Clinical studies are needed to clarify the use of direct oral anticoagulants (DOACs) in breastfeeding women. To support emerging clinical studies on investigating DOAC's transfer into breast milk, an ultra-high-performance liquid chromatography/tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for quantifying three DOACs - apixaban, edoxaban and rivaroxaban in human plasma and breast milk. Protein precipitation with methanol was performed for sample preparation. Chromatographic analysis was performed using a C18 column. The MS detection was performed in MRM mode. The method was validated in accordance with the European Guideline (EMA). The calibration range was 5-500 ng/mL in plasma and 5-250 ng/mL in breast milk. The within-batch and between-batch variability remained <9%. Recoveries ranged from 106.13% to 109.05% in plasma and from 93.40% to 107.91% in breast milk. The lot-to-lot matrix variability was within ±15% among a range of samples originating from many different subjects. All analytes were stable when stored for 24 h at room temperature, 7 days at 2-8 °C, and at least 5 weeks at -20 °C in both plasma and breast milk. The developed method fulfilled the EMA bioanalytical method validation guideline and was shown to be simple, fast, accurate and will now be used in a clinical trial evaluating the transfer of apixaban and rivaroxaban into human breast milk.

中文翻译：

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UHPLC-MS / MS方法可同时定量人血浆和母乳中的apixaban，edoxaban和rivaroxaban：用于新兴泌乳研究。

需要进行临床研究以阐明在母乳喂养妇女中使用直接口服抗凝剂（DOAC）。为了支持有关研究DOAC转移至母乳中的新兴临床研究，开发了一种超高效液相色谱/串联质谱（UHPLC-MS / MS）方法，并验证了该方法用于定量人血浆中的3种DOAC（阿哌沙班，依多沙班和利伐沙班）和母乳。用甲醇进行蛋白质沉淀以制备样品。色谱分析使用C18色谱柱进行。MS检测在MRM模式下执行。该方法已根据欧洲指南（EMA）进行了验证。血浆的校准范围是5-500 ng / mL，母乳的校准范围是5-250 ng / mL。批内和批间差异保持<9％。回收率从106.13％到109不等。血浆中05％，母乳中93.40％至107.91％。在来自许多不同受试者的一系列样品中，批次之间的矩阵变异性在±15％以内。在血浆和母乳中，所有分析物在室温下保存24小时，在2-8°C下保存7天以及在-20°C下保存至少5周时都是稳定的。所开发的方法符合EMA生物分析方法验证指南，并且被证明简单，快速，准确，现在将用于评估apixaban和rivaroxaban向人母乳中转移的临床试验。并且在-20°C的血浆和母乳中至少放置5周。所开发的方法符合EMA生物分析方法验证指南，并且被证明简单，快速，准确，现在将用于评估阿哌沙班和利伐沙班向母乳中转移的临床试验。并且在-20°C的血浆和母乳中至少放置5周。所开发的方法符合EMA生物分析方法验证指南，并且被证明简单，快速，准确，现在将用于评估apixaban和rivaroxaban向人母乳中转移的临床试验。