Bai Feng, Minkin Patton, Fraga Charles H, O'Shaughnessy Melinda A, Gururangan Sri, Stewart Clinton F
Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, TN 38105, and The Preston Robert Tisch Brain Tumor Center, Duke University Medical Center, Durham, NC 27710, USA.
J Chromatogr B Analyt Technol Biomed Life Sci. 2007 Jun 15;853(1-2):97-104. doi: 10.1016/j.jchromb.2007.02.062. Epub 2007 Mar 24.
A sensitive method for the determination of Cloretazine (VNP40101M) and its metabolite (VNP4090CE) with an internal standard (ISTD) in human plasma was developed using high-performance liquid chromatographic separation with tandem mass spectrometric detection. Acidified plasma samples (500 microL) were prepared using solid phase extraction (SPE) columns, and 25 microL of the reconstituted sample was injected onto an Ascentis C18 HPLC column (3 microm, 5 cmx2.1 mm) with an isocratic mobile phase. Analytes were detected with an API-3000 LC-MS/MS System at unit (Q1) and low (Q3) resolution in negative multiple reaction monitoring mode: m/z 249.0 (precursor ion) to m/z 114.9 (product ion) for both Cloretazine (at 3.64 min) and VNP4090CE (at 2.91 min), and m/z 253.0 (precursor ion) to m/z 116.9 (product ion) for the ISTD. The mean recovery for Cloretazine (VNP40101M) and its metabolite (VNP4090CE) was greater than 87% with a lower limit of quantification of 1.0 ng/mL for Cloretazine (S/N=9.7, CV<or=12%) and 0.5 ng/mL for VNP4090CE (S/N=11.3, CV<or=9.7%). This method was validated over a linear range of 1.0-1000 ng/mL for Cloretazine and 0.5-100 ng/mL for VNP4090CE, and results from a five day validation study demonstrated good within-day and between-day precision and accuracy. This method has been used to measure plasma Cloretazine and its metabolite concentrations in a Phase I study in children with recurrent progressive or refractory primary brain tumors.
建立了一种采用高效液相色谱分离-串联质谱检测法，以内标物（ISTD）测定人血浆中氯雷他嗪（VNP40101M）及其代谢物（VNP4090CE）的灵敏方法。使用固相萃取（SPE）柱制备酸化血浆样品（500微升），取25微升复溶后的样品注入到配备等度流动相的Ascentis C18高效液相色谱柱（3微米，5厘米×2.1毫米）上。采用API-3000液相色谱-串联质谱系统，在负离子多反应监测模式下，于单位（Q1）和低（Q3）分辨率下对分析物进行检测：氯雷他嗪（在3.64分钟处）和VNP4090CE（在2.91分钟处）均为m/z 249.0（母离子）到m/z 114.9（子离子），内标物为m/z 253.0（母离子）到m/z 116.9（子离子）。氯雷他嗪（VNP40101M）及其代谢物（VNP4090CE）的平均回收率大于87%，氯雷他嗪的定量下限为1.0纳克/毫升（信噪比=9.7，变异系数≤12%），VNP4090CE的定量下限为0.5纳克/毫升（信噪比=11.3，变异系数≤9.7%）。该方法在氯雷他嗪1.0 - 1000纳克/毫升和VNP4090CE 0.5 - 100纳克/毫升的线性范围内得到验证，为期五天的验证研究结果表明日内和日间精密度及准确度良好。该方法已用于在一项针对复发性进展性或难治性原发性脑肿瘤儿童的I期研究中测量血浆氯雷他嗪及其代谢物浓度。
