Simultaneous determination of retinol and α-tocopherol by high pressure liquid chromatography in micro-volumes of...

The lack of biochemical data to indicate the prevalence of marginal vitamin A deficiency (VAD) in the country is largely because of the difficulty of obtaining enough serum for analysis, especially from malnourished children. Efforts were therefore made to establish a high-pressure liquid chromatography system (HPLC) for the determination of retinol in micro-volumes of serum. Since our previous studies showed that tocopherol could be simultaneously determined in the same system, studies have been carried out for the determination of both vitamins using retinol acetate and -tocopherol acetate as internal standards. Trials were carried out to determine the most suitable sample treatment procedures and chromatographic system including composition of the mobile phase for handling 20 ml of serum. The HPLC system proposed enables successful separation and quantitation of retinol and α-tocopherol and their respective internal standards, retinol acetate and -tocopherol in less than 14 minutes. Reproducibility studies carried out with pooled sera showed a within day and between day variation of less than 8% and 13% respectively for retinol, whilst variations for α-tocopherol were higher, ranging from 8-16%. The proposed method is currently being applied to the determination of retinol and α-tocopherol in a group of malnourished children under six years of age.