Kushwaha Hari Narayan, Gautam Nagsen, Singh Shio Kumar
Pharmacokinetics and Metabolism Division, Central Drug Research Institute, CSIR, Lucknow, India.
Arzneimittelforschung. 2011;61(7):425-32. doi: 10.1055/s-0031-1296222.
A sensitive, selective and specific LC-MS/ MS assay for simultaneous quantification of compound 97/78 and its active in vivo metabolite 97/63, a novel 1,2,4-trioxane antimalarial, in human plasma has been developed and validated using alpha-arteether as internal standard (IS). Extraction from plasma involves a simple protein precipitation method. The analytes were chromatographed on a Columbus C18 column with guard by isocratic elution with acetonitrile:ammonium acetate buffer (10 mM, pH 4.0) (80:20 v/v) as mobile phase at a flow rate of 0.45 mL min(-1) and analyzed in multiple reaction-monitoring (MRM) positive ion mode. The chromatographic run time was 4.0 min. The weighted (1/x2) calibration curves were linear over a range of 1.56-200 ng mL(-1) with correlation coefficients > 0.998. For both analytes, the limit of detection (LOD) and lower limit of quantification (LLOQ) were 0.5 ng mL(-1) and 1.56 ng mL(-1), respectively. The recovery of 97/78, 97/63 and IS from spiked control samples were > 90% and their matrix suppression obtained were < 8 %. The accuracy (% bias) and precision (%RSD) for both analytes were < 6.78%. Both analytes were stable after three freeze-thaw cycles (% deviation < 12.80), long-term for 30 days in plasma at -60 degrees C (% deviation < 14.38), for 8 h on bench top in plasma at ambient temperature (% deviation < 1.52) and also in the auto-sampler for 12 h (% deviation < 3.9%). The validated method was successfully applied to a protein binding study of compound 97/78 and metabolite 97/63 in human plasma. Furthermore, the validated method will be applicable to pharmacokinetics, bioavailability and metabolism in various clinical phases and in drug interaction studies.
已开发并验证了一种灵敏、选择性强且特异的液相色谱-串联质谱（LC-MS/MS）分析法，用于同时定量测定人血浆中新型1,2,4-三氧杂环己烷抗疟药化合物97/78及其活性体内代谢物97/63，采用α-蒿乙醚作为内标（IS）。血浆提取采用简单的蛋白沉淀法。分析物在配有保护柱的哥伦布C18柱上进行色谱分离，以乙腈:醋酸铵缓冲液（10 mM，pH 4.0）（80:20 v/v）作为流动相进行等度洗脱，流速为0.45 mL min(-1)，并在多反应监测（MRM）正离子模式下进行分析。色谱运行时间为4.0分钟。加权（1/x2）校准曲线在1.56 - 200 ng mL(-1)范围内呈线性，相关系数>0.998。对于两种分析物，检测限（LOD）和定量下限（LLOQ）分别为0.5 ng mL(-1)和1.56 ng mL(-1)。加标对照样品中97/78、97/63和内标的回收率>90%，获得的基质抑制<8%。两种分析物的准确度（%偏差）和精密度（%RSD）<6.78%。两种分析物在三个冻融循环后稳定（%偏差<12.80），在-60℃血浆中可长期保存30天（%偏差<14.38），在室温下血浆中在台式上放置8小时稳定（%偏差<1.52），在自动进样器中放置12小时也稳定（%偏差<3.9%）。该验证方法已成功应用于化合物97/78和代谢物97/63在人血浆中的蛋白结合研究。此外，该验证方法将适用于各个临床阶段的药代动力学、生物利用度和代谢研究以及药物相互作用研究。
