A rapid HPLC column switching method for sample preparation and determination of β-carotene in food supplements

Abstract A simple and automated HPLC column-switching method with rapid sample pretreatment has been developed for quantitative determination of β-carotene in food supplements. Commercially samples of food supplements were dissolved in chloroform with help of saponification with 1 M solution of sodium hydroxide in ultrasound bath. A 20-min sample dissolution/extraction step was necessary before chromatography analysis to transfer β-carotene from solid state of food supplements preparations (capsules, tablets) to chloroform solution. Sample volume – 3 μL of chloroform phase was directly injected into the HPLC system. Next on-line sample clean-up was achieved on the pretreatment precolumn Chromolith Guard Cartridge RP-18e (Merck), 10 × 4.6 mm, with a washing mobile phase (methanol:water, 92:8, (v/v)) at a flow rate of 1.5 mL/min. Valve switch to analytical column was set at 2.5 min in a back-flush mode. After column switching to the analytical column Ascentis Express C-18, 30 × 4.6 mm, particle size 2.7 μm (Sigma Aldrich), the separation and determination of β-carotene in food supplements was performed using a mobile phase consisting of 100% methanol, column temperature at 60 °C and flow rate 1.5 mL/min. The detector was set at 450 nm. Under the optimum chromatographic conditions standard calibration curve was measured with good linearity – correlation coefficient for β-carotene ( r 2  = 0.999014; n  = 6) between the peak areas and concentration of β-carotene 20–200 μg/mL. Accuracy of the method defined as a mean recovery was in the range 96.66–102.40%. The intraday method precision was satisfactory at three concentration levels 20, 125 and 200 μg/mL and relative standard deviations were in the range 0.90–1.02%. The chromatography method has shown high sample throughput during column-switching pretreatment process and analysis in one step in short time (6 min) of the whole chromatographic analysis.