Development and validation of a ultraperformance liquid chromatography–tandem mass spectrometry method for quantifying free and total dabigatran in human plasma: An application for a bioequivalence study

Abstract

Dabigatran etexilate mesylate (DABE), a prodrug, quickly changes into dabigatran (DAB) after its oral administration. Accordingly, detecting DABE in plasma is practically unmanageable. An ultraperformance liquid chromatography–tandem mass spectrometry (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 applied for optimizing variables affecting the cleavage of glucuronide bond. In addition, the pharmacokinetic parameters of generic medication (okanadab) were determined, and the obtained outcomes were compared with those of the branded drug (pradaxa). The sample preparation was done using methanol as a protein precipitant and the separation was achieved using an ACQUITY UPLC BEH C18 column (2.1 × 50 mm, 1.7 μm). The elution was isocratically conducted using 10 mM ammonium formate:methanol (72:28, v/v) as a mobile phase and the flow rate was 0.25 mL/min. Multiple reaction monitoring and positive electrospray ionization were used. The determination was performed within 1 min, and the calibration growth curve was established over a range of 1.19–475 ng/mL using DAB‐d3 as a tagged internal standard. Bioequivalence research was validated following the US Food and Drug Administration (US FDA) guidelines for bioanalytical procedures and acceptable outcomes were achieved. The outcomes for okanadab and pradaxa did not differ significantly.

What is the role of current mass spectrometry in pharmaceutical analysis?

“…The highly sensitive detection has been recently demonstrated by UHPLC/HPLC‐QqQ methods used for the bioequivalence study of dabigatran, which is the active metabolite of the prodrug dabigatran etexilate mesylate (Abd Allah et al, 2022), triamterene and hydrochlorothiazide (Margaryan et al, 2019), sumatriptan (Wichitnithad et al, 2020), and beraprost sodium (Prasaja et al, 2019). MS is also needed when the API does not contain chromophores for UV‐VIS detection, for example, bioequivalence study of isosorbide‐5‐mononitrate (Zhou et al, 2020) or the total amount of the drug in plasma and the unbound fractions are required, for example, bioequivalence study of paclitaxel protein‐bound particles for injectable suspension (Li et al, 2019).…”

“…Bioequivalence studies must be demonstrated for the parent drug, and the determination of active metabolite is not recommended. The exceptions are prodrugs with very fast metabolization, for example, dabigatran etexilate mesylate (Abd Allah et al, 2022).…”