Korecka Magdalena, Shaw Leslie M
Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA.
Methods Mol Biol. 2010;603:469-77. doi: 10.1007/978-1-60761-459-3_46.
A multiple reaction monitoring positive ion HPLC method with tandem mass spectrometric detection (MS-MS) for determination of sirolimus in human blood samples is described. This method utilizes an online cleanup step that provides simple and rapid sample preparation with a switching valve technique. This procedure includes: instrumentation, API 3000 triple quadrupole with turbo-ion spray (Applied Biosystems, Foster City, CA); HPLC system (Agilent Technologies series 1100, Wilmington, DE); two position switching valve (Valco, Houston, TX); 10 mm guard cartridge (C(18)) used as an extraction column (Perkin Elmer, Norwalk, CT); analytical column (Nova-Pak C(18) column, 2.1 x 150 mm I.D., 4 microm, Waters Corp, Milford, MA) maintained at 65 degrees C; extraction solution, ammonium acetate (30 mM, pH 5.2), flow rate 1.0 mL/min; eluting solution, methanol:30 mM ammonium acetate buffer (pH 5.2, 97:3 v/v), flow rate 0.8 mL/min with 1/3 of the flow split post-column into the MS-MS; total run-time 3.5 min. Sample preparation is based on simple protein precipitation with a mixture of methanol and zinc sulfate (7:3, v/v) followed by online sample cleanup. This procedure provides a decreased sample preparation time by a factor of four compared to a method that uses an SPE column. The first and third quadrupoles were set to detect the ammonium adduct ion and a high mass fragment of sirolimus (m/z 931.8-->864.6), and of an internal standard (ascomycin) (m/z 809.5-->756.5). The lower limit of quantification of this method is 2.5 microg/L. The quantification of drug is made from standard curve using peak-area ratio of analyte vs. internal standard. Calibration curve is constructed using non-weighted linear through zero regression.
本文描述了一种采用串联质谱检测（MS-MS）的多反应监测正离子HPLC方法，用于测定人血样中的西罗莫司。该方法利用在线净化步骤，通过切换阀技术实现简单快速的样品制备。该程序包括：仪器设备，配备涡轮离子喷雾的API 3000三重四极杆质谱仪（应用生物系统公司，加利福尼亚州福斯特城）；HPLC系统（安捷伦科技1100系列，特拉华州威尔明顿）；二位切换阀（瓦尔科公司，得克萨斯州休斯顿）；10 mm保护柱（C(18)）用作萃取柱（珀金埃尔默公司，康涅狄格州诺沃克）；分析柱（Nova-Pak C(18)柱，内径2.1 x 150 mm，4μm，沃特世公司，马萨诸塞州米尔福德），柱温保持在65℃；萃取溶液，醋酸铵（30 mM，pH 5.2），流速1.0 mL/min；洗脱溶液，甲醇:30 mM醋酸铵缓冲液（pH 5.2，97:3 v/v），流速0.8 mL/min，柱后1/3流速分流进入MS-MS；总运行时间3.5分钟。样品制备基于用甲醇和硫酸锌混合物（7:3，v/v）进行简单的蛋白质沉淀，随后进行在线样品净化。与使用固相萃取柱的方法相比，该程序将样品制备时间缩短了四倍。第一和第三四极杆设置为检测西罗莫司的铵加合离子和高质量碎片（m/z 931.8-->864.6）以及内标（子囊霉素）（m/z 809.5-->756.5）。该方法的定量下限为2.5μg/L。药物定量通过使用分析物与内标的峰面积比从标准曲线进行。校准曲线采用非加权线性过零回归构建。
