Blaich Cornelia, Müller Carsten, Michels Guido, Wiesen Martin H J
Clin Chem Lab Med. 2015 Nov;53(12):1981-90. doi: 10.1515/cclm-2014-1108.
The non-vitamin K antagonist oral anticoagulants (NOACs) apixaban, dabigatran, and rivaroxaban are being administered in fixed doses without routine monitoring of anticoagulant activities. Despite this key advantage over vitamin K antagonists (VKAs), assessment of anticoagulant intensities is required in various clinical circumstances. We developed a multi-analyte approach for mass spectrometric analysis of NOACs in human plasma.
Plasma samples were precipitated with acetonitrile. Separation was achieved by liquid chromatography using a C18 column and a gradient elution within a run time of 2.5 min. Positive electrospray ionization was used and ion transitions monitored by a triple quadrupole mass spectrometer. Stable-isotope-labeled analogues of analytes were employed as internal standards for quantitative analysis. Certified external quality control samples were obtained for external validation.
For all analytes, linearity could be demonstrated over the concentration range of 1-500 μg/L (R2>0.999), and the calculated limits of quantification were <1 μg/L. Results for inter- and intra-day assay precision and trueness were obtained using internal quality control samples and remained within the acceptance criterion of ±15%. External quality control samples were measured at the specified nominal values with inter- and intra-day precisions <14%. Matrix effects were fully compensated by co-eluting internal standards, which in turn did not relevantly influence ionization efficiency.
The method enables rapid and reliable simultaneous determination of NOAC concentrations in human plasma. It was successfully introduced into clinical practice; a case with rivaroxaban overdose is presented to exemplify the method's applicability.
非维生素K拮抗剂口服抗凝药（NOACs）阿哌沙班、达比加群和利伐沙班采用固定剂量给药，无需常规监测抗凝活性。尽管相对于维生素K拮抗剂（VKAs）具有这一关键优势，但在各种临床情况下仍需要评估抗凝强度。我们开发了一种多分析物方法，用于质谱分析人血浆中的NOACs。
血浆样本用乙腈沉淀。使用C18柱通过液相色谱进行分离，并在2.5分钟的运行时间内进行梯度洗脱。采用正电喷雾电离，并用三重四极杆质谱仪监测离子跃迁。分析物的稳定同位素标记类似物用作定量分析的内标。获得经认证的外部质量控制样本用于外部验证。
对于所有分析物，在1 - 500μg/L的浓度范围内均可证明线性关系（R2>0.999），计算出的定量限<1μg/L。使用内部质量控制样本获得了日内和日间分析精密度及准确性的结果，且均保持在±15%的可接受标准范围内。外部质量控制样本在指定的标称值下进行测量，日内和日间精密度<14%。共洗脱的内标完全补偿了基质效应，而内标又不会对电离效率产生显著影响。
该方法能够快速、可靠地同时测定人血浆中NOAC的浓度。它已成功引入临床实践；通过一个利伐沙班过量的病例举例说明该方法的适用性。
