Reversed-phase Salt Assisted Liquid-liquid Extraction: A New Technique for Preconcentration and Determination of Crocin in Herbal Medicines by HPLC

A reversed-phase salt assisted liquid-liquid extraction (RP-SALLE) method was developed for the preconcentration and determination of trace crocin in herbal medicines by high-performance liquid chromatography (HPLC). In developed method, the roles of the aqueous and organic phases are inverted so that the analyte is extracted from the organic into the aqueous phase after salt addition and phase separation. In this work, a mixture of methanol-acetonitrile (1:6 volume ratio) containing the extracted crocin from the powdered sample was used as the organic phase. Upon addition of sodium chloride to a mixture of methanol-acetonitrile-water and vortexing, crocin was enriched into the aqueous phase and sedimented at the bottom. A portion of the aqueous phase containing the analyte was then injected into the HPLC column for analysis. The RP-SALLE conditions including volume ratio of the solvents, pH of sample, salt concentration, time of vortex, type of salt and temperature as variable factors were carefully optimized. Under the optimized conditions, a linear calibration graph within 0.06 – 100 mg L-1 with an R2 exciding 0.999, a detection limit of 0.02 mg L-1 and a relative standard deviation of 3.5% for six replicates were obtained.