LC‐MS/MS Assay for Quantification of DA‐1241, a Novel GPR119 Agonist, in Animal and Human Plasma: Application to Preclinical Pharmacokinetic Studies

DA-1241 is a novel GPR119 receptor agonist under development as a therapeutic candidate for metabolic dysfunction-associated steatohepatitis. To enable precise quantification of DA-1241 in mouse, rat, dog, and human plasma, a simple and highly sensitive LC-MS/MS approach was designed and thoroughly evaluated. Plasma samples were processed using protein precipitation with acetonitrile, followed by chromatographic separation on a reverse-phase C₁₈ column (3.0-mm i.d. × 50 mm, 2.7 μm). Isocratic elution was achieved using a mobile phase composed of 1-mM ammonium formate and 2-mM formic acid in water and acetonitrile (13:87, v/v) operated at a flow setting of 0.3 mL/min. Mass spectrometry analysis was conducted using an Agilent 6460 triple quadrupole system operated in multiple reaction monitoring with positive electrospray ionization. Ion transitions were m/z 472.1 → 135.9 for DA-1241 and m/z 480.2 → 404.1 for DA-1241-d₈ (internal standard). The total chromatographic run time was 4.5 min. Linearity of the calibration curves was confirmed throughout the range of 1-10 000 ng/mL, with no carry-over observed. Precision and accuracy for intra-assay and interassay were within 14.3%, except at the lower limit of quantification, where values remained within 16.0%. The method met all the other validation criteria for matrix effect, recovery, and stability. The established method was successfully implemented on plasma protein binding assessments and preclinical pharmacokinetic studies of DA-1241 after a single oral administration in mice, rats, and dogs.