Enzyme-linked immunosorbent assay of pravastatin, a HMG-CoA reductase inhibitor, in human plasma.
An enzyme-linked immunosorbent assay (ELISA) was developed for sensitive and specific determination of pravastatin (PS) sodium, a HMG-CoA reductase inhibitor. Preparation of immunogens to obtain antisera was carried out using chemically modified PS; beta-alanine derivative of PS (for ELISA-1) and 5-deoxy- PS (for ELISA-2) were linked to bovine serum albumin via its terminal carboxylic acid by the N-succinimidyl ester method, to avoid intramolecular lactonization of PS. Enzyme-labeled antigens were prepared similarly by coupling with horseradish peroxidase, and were used by homogeneous combination of antisera. The enzymic activity was determined using a microtiter plate coated with second antibody 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-reactivties with main metabolites in plasma, which differed from PS in decaline moiety, were less than a few percent. When ELISA-1 was applied to the determination of PS in human plasma directly after dilution with the ELISA buffer, the detection limit and the intra-assay coefficient (5 ng/ml of PS) were 500 pg/ml and 4.5%, respectively. Further, ELISA-1 was validated by gas chromatography-mass spectrometry with the determination of PS in human plasma after oral administration at a dose of 10 mg/body.
Muramatsu
S
S
Analytical and Metabolic Research Laboratories, Sankyo Co., Ltd., Tokyo, Japan.
Takasaki
W
W
Uchiyama
M
M
Komokata
Y
Y
Tanaka
Y
Y
Takahagi
H
H
D000284:Administration, Oral; D000924:Anticholesteremic Agents / Q000008:administration & dosage / Q000097:blood* / Q000493:pharmacokinetics; D000941:Antigens / Q000737:chemistry / Q000276:immunology; D003429:Cross Reactions; D004791:Enzyme Inhibitors / Q000008:administration & dosage / Q000097:blood* / Q000493:pharmacokinetics; D004797:Enzyme-Linked Immunosorbent Assay; D005069:Evaluation Studies as Topic; D008401:Gas Chromatography-Mass Spectrometry; D006241:Haptens; D006801:Humans; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D008297:Male; D015394:Molecular Structure; D017035:Pravastatin / Q000008:administration & dosage / Q000031:analogs & derivatives / Q000097:blood* / Q000493:pharmacokinetics; D012710:Serum Albumin, Bovine
D000924:Anticholesteremic Agents; D000941:Antigens; D004791:Enzyme Inhibitors; D006241:Haptens; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D012710:Serum Albumin, Bovine; D017035:Pravastatin