Rapid separation and quantitation of curcuminoids combining pseudo two-dimensional liquid flash chromatography and NMR spectroscopy.

Rapid separation, characterization and quantitation of curcuminoids are important owing to their numerous pharmacological properties including antimicrobial, antiviral, antifungal, anticancer, and anti-inflammatory activities. In the present study, pseudo two-dimensional liquid flash chromatography was used for the separation of four curcuminoids (curcumin, demethoxy curcumin, bisdemethoxy curcumin and dihydro bisdemethoxy curcumin) for the first time. Silica and diol columns were used for separation of curcuminoids using gradient mobile phase. The separated peaks were monitored at 244, 360nm to obtain four compounds. The purity of compounds were determined by rapid quantitative (1)H NMR (qNMR) using 3-(trimethylsilyl) propionic-(2,2,3,3-d4) acid sodium salt (TSP-d4) (0.012%) in D2O. These results were compared with those obtained by HPLC method. The purity of isolated curcuminoids using pseudo 2D chromatography was found to be in the range of 92.4-95.45%. The structures of these compounds were characterized unambiguously using (13)C (APT) NMR spectra. The developed pseudo 2D separation technique has the advantage of simplified automation with shorter run time compared to conventional separation techniques. The method that combines rapid pseudo 2D separation and simple quantitation using qNMR reported herein can be of wide utility for routine analysis of curcuminoids in complex mixtures.