Wiesen Martin H J, Blaich Cornelia, Streichert Thomas, Michels Guido, Müller Carsten
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Clin Chem Lab Med. 2017 Aug 28;55(9):1349-1359. doi: 10.1515/cclm-2016-0888.
Assessment of the anticoagulant activity of direct oral anticoagulants (DOACs) is justified in special clinical situations. Here, we evaluated two independent extraction methods and developed a multi-analyte ultra-high performance liquid chromatography tandem mass (UHPLC-MS/MS) method for the quantification of apixaban, dabigatran, edoxaban and rivaroxaban in human plasma.
Routine extraction based on protein precipitation with acetonitrile and subsequent centrifugation was compared to sample clean-up using commercial paramagnetic micro-particles and subsequent magnetic depletion. Stable isotope-labeled analogs of all analytes were employed as internal standards. The method was validated according to international guidelines in terms of linearity, precision, trueness, sensitivity, recovery and matrix effects. The performances of both extraction methods were assessed in clinical samples obtained from patients treated with either apixaban or rivaroxaban. Additionally, we report on a patient with nonadherence to rivaroxaban treatment and fulminant pulmonary embolism.
The method was linear from 2 to 500 ng/mL for all analytes, and quantification of DOACs was established within a run time of 2.0 min. Based on MS/MS analyte responses, relative matrix effects were better controlled for dabigatran after extraction with paramagnetic micro-particles. Internal standards fully compensated for recovery and matrix effects in all assays, yielding equivalent results for both methods. Apixaban and rivaroxaban concentrations determined in clinical samples after extraction with both methods were in good agreement (R2=0.990).
A rapid and accurate multi-component UHPLC-MS/MS method for the quantification of four DOACs in human plasma was established. Paramagnetic micro-particles appear suitable for clean-up of plasma samples for LC-MS/MS-based therapeutic drug monitoring purposes.
在特殊临床情况下，对直接口服抗凝剂（DOACs）的抗凝活性进行评估是合理的。在此，我们评估了两种独立的提取方法，并开发了一种多分析物超高效液相色谱串联质谱（UHPLC-MS/MS）方法，用于定量人血浆中的阿哌沙班、达比加群、依度沙班和利伐沙班。
将基于乙腈沉淀蛋白质并随后离心的常规提取方法与使用商业顺磁性微粒进行样品净化并随后进行磁性去除的方法进行比较。所有分析物的稳定同位素标记类似物均用作内标。该方法根据国际指南在线性、精密度、准确性、灵敏度、回收率和基质效应方面进行了验证。在从接受阿哌沙班或利伐沙班治疗的患者获得的临床样本中评估了两种提取方法的性能。此外，我们报告了一名不依从利伐沙班治疗并发生暴发性肺栓塞的患者。
所有分析物在2至500 ng/mL范围内呈线性，并且在2.0分钟的运行时间内完成了DOACs的定量。基于MS/MS分析物响应，在用顺磁性微粒提取后，达比加群的相对基质效应得到了更好的控制。内标在所有测定中充分补偿了回收率和基质效应，两种方法产生了等效结果。用两种方法提取后在临床样本中测定的阿哌沙班和利伐沙班浓度具有良好的一致性（R2 = 0.990）。
建立了一种快速、准确的多组分UHPLC-MS/MS方法，用于定量人血浆中的四种DOACs。顺磁性微粒似乎适用于基于LC-MS/MS的治疗药物监测目的的血浆样品净化。
