Liquid chromatography-tandem mass spectrometric assay for determination of unstable arecoline in rat plasma and its application

Arecoline, a predominant alkaloid in areca nut, shows several pharmacological and toxicological effects. In present study, a sensitive and accurate liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed and fully validated for quantification of arecoline in rat plasma. Importantly, our group found that arecoline was highly unstable in rat plasma samples, which brought challenges to accurately quantify in vivo. The hydrolysis of ester moiety of arecoline by carboxylesterases was responsible for its instability, and arecoline was hydrolyzed to arecaidine. The degradation of arecoline was completely inhibited using 5% formic acid as a stabilizer, which was immediately added to freshly collected rat plasma samples. EDTA was adopted as the anticoagulant to also reduce the degradation during blood collection and plasma separation owing to its anti-esterase effect. The plasma sample was separated by a C18 analytical column with a mobile phase of 0.1% formic acid and methanol (95:5   v/v) at an isocratic flow rate of 300   μL/min. The analyte was monitored on a tandem mass spectrometer using the multiple reaction monitoring scan in a positive electrospray ionization mode. The method exhibited high sensitivity and a good linearity rang of 1–1000   ng/mL. The developed analytical method was employed in a pilot pharmacokinetic study of arecoline in rats. Arecoline was rapidly eliminated within 45   min in rats after oral treatment of 150   mg/kg arecoline.