Abstract
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.
Similar content being viewed by others
References
Sierra C, Pastor MC, de Ramón M (1992) Clin Chim Acta 208:119–126
Gonzalez-Corbella MJ, Lloberas-Blanch N, Castellote-Bargallo AI, Lopez-Sabater MC, Rivero-Urgell M (1994) J Chromatogr B 660:395–400
Takeda H, Shibuya T, Yanagawa K, Kanoh H, Takasaki M (1996) J Chromatogr A 722:287–294
Rupérez FJ, Martín D, Herrera E, Barbas C (2001) J Chromatogr A 935:45–69
Bourgeosis C (1992) Determination of vitamin E: tocopherols and tocotrienols. Elsevier, New York
De Leenheer AP, Lambert WE, DeRuyter MGM (1985) Modern chromatographic analysis of vitamins. Marcel Dekker, New York
Machlin LJ (1984) Handbook of vitamins: nutritional, biochemical and clinical aspects. Marcel Dekker, New York
Drotleff AM, Ternes W (2001) J Chromatogr A 909:215–223
Kamal-Eldin A, Görgen S, Petterson J, Lampi AM (2000) J Chromatogr A 881:217–227
Vatassery GT, Smith Wed, Quach H (1993) Anal Biochem 214:426–430
Acknowledgements
The authors wish to express their gratitude to the IGA Ministry of Health Czech Republic (grants No. NG/6770-3, NB/6043-3 and NB/6822-3) and Ministry of Education Czech Republic (grant VZ 253100002) for financial support of this work.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Solichová, D., Korecká, L., Svobodová, I. et al. Development and validation of HPLC method for the determination of α-tocopherol in human erythrocytes for clinical applications. Anal Bioanal Chem 376, 444–447 (2003). https://doi.org/10.1007/s00216-003-1886-1
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00216-003-1886-1