HPLC-APCI-MS/MS法同时测定猪苓颗粒中麦角甾酮与麦角甾醇的含量
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摘要
目的:建立同时测定猪苓配方颗粒中麦角甾酮与麦角甾醇的液相色谱-串联质谱法。方法:采用超声提取方法制备供试品溶液,以Zorbax Eclipse XDB-C18(2.1 mm×150 mm,3.5μm)色谱柱进行分离,流动相为甲醇-10 mmol·L-1醋酸铵缓冲液(98∶2,V/V),流速0.4 mL·min-1。试样在三重四极杆串联质谱仪中经过大气压化学离子化(APCI)离子源,在正离子方式下采用多反应离子监测(MRM)模式进行检测。结果:麦角甾酮与麦角甾醇浓度在1~3000 ng·mL-1范围内与峰面积线性关系良好,麦角甾酮和麦角甾醇的平均加样回收率分别为100.8%和100.7%,日内和日间变异(RSD)均小于6.5%,色谱峰保留时间分别为4.2 min和4.6 min。结论:本实验建立的HPLC-APCI-MS/MS法用时短、专属性强,可以准确、灵敏、快速地检测猪苓配方颗粒中麦角甾酮与麦角甾醇的含量,对猪苓颗粒质量控制和评价具有一定意义,为下一步深入研究其活性成分的药理机制奠定基础。
Objective: To develop a liquid chromatography-tandem mass spectrometry for the simultaneous determination of ergot sterone and ergosterol in Polyporus umbellatus particles. Methods: The test sample solution was prepared with methanol by ultrasonic extraction system. Ergot sterone and ergosterol were analyzed on a Zorbax Eclipse XDB-C18column( 2. 1 mm × 150 mm,3. 5 μm) with the mobile phase of methanol-10 mmol·L-1ammonium acetate buffer( 98∶ 2,V / V) at a flow rate of 0. 4 mL·min-1. The sample was injected into a triple quadrupole LC-MS with APCI probe and analyzed by the multi reaction monitoring( MRM) in the positive ion detection mode. Results: The assay was linear over the entire range of the calibration standard i. e.,a concentration range of 1 ng· mL-1to 3000 ng·mL-1which had good relationship with the peak area. The average recoveries of ergot sterone and ergosterol after ultrasonic extraction were 100. 8% and 100. 7%. The intra-day and inter-day variations( RSD) were less than 6. 5%. And the chromatographic peak retention time of ergot sterone was 4. 2 min. The retention time of ergosterol was 4. 6 min. Conclusion: The method established in the experiment is characterized by short analysis time and strong specificity. It can be applied to simultaneous determination of the levels of ergot sterone and ergosterol in the same sample accurately,sensitively and rapidly. Therefore the developed method has significance for quality control and evaluation of Polyporus umbellatus particles,laying the foundation for further research on the pharmacological mechanism of the active components.
Objective: To develop a liquid chromatography-tandem mass spectrometry for the simultaneous determination of ergot sterone and ergosterol in Polyporus umbellatus particles. Methods: The test sample solution was prepared with methanol by ultrasonic extraction system. Ergot sterone and ergosterol were analyzed on a Zorbax Eclipse XDB-C18column( 2. 1 mm × 150 mm,3. 5 μm) with the mobile phase of methanol-10 mmol·L-1ammonium acetate buffer( 98∶ 2,V / V) at a flow rate of 0. 4 mL·min-1. The sample was injected into a triple quadrupole LC-MS with APCI probe and analyzed by the multi reaction monitoring( MRM) in the positive ion detection mode. Results: The assay was linear over the entire range of the calibration standard i. e.,a concentration range of 1 ng· mL-1to 3000 ng·mL-1which had good relationship with the peak area. The average recoveries of ergot sterone and ergosterol after ultrasonic extraction were 100. 8% and 100. 7%. The intra-day and inter-day variations( RSD) were less than 6. 5%. And the chromatographic peak retention time of ergot sterone was 4. 2 min. The retention time of ergosterol was 4. 6 min. Conclusion: The method established in the experiment is characterized by short analysis time and strong specificity. It can be applied to simultaneous determination of the levels of ergot sterone and ergosterol in the same sample accurately,sensitively and rapidly. Therefore the developed method has significance for quality control and evaluation of Polyporus umbellatus particles,laying the foundation for further research on the pharmacological mechanism of the active components.
引文
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[8]ZHAO Ying-yong(赵英永),CHENG Xian-long(程显隆),ZHANG Ping(张萍),et al.HPLC determination of ergosta-4,6,8(14),22-tetraen-3-one in Polyporus umbellatus(HPLC法测定猪苓中麦角甾酮的含量)[J].Chin J Pharm Anal(药物分析杂志),2009,2(9):1579
[9]Zhao YY,Zhao Y,Zhang YM,et al.Qualitative and quantitative analysis of diuretic component ergone in Polyporus umbellatus by HPLC with fluorescence detector and HPLC-APCI-MS/MS[J].Pharmazie,2009,64(6):366
[10]Zhao YY,Qin XY,Cheng XL,et al.Rapid resolution liquid chromatography-mass spectrometry and high-performance liquid chromatography-fluorescence detection for metabolism and pharmacokinetic studies of ergosta-4,6,8(14),22-tetraen-3-one[J].Anal Chim Acta,2010,675(2):199
[11]ZHAO Ying-yong(赵英永),ZHAO Ye(赵晔),LIN Rui-chao(林瑞超),et al.RP-HPLC determination of ergosterol in Polyporus umbellatus(RP-HPLC法测定猪苓中麦角甾醇的含量)[J].Chin J Pharm Anal(药物分析杂志),2009,29(6):898
[12]Zhao YY,Cheng XL,Zhang Y,et al.Simultaneous determination of eight major steroids from Polyporus umbellatus by high-performanceliquid chromatography coupled with mass spectrometry detections[J].Biomed Chromatogr,2010,24(2):222
[2]Ohsawa T,Yukawa M,Takao C,et al.Studies on constituents of fruit body of Polyporus umbellatus and their cytotoxic activity[J].Chem Pharm Bull,1992,40(1):143
[3]Sun Y,Yasukawa K.New anti-infla mmatory ergostane-type ecdysteroids from the sclerotium of Polyporus umbellatus[J].Bioorg Med Chem Lett,2008,18(11):3417
[4]Zheng SZ,Yang HP,Ma XM,et al.Two new polyporusterones from Polyporus umbellatus[J].Nat Prod Res,2004,18(5):403
[5]Zhou WW,Lin WH and Guo SX.Two new polyporusterones isolated from the sclerotia of Polyporus umbellatus[J].Chem Pharm Bull,2007,55(8):1148
[6]Yuan D,Yamamoto KI,Bi KS,et al.Studies on the marker compounds for standardization of traditional Chinese medicine‘Polyporus Sclerotium’[J].Yakugaku Zasshi,2003,123(2):53
[7]Yuan D,Mori J,Komatsu KI,et al.An anti-aldosteronic diuretic component(drain dampness)in Polyporus sclerotium[J].Biol Pharm Bull,2004,27(6):867
[8]ZHAO Ying-yong(赵英永),CHENG Xian-long(程显隆),ZHANG Ping(张萍),et al.HPLC determination of ergosta-4,6,8(14),22-tetraen-3-one in Polyporus umbellatus(HPLC法测定猪苓中麦角甾酮的含量)[J].Chin J Pharm Anal(药物分析杂志),2009,2(9):1579
[9]Zhao YY,Zhao Y,Zhang YM,et al.Qualitative and quantitative analysis of diuretic component ergone in Polyporus umbellatus by HPLC with fluorescence detector and HPLC-APCI-MS/MS[J].Pharmazie,2009,64(6):366
[10]Zhao YY,Qin XY,Cheng XL,et al.Rapid resolution liquid chromatography-mass spectrometry and high-performance liquid chromatography-fluorescence detection for metabolism and pharmacokinetic studies of ergosta-4,6,8(14),22-tetraen-3-one[J].Anal Chim Acta,2010,675(2):199
[11]ZHAO Ying-yong(赵英永),ZHAO Ye(赵晔),LIN Rui-chao(林瑞超),et al.RP-HPLC determination of ergosterol in Polyporus umbellatus(RP-HPLC法测定猪苓中麦角甾醇的含量)[J].Chin J Pharm Anal(药物分析杂志),2009,29(6):898
[12]Zhao YY,Cheng XL,Zhang Y,et al.Simultaneous determination of eight major steroids from Polyporus umbellatus by high-performanceliquid chromatography coupled with mass spectrometry detections[J].Biomed Chromatogr,2010,24(2):222