A sensitive and specific liquid chromatography/tandem mass spectrometry method for determination of echinacoside and its pharmacokinetic application in rats

A rapid and sensitive method based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the determination of echinacoside in rat plasma was established and fully validated. A single step of liquid-liquid extraction with n-butanol was utilized. Chromatographic separation of the analyte and the internal standard (IS), chlorogenic acid, from the sample matrix was performed using a Capcell-MG C₁₈ analytical column (100 × 2.0 mm × 5 μm), with a gradient of acetonitrile and water containing 0.1% acetic acid as the mobile phase. Detection was performed on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization source operated in negative ion selected reaction monitoring mode. The method was linear in the concentration range 10-2500 ng/mL. The deviations of both intra- and inter-day precisions (RSD) were 7.1% and the assay accuracies were within 99.2-106.5%. Echinacoside proved to be stable during sample storage, preparation and analysis when an antioxidant solution was used. The method was successfully applied to a pharmacokinetic study in rats after an intragastric administration of echinacoside (100 mg/kg). With the lower limit of quantification at 10 ng/mL, this method proved to have sufficient selectivity, sensitivity and reproducibility for the pharmacokinetic study of echinacoside.