Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 646 Songtao Road, Shanghai 201203, China.
J Pharm Biomed Anal. 2010 Aug 1;52(4):571-9. doi: 10.1016/j.jpba.2010.01.040. Epub 2010 Feb 1.
An ion-pair reversed phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed for the quantification of a phosphorothioate oligonucleotide (PS-OGN) PF-ODN and its metabolites 5'N-1/3'N-1, 5'N-2 and 5'N-3 in rat plasma. Plasma samples were prepared with an initial phenol/dichloromethane liquid-liquid extraction followed by a solid phase extraction. Chromatographic separation was performed with a gradient system on a Phenomenex Gemini C(18) column using hexafluoro-2-propanol/triethylamine buffer and methanol as the mobile phase at a flow rate of 0.5mL/min. Except for 5'N-1 and 3'N-1, which were coeluted and could not be differentiated by mass spectrometer, the other analytes were separated chromatographically and mass spectrometrically. The detection was carried out in multiple reaction monitoring (MRM) mode using a negative electrospray ionization (ESI) interface. The lower limit of quantification (LLOQ) achieved was 4.0ng/mL for PF-ODN and its four metabolites with acceptable precision and accuracy. Inter-day and intra-day RSD for three quality control (QC) levels across validation runs were less than 12.0% and the accuracy ranged from -9.6% to 6.0% for the analytes. This validated LC-MS/MS method was applied to a preliminary pharmacokinetic study of PF-ODN in rats.
已开发出一种用于定量检测大鼠血浆中硫代磷酸寡核苷酸（PS-OGN）PF-ODN 及其代谢物 5'N-1/3'N-1、5'N-2 和 5'N-3 的离子对反相液相色谱-串联质谱（LC-MS/MS）方法。血浆样品采用初始苯酚/二氯甲烷液液萃取，然后固相萃取进行预处理。采用 Phenomenex Gemini C(18)柱，以六氟-2-丙醇/三乙胺缓冲液和甲醇为流动相，在 0.5mL/min 的流速下进行梯度洗脱，实现色谱分离。除 5'N-1 和 3'N-1 因质谱仪不能区分而共洗脱外，其他分析物均实现了色谱和质谱的分离检测。采用负电喷雾电离（ESI）接口，以多反应监测（MRM）模式进行检测。PF-ODN 及其四种代谢物的定量下限（LLOQ）均达到 4.0ng/mL，具有可接受的精密度和准确度。三个验证运行的 QC 水平的日内和日间 RSD 均小于 12.0%，分析物的准确度范围为-9.6%至 6.0%。该经验证的 LC-MS/MS 方法已应用于大鼠 PF-ODN 的初步药代动力学研究。
