ENZYME-LINKED-IMMUNOSORBENT-ASSAY OF PRAVASTATIN, A HMG-COA REDUCTASEINHIBITOR, IN HUMAN PLASMA

An enzyme-linked immunosorbent assay (ELISA) was developed for sensiti ve and specific determination of pravastatin (PS) sodium, a HMG-CoA re ductase inhibitor. Preparation of immunogens to obtain antisera was ca rried out using chemically modified PS; beta-alanine derivative of PS (for ELISA-1) and 5-deoxy- PS (for ELISA-2) were linked to bovine seru m albumin via its terminal carboxylic acid by the N-succinimidyl ester method, to avoid intramolecular lactonization of PS. Enzyme-labeled a ntigens were prepared similarly by coupling with horseradish peroxidas e, and were used by homogeneous combination of antisera. The enzymic a ctivity was determined using a microtiter plate coated with second ant ibody and tetramethylbenzidine as a chromogenic substrate. Both of the ELISA systems enabled the determination of PS in a range of 5 to 500 pg/well, with an IC50 of 36 to 130 pg/well. Cross-reactivities with ma in metabolites in plasma, which differed from PS in decaline moiety, w ere less than a few percent. When ELISA-1 was applied to the determina tion of PS in human plasma directly after dilution with the ELISA buff er, tile detection limit and the intra-assay coefficient (5 ng/ml of P S) were 500 pg/ml and 4.5%, respectively. Further, ELISA-1 was validat ed by gas chromatography-mass spectrometry with the determination of P S in human plasma after oral administration at a dose of 10 mg/body.