Importance of measuring pharmacologically active metabolites of edoxaban: development and validation of an ultra-high-performance liquid chromatography coupled with a tandem mass spectrometry method


Although DOACs do not require regular measurements of their blood concentrations, clinical situations may require an assessment of their concentration. Among the factor Xa inhibitors, edoxaban is the only compound for which some metabolites (e.g. edoxaban-M4) are reported to be pharmacologically active. Therefore, their contribution could interfere with assays used for the estimation of edoxaban concentration. In addition, drug interactions may alter the metabolite/parent compound ratio making the sole estimation of edoxaban concentration, a poor assessment of the overall anticoagulation. To develop a validated UHPLC–MS/MS method to quantify simultaneously edoxaban and its more relevant M4-metabolite in human plasma. Electrospray ionization and chromatographic separation were optimized for the simultaneous dosage of edoxaban and edoxaban-M4. The method was validated according to regulatory guidelines for bioanalytical method validation. The total run time was 6 min. The method was validated for calibration curves, precision, accuracy, carry-over, selectivity, matrix effect and short-time stability. This method permits quantification of edoxaban and edoxaban-M4 providing complementary information about the inhibitory effect of this active metabolite in chronometric or chromogenic assays. Although patients treated with edoxaban exhibits usually low concentrations of active metabolites, the measurement of edoxaban-M4 is interesting; especially in case of drug interactions. Indeed, concomitant prescriptions of edoxaban and carbamazepine or rifampicin is frequent and may lead to disturbance of the estimations of edoxaban concentration by chromogenic anti-Xa assays. Therefore, patients are at risk of having inadequate control of anticoagulation supporting the need of measuring the most representative edoxaban metabolite concomitantly to the parent compound.

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Fig. 1
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Fig. 3



Percentage of relative standard deviation


Coefficient of variation


Dimethyl sulfoxide


Direct oral anticoagulants


European Medicines Agency


Electro spray ionization

ESI + :

Electro spray on positive ionization mode


Food and Drug Administration


International Council of Harmonization


Internal standard


Liquid chromatography


Lower limit of quantification


Matrix effect


Multiple reaction monitoring


Mass spectrometry


Normal pooled plasma


Phosphate buffer saline


Platelet poor plasma


Quality controls


Ultra-high-performance liquid chromatography coupled with mass spectrometry


Upper limit of quantification


Vitamin K antagonists


Venous thromboembolisms


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The authors would like to acknowledge Christelle Vancraeynest, Vincent Maloteau, Lionel Pochet and Jonathan Evrard for their contribution to this work. The authors would also like to thank the NAB-X biobank from the University of Namur (UNamur) for the management and storage of plasma samples.

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Correspondence to Romain Siriez.

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The other authors have no conflicts of interest to disclose.

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Among the authors, J. Douxfils is CEO and founder of QUALIblood s.a. and reports personal fees from Portola Pharmaceuticals, Stago, Roche, Roche Diagnostics and Daiichi-Sankyo, outside the submitted work.

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Siriez, R., Alpan, L., Elasaad, K. et al. Importance of measuring pharmacologically active metabolites of edoxaban: development and validation of an ultra-high-performance liquid chromatography coupled with a tandem mass spectrometry method. J Thromb Thrombolysis (2020).

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  • Edoxaban
  • Edoxaban-M4
  • Ultra-high-performance liquid chromatography/tandem mass spectrometry
  • Metabolite
  • Direct oral anticoagulant