SIMULTANEOUS DETERMINATION OF VITAMIN-A AND VITAMIN-E AND CAROTENOIDSIN PLASMA BY REVERSED-PHASE HPLC IN ELDERLY AND YOUNGER SUBJECTS

A reversed-phase high-performance liquid-chromatographic method for th e simultaneous determination of retinol, alpha-tocopherol, alpha-carot ene, beta-carotene, cryptoxanthin, lutein/zeaxanthin, and lycopene is described. This method was applied to plasma measurements in healthy y oung and elderly subjects. The plasma, deproteinized with ethanol, is extracted twice with n-hexane. After evaporation, the residue is disso lved in 50 muL of tetrahydrofuran and made up to 200 muL with ethanol. Samples (50 muL) are injected onto a 250 x 4.6 mm column of 5-mum-par ticle Spherisorb ODS1 (Phase Separations) that had been equilibrated w ith solvent mixture A:B (90:10 by vol) [A = 100 mmol/L ammonium acetat e in methanol: acetonitrile (80:20 by vol) and B = 100 mmol/L ammonium acetate in water] at 2 mL/min. The analytes are eluted by running a 1 2-min linear gradient to 100% A; solvent A is then maintained for 10 m in. Intrabatch CVs were 2.3%,3.3%,2.8%,3.6%,3.6%, and 3.0% for retinol , alpha-tocopherol, lutein/zeaxanthin, cryptoxanthin, lycopene, and be ta-carotene, respectively. The corresponding interbatch CVs were 4.9%, 5.8%, 12.3%, 6.5%, 8.0%, and 3.4%.