Szultka-Mlynska Malgorzata, Buszewski Boguslaw
Department of Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University, Gagarin 7, 87-100, Torun, Poland.
Center for Modern Interdisciplinary Technologies, Nicolaus Copernicus University, Wilenska 4, 87-100, Torun, Poland.
Anal Bioanal Chem. 2016 Nov;408(29):8273-8287. doi: 10.1007/s00216-016-9929-6. Epub 2016 Oct 4.
High performance liquid chromatography coupled with triple-quadrupole mass spectrometry was applied in the determination of in vitro metabolism products of selected antibiotic drugs (cefotaxime, ciprofloxacin, fluconazole, gentamicin, clindamycin, linezolid, and metronidazole). The analytes were separated on a reversed phase C18 column, with acetonitrile and 0.1 % aqueous formic acid as the mobile phase. Tandem mass spectrometry with positive electrospray ionization was used to facilitate the structural characterization of the potential metabolites. Metabolism studies on human liver microsomes were performed via cytochromes P450 (phase I) and via NADPH/UDP-glucuronosyltransferase (phase II) mediated reactions. LC-MS/MS experiments allowed potential metabolite peaks, including sum formulae suggestions, to be identified; high resolution MS/MS experiments led to the identification of various oxidative and reductive modifications of target compounds in phase I biotransformation, and conjugation products with glucuronic acid in phase II reactions. A total of 11 potential metabolites and their proposed structures were characterized during the incubation of human liver microsomes by comparing their retention times and spectral patterns with those of the parent drug. Dehydrogenation and reactions of side chains such as hydroxylation and hydrolysis of ester bonds constituted the major metabolic pathways. Finally, LC-MS/MS spectrometry was revealed to be a suitable analytical tool to procure a feasible analytical base for the envisioned in vivo experiments. Graphical Abstract Workflow overview of in vitro drug metabolism studies.
采用高效液相色谱-三重四极杆质谱联用技术测定了选定抗生素药物（头孢噻肟、环丙沙星、氟康唑、庆大霉素、克林霉素、利奈唑胺和甲硝唑）的体外代谢产物。分析物在反相C18柱上分离，以乙腈和0.1%甲酸水溶液为流动相。采用正电喷雾电离串联质谱法对潜在代谢产物进行结构表征。通过细胞色素P450（I相）和NADPH/ UDP-葡萄糖醛酸转移酶（II相）介导的反应，对人肝微粒体进行代谢研究。液相色谱-串联质谱实验可鉴定潜在的代谢产物峰，包括分子式建议；高分辨率串联质谱实验可鉴定I相生物转化中目标化合物的各种氧化和还原修饰，以及II相反应中与葡萄糖醛酸的缀合产物。通过将人肝微粒体孵育过程中的保留时间和光谱模式与母体药物进行比较，共鉴定出11种潜在代谢产物及其推测结构。脱氢以及侧链反应，如羟基化和酯键水解，构成了主要代谢途径。最后，液相色谱-串联质谱法被证明是一种合适的分析工具，可为设想的体内实验提供可行的分析基础。图形摘要体外药物代谢研究工作流程概述。
