A UHPLC-MS/MS method to simultaneously quantify apixaban, edoxaban and rivaroxaban in human plasma and breast milk: For emerging lactation studies

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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.

Original languageEnglish
Article number122095
JournalJournal of Chromatography B: Analytical Technologies in the Biomedical & Life Sciences
Publication statusPublished - 1 May 2020


  • Breastfeeding
  • Direct oral anticoagulants
  • Liquid chromatography
  • Tandem mass spectrometry
  • Venous thromboembolism

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