A Fast, Reliable and Low-cost Saponification Protocol for Analysis of Carotenoids in Vegetables

Carotenoids are mainly present in vegetables and fruits and display several important biological actions (provitamin A, antioxidant and anticarcinogenic activities and immunomodulation). To assess the intake of carotenoids and their potential health benefits in humans, it is necessary to have reliable information about carotenoid content in food. Saponification is commonly used during analysis of carotenoids in vegetables and fruits to release esterified xanthophylls and to eliminate interfering substances. It is a time-consuming step and involves the use of considerable volumes of solvents in the partition. To reduce the time and costs of the saponification process, we compare the reliability of a “shortcut” (small volumes, vortex 3 min, 20% potassium hydroxide, hexane/methylene chloride extraction) with that of a “standard” protocol (stirring 30 min, 5% potassium hydroxide, petroleum ether/diethyl ether extraction twice plus washings). Accuracy and precision were assessed according to studies of certified reference materials for carotenoid analysis in foods. For lutein, zeaxanthin, α -cryptoxanthin, β -cryptoxanthin, lycopene, α -carotene andβ -carotene, the “shortcut” showed within-day and between-day variability similar to or lower than that of the “standard” protocol, comparable deviation from the assigned values and higher recovery for carotenes, especially for lycopene. Advantages of the “shortcut” include considerably reduced time, higher overall recoveries and lower solvent expenditure, resulting in a reduction in time and total cost of up to 80–90% without loss of accuracy or precision.