AIM: To study metabolites of guanfu base A hydrochloride (GFA) in rat bile. METHODS: An analytical methodwas developed to identify guanfu base A and its metabolites by liquid chromatography mass spectrometry (LC-MS)and electrospray ionization tandem mass spectrum (MS-MS). Rat bile was collected after iv injection of GFA.Phase I metabolite was identified by comparison with authentic standard for their retention time, molecular ionpeaks, fragment ions, and UV spectrums. In order to identified phase Ⅱ conjugates, the aglycones were identifiedafter rat bile was treated with either glucuronidase or sulfatase firstly. Phase Ⅱ conjugates were also separated anddetermined for their molecular ions by LC-MS, at last they were verified by identifying characteristic product ionsor precursor ions by MS-MS. RESULTS: Phase I metabolite, guanfu base I (GFI), was identified in rat bile. Afterphase Ⅱ conjugates were treated with glucuronidase or sulfatase, GFA and GFI occurred in chromatograms. Quasimolecular ions m/z 606 and 510 were separated and detected in rat bile. They were indicated to be GFA glucuronideand GFA sulfate, respectively. Furthermore, GFA glucuronide was confirmed to exist in rat bile by identifying twocharacteristic ions, m/z 177, [M+H]+ of glucuronic acid, and m/z 430, [M+H]+ of GFA, as product ions of m/z 606.CONCLUSION: Phase I metabolite GFI and phase Ⅱ conjugates, GFA glucuronide and sulfate, GFI glucuronideand sulfate, were identified in rat bile.%目的:研究盐酸关附甲素在大鼠胆汁中的代谢产物.方法:建立了液相质谱和串联质谱法(LC-MS)对关附甲素及其代谢产物鉴定的方法.大鼠静脉注射盐酸关附甲素后采集胆汁,通过与对照化合物的色谱保留时间、分子离子峰、碎片离子峰和紫外图谱对照从而鉴定Ⅰ相代谢物.胆汁经过葡萄糖醛酸酶或硫酸酯酶水解,鉴定其水解产物(苷元),从而确定Ⅱ相结合物,再通过LC-MS分离和确定分子离子峰,最后利用MS-MS寻找特征子离子和母离子的方法进行验证.结果:大鼠胆汁中存在Ⅰ相代谢物关附壬素;Ⅱ相结合物经葡萄糖醛酸酶和硫酸酯酶酶解后,出现关附甲素和关附壬素;LC-MS检测发现胆汁中m/z 606和510两个准分子离子峰,推测分别为关附甲素葡萄糖醛酸苷和关附甲素硫酸酯;经MS-MS鉴定出m/z606特征子离子m/z177和m/z430,进一步确证大鼠胆汁中存在关附甲素葡萄糖醛酸苷.结论:大鼠胆汁中存在Ⅰ相代谢产物关附壬素,以及Ⅱ相结合物关附甲素葡萄糖醛酸和硫酸结合物、关附壬素葡萄糖醛酸和硫酸结合物.
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