Development of an UHPLC-UV-Method for Quantification of Direct Oral Anticoagulants: Apixaban, Rivaroxaban, Dabigatran, and its Prodrug Dabigatran Etexilate in Human Serum

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Direct oral anticoagulants currently have no indication for monitoring even though there are data that imply that individual dosing can improve and add safety to the therapy.


An ultra-high performance liquid chromatography method with ultra violet detection has been developed and validated for apixaban, dabigatran, dabigatran etexilate, and rivaroxaban. Protein precipitation with methanol (1:3 vol/vol) was used as sample preparation. Analyses were performed on an Agilent 1290 ultra-high performance liquid chromatography system with an Agilent Poroshell 120 EC-C18-RP column using eluents [A] H2O and [B] methanol with 0.1% formic acid added to each. A gradient run was performed with a flow of 0.7 mL/min at 35°C. Apixaban was detected at 280 nm, dabigatran at 294 nm, dabigatran etexilate at 340 nm, and rivaroxaban at 249 nm.


Retention times were 1.83 minutes for dabigatran, 4.10 minutes for rivaroxaban, 4.30 minutes for apixaban, and 6.10 minutes for dabigatran etexilate within a total run time of 7 minutes. Linearity was given over a range from 20 to 300 ng/mL with r2 >0.999. The limit of detection ranged from 4 to 5 ng/mL and the limit of quantification from 15 to 19 ng/mL, respectively. Usability in daily routine was demonstrated in 27 samples from patients receiving rivaroxaban and 11 samples from patients receiving apixaban. In the absence of validated therapeutic ranges, we estimated “assumed therapeutically effective concentrations” from dose-related ranges for the respective licensed dosages.


The method offers a fast, reliable, and low-cost way to quantify direct oral anticoagulants in daily routine even in smaller laboratories without access to liquid chromatography-mass spectrometry.

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