Department of Pharmaceutical Analysis, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar 160062, Punjab, India.
J Pharm Biomed Anal. 2010 Jul 8;52(3):410-5. doi: 10.1016/j.jpba.2009.05.033. Epub 2009 Jun 6.
An ultra-high performance liquid chromatographic tandem mass spectroscopy (UHPLC-MS/MS) method was developed and validated for the quantification of an investigational anti-malarial entity, 2-tert-butylprimaquine (NP-96), in rat plasma. Simple protein precipitation by acetonitrile was used for the sample preparation. Effective separation of NP-96, internal standard (IS) and matrix components were achieved on an UHPLC column (Hypersil Gold C18, 50mmx2.1mm, 1.9microm) using a mobile phase composed of acetonitrile and 20mM ammonium acetate, which was pumped in a gradient mode at a flow rate of 450microl/min. Selective reaction monitoring (SRM) was utilized for quantitation of the molecules. To increase sensitivity of the method, two ions of m/z 299 and m/z 231 were selected for NP-96, while IS was monitored for an ion of m/z 489. The method was validated according to FDA guideline on bioanalytical method validation and showed good compliance. The intra-day and inter-day precision expressed as R.S.D. was lower than 15% at all the tested quality control levels, including upper and lower limits of quantification. The calibration range was 2.5-500ng/ml. Total runtime for the method was 5min, which was suitable to produce high-throughput results for pharmacokinetic evaluation.
建立并验证了一种超高效液相色谱-串联质谱法(UHPLC-MS/MS),用于定量检测研究用抗疟实体化合物 2-叔丁基伯氨喹(NP-96)在大鼠血浆中的浓度。采用乙腈沉淀蛋白法进行样品前处理。在 UHPLC 柱(Hypersil Gold C18,50mm×2.1mm,1.9μm)上,采用乙腈和 20mM 乙酸铵组成的流动相,以梯度模式在 450μL/min 的流速下进行洗脱,实现了 NP-96、内标(IS)和基质成分的有效分离。采用选择反应监测(SRM)定量检测分子。为了提高方法的灵敏度,选择 m/z 299 和 m/z 231 两个离子用于 NP-96,而 IS 则监测 m/z 489 的离子。该方法按照美国 FDA 生物分析方法验证指南进行了验证,符合要求。在所有测试的质控水平(包括定量下限和上限),日内和日间精密度(RSD)均低于 15%。校准范围为 2.5-500ng/ml。方法总运行时间为 5min,适用于产生高通量的药代动力学评价结果。