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

Solichová, Dagmar; Korecká, Lucie; Svobodová, Iveta; Musil, František; Bláha, Vladimír; Žd'ánský, Petr; Zadák, Zdeněk
June 2003
Analytical & Bioanalytical Chemistry;Jun2003, Vol. 376 Issue 4, p444
Academic Journal
In this work, a simple isocratic reversed-phase HPLC method for determination of α-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 μL of n -hexane was added to 500 μL of erythrocytes. After 2 min this mixture was deproteinized by addition of cool ethanol (500 μL, 5 min) denatured with 5% methanol containing α-tocopherol acetate (20 μmol L-1), as internal standard, and then extracted for 5 min by vortex mixing. After centrifugation (10 min, 1600×g) an aliquot (2000 μL) of the clean extract was separated and evaporated under nitrogen. The residue was dissolved in 400 μL methanol and analysed by reversed-phase HPLC on a 4.6 mm×150 mm, 5 μm Pecosphere C18 column; the mobile phase was 100% methanol, flow rate 1.2 mL min-1. The volume injected was 100 μL and detection was by diode-array detector at a wavelength of 295 nm. The extraction recovery of α-tocopherol from human erythrocytes was 100.0±2.0%. The detection limit was 0.1 μmol L-1 and a linear calibration plot was obtained in the concentration range 0.5–20.0 μmol 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.


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