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.

Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries, such as Australia. Lutein and zeaxanthin are the only two carotenoids found in the macular region of the eye. Studies have shown that an intake of 10 mg and 2 mg per day of lutein and zeaxanthin, respectively, can reduce the rate of progression of AMD. The supply of these carotenoids can only be met through dietary sources or supplements, as these compounds cannot be synthesised by humans. Although lutein is relatively abundant in dietary sources, zeaxanthin has limited sources. In this study, eight orange and three red capsicum varieties were analysed for their carotenoid profiles by UHPLC-DAD-APCI-MS. It was observed that the principal carotenoid for seven of the orange varieties was zeaxanthin, and capsanthin for the three red varieties. One orange variety, which had a darker orange hue, had capsanthin and violaxanthin as its principal carotenoids instead of zeaxanthin. Zeaxanthin concentration (the principal carotenoid) in the seven orange varieties varied from 2.6 ± 0.5 mg/100 g to 25.27 ± 9.4 mg/100 FW, suggesting that as little as 7 g of the high-zeaxanthin line could meet the recommended daily dietary intake of 2 mg/person/day.

Zeaxanthin is a dietary carotenoid accumulated in the macula in order to reduce photoreceptor oxidation by blue light. Damage caused to photoreceptor cells in the human eye leads to macular degeneration, which is the leading cause of blindness in developed countries. Zeaxanthin, an orange pigment, is rarer in western diets as compared to the yellow pigment, lutein, the other important macular carotenoid. Orange capsicums (Capsicum annuum) have been reported to be an excellent source of zeaxanthin, but there are limited reports about its occurrence both within, and in other closely related species (C. baccatum, C. chinense). In the current investigation, yellow, orange and red coloured accessions of C. annuum, C. chinense and C. baccatum were analysed for their carotenoid profiles to identify high zeaxanthin accessions. A carotenoid extraction protocol and ultra-high-performance liquid chromatography-photometric diode array-mass spectrometry (UHPLC-PDA-MS) analysis was optimised to identify and quantify carotenoids in the capsicum accessions both before and after saponification. Interestingly, out of 22 varieties tested, only the ‘Orange Belle’ orange capsicum demonstrated a high accumulation of zeaxanthin. Other accessions exhibiting orange colour accumulated different orange carotenoid pigments to zeaxanthin, such as violaxanthin, beta-carotene, and beta-cryptoxanthin. Yellow coloured accessions accumulated a high concentration of lutein or alpha-carotene, while red cultivars were highest in capsanthin and capsorubin. The present findings suggests a need for further studies to identify high zeaxanthin germplasm which can be cross-bred with orange capsicums for future biofortification, in order to help increase the daily dietary intake of zeaxanthin in western diets.