Development and validation of an analytical method for the determination of direct oral anticoagulants (DOAC) and the direct thrombin-inhibitor argatroban by HPLC–MS/MS

Abstract

Since direct oral anticoagulants (DOAC) are administered frequently to an elderly, co-morbid population, medical emergencies including trauma, acute bleeding or organ failure are not uncommon. In these situations, the type, dosage or the time of last intake of anticoagulants is often unknown and single substance analysis by functional tests is only possible if the substance contained in the sample is known. A reliable and validated toxicology screen of DOAC and argatroban would be helpful inform not only attending physicians in the emergency department but also law enforcement and courts of justice. After precipitation with acetone, HPLC separation was achieved on a Phenomenex Luna Pentafluorophenyl Colum using acetonitrile–water (90:10, v/v) as mobile phase system. Detection was performed using a 3200 Q Trap mass spectrometer (AB Sciex). For analysis MRM Scans (MS/MS) with positive ionization were chosen. The method was validated for blank serum as the matrix of choice. Limits of detection are between 0.5 and 1.0 ng/mL, limits of quantification are between 1.9 and 3.6 ng/mL and recoveries are above 60%. The applicability of the method was demonstrated by the determination of DOAC in body fluids from forensic cases and in therapeutic drug monitoring. The rapid simultaneous detection and quantification of apixaban, argatroban, dabigatran etexilate, dabigatran, edoxaban and rivaroxaban in body fluids by HPLC–MS/MS closes an important gap in emergency toxicology.

Point-of-Care Assessment of Direct Oral Anticoagulation in Acute Ischemic Stroke: Protocol for a Prospective Observational Diagnostic Accuracy Study

“…The HPLC-MS/MS method for the simultaneous determination of DOACs that will be used this study has recently been developed and validated by members of our group. 52 …”

“…12 High performance liquid chromatography-tandem mass-spectrometry 500 µl of every blood sample taken in the acute setting will be frozen and multiple samples will be analyzed in by HPLC-MS/MS using a 1260 Infinity Quaternary LC System (Agilent Technologies, Santa Clara, California, USA), equipped with a Phenomenex Luna Pentafluorophenyl Column (length: 150 mm, internal diameter: 2 mm, particle size: 5 µm), and a 3200 Q Trap (AB Sciex Germany GmbH, Darmstadt, Germany) mass spectrometer. 52 Calibration reagents encompass apixaban, edoxaban and rivaroxaban supplied by Cayman Chemical Company (Ann Arbor, USA), dabigatran etexilate supplied by Sigma Aldrich (St. Louis, USA) and dabigatran supplied by Biosynth/Carbosynth (Thal, Switzerland). Apixaban-13C-d3 (Cayman Chemical Company, Ann Arbor, USA) is used as internal standard.…”

Direct oral anticoagulants (DOACs): From the laboratory point of view

“…This methodology has a high degree of specificity and sensitivity, with a limit of detection (LOD) and a limit of quantitation (LOQ) between 0.025 and 3 ng mL -1 , depending on the drug and technology (4). Bruckner et al (72) developed and validated an analytical method for the determination of DOAC by HPLC-MS/MS. It simultaneously determines three DOACs with limits of quantification of 0.57 ng mL -1 for dabigatran, 0.79 ng mL -1 for rivaroxaban, and 0.22 ng mL -1 for apixaban.…”

Pearls and Pitfalls in the Measurement of Direct Oral Anticoagulants

“…Accurate quantification of the plasma concentration of DOAC is currently possible using a range of separation techniques, most of which are LC methods, as recently reported by Gouveia et al 26 Some of these assays have also been validated for the simultaneous screening and quantification of several DOACs. 27 Although analytical techniques based on LC, usually coupled with MS based on the mass-to-charge ratio for direct quantification of the specific analyte (i.e., specific DOAC) and thus allowing higher selectivity and sensitivity, are still considered the benchmark for accurate quantification of plasma concentrations of DOACs, some important considerations must be made. First and foremost is the fact that DOACs are active drugs that have a strong inhibitory effect on either FIIa or FXa.…”