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.