A rapid HPLC column switching method for sample preparation and determination of beta-carotene in food supplements
Abstract
A simple and automated HPLC column-switching method with rapid sample pretreatment has been developed for quantitative determination of beta-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 beta-carotene from solid state of food supplements preparations (capsules, tablets) to chloroform solution. Sample volume - 3 mu L 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 x 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 x 4.6 mm, particle size 2.7 mu m (Sigma Aldrich), the separation and determination of beta-carotene in food supplements was performed using a mobile phase consisting of 100% methanol, column temperature at 60 degrees 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 beta-carotene (r(2) = 0.999014; n = 6) between the peak areas and concentration of beta-carotene 20-200 mu g/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 mu g/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.