RESUMO
Objective: A high-performance liquid chromatographic (HPLC) procedure with a fluorescence detector was developed to rapidly separate ?,?,?,?- tocopherol isomers in human serum Methods: The HPLC system consisted of Inertsil silica column (100-A, 3?m,4.6mm?250mm) and 7% (v/v) methyl-tert- butyl ether in n-hexane as mobile phase . Prior to HPLC, the serum sample wa deproteined by ethanol (BHT 0.0625%) and the tocopherol isomers were efficiently extracted in thei original isomeric conformations using n-hexane-ethyl acetate (5:1) in the presence of 2,6-bi-buty p-methylphenol (BHT). Result: The quantification limits, defined as the lowest quantitatively measurable concentration of the different compounds (ng/ml) are calculated according to the experiment:?-tocophero 1.0,?-tocopherol 1.0,?-tocopherol 0.5,?-tocopherol 0.5. The recovery rates are between 95%~105% Correlation coefficients are over 0.999 when the concentration is between 5 ng/ml~5 ?g/ml. Conclusion This technique is suitable for assay of tocopherol isomers in human serum at all ages.
RESUMO
A method for the determination of B6-pyridoxamine (PAM), pyridoxal (PAL) and pyridoxine (POL) by RPLC was proposed. The procedure included the addition of 0.1M H2SO4 to the sample, hydrolysis for 30 min at 120℃, centrifuging, filtration and direct analysis by ODS Cl8 column. PAM, PAL and POL were completely separated, when the flow rate of the mobile phase (pH2.0-2.1) was 1.0-1.5ml/min. Quantitation by a fluorescent detector was performed with Exc: 293 nm and Em: 395 nm. The recovery ranged from 99 to 103% with CV 5.0-5.3%. The method was simple, rapid, sensitive and reproducible.