Pharmacokinetic Analysis of Two N-Substituted Rosmarinic Acid Analogs in Rat Plasma by Liquid Chromatography-Tandem Mass Spectrometry.

ABSTRACT

This study reports the development of an effective high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the quantification of two analogs of rosmarinic acid (RA) in rat plasma, namely methyl (E)-2-(3-(3,4-difluorophenyl)acrylamido)-3-(3,4-dihydroxyphenyl)propanoate (A11) and methyl (E)-3-(3,4-dihydroxyphenyl)-2-(3-(3,4-dihydroxyphenyl)acrylamido)propanoate (A2). These analogs, featuring N atoms instead of O atoms, exhibit enhanced bioavailability and distinct pharmacological activities compared with RA. The HPLC separation was carried out on a C18 column (1.9 µm, 2.1 mm × 100 mm) coupled with a security guard C18 column (5 µm, 2.1 mm × 10 mm). A triple-quadrupole mass spectrometer equipped with an electrospray ionization ion source was utilized for ion generation. Pseudoephedrine hydrochloride was utilized as a standard, and a single-step protein precipitation method using isopropanolethyl acetate (v/v, 2080) was employed for sample pretreatment. The developed method demonstrated excellent linearity over the concentration range of 5-750 ng/ml for both A11 and A2, with relative standard deviations of <15% and relative errors within 15% during daily course analysis. The method allowed for the unambiguous quantification and identification of A11 and A2 in vivo. The results of this study provide a meaningful foundation for evaluating the clinical applications of these analogs.