Determination of Zofenopril and Its Active Metabolite in Human Plasma Using High-Performance Liquid Chromatography Combined With a Triple-Quadruple Tandem Mass Spectrometer

Journal of Chromatographic Science 2015;53:253– 262 doi:10.1093/chromsci/bmu050 Advance Access publication June 16, 2014 Article Determination of Zofenopril and Its Active Metabolite in Human Plasma Using High-Performance Liquid Chromatography Combined With a Triple-Quadruple Tandem Mass Spectrometer Yuan Tian1, Jing Cao1, Lifang Luo1, Zunjian Zhang1,2* and Pengcheng Ma3 1 Key Laboratory of Drug Quality Control and Pharmacovigilance, Center for Instrumental Analysis, China Pharmaceutical University, Ministry of Education, 24 Tongjia Lane, Nanjing, Jiangsu 210009, P. R. China, 2State Key Laboratory of Natural Medicines China Pharmaceutical University, Nanjing, Jiangsu 210009, P. R. China, and 3Institute of Dermatology, Chinese Academy of Medical Sciences, Perking Union Medical Sciences, Perking Union Medical College, Nanjing 210042, P. R. China *Author to whom correspondence should be addressed. Email: Received 14 July 2013; revised 18 March 2014 A simple, selective and sensitive LC–MS-MS method has been developed and validated to simultaneously quantify zofenopril and its active metabolite zofenoprilat in human plasma, using diazepam as internal standard. 1,4-Dithiothreitol was used as a reducer to release and stabilize the thiol group of zofenoprilat from dimer and mixed forms with endogenous thiols in the treatment of plasma samples. After a liquid – liquid extraction with methyl tert-butyl ether under acidic conditions, the post-treatment samples were analyzed on an Agilent ZORBAX Eclipse XDB-C8 column interfaced with a triple-quadruple tandem mass spectrometer using positive electrospray ionization. A solution of methanol and 0.1% formic acid solution (85 : 15, v/ v) was used as the isocratic mobile phase with a flow rate of 0.2 mL/ min. The method was validated to demonstrate the specificity, lower limit of quantitation, accuracy and precision of measurements. The validated LC – MS-MS method has been successfully applied to study the pharmacokinetics of zofenopril calcium in healthy Chinese volunteers. Introduction Zofenopril calcium, [1(S), 4(S)]-1(3-mercapto-2-methyl-1oxopropyl) 4-phenyl-thio-L-proline-S-benzoylester, is a new sulfhydryl-group-containing ACE inhibitor with antihypertensive, remarkable antioxidant and cardioprotective properties, including the ability to improve endothelial function and protect against ischemia (1, 2). These peculiar characteristics are mainly due to the presence of a sulfhydryl group and the highly lipophilic nature of the compound. Zofenopril (Figures 1 and 2), as most compounds of this class, is a prodrug that is deesterified to the active metabolite, the sulfhydryl group containing compound, zofenoprilat (Figure 1). Thiol compounds can be easily oxidized to disulfides either as dimer or mixed forms with endogenous thiols in biological matrices (3). So, it is difficult to determine zofenoprilat directly in human plasma because of its active thiol group. The measurement of free or unchanged zofenoprilat concentration needs to be preceded by the addition of chemical stabilizer or by molecule derivatization of biological samples in order to prevent zofenoprilat disulfide formation. Several analytical methods have been reported with complicated sample preparation. Jemal et al. (4) used solid-phase extraction after liquid –liquid extraction for isolation and purification, followed by methylation and # Crown copyright 2014. reconstitution with tetramethylbenzene before GC–MS analysis. Dal Bo et al. (5) described an LC–MS-MS method for simultaneous analysis of zofenopril and zofenoprilat with N-ethylmaleimide as derivatization reagent. Gao et al. (2) used p-bromophenacyl bromide as a derivatization reagent for the simultaneous determination of zofenopril and zofenoprilat in human plasma by LC – MS-MS. These methods involved derivatization and/or specialized and expensive equipment, which appeared to show low reproducibility and time-consuming sample preparation. Although Jiang et al. (6) proposed a relatively simple and fast method for the simultaneous determination of zofenopril and zofenoprilat by LC – MS-MS; 2-mercaptoethanol was used as the stabilization agent which carried the disadvantage of being toxic. Therefore, it was considered necessary to use a safer derivatization reagent. In this paper, a relatively simple, fast and sensitive HPLC – MS-MS method for direct simultaneous determination of zofenopril and zofenoprilat in human plasma is proposed. In the sample preparation process, 1,4-dithiothreitol (DTT) was used for the stabilization to revert the converted disulfide dimers (or conjugates) into zofenoprilat as well as to prevent the formation of the disulfide dimers in plasma. In this investigation, the validated HPLC – MS-MS method has been successfully used to a clinical pharmacokinetic study of zofenopril calcium tablets in healthy Chinese volunteers. Experimental Instrumentation and reagents Liquid chromatography was performed using the FinniganTM TSQ Quantum Discovery MAXTM LC – MS-MS system consisting of a FinniganTM Surveyor LC pump, a FinniganTM Surveyor autosampler and combined with a triple-quadrupole TSQ Quantum mass spectrometer (Thermo Electron Corporation). The zofenopril calcium reference standard (Batch No.: 20090110, 99.15% purity) was identified and supplied by Hefei Xinfeng Co. Ltd (Hefei, P. R. China); the zofenoprilat reference standard (Batch No.: 080701, 99.2% purity) was obtained from Shanghai Haini Pharmaceutical Co. Ltd (Shanghai, P. R. China); the diazepam reference standard (Batch No.: 1230-9601, 99.0% purity) was purchased from National Institute for the Control of Pharmaceutical and Biological Products of China (Beijing, P. R. China). HPLC grade methanol was purchased from Merck (Merck Company, Germany). DTT was of analytical grade purity 200 mg/mL (based on zofenopril). Standard stock solutions of zofenoprilat were prepared in methanol at a concentration of 500 mg/mL. Subsequently, 1 mL of zofenopril stock solution and 1 mL of zofenoprilat stock solution were accurately transferred into a 5 mL brown volumetric flask and diluted with methanol to 5 mL. Before adjusting the volume, 100 mL of a 200 mM DTT solution were added to the flask to obtain stable stock solutions. The IS stock solutions were also prepared in methanol at a concentration of 1.0 mg/mL. All stock solutions were stored at 48C. Working solutions of zofenopril and zofenoprilat were prepared daily in methanol: 0.1% formic acid solution (85 : 15, v/v) by appropriate dilution at 0.01, 0.02, 0.04, 0.1, 0.4, 1, 4, 10 and 40 mg/mL for zofenopril and 0.025, 0.05, 0.1, 0.25,1, 2.5, 10, 25 and 100 mg/mL for zofenoprilat. The IS working solution was prepared by diluting the stock solution to 500 ng/mL. Figure 1. Chemical structure of zofenopril (A) and zofenoprilat (B). and purchased from Aladdin Chemistry Co. Ltd. (Shanghai, P. R. China); other reagents of analytical grade such as methyl tert-butyl ether, formic acid, hydrochloric a (...truncated)