Determination of Retinol and Tocopherols in Human Serum using Ultra-Performance Liquid Chromatography with Photodiode Array

Method that can simultaneously determine retinol, γ-tocopherol and a-tocopherol in human serum was developed utilizing ultra-performance liquid chromatography. Retinyl and tocopheryl acetates were employed as internal standards. The reverse-phased method utilizes isocratic elution with a mobile phase consisting of 20% acetonitrile and 80% methanol at a flow rate of 0.800 mL/minute. Separation was attained using an ethylene bridged hybrid (BEH) C18 column. Retinols and tocopherols were detected by photodiode array at wavelengths 325 nm and 295 nm, respectively. The retention times for retinol and retinyl acetate were 0.42 and 0.49 minutes respectively. γ-Tocopherol, α-tocopherol and tocopheryl acetate eluted at 0.86, 0.94 and 1.1 min, respectively. The limits of quantification were determined and found to be 0.025 μg/mL, 0.50 μg/mL and 1.0 μg/mL for retinol, γ-tocopherol and α-tocopherol, respectively. The method has been found to be suitable for the determination of retinol and tocopherols in human serum.