Qualitative and quantitative determination of carotenoid stereoisomers in a variety of spinach samples by use of MSPD before HPLC-UV, HPLC-APCI-MS, and HPLC-NMR on-line coupling

Abstract

The carotenoids lutein and zeaxanthin have been identified as the macular pigments of the human retina. Nutritional epidemiological reports indicate that high consumption of fruits and vegetables rich in these carotenoids is correlated with reduced risk of some illnesses, e.g. agerelated macular degeneration (AMD). Because carotenoids are extremely sensitive to UV light and oxygen, they occur in nature as several Z/E stereoisomers which can differ considerably in their biological effectiveness. With particular regard to dietary supplementation we have focussed on identification and quantification of all the carotenoid stereoisomers occurring in a variety of raw and processed homegrown and commercial spinach samples, a natural source of carotenoids. Isolation of the unstable carotenoid stereoisomers from biological tissues without sample-preparation artifacts requires a mild, rapid, complete, and reproducible extraction technique such as matrix solid-phase dispersion (MSPD). Separation and unequivocal structural elucidation of the main carotenoid stereoisomers was achieved by use of hyphenated analytical techniques and exclusion of light and oxygen. HPLC analysis with highly selective C30 columns was used for quantitative determination of the main Z/E carotenoid stereoisomers and HPLC-APCI-MS and HPLC-NMR on-line coupling was used for unequivocal structural elucidation. Whereas HPLC-APCI-MS can distinguish between the carotenoids lutein and zeaxanthin, HPLC-NMR enables identification of all the main Z/E stereoisomers.