Determination of rosmarinic acid and its N-substituted analog A1 in rat plasma using high-performance liquid chromatography-tandem mass spectrometry and its application in pharmacokinetics.

Background: The aim of this study was to determine the concentration of rosmarinic acid (RA) and its analog (E)-3-(3,4-dihydroxyphenyl)-2-(3-(3,4-dihydroxyphenyl)acrylamido)propanoic acid (A1) in rat plasma following oral administration. The significance of this study lies in the development of a rapid, sensitive, and alternative method using liquid chromatography-tandem mass spectrometry for the accurate quantification and identification of RA and A1 in vivo.

Methods: Liquid chromatography-tandem mass spectrometry was employed to analyze RA and A1 in rat plasma. A C18 column (1.9 µm, 2.1 × 100 mm) with a C18 guard column (5 µm, 2.1 × 10 mm) and a triple-quadrupole mass spectrometer combined with an electrospray ionization source were utilized. Sample pretreatment involved a one-step protein precipitation using isopropanol:ethyl acetate (20:80, v/v) as the solvent. Pseudoephedrine hydrochloride served as a standard.

Results: The developed method exhibited a linear relationship within the concentration ranges of 5-750 ng/ml for both RA and A1. Relative standard deviations in daily courses were less than 15%, and the relative errors recorded were within 15%. This is the first study to concentrate on determining A1 and RA in rat plasma through oral administration.

Conclusions: The liquid chromatography-tandem mass spectrometry method developed in this study offers a rapid, sensitive, and alternative approach for the accurate quantification and identification of RA and A1 in vivo. The findings serve as a significant foundation for evaluating the clinical applications of the medicine.