacetic acid (6-MNA, the active metabolite of nabumetone ). The sample preparation involved a simple but effective protein precipitation procedure. Reversed-phase liquid chromatography was optimised, and full resolution between the analyte and endogenous matrix peaks achieved in a chromatographic runtime of five minutes. The assay method was validated over a range of plasma concentrations between 0.070 and 145 µg/ml. 1242 Plasma samples generated during a comparative bioequivalence study were then assayed and the performance of the assay method shown to be well within accepted international norms. The coefficient of variation for quality control standards over the range of 0.18 to 39 µg/ml processed during the assaying of the study samples, varied between 3.6 and 8.1 %. In the second instance, a novel method for the determination of piroxicam in four biological matrices (sub-cutaneous tissue (SCT), synovial fluid (SF), synovial capsule (SC) and plasma) was developed. A double back-extraction procedure was followed by reversed-phase liquid chromatography and electrochemical detection (ECD). Extracts from all four biological matrices were injected onto a single HPLC system. Ratios between plasma and the three remaining matrices were used to characterise transdermal absorption of two topical preparations of piroxicam when applied to the knee. Low systemic levels associated with topical formulations necessitated the development and validation of a highly sensitive assay method. Plasma was used as a surrogate matrix for all the processed tissue samples and the assay method was validated over a range of plasma concentrations between 1.24 and 600 ng/ml. 168 Samples generated during a multi-centre study involving knee replacement surgery, were assayed and the performance of the assay method shown to be well within accepted international norms. The coefficient of variation for quality control standards over the range of 1.74 to 300 ng/ml processed during the assaying of the study samples, varied between 7.7 and 13.5 % which can be considered excellent in the light of the complexity of the sample preparation process. Analytical data generated during the above-mentioned two research projects are discussed, with novelties and improvements to existing assay methods being elucidated. Both assay methods were presented and accepted for publication in peer reviewed scientific journals. Both full-length publications are included in an appendix in this dissertation, together with the correspondence entered into with journal editors and referees. Furthermore, a section containing copies of the slides used to present the latter HPLC assay method as an oral presentation at the 1998 Annual Congress of the South African Pharmacological Society, is included.