A Modified Extraction and Saponification Method for the Determination of Carotenoids in the Fruit of Capsicum annuum

Quantification of free and bound carotenoids in pigmented fruit and vegetable matrices has previously been challenging due to carotenoid instability, degradation during extraction, and the prevalence of predominant carotenoid esters. The aim of the present study was to develop an optimized extraction procedure that minimises the loss of free and bound carotenoids by utilising a combination of extraction solutions, followed by an improved saponification process. A mixture of hexane, dichloromethane, ethanol and water achieved the highest extraction efficiency (>97%) from the chili/capsicum matrix. The study also addressed the previously unexplained loss of carotenoids during saponification by adding phosphate buffer to the sample–extract mixture, which prevented soap micelle formation. Additionally, the duration and temperature of the saponification procedure and pH of the final extraction solution were further optimised to achieve a higher total carotenoid recovery. A total of 48 free and bound carotenoids were identified in the capsicum fruit samples using UHPLC-DAD-MS/MS. The total carotenoid content within six bell pepper and chili fruits ranged between 1.63 (green bell capsicum) and 32.08 mg/100 g fresh weight (sweet red baby capsicum). The current methodology potentially could be used in a broad range of different carotenoid-containing matrices and commodities.