甲基原薯蓣皂苷静脉注射给予大鼠后体内代谢物的LC-MSn 分析

摘要

A high sensitive performance liquid chromatography-electrospray ion trap-mass spectro-metric(LC-MSn)method was developed for the determination of the major methyl protodioscin me-tabolites(MPD)in rats plasma,urine,bile and feces in order to discover its in vivo metabolism pathway. The Sprague -Dawley rats were given a single intravenous injection of 40 mg/kg MPD, and 30 min blood sample,24 hour bile sample,48 hour urine sample and feces samples after admin-istration were collected. The in vivo samples were extracted by solid phase extraction( SPE). The extracted samples were separated with Phenomenex Gemini C18 column,using methanol( B)-water ( A)as mobile phase by elution gradient. The flow rate was set at 1. 0 mL/min. The column temper-ature was 30 ℃. Total of fourteen metabolites in rat biosamples were detected under negative and positive modes . Six of them were identified by comparing HPLC retention times and mass spectra with those of authentic standards. They were protodioscin(M3),26-O-β-D-glucopyrannosyl-(25R)-furan-5-ene-3β,22α, 26-trihydroxy-3-O-α-L-rhamnopyranosyl-( 1→2 or 4 )-β-D-glucopyranoside (M5),MPD(M0),Pseudoprotodioscin(M7),26-O-β-D-glucopyrannosyl(25R)-furan-5-ene-3β, 26-dihydroxy-22-methoxy-3-O-α-L-rhamnopyranosyl-(1→ 4)-β-D-glucopyranoside(M9),26-O-β-D-glucopyrannosyl(25R)-furan-5-ene-3β,26-dihydroxy-22-methoxy-3-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside(M10)and dioscin(M11). Five of them were proposed based on the MS fragmentation characteristics of metabolite contrast. The results indicated that protodioscin and dioscin were two primary metabolites,and the formation routes of these two metabolites were the major meta-bolic pathways of MPD in rat. Deglycosylation was found to be the primary reaction. Most of the me-tabolites belong to phase I,except 2 metabolites belong to phaseⅡreaction and most of the structure changes took place in sugar part rather than in mother nucleus.%采用液相色谱-多级离子阱质谱法（ LC-MSn ）法，检测大鼠生物样本中的甲基原薯蓣皂苷（ MPD）及其代谢产物，以推测MPD在大鼠体内的代谢途径。对大鼠静脉注射给予40 mg/kg的MPD，并收集尿液、血浆、胆汁和粪便等样本，经固相萃取（ SPE）净化提取后，采用Phenomenex Gemini C18色谱柱，以甲醇（ B）-水（ A）为流动相进行梯度洗脱，流速1.0 mL/min。采用正负离子检测。在生物样本中共检测到14个代谢产物。通过与对照品的色谱行为和多级质谱特征相比对，鉴定了其中6个代谢产物，分别为Protodioscin（ M3），26-O-β-D-Glucopyrannosyl-（25R）-furan-5-ene-3β，22α，26-trihydroxy-3-O-α-L-rhamnopyranosyl-（1→2 or 4）-β-D-glucopyranoside（M5），MPD（M0），Pseudoprotodioscin（M7），26-O-β-D-Glucopyrannosyl（25R）-furan-5-ene-3β，26-dihydroxy-22-methoxy-3-O-α-L-rhamnopyranosyl-（1→4）-β-D-glucopyranoside（M9），26-O-β-D-Glucopyran-nosyl（25R）-furan-5-ene-3β，26-dihydroxy-22-methoxy-3-O-α-L-rhamnopyranosyl-（1→2）-β-D-glucopyranoside（M10）和Dioscin（M11）。根据已有对照品的质谱裂解规律，用LC-MSn 法推测了另外5个代谢物。其中Protodioscin （ PD）和Dioscin是两个主要的代谢产物，其对应的生成途径是MPD在大鼠体内的主要代谢途径。代谢反应总体以一相代谢为主，主要是水解脱糖，同时检出2个二相代谢产物；代谢物结构变化主要发生在糖上，母核均无明显变化。