Assay of zofenopril and its active metabolite zofenoprilat by liquid chromatography coupled with tandem mass spectrometry

Zofenopril is a pro-drug designed to undergo metabolic hydrolysis yielding the active free sulfhydryl compound zofenoprilat, which is an angiotensin c onverting enzyme (ACE) inhibitor, endowed also with a marked cardioprotecti ve activity. A simple, highly sensitive specific LC-MS-MS method was develo ped for the determination of zofenopril and zofenoprilat in human plasma. I n order to prevent oxidative degradation of zofenoprilat and its internal s tandard, their free sulfhydryl groups were protected by treatment with N-et hylmaleimide (NEM), which produced the succinimide derivatives. The compoun ds and their corresponding fluorine derivatives, used as internal standards , were extracted from plasma with toluene. The reconstituted dried extracts were chromatographed and then monitored by a triple-stage-quadrupole instr ument operating in the negative ion spray ionization mode. The method was v alidated over the concentration range of 1-300 ng/ml for zofenopril and 2-6 00 ng/ml for zofenoprilat, Inter- and intra-assay precision and accuracy of both zofenopril and zofenoprilat were better than 10%. The limit of quanti tation was 1 ng/ml with zofenopril and 2 ng/ml with zofenoprilat. Extractio n recovery proved to be on average 84.8% with zofenopril and 70.1% with zof enoprilat. Similar recoveries were shown by the above two internal standard s. The method was applied to measure plasma concentrations of zofenopril an d zofenoprilat in 18 healthy volunteers treated orally with zofenopril calc ium salt at the dose of 60 mg. (C) 2000 Elsevier Science B.V. All rights re served.