Development of an innovative phytosterol derivatization method to improve the HPLC-DAD analysis and the ESI-MS detection of plant sterols/stanols.

HPLC analyses of phytosterols are associated with the issues of sensitivity due to their high lipophilicity and their lack of chromophore. These problems could be solved through chemical modifications of plant sterols/stanols structures. Therefore, the present study aims to develop a new method for phytosterols derivatization. This method was performed using dansyl chloride (4 mg ml-1) as derivatizing agent and different reaction parameters have been optimized. The highest yields of phytosterol derivatization were obtained with 4-dimethylaminopyridine (DMAP) as catalyst at a concentration of 8 mg ml-1 and dichloromethane as reaction solvent. In addition, 40 ˚C was the best reaction temperature for 30 min as the best reaction time. This derivatization method presented a high reproducibility (%RSD = 1.2-2.7%) and a good linearity (R2 = 0.9982-0.9999). The UV absorption intensities after derivatization showed a 23-fold increment for plant sterols and a 400-fold increment for plant stanols. Moreover, this derivatization method allowed the use of high and more selective wavelengths of detection and improved the chromatographic separation of phytosterols. Furthermore, the developed method allowed the ESI-MS ionization and analysis of phytosterols. This method can therefore contribute to the improvement of the HPLC analyses of plant sterols/stanols.