Verweij-van Wissen Corrien P W G M, de Graaff-Teulen Marga J A, de Kanter Clara T M M, Aarnoutse Rob E, Burger David M
Department of Pharmacy, Radboud University Medical Center, Nijmegen, the Netherlands.
Ther Drug Monit. 2015 Oct;37(5):626-33. doi: 10.1097/FTD.0000000000000189.
Telaprevir is a protease inhibitor used in the treatment of hepatitis C virus infection. Analytical methods for telaprevir should separate the compound from its R-diastereomer VRT-127394, which is 30-fold less active. The objective of this work was to develop liquid chromatography-tandem mass spectrometer (LC-MS/MS) assays for telaprevir both in plasma and in dried blood spot (DBS), capable of stabilizing the equilibrium and chromatographically separating the 2 epimers.
Human plasma was acidified with formic acid and frozen within 1 hour after collection to stabilize the equilibrium between the 2 telaprevir diastereomers ex vivo in plasma. After protein precipitation, the sample was analyzed with LC-MS/MS. For the DBS assay, sampling paper was impregnated with citric acid solution to achieve stabilization of the epimers on the sampling paper. DBS samples were extracted before LC-MS/MS analysis. LC-MS/MS analysis comprised online solid-phase extraction and separation on a C18 column, with the mass spectrometer operating in TurboIonSpray-negative ionization mode and performing multiple reaction monitoring.
The assays were linear over the concentration range of 0.1-10 mg/L in plasma and DBS. Accuracies ranged from 97% to 106% in plasma and from 93% to 99% in DBS. Within- and between-day coefficients of variation were <7.9% in plasma and <9.3% in DBS. Human whole blood samples with hematocrit values of 27%-47% gave reproducible quantitation results in the DBS assay, and spot volume did not affect results of the DBS assay either. Acidified plasma with telaprevir was stable for 5 hours at 20°C, and telaprevir on impregnated DBS paper was stable for at least 3 months at 4°C or at 20°C.
An assay was developed and validated for the determination of telaprevir in human plasma, separating telaprevir from its R-diastereomer VRT-127394. In addition, a DBS assay was developed, which avoids immediate centrifuging, acidification, and freezing of patient samples to stabilize the equilibrium between the 2 telaprevir diastereomers.
特拉匹韦是一种用于治疗丙型肝炎病毒感染的蛋白酶抑制剂。特拉匹韦的分析方法应将该化合物与其活性低30倍的R-非对映异构体VRT-127394分离。本研究的目的是开发用于血浆和干血斑（DBS）中特拉匹韦的液相色谱-串联质谱（LC-MS/MS）测定法，该方法能够稳定平衡并通过色谱法分离这两种差向异构体。
用甲酸将人血浆酸化，并在采集后1小时内冷冻，以在体外血浆中稳定两种特拉匹韦非对映异构体之间的平衡。蛋白质沉淀后，用LC-MS/MS分析样品。对于DBS测定，将采样纸用柠檬酸溶液浸渍，以实现差向异构体在采样纸上的稳定。DBS样品在LC-MS/MS分析前进行提取。LC-MS/MS分析包括在线固相萃取和在C18柱上的分离，质谱仪在TurboIonSpray负电离模式下运行并进行多反应监测。
该测定法在血浆和DBS中的浓度范围为0.1 - 10 mg/L时呈线性。血浆中的准确度范围为97%至106%，DBS中的准确度范围为93%至99%。血浆中的日内和日间变异系数<7.9%，DBS中的<9.3%。血细胞比容值为27% - 47%的人全血样品在DBS测定中给出了可重复的定量结果，且点样体积也不影响DBS测定结果。含有特拉匹韦的酸化血浆在20°C下稳定5小时，浸渍在DBS纸上的特拉匹韦在4°C或20°C下稳定至少3个月。
开发并验证了一种用于测定人血浆中特拉匹韦的方法，可将特拉匹韦与其R-非对映异构体VRT-127394分离。此外，还开发了一种DBS测定法，该方法避免了对患者样品立即进行离心、酸化和冷冻，以稳定两种特拉匹韦非对映异构体之间的平衡。
