Abstract: A high performance liquid chromatography (HPLC) method was developed for the determination of capsanthin and capsaicin in chili peppers by using a three-phase salting-out extraction system (petroleum ether/acetonitrile/dipotassium hydrogen phosphate). The composition of the system was determined, and the effects of temperature, sonication time and mixing sequence on the separation of the target products were further investigated. Based on the yields of capsaicin and capsaicin, the optimal extraction conditions were determined by response surface methodology as follows: temperature 45 ℃, sonication time 10 min, distilled water 44%, dipotassium hydrogen phosphate 20%, acetonitrile 20%, and petroleum ether 16%. After the capsicum sample powder was treated by the three-phase mixed solvent, capsanthin and capsaicin were concentrated in the upper and middle phases, respectively, and the concentrations of the target products in either phase were analyzed by HPLC. The results showed that the highest yields of capsanthin and capsaicin under optimal conditions were 0.263 mg/g and 1.412 mg/g, respectively. The proposed method could extract both capsaicin and capsaicin from chili peppers quickly and efficiently. The contents of capsanthin and capsaicin in 33 different varieties of chili peppers were determined, and these varieties were evaluated by cluster analysis, aiming to provide a theoretical basis for the identification and cultivation of chili varieties.

Key words: capsanthin; capsaicin; salting-out extraction; ultrasound-assisted extraction; response surface analysis; high performance liquid chromatography; cluster analysis