A simple, sensitive HPLC-DAD method for simultaneous determination of carotenoids, chlorophylls and α-tocopherol in leafy vegetables

Leafy vegetables are the important components of our diet and are the source of several beneficial phytochemicals. A sensitive, simple analytical method is therefore required to precisely measure the phytochemical composition. A validated reversed phase high performance liquid chromatography with diode array detection (HPLC-DAD) method was developed to determine simultaneously carotenoids, chlorophylls and α-tocopherol composition of six leafy vegetables (B. compestris, B. rapa, B. juncea, M. neglecta, and two spinach varieties). Carotenoids were extracted and efficiently separated using a tertiary mobile gradient system of methanol–water, water and MTBE in 40 min on a reversed phase C18 column. The method was simple, precise, accurate and highly reproducible. Twelve carotenoids namely lutein and its three isomers, β-carotene-5,6-epoxide, neoxanthin, violaxanthin, two cis-isomers of zeaxanthin, 8-apo-carotenal, all-trans-β-carotene and its 13-cis-isomer; one fatty acid ester (β-cryptoxanthin ester); α-tocopherol and chlorophyll a & b were quantified in vegetable leaves. α-Tocopherol, neoxanthin, violaxanthin, lutein, 8-apo-carotenal, chlorophyll a and all-trans-β-carotene were present in higher amounts. Significant variations in the major compounds were observed in the selected vegetables. It was concluded that the developed method was highly sensitive, accurate and can be used to analyze carotenoids, chlorophylls and α-tocopherol simultaneously in leafy vegetables as well as in other plant leaves.