Chen Xinhui, Bushman Lane R, McAllister Kevin J, Anderson Peter L, Kiser Jennifer J
University of Colorado Denver, School of Pharmacy, Department of Pharmaceutical Sciences, Aurora, Colorado, USA.
Biomed Chromatogr. 2014 Dec;28(12):1714-21. doi: 10.1002/bmc.3211. Epub 2014 Apr 24.
The purpose of this study was to validate a reversed-phase high-performance liquid chromatographic (HPLC), tandem mass spectrometry (MS/MS) assay for the determination of telaprevir and its R-diastereomer (VRT-127394) in acidified and nonacidified human plasma. The chromatographic baseline separation of telaprevir and telaprevir-R was performed on a Waters XBridge(TM) BEH Shield C18 , 2.1 × 75 mm column with a 2.5 µm particle size, under isocratic conditions consisting of a mobile phase of 50:45:5 water-acetonitrile-isopropanol with 1% ammonia at 0.2 mL/min. This method utilized a stable isotope internal standard with 11 deuterium atoms on the structure of the telaprevir molecule (telaprevir-d11). An internal standard for the telaprevir-R (telaprevir-R-d11) was also prepared by incubating telaprevir-d11 in basic solution, which facilitated isomer inter-conversion. The detection and quantitation of telaprevir, telaprevir-R, telaprevir-IS and telaprevir-R-IS was achieved by positive ion electrospray (ESI+) MS/MS detection. The assay quantifiable limit was 5.0 ng/mL when 0.100 mL of acidified human plasma was extracted. Accuracy and precision were validated over the calibration range of 5.0-5000 ng/mL. It was demonstrated using patient samples that, contrary to previous recommendations, quantitation of telaprevir does not require acidified plasma.
本研究的目的是验证一种反相高效液相色谱（HPLC）-串联质谱（MS/MS）分析法，用于测定酸化和未酸化人血浆中的特拉匹韦及其R-非对映体（VRT-127394）。在Waters XBridge™ BEH Shield C18 2.1×75 mm、粒径2.5 µm的色谱柱上，在由50:45:5水-乙腈-异丙醇与1%氨水组成的流动相、流速0.2 mL/min的等度条件下，实现了特拉匹韦和特拉匹韦-R的色谱基线分离。该方法使用了在特拉匹韦分子结构上带有11个氘原子的稳定同位素内标（特拉匹韦-d11）。还通过将特拉匹韦-d11在碱性溶液中孵育制备了特拉匹韦-R的内标（特拉匹韦-R-d11），这有助于异构体的相互转化。通过正离子电喷雾（ESI+）MS/MS检测实现对特拉匹韦、特拉匹韦-R、特拉匹韦内标和特拉匹韦-R内标的检测和定量。当提取0.100 mL酸化人血浆时，该分析方法的定量下限为5.0 ng/mL。在5.0 - 5000 ng/mL的校准范围内验证了准确性和精密度。使用患者样本证明，与先前的建议相反，特拉匹韦的定量不需要酸化血浆。
