Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Method for Quantification of 3,14,19-Triacetyl Andrographolide in Rat Plasma: Application to Preclinical Pharmacokinetic Studies.

3,14,19-triacetyl andrographolide (ADA), a derivative of andrographolide, has emerged as a promising anti-inflammatory compound. In the present study, we established a rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometry method for the quantification of ADA in rat plasma. ADA and celecoxib (internal standard, IS) were separated using an ACQUITY UPLC BEH C18 chromatographic columns with a gradient mobile phase of acetonitrile (containing 0.1% formic acid)/water (containing 0.1% formic acid) at a flow rate of 0.3 mL/min. Quantification was performed using the multiple reaction monitoring transitions to m/z 499.1 → 439.1 for ADA, m/z 381.9 → 361.9 for celecoxib, with plasma samples undergoing liquid-liquid extraction. The method effectively detected ADA in plasma and exhibited excellent linearity (R 2 > 0.9921) over concentrations ranging from 5 to 2000 ng/mL. Both within-run and between-run precision (%RSD) were less than 6.88%, and accuracy ranged from 94.86% to 107.61%. Matrix effects were 87.53% and 112.78%, with recoveries ranging from 98.60% to 104.39%. ADA was proven to be stable under different storage conditions (4°C and -70°C) in plasma. The validated method was successfully applied to determine ADA concentrations in rat plasma. The results demonstrated that ADA exhibited a noticeable improvement in bioavailability compared with andrographolide.