Development and validation of HPLC method for the determination of α-tocopherol in human erythrocytes for clinical applications

In this work, a simple isocratic reversed-phase HPLC method for determination of alpha-tocopherol in human erythrocytes has been developed and validated. After separation of plasma the erythrocytes were washed three times with 0.9% sodium chloride containing 0.01% butylated hydroxytoluene (BHT) as antioxidant and then were diluted 1:1 (v/v) with the same solution. In the liquid-liquid extraction (LLE) procedure, 2500 muL of n-hexane was added to 500 muL of erythrocytes. After 2 min this mixture was deproteinized by addition of cool ethanol (500 muL, 5 min) denatured with 5% methanol containing alpha-tocopherol acetate (20 mumol L-1), as internal standard, and then extracted for 5 min by vortex mixing. After centrifugation (10 min, 1600xg) an aliquot (2000 muL) of the clean extract was separated and evaporated under nitrogen. The residue was dissolved in 400 muL methanol and analysed by reversed-phase HPLC on a 4.6 mmx150 mm, 5 mum Pecosphere C18 column; the mobile phase was 100% methanol, flow rate 1.2 mL min(-1). The volume injected was 100 muL and detection was by diode-array detector at a wavelength of 295 nm. The extraction recovery of alpha-tocopherol from human erythrocytes was 100.0+/-2.0%. The detection limit was 0.1 mumol L-1 and a linear calibration plot was obtained in the concentration range 0.5-20.0 mumol L-1. Within determination precision was 5.2% RSD (n=10), between determination precision was 6.1% RSD (n=10). The method was applied successfully in a clinical study of patients with acute pancreatitis and for determination of the reference values in the healthy Czech population.