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 converting enzyme (ACE) inhibitor, endowed also with a marked cardioprotective activity. A simple, highly sensitive specific LC–MS–MS method was developed for the determination of zofenopril and zofenoprilat in human plasma. In order to prevent oxidative degradation of zofenoprilat and its internal standard, their free sulfhydryl groups were protected by treatment with N-ethylmaleimide (NEM), which produced the succinimide derivatives. The compounds 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 instrument operating in the negative ion spray ionization mode. The method was validated over the concentration range of 1–300 ng/ml for zofenopril and 2–600 ng/ml for zofenoprilat. Inter- and intra-assay precision and accuracy of both zofenopril and zofenoprilat were better than 10%. The limit of quantitation was 1 ng/ml with zofenopril and 2 ng/ml with zofenoprilat. Extraction recovery proved to be on average 84.8% with zofenopril and 70.1% with zofenoprilat. Similar recoveries were shown by the above two internal standards. The method was applied to measure plasma concentrations of zofenopril and zofenoprilat in 18 healthy volunteers treated orally with zofenopril calcium salt at the dose of 60 mg.     

CALB-catalyzed kinetic resolution of (RS)-3-benzoylthio-2-methylpropyl azolides: kinetic and thermodynamic analysis

Abstract

Zofenopril as an ACE inhibitor expired recently was found to have a favourable safety profile in comparison with other ACE inhibitors in treating high blood pressure, congestive heart failure, and acute myocardial infarction. It can be synthesised from the key building blocks of (S)-3-benzoylthio-2-methylpropanoic acid and (4S)-phenylthio-L-proline. In this report, an efficient hydrolytic resolution via Candida antarctic lipase B (CALB) for preparing the former block in isopropyl ether (IPE) containing (RS)-3-benzoylthio-2-methylpropyl pyrazolide (1) and water was developed. Quantitative improvements of the enzyme activity and enantioselectivity in terms of k_2SK_mS^?1?=?5.726?L h^?1 g^?1 and E?=?217 at 45?°C were found from the kinetic analysis. Insights into the CALB performance via thermodynamic analysis were then addressed and compared with those by using (RS)-3-benzoylthio-2-methylpropyl 1,2,4-triazolide (2) as the substrate. A putative thermodynamic model was moreover hypothesised for elucidating the more enthalpy reduction of 68.92-70.86?kJ mol^?1 from the acyl part of (S)-1 and (S)-2 as well as that of 23.69-25.63?kJ mol^?1 from the triad imidazolium to Ser105 and leaving 1,2,4-triazole moiety of (R)-2 and (S)-2 on stabilising the corresponding transition states.