Effects of extraction and high-performance liquid chromatographic conditions on the determination of lutein in spinach

A major factor in the direct determination of lutein in spinach extracts proved to be obtaining reproducible and stable chromatography of lutein. This was achieved on a C30 column with the mobile phase acetone–0.1 M triethylammonium acetate (TEAA) buffer (pH 7) 9:1 (v/v). Extraction of 10 mg of lyophilized spinach with 10 mL of extraction solvent (ethanol, acetone, ethanol–ethyl acetate 1:1 (v/v), methanol–THF 1:1 (v/v)) for 15 min with magnetic stirring under nitrogen resulted in equal yields of lutein. The yields were enhanced by addition of 15% of 1 M TEAA buffer pH 7 to all four extraction solvents. As confirmed by recovery experiments, no loss of lutein occurred during the extraction. The relative standard deviation from triplicate extractions was less than 5%. The addition of 15% TEAA pH 7 to acetone enhanced the extraction yield of lutein also from unlyophilized spinach. The content of lutein in different spinach samples ranged from 5 to 15 mg/100 g of fresh weight. The first separation is reported of all the carotenoids and chlorophylls on a C18 core–shell column and the addition of 15% of 1 M TEAA buffer pH 7 to acetone also enhanced the extraction yield of β-carotene compared to the yield produced by pure acetone.

► Triethylammonium acetate buffer pH 7 in the aqueous acetone for HPLC of carotenoids.
► The same mixture improved extraction of lutein from spinach compared to pure acetone.
► Separation of all spinach pigments in less than 15 min on core–shell C18 column.