Abstract
In this contribution, the rapid and simple determination of vitamins A and E, and vitamin E acetate as internal standard in human plasma by high-performance liquid chromatography with a column-switching technique was proposed. Restricted Access Material - RAM column 25 x 4 mm containing 25 μm C18 alkyl-diol silica support was integrated into lab-made column-switching HPLC system. A monolithic column Chromolith Performance RP-18e, 100 x 4.6 mm, (Merck) was used for simple separation of vitamins in gradient mode. The mobile phase I: methanol – 20 mM water solution of dodecyl sulfate sodium salt (30:70, v/v) was used for protein matrix elution, flow rate 1.3 ml min-1. Valve switching time was determined to 4 minutes after sample injection. The gradient elution of mobile phase II: methanol – water 90:10 (v/v) → 100% methanol, flow rate 1.0 ml min-1 was used as optimal condition of vitamins separation in relatively short time. Analysis time from raw sample to peaks evaluation was less than 15 min. UV detection was carried out at wavelength 285 nm. The recoveries of vitamins from spiked human plasma were 102.5% for vitamin A and 91.4% for vitamin E. Limits of detection were 0.24 µmol l-1 and 0.84 μmol l-1 for vitamin A and E, respectively.
Keywords: Vitamin A, Vitamin E, Vitamin E acetate, Column-switching, Restricted Access Material (RAM), HPLC
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