LC-MS/MS鉴定木兰花碱在大鼠体内外主要代谢产物

Identification of major metabolites of magnoflorine in vivo and in vitro of rats by LC-MS/MS

目的研究木兰花碱在大鼠体内外的主要代谢产物及代谢途径。方法SD大鼠ig木兰花碱(50 mg/kg),收集0~24 h尿液和粪便,0~6 h胆汁以及1、2、4、6、8 h血浆;体外代谢采用肝微粒体温孵系统和肠菌培养液。利用LC-MS/MS对生物样品中的原型药及代谢产物进行鉴定。采用Agilent TC-C18色谱柱(150 mm×4.6 mm,5μm),以乙腈-0.1%甲酸水溶液为流动相梯度洗脱,体积流量1.0 mL/min,柱温30℃。质谱采用电喷雾电离源(ESI),正离子采集模式;扫描范围m/z100~1000。根据药物体内代谢规则,结合木兰花碱的色谱保留时间和多级质谱碎片离子特征,推测其代谢产物的结构。结果给药后生物样品中共鉴定出12个代谢产物,其中Ⅰ相代谢产物8个,Ⅱ相代谢产物4个。主要的代谢途径为羟基化、去甲基化、脱氢作用、酮基化、葡萄糖化、葡萄糖醛酸化及硫酸酯化。结论木兰花碱在体内可发生Ⅰ相和Ⅱ相代谢,肠道菌群和肝药酶可催化木兰花碱发生Ⅰ相代谢转化,Ⅱ相代谢存在于肠道以外部位,最有可能的部位是肝脏。

Objective To study the main metabolites and metabolic pathway of magnoflorine in vitro and in vivo.Methods SD rats were given magnolithine by single gavage at a dose of 50 mg/kg.Urine and feces form 0 h to 24 h,bile form 0 h to 6 h,and plasma samples at 1,2,4,6,and 8 h after administration were collected.In vitro metabolism was incubated with rat liver microsome and intestinal flora.The metabolites were analyzed and identified by the high-resolution HPLC-MS/MS technique.Chromatographic separation was achieved on Agilent TC-C18 chromatograph column(150 mm×4.6 mm,5μm).The mobile phase consisted of 0.1%formic acid-acetonitrile with a flow rate of 1.0 mL/min,and the column temperature was 30℃.The mass spectra were obtained in positive ion modes with electrospray ionization(ESI),the scanning range was m/z 100—1000.The structures of metabolites were elucidated by the metabolic rules of drugs in vivo,and combined with the chromatographic retention time of magnoflorine and the characteristics of fragment ions of MS.Results A total of 12 metabolites were identified in each sample,including 8 phaseⅠmetabolites and 4 phaseⅡmetabolites.The pathways to these metabolites were hydroxylation,demethylation,dehydrogenation,ketoylation,gluconylation,glucuronide conjugation,and sulfation.Conclusion Magnoflorine could produce metabolic reaction of phaseⅠand phaseⅡin rat.Intestinal flora and liver drug enzymes could catalyze the phaseⅠmetabolism of magnoflorine,and phaseⅡmetabolism exists outside the intestine,and the most likely site is the liver.