Unit of Infectious Diseases, University of Turin, Department of Medical Sciences, Amedeo di Savoia Hospital, Turin, Italy.
J Pharm Biomed Anal. 2013 May 5;78-79:217-23. doi: 10.1016/j.jpba.2013.02.025. Epub 2013 Feb 27.
HCV infection affects over 170 milions people in the world. Up to now standard-of-care therapy consisted in ribavirin plus interferon-α 2a or 2b. Recently, two new Direct Acting Antivirals (DAA), inhibitors of NS3 HCV protease, have been developed and approved for the routine use on HCV genotype 1 infected patients: boceprevir and telaprevir. Protease inhibitors show complex pharmacokinetics (strong metabolism of both drugs by CYP3A and drug interactions), they require a TID dosage and, furthermore, they are present in plasma patients in two different isomeric forms. In this work, we developed and validated an UPLC-tandem mass spectrometry assay method capable of monitoring the telaprevir and boceprevir concentrations in plasma, discriminating each isomer of both drugs. Blank plasma used for Standards and QCs preparation, was obtained from blood of healthy donors. The calibration curves for each isomer of telaprevir and boceprevir were linear in a range from 46.87 ng/mL to 6000 ng/mL and from 23.43 to 3000 ng/mL, respectively (mean r(2)>0.998 for all analytes). QCs at three different concentration were prepared: High, Medium and Low. Each Standard, QC and patient sample was treated with a protein precipitation protocol with a solution containing acidified acetonitrile and Internal Standard (6,7-Dimethyl-2,3-di(2-pyridyl)quinoxaline). An aliquot of supernatant was diluted and then directly analyzed by reverse-phase UPLC-MS/MS. Accuracy (mean 98.47%), intra-day (mean <5.21%) and inter-day (mean <7.57%) precision for telaprevir and boceprevir quality controls fitted all FDA guidelines. All compounds resulted stable in our method conditions and the extraction procedure showed a recovery near to 100%. Finally, we tested this method by monitoring plasma concentrations in 9 HCV+ patients, for each triple combined therapy, with good results. The observed median ratio between S and R isomers for boceprevir and telaprevir were 1.22 (IQR 1.10-1.33) and 1.52 (IQR 1.21-1.67), respectively. The use of this simple assay method could be an important tool for management of HCV-1 DAAs treated patients.
丙型肝炎病毒（HCV）感染影响着全球超过 1.7 亿人。目前，标准治疗方案包括利巴韦林联合干扰素-α 2a 或 2b。最近，两种新的直接作用抗病毒药物（DAA），HCV 蛋白酶 NS3 的抑制剂，已被开发并批准用于治疗 HCV 基因型 1 感染患者：博赛泼维（boceprevir）和特拉泼维（telaprevir）。蛋白酶抑制剂表现出复杂的药代动力学（两种药物均通过 CYP3A 强烈代谢和药物相互作用），需要每日三次剂量，此外，它们在血浆中的两种不同异构体形式存在。在这项工作中，我们开发并验证了一种 UPLC-串联质谱检测方法，能够监测血浆中特拉泼维（telaprevir）和博赛泼维（boceprevir）的浓度，并区分两种药物的每个异构体。用于标准和质控品制备的空白血浆取自健康供体的血液。每个异构体的特拉泼维（telaprevir）和博赛泼维（boceprevir）的校准曲线均呈线性，范围分别为 46.87ng/mL 至 6000ng/mL 和 23.43ng/mL 至 3000ng/mL（所有分析物的平均 r（2）>0.998）。以高、中、低三个不同浓度制备质控品。每个标准品、质控品和患者样本均采用含酸化乙腈和内标（6,7-二甲基-2,3-二（2-吡啶基）喹喔啉）的蛋白沉淀方案处理。取上清液的一部分稀释，然后直接通过反相 UPLC-MS/MS 进行分析。特拉泼维（telaprevir）和博赛泼维（boceprevir）质控品的准确度（平均 98.47%）、日内（平均<5.21%）和日间（平均<7.57%）精密度均符合 FDA 指南。所有化合物在我们的方法条件下均稳定，提取过程的回收率接近 100%。最后，我们通过监测 9 例 HCV+患者的血浆浓度，对每种三联联合治疗方法进行了测试，结果良好。博赛泼维（boceprevir）和特拉泼维（telaprevir）的 S 和 R 异构体的观察中位数比值分别为 1.22（IQR 1.10-1.33）和 1.52（IQR 1.21-1.67）。这种简单检测方法的使用可能是管理 HCV-1 DAA 治疗患者的重要工具。
