LC-MS/MS Bioanalytical Approach for the Quantitative Analysis of Dabigatran in Biological Fluids

Abstract

A novel bioanalytical methodology has been developed to establish a rapid and sensitive procedure for the detection of dabigatran in biological fluid, specifically human plasma. In this study, dabigatran 13C6 was utilized as an internal standard (IS). The solid phase extraction method was employed to extract dabigatran and internal standard (IS) from a plasma sample. The separated components are determined by utilizing a eluting mixture composed of 2 mM ammonium formate:methanol:acetonitrile (20:40:40 v/v/v). The procedure utilized a GL Sciences Ace C18 stationary phase with dimensions of 150 mm × 4.6 mm; 5 µm. The identification of dabigatran and IS was carried out using the MRM mode, which was chosen to minimize interference from the surrounding matrix. The measured m/z of the protonated precursor ion of dabigatran was 472.20, while the corresponding product ion has a m/z value of 289.10. The precursor ion with a m/z of 478.20 and the product ion with a m/z of 295.20 were generated by the internal standard (IS). Linearity was observed within the range of 1.016-304.025 ng/mL; r2 of 0.9956 and a sample size (n) of 5. The stability of the drug in plasma was evaluated through a series of tests. These tests included a 17 h period on a laboratory bench, 24 h in an injector, six cycles of freeze-thaw and a storage period of 7 days and 22 h at -20 ± 5 ºC. The results of these stability studies indicated that the drug remained stable in plasma. The method was subjected to validation and was determined to be qualified. The proposed analytical approach demonstrates potential for the routine analysis and pharmacokinetic study of dabigatran in biological fluids.