Determination of egg yolk xanthophylls by isocratic high-performance liquid chromatography

•An isocratic HPLC method for the determination of eight xanthophylls in egg yolks was developed.•Three new mobile phases were proposed for the separation on C30 column.•The influence of column temperature on the separation was evaluated.•The addition of triethylammonium acetate buffer pH 7 to the mobile phase enhanced the peak areas.•A simple yolk test solution preparation by acetone extraction under nitrogen was used.

An isocratic HPLC method was developed for the determination of eight xanthophylls (lutein, capsanthin, zeaxanthin, canthaxanthin, β-apo-8′-carotenal, ethyl-8′-apo-β-carotene-8′-oate, citranaxanthin and β-cryptoxanthin; registered as additives in poultry feeding) in egg yolks. Optimum separation of all-E-isomers of these xanthophylls was achieved in less than 18 min on a ProntoSIL C30 column at 27 °C using acetone–methanol–0.5 M triethylammonium acetate buffer pH 7 14:5:1 (v/v) as the mobile phase with a flow rate of 1 mL/min using spectrophotometric detection at 450 nm. Other mobile phases were also found suitable, including acetone–water 93:7 (v/v) and acetone–methanol 1:4 (v/v) and the influences of column temperature on the separation and addition of triethylammonium acetate buffer pH 7 to the mobile phase on enhancement of the peak areas were evaluated. Preparation of test solution from yolks included a short vortexing of 0.5 g of yolk in 10 mL of acetone, followed by 15 min magnetic stirring under nitrogen and centrifugation. The method was validated for 5 analytes. The calibration range was between 0.04 and 2 μg/mL and the mean recovery of the analytes (95%) and the intra-day precision of the method (RSD less than 5%) on three levels in triplicates were obtained. Analyses of eggs from four husbandry classes showed the presence of up to four xanthophylls (lutein, zeaxanthin, canthaxanthin and ethyl-8′-apo-β-carotene-8′-oate) and traces of β-cryptoxanthin.