Rapid isolation and characterization of crocins, picrocrocin, and crocetin from saffron using centrifugal partition chromatography and LC–MS

Abstract This study demonstrates a simple method for one‐step isolation of the main secondary metabolites of a hydroalcoholic extract of Crocus sativus stigmas (saffron) using step‐gradient centrifugal partition chromatography. The analysis was performed in dual and elution−extrusion mode, using five biphasic systems of the solvents heptane/ethyl acetate/butanol/ethanol/water in ratios of 4:10:0:4:10, 1:13:0:4:10, 1:12:1:4:10, 1:10:3:4:10, and 1:7:6:4:10. Five major crocins, picrocrocin, and crocetin were directly isolated in one step. Scaling up to preparative level, allowed the recovery of significantly high quantities of pure compounds, especially trans ‐crocin‐4, saffron's principal crocin. Comparing dual‐mode and elution‐extrusion, in dual‐mode, the trans ‐crocin‐4 containing fractions were co‐eluted with a high amount of free β ‐ d ‐glucose. In contrast, absence of free β ‐ d ‐glucose was observed in the corresponding trans ‐crocin‐4 fractions obtained by the second method denoting its superiority against dual‐mode. Initiating analysis with the 4 th solvent‐system afforded selective isolation of trans ‐crocin‐4, with reduction in experimental time and solvent consumption. Structure elucidation was performed by nuclear magnetic resonance spectroscopy, liquid chromatography with mass spectrometry, and high‐resolution tandem mass spectrometry. The proposed methodology comprises an integrated approach for the purification and characterization of biologically active saffron components in a fast, selective, and environmentally friendly manner.