采用超高效液相色谱-串联质谱法（UPLC-MS/MS）和凝血检测法测定新型直接口服抗凝剂的达比加群、利伐沙班和阿哌沙班，用于治疗监测。

Determination of dabigatran, rivaroxaban and apixaban by ultra-performance liquid chromatography - tandem mass spectrometry (UPLC-MS/MS) and coagulation assays for therapy monitoring of novel direct oral anticoagulants.

机构信息

Clinical Laboratory, Catharina Hospital, Eindhoven, The Netherlands; Laboratory of Chemical Biology and Institute of Complex Molecular Systems, Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands; Expert Center Clinical Chemistry Eindhoven, Eindhoven, The Netherlands.

出版信息

J Thromb Haemost. 2014 Oct;12(10):1636-46. doi: 10.1111/jth.12702.

DOI:10.1111/jth.12702

PMID:25142183

Abstract

BACKGROUND

Three novel direct oral anticoagulants (DOACs) have recently been registered by the Food and Drug Administration and European Medicines Agency Commission: dabigatran, rivaroxaban, and apixaban. To quantify DOACs in plasma, various dedicated coagulation assays have been developed.

OBJECTIVE

To develop and validate a reference ultra-performance liquid chromatography - tandem mass spectrometry (UPLC-MS/MS) method and to evaluate the analytical performance of several coagulation assays for quantification of dabigatran, rivaroxaban, and apixaban.

METHODS

The developed UPLC-MS/MS method was validated by determination of precision, accuracy, specificity, matrix effects, lower limits of detection, carry-over, recovery, stability, and robustness. The following coagulation assays were evaluated for accuracy and precision: laboratory-developed (LD) diluted thrombin time (dTT), Hemoclot dTT, Pefakit PiCT, ECA, Liquid anti-Xa, Biophen Heparin (LRT), and Biophen DiXal anti-Xa. Agreement between the various coagulation assays and UPLC-MS/MS was determined with random samples from patients using dabigatran or rivaroxaban.

RESULTS

The UPLC-MS/MS method was shown to be accurate, precise, sensitive, stable, and robust. The dabigatran coagulation assay showing the best precision, accuracy and agreement with the UPLC-MS/MS method was the LD dTT test. For rivaroxaban, the anti-factor Xa assays were superior to the PiCT-Xa assay with regard to precision, accuracy, and agreement with the reference method. For apixaban, the Liquid anti-Xa assay was superior to the PiCT-Xa assay.

CONCLUSIONS

Statistically significant differences were observed between the various coagulation assays as compared with the UPLC-MS/MS reference method. It is currently unknown whether these differences are clinically relevant. When DOACs are quantified with coagulation assays, comparison with a reference method as part of proficiency testing is therefore pivotal.

摘要

背景

三种新型直接口服抗凝剂（DOAC）最近已被美国食品和药物管理局及欧洲药品管理局批准上市：达比加群、利伐沙班和阿哌沙班。为了定量检测血浆中的 DOAC，已经开发了各种专用的凝血检测方法。

目的

开发和验证一种参考超高效液相色谱-串联质谱（UPLC-MS/MS）方法，并评估几种凝血检测方法用于定量检测达比加群、利伐沙班和阿哌沙班的分析性能。

方法

通过测定精密度、准确度、特异性、基质效应、检测限、交叉污染、回收率、稳定性和耐用性来验证所开发的 UPLC-MS/MS 方法。对实验室自建（LD）稀释凝血酶时间（dTT）、Hemoclot dTT、Pefakit PiCT、ECA、液体抗-Xa、LRT 和 DiXal 抗-Xa 等凝血检测方法的准确性和精密度进行了评估。使用达比加群或利伐沙班的患者随机样本，确定各种凝血检测方法与 UPLC-MS/MS 之间的一致性。

结果

UPLC-MS/MS 方法被证明具有准确性、精密度、灵敏度、稳定性和耐用性。在与 UPLC-MS/MS 方法的准确性和一致性方面，达比加群凝血检测中，LD dTT 检测具有最佳的精密度、准确度。对于利伐沙班，抗 Xa 因子检测在精密度、准确度和与参考方法的一致性方面优于 PiCT-Xa 检测。对于阿哌沙班，液体抗-Xa 检测在精密度和与 PiCT-Xa 检测的一致性方面优于后者。