基于UPLC-Q-TOF-MS技术的大鼠肠内菌转化间尼索地平对映体的体外代谢产物研究

Studies on in vitro metabolites of m-nisoldipine enantiomers in rat intestinal flora transformation by UPLC-Q-TOF-MS

目的:建立肠内菌转化研究间尼索地平对映异构体(R,S-MNS)代谢产物的方法,确定代谢物的结构及转化途径。方法:将R,S-MNS分别与大鼠肠内菌于体外厌氧温孵培养2 h,用正己烷-乙醚(1∶1)萃取,离心后取上清液氮气吹干,复溶后进行UPLC-Q-TOF-MS分析。色谱条件:采用Poroshell 120 EC-C_(18)色谱柱(2.1 mm×100 mm,2.7μm)色谱柱,以0.5%甲酸水-乙腈为流动相,梯度洗脱,流速400μL·min^(-1),柱温40℃;质谱条件:电喷雾离子源(ESI源),负离子模式检测。比较样品和相应的菌空白、药物空白的数据,检测推断其代谢产物结构。结果:在对映体温孵液中检测到原药及其1,4-二氢-2,6-二甲基-4-(3-氨基苯基)-3,5-吡啶二羧酸异丁基甲酯、1,4-二氢-6-甲基-4-(3-硝基苯基)-3,5-吡啶二羧酸异丁基甲酯和1,4-二氢-2,6-二甲基-4-(3-硝基苯基)-3,5-吡啶二羧酸二甲酯3种代谢产物,并推断MNS的肠内菌转化途径。结论:MNS在肠道菌从中发生生物转化而形成代谢产物,对映体的代谢产物种类相同,代谢途径主要为还原和氧化反应。

Objective： To establish a rat intestinal flora transformation method of m-nisoldipine enantiomers （ R, S-MNS ）, so as to identify the chemical structures of the metabolites and propose the possible transformation pathways of m-nisoldipine （ MNS ）. Methods： R, S-MNS were anaerobically incubated with rat intestinal flora in vitro for 2 h respectively, and then extracted by hexane-ether （ 1 ： 1 ）. The supernatant liquid after centrifugation was dried by nitrogen and analyzed using the UPLC-Q-TOF-MS. Chromatographic conditions： analytes were separated on a Poroshell 120 EC-Cls column （2. 1 mm × 100 mm, 2.7 μ m ）with gradient elution. The mobile phase consisting of 0.5% aqueous formic acid and acetonitrile, the flow rate was 400 μ L ·min^-1, and the column temperature was 40 ℃ . Detection and quantitation was performed by electrospray ionization （ ESI ） in the negative ion mode. Finally, the metabolites were discovered by comparing the data of samples with the corresponding blanks. Results： The three metabolites were 1,4-dihydro-2, 6-dimethyl-4- （ 3-aminophenyl ） -3,5-pyridinedicarboxylic acid isobutyl methyl ester, 1,4-dihydro-6-methyl-4- （ 3-nitrophenyl ） -3,5-pyridinedicarboxylic acid isobutyl methyl ester and 1, 4-dihydro-2, 6-dimethyl-4-（ 3-nitrophenyl ）-3, 5-pyridinedicarboxylic acid dimethyl ester. The drug and three metabolites were found in the incubation solution of enantiomers and possible microbial transformation pathways of MNS by rat intestinal flora were inferred. Conclusion： Rat intestinal flora can metabolize R, S-MNS into the same metabolites. The main metabolic pathways of enantiomers were reduction and oxidation.