Vol. 43, No. 2, 1999
THE METHODOLOGY FOR THE SIMULTANEOUS ANALYSIS OF VITAMINS A, E, AND BETA-CAROTENE IN ANIMAL BLOOD PLASMA BY GRADIENT AND ISOCRATIC METHODS OF HPLC
Tučková, M. Kaštel, R.
University of Veterinary Medicine
Komenskeho 73
041 81 Kosice
The Slovak Republic
Summary
The isocratic and gradient high performance liquid chromatography (HPLC) methods for analysing of vitamins A, E, and beta-carotene in animal blood plasma were compared. In the isocratic procedure the mobile phase consisted of methanol and water (94.3 : 5.7, v/v) with a flow rate of 1.5 ml/min and a column pressure of 16 MPa. A reversed phase C-18 column was used. The separated components were detected at 294 nm. Retinol acetate and tocopherol acetate were used as internal standards. The separation by the gradient method was carried out on a reversed phase C-18 column with phase A = H2O and a linear gradient of phase B = methanol/ethyl acetate from 88-100 % over 10 min with a flow rate of 1.5 ml/min, and a column pressure of 14.1 MPa after the preseparation of a sample on the reversed phase of Silipor C-18. The optimal wavelength for detection was 280 nm. The gradient method is considered to be better for the simultaneous determination of vitamins A, E, and beta-carotene in biological materials. The isocratic method described can be recommended for the determination of vitamins A and E without beta-carotene.
Key words: vitamins A, E; beta-carotene; animal blood plasma; HPLC
References
Folia Veterinaria / 1999