An improved extraction protocol for therapeutic dabigatran monitoring using HPLC-MS/MS.

A new sample extraction protocol was developed for pharmacokinetic studies of dabigatran with high-performance liquid chromatography separation - electrospray ionization time-of-flight mass spectrometry analysis. After protein precipitation with acetonitrile, free dabigatran and its metabolites are separated into water phase by water-dichloromethane liquid-liquid extraction to purify the sample from proteins and endogenous lipophilic compounds. Chromatographic separation was achieved on an Agilent Zorbax SB-CN column (150 × 4.6 mm, 5 µm)) using 0.1% aqueous solution of formic acid and acetonitrile (80:20) as the mobile phase. Agilent Zorbax SB-CN column was selected to improve sample resolution and to avoided early elution of dabigatran previously seen when using a C18 column. The extended calibration curve was constructed from 5 to 1000 ng/L while precision and accuracy were assessed at four levels across the linear dynamic ranges. Within-run precision was <5.6% and the between-run precision was <3.9%. The method accuracy ranged from 89.8% to 104.4%. The developed method was successfully applied to 30 patient samples to evaluate antithrombotic efficacy and anticoagulant activity of dabigatran following knee endoprosthesis surgery.

The adsorption of dabigatran is as efficient as addition of idarucizumab to neutralize the drug in routine coagulation assays.

INTRODUCTION: The direct thrombin inhibitor dabigatran interferes with thrombophilia screening and with the diagnosis of hemostasis disorders that develop during treatment with the anticoagulant. In vitro addition of idarucizumab, a humanized antibody fragment that binds dabigatran, to plasma samples containing dabigatran fully neutralizes the drug. This study was carried out to determine whether binding of dabigatran on selected insoluble commercial adsorbent material, DOAC-STOPR , was as efficient as idarucizumab to neutralize the anticoagulant activity of the drug in vitro. METHODS: Coagulation assays sensitive to dabigatran were carried out with patient and control plasma samples spiked with dabigatran and supplemented with idarucizumab or incubated with adsorbent material. RESULTS: In samples containing upto 10 000 ng/mL dabigatran, the adsorption procedure was at least as efficient as the addition of idarucizumab to neutralize the activity of the anticoagulant drug. Neither the adsorption procedure nor the addition of idarucizumab did impair routine coagulation assays carried out with plasma devoid of dabigatran, such as the activated partial thromboplastin time, prothrombin time, fibrinogen Clauss, and the thrombophilia screening assays used to detect antiphospholipid antibodies or activated protein C resistance. In addition, the adsorption procedure did not interfere with the detection of lupus anticoagulant samples. CONCLUSIONS: Adsorption of dabigatran in plasma samples containing the drug neutralizes its activity as efficiently as the addition of idarucizumab. This method allows the evaluation of thrombophilia markers without interruption of anticoagulation therapy or the detection of hemostasis disorders in patients treated with the drug.

The effect of ultrafiltration with cardiopulmonary bypass on the removal of dabigatran from the circulation of adult pigs.

OBJECTIVE: Dabigatran etexilate is a direct thrombin inhibitor approved for use in patients with non-valvular atrial fibrillation. There is no currently available pharmacological therapy to reverse this renally cleared anticoagulant. Dabigatran has a low level of plasma protein binding and has been considered dialyzable. We used a pig model with renal artery ligation to exclude intrinsic drug excretion to examine the efficacy of ultrafiltration (UF) during cardiopulmonary bypass (CPB) for dabigatran removal. METHOD: Dabigatran was intravenously infused (20 mg) in Yorkshire pigs (male, n=7, 70±1 kg) following renal artery ligation. CPB with UF was initiated after heparinization and continued until a total volume of 6 liters of UF effluent was removed. Serial labs, including dabigatran concentration, activated coagulation times (ACT), hematocrit and creatinine were drawn at intervals before the start of CPB and then incrementally during UF (0, 2, 4 and 6 L removed). Hemodialysis (HD) was performed on one animal following UF. RESULTS: Dabigatran concentration (ng/mL) rose from undetectable levels at baseline to 296±70 (p<0.05) at the conclusion of infusion, but dropped significantly upon administration of heparin (178±40, p<0.05). A further decrement in dabigatran concentration was observed from the administration of heparin to the start of CPB (to 135±28, p<0.05). Once on CPB, dabigatran remained stable, with the end UF (eUF) dabigatran concentration being 133±34. Dabigatran concentration in the UF effluent was measured in one animal and was 98.8, with 6 L of effluent having been removed. The total recovery of dabigatran was calculated to be less than 5%. Dabigatran concentrations also did not decrease appreciably with HD on CPB following UF. CONCLUSIONS: UF in conjunction with CPB was ineffective at removing dabigatran. Heparin demonstrated a dabigatran-lowering effect, suggesting a possible drug interaction or assay impairment. Based on these findings, emergent cardiac surgery with UF on cardiopulmonary bypass to remove dabigatran is not advisable. Alternative forms of drug removal or reversal must be identified.