Innovation & Development Laboratory, Service of Biomedicine, Centre Hospitalier Universitaire Vaudois, Switzerland.
J Mass Spectrom. 2013 May;48(5):616-25. doi: 10.1002/jms.3200.
Because of the large variability in the pharmacokinetics of anti-HIV drugs, therapeutic drug monitoring in patients may contribute to optimize the overall efficacy and safety of antiretroviral therapy. An LC-MS/MS method for the simultaneous assay in plasma of the novel antiretroviral agents rilpivirine (RPV) and elvitegravir (EVG) has been developed to that endeavor. Plasma samples (100 μL) extraction is performed by protein precipitation with acetonitrile, and the supernatant is subsequently diluted 1:1 with 20-mM ammonium acetate/MeOH 50:50. After reverse-phase chromatography, quantification of RPV and EVG, using matrix-matched calibration samples, is performed by electrospray ionization-triple quadrupole mass spectrometry by selected reaction monitoring detection using the positive mode. The stable isotopic-labeled compounds RPV-(13) C6 and EVG-D6 were used as internal standards. The method was validated according to FDA recommendations, including assessment of extraction yield, matrix effects variability (<6.4%), as well as EVG and RPV short and long-term stability in plasma. Calibration curves were validated over the clinically relevant concentrations ranging from 5 to 2500 ng/ml for RPV and from 50 to 5000 ng/ml for EVG. The method is precise (inter-day CV%: 3-6.3%) and accurate (3.8-7.2%). Plasma samples were found to be stable (<15%) in all considered conditions (RT/48 h, +4°C/48 h, -20°C/3 months and 60°C/1 h). Selected metabolite profiles analysis in patients' samples revealed the presence of EVG glucuronide, that was well separated from parent EVG, allowing to exclude potential interferences through the in-source dissociation of glucuronide to parent drug. This new, rapid and robust LCMS/MS assay for the simultaneous quantification of plasma concentrations of these two major new anti-HIV drugs EVG and RPV offers an efficient analytical tool for clinical pharmacokinetics studies and routine therapeutic drug monitoring service.
由于抗 HIV 药物的药代动力学存在很大的可变性，因此对患者进行治疗药物监测可能有助于优化抗逆转录病毒治疗的整体疗效和安全性。为了实现这一目标，我们开发了一种同时测定新型抗 HIV 药物利匹韦林（RPV）和艾维雷格（EVG）在血浆中浓度的 LC-MS/MS 方法。通过乙腈沉淀蛋白提取血浆样品（100μL），然后用 20-mM 乙酸铵/甲醇 50:50 稀释上清液 1:1。反相色谱后，采用电喷雾电离-三重四极杆质谱，通过正离子模式选择反应监测检测，使用基质匹配校准样品对 RPV 和 EVG 进行定量。稳定同位素标记化合物 RPV-（13）C6 和 EVG-D6 用作内标。该方法按照 FDA 的建议进行了验证，包括评估提取效率、基质效应变异性（<6.4%），以及 EVG 和 RPV 在血浆中的短期和长期稳定性。RPV 的校准曲线在 5 至 2500ng/ml 的临床相关浓度范围内，EVG 的校准曲线在 50 至 5000ng/ml 的范围内进行了验证。该方法具有良好的精密度（日内 CV%：3-6.3%）和准确度（3.8-7.2%）。在所有考虑的条件下（RT/48h、+4°C/48h、-20°C/3 个月和 60°C/1h），血浆样品均稳定（<15%）。对患者样本中代谢物特征的分析表明，存在 EVG 葡萄糖醛酸苷，其与母体 EVG 很好地分离，通过葡萄糖醛酸苷在源内裂解为母体药物，可排除潜在的干扰。这种新的、快速而强大的 LCMS/MS 测定法可同时定量测定这两种新型主要抗 HIV 药物 EVG 和 RPV 在血浆中的浓度，为临床药代动力学研究和常规治疗药物监测服务提供了一种有效的分析工具。
