浅析HPLC-MS-MS法测定兔血浆中黄芪甲苷的含量.docVIP

浅析HPLC-MS/MS法测定兔血浆中黄芪甲苷的含量 【摘要】 目的建立兔含药血浆中黄芪甲苷含量快速测定的新方法。方法固相萃取法富集血浆中的黄芪甲苷;Agilent SB-C18反相色谱进行分离;采用离子阱质谱多级反应模式，以m/z 807.5→627.1为选择离子对，内标法测定含量。结果血浆中黄芪甲苷的检测限为0.1ng/mL，线性范围为0.5～50.0μg/mL，平均方法回收率在96.3%～104.7%范围内。结论该方法具有灵敏度高、稳定性好和快速的特点，符合生物样品的分析要求，有望用于黄芪甲苷的药代动力学研究。 【关键词】 黄芪;黄芪甲苷;固相萃取;HPLC-MS/MS 　ABSTRACT: ObjectiveTo establish a new method for the determination of astragaloside in rabbit plasma by high performance liquid chromatography-ion trap mass spectrometry (HPLC-MS/MS).MethodsAstragaloside Ⅳ in plasma samples was enriched by solid phase extraction. The resulted samples were further separated by reverse phase chromatography with Agilent SB-C18 column. Multiple reaction mode of MS/MS was employed to determine the concentration of astragaloside Ⅳ by internal standard with m/z 807.5→627.1 as the selected ion pair.ResultsThe detection limit of astragaloside Ⅳ in plasma samples was 0.1ng/mL, the linear range was 0.5-50.0μg/mL, and the average recovery rate of the method was within the range of 96.3%-104.7%.　ConclusionThe proposed method has the characteristics of high sensitivity, good stability and high speed. These properties can meet the requirements of the analytical method for biological samples, which indicates that the method may be used in pharmacokinetic study of astragaloside Ⅳ.KEYWORDS: Radix Astragali; astragaloside Ⅳ; solid phase extraction; high performance liquid chromatography-ion trap mass spectrometry (HPLC-MS/MS) 　　黄芪为中医临床广泛应用的益气中药之一，味甘、性温，具有补气固表、脱毒和利尿等功效。中华人民共和国规定临床所用黄芪药材为豆科植物蒙古黄芪Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao或膜荚黄芪Astragalus membranaceus (Fisch.) Bge.的干燥根[1]。黄芪甲苷为黄芪的主要有效成分之一，其含量测定方法主要包括薄层扫描法[2]和以紫外[3]和蒸发光散射[4]为检测方法的高效液相色谱法。由于黄芪甲苷为紫外末端吸收物质，上述方法虽能用于黄芪药材及制剂中黄芪甲苷的含量测定，但灵敏度方面难以满足生物样品中黄芪甲苷的测定要求。本文采用固相萃取法对兔含药血浆中的黄芪甲苷进行富集，高效液相色谱-质谱法(HPLC-MS/MS)进行分离测定，旨在建立可用于黄芪甲苷药代动力学及在其他生物样品中含量测定的新方法。 　　1材料与方法 　　1.1药品与试剂黄芪甲苷对照品(中国药品生物制品检定所)，地高辛对照品(中国药品生物制品检定所)。黄芪药材购自陕西省中药材公司，经陕西省药品检验所鉴定为蒙古黄芪。乙腈(色谱纯，美国Fisher公司)，水为超纯水(自制)，其他试剂均为分析纯。 　　1.2