Development and Validation of UPLC-MS/MS Method for Quantifying of Free and Total Dabigatran in Human Plasma: An Application for a Bioequivalence Study.

Dabigatran etexilate mesylate (DABE), a prodrug, quickly changes in our bodies after its oral administration into dabigatran (DAB). Accordingly, detecting DABE in plasma is practically unmanageable. A UPLC-MS/MS technique was developed and validated to compute free DAB in participants. For the first time, the central composite design- a type of response surface methodology- was utilized for optimizing variables affecting the cleavage of glucuronide bond. Additionally, the pharmacokinetic parameters of generic medication (okanadab) were determined, and the obtained outcomes were compared to those of branded drug (pradaxa®). The sample preparation was done using methanol as a protein precipitant and the separation was achieved via ACQUITY UPLC BEH C18 column (2.1x50mm, 1.7μm). The elution was isocratically conducted using 10mM ammonium formate: methanol (72:28, v/v) as a mobile phase (MP) and the flow rate was 0.25mL/min. Multiple reaction monitoring (MRM) and positive electrospray ionization (ESI) were used. The determination was performed within 1min, and the calibration growth curve was established over a range of (1.19 - 475) ng/mL using dabigatran-d3 as a tagged internal standard (IS). Bioequivalence research was validated following FDA guidelines for bio-analytical procedures and acceptable outcomes were achieved. The outcomes for okanadab and pradaxa® did not differ significantly.