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

[摘要] English: The development and validation of bioanalytical assay methods suitable for the quantitationof drugs in biological matrices is discussed, as well as general principles applicable to thisparticular aspect of drug development. Relevant literature is consulted, with a view toexemplifying what constitutes good assay method development strategy, as well as to reflectcurrent international policy in this field, with particular reference to bioequivalence studies.Comparisons are made between international practices and those in place at FARMOVSPAREXELBioanalytical Services Division®. Attention is given inter alia to detectorselection, chromatographic optimisation, extraction procedures and method validation, withreference to new assay methods for two drugs in particular that have been developed andvalidated according internationally acceptable standards. In the first instance, a highperformanceliquid chromatographic method with ultraviolet detection was developed for thedetermination of 6-methoxy-2-naphthylacetic acid (6-MNA, the active metabolite ofnabumetone ). The sample preparation involved a simple but effective protein precipitationprocedure. Reversed-phase liquid chromatography was optimised, and full resolutionbetween the analyte and endogenous matrix peaks achieved in a chromatographic runtime offive minutes. The assay method was validated over a range of plasma concentrationsbetween 0.070 and 145 µg/ml. 1242 Plasma samples generated during a comparativebioequivalence study were then assayed and the performance of the assay method shown tobe 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 studysamples, varied between 3.6 and 8.1 %.In the second instance, a novel method for the determination of piroxicam in four biologicalmatrices (sub-cutaneous tissue (SCT), synovial fluid (SF), synovial capsule (SC) andplasma) was developed. A double back-extraction procedure was followed by reversed-phaseliquid chromatography and electrochemical detection (ECD). Extracts from all fourbiological matrices were injected onto a single HPLC system. Ratios between plasma and thethree remaining matrices were used to characterise transdermal absorption of two topicalpreparations of piroxicam when applied to the knee. Low systemic levels associated withtopical formulations necessitated the development and validation of a highly sensitive assaymethod. Plasma was used as a surrogate matrix for all the processed tissue samples and theassay method was validated over a range of plasma concentrations between 1.24 and 600ng/ml. 168 Samples generated during a multi-centre study involving knee replacementsurgery, were assayed and the performance of the assay method shown to be well withinaccepted international norms. The coefficient of variation for quality control standards overthe range of 1.74 to 300 ng/ml processed during the assaying of the study samples, variedbetween 7.7 and 13.5 % which can be considered excellent in the light of the complexity ofthe 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 assaymethods were presented and accepted for publication in peer reviewed scientific journals.Both full-length publications are included in an appendix in this dissertation, together withthe correspondence entered into with journal editors and referees. Furthermore, a sectioncontaining copies of the slides used to present the latter HPLC assay method as an oralpresentation at the 1998 Annual Congress of the South African Pharmacological Society, isincluded.

[发布机构] University of the Free State