Harahap Yahdiana, Mulyadi Callista Andinie, Muliawan Hary Sakti, Wahab Habibah A
Faculty of Pharmacy, Universitas Indonesia, Depok, 16424, Indonesia.
Faculty of Military Pharmacy, the Republic of Indonesia Defense University, Bogor, 16810, Indonesia.
Heliyon. 2024 Jul 11;10(14):e34500. doi: 10.1016/j.heliyon.2024.e34500. eCollection 2024 Jul 30.
This study aims to develop and validate bioanalytical method for quantifying warfarin in VAMS samples using liquid chromatography tandem mass spectrometry (LC-MS/MS), directly implementing the method to patients receiving warfarin therapy.
The UPLC-MS/MS method was developed and optimized, with quercetin as the internal standard. Sample preparation was carried out using protein precipitation with methanol-acetonitrile (1:3 v/v).
Chromatographic separation was achieved using Acquity® UPLC BEH C18 column with 0.1 % formic acid-acetonitrile-methanol (30:69:1 v/v) as mobile phase, in isocratic elution. Multiple Reaction Monitoring (MRM) detection was done using / values of 307.10 → 161.06 for warfarin and 301.03 → 150.98 for quercetin as internal standard, using Electrospray Ionization (ESI) negative ion source. The clinical application of the bioanalytical method was carried out on 25 patients receiving warfarin therapy at Universitas Indonesia Hospital and warfarin levels were well within the calibration range from 6.05 to 431.39 ng/mL.
A novel method has been developed to analyze warfarin in VAMS samples. This method has been fully validated according to guideline from FDA 2022 and is linear in the range of 5-500 ng/mL and the value of r ≥ 0.9977, and successfully applied for the analysis of warfarin in VAMS samples of clinical patients.
本研究旨在开发并验证一种使用液相色谱串联质谱法（LC-MS/MS）定量测定VAMS样本中华法林的生物分析方法，并将该方法直接应用于接受华法林治疗的患者。
以槲皮素为内标，开发并优化了超高效液相色谱-质谱/质谱法。采用甲醇-乙腈（1:3 v/v）蛋白沉淀法进行样品制备。
使用Acquity® UPLC BEH C18色谱柱，以0.1%甲酸-乙腈-甲醇（30:69:1 v/v）为流动相进行等度洗脱，实现了色谱分离。使用电喷雾电离（ESI）负离子源，以华法林的m/z值307.10 → 161.06和槲皮素作为内标的m/z值301.03 → 150.98进行多反应监测（MRM）检测。该生物分析方法在印度尼西亚大学医院对25例接受华法林治疗的患者进行了临床应用，华法林水平完全在校准范围内，为6.05至431.39 ng/mL。
已开发出一种分析VAMS样本中华法林的新方法。该方法已根据FDA 2022指南进行了全面验证，在5-500 ng/mL范围内呈线性，r值≥0.9977，并成功应用于临床患者VAMS样本中华法林的分析。
