High-performance liquid chromatographic (HPLC) separation and determination of four vitamins in some Kenyan foods
Techniques utilizing high-performance liquid chromatography (HPLC) were developed for the determination of four vitamins, -carotene, -tocopherol, thiamine and riboflavin, in some commonly consumed foods in Kenya. The techniques involve the simultaneous extraction and determination of -carotene and -tocopherol on one hand, and thiamine and riboflavin on the other hand. The technique for the simultaneous determination of -carotene and -tocopherol involved sample saponification in an ethanolic alkaline solution and the extraction of the unsaponifiable matter with n-hexane. After evaporation of the solvent under vacuum, the residue was dissolved in ethanol and then assayed on a reversed phase HPLC system. The HPLC system used for the separation of the vitamins and various forms of carotene and tocopherol from each other as well as from ingredients in the sample matrix consisted of a Bondapack C18 reversed phase column and a mobile phase composed of methanol-acetonitrile-chloroform-water in the ratio 46:30:18:6. The vitamins were detected from their ultraviolet (UV) absorbance at 297nm. The method was shown to be reproducible for both vitamins with coefficients of variation between 2.0 and 7.3%. The average recovery for -carotene was 95.0% and that for -tocopherol was 94.7%. Detection limits were 1.0 g/ml and 2.0 g/ml for -carotene and -tocopherol respectively. Thiamine and riboflavin determination involved extraction of the vitamins by hydrolysis in a limited amount of dilute hydrochloric acid followed by enzymatic incubation for 2.5 hours. Then the mixture was filtered and subsequently injected into a Bondapak C18 reversed phase column without further cleanup. The mobile phase used for separation consisted of 0.01M phosphate buffer plus 1-hexanesulfonic acid sodium salt as counter ion (PH 7.0), methanol and acetonitrile in ratio 80:12:8. Detection of the vitamins was from their UV absorbance at 265nm. The coefficients of variation of thiamine and riboflavin, between 4.4 and 9.0%, and 3.1 and 5.6% respectively, indicated that the method was reproducible. The average recoveries of thiamine and riboflavin were 92.0% and 93.7% respectively. Detection limits were 1.0 g/ml for thiamine and 0.5 g/ml for riboflavin. The methods developed were applied to the analysis of some foods. The results indicate that indigenous green vegetables are good sources of -carotene and -tocopherol in comparison to the exotic vegetables such as cabbage and lettuce. The levels of both -carotene and -tocopherol in the indigenous green vegetables are between 3000 and 10650 g per 100g of the vegetables, while those of exotic vegetables were between 30 to 230 g per 100g. Indigenous green vegetables were also found to contain high amounts of riboflavin. Legumes are good sources of thiamine and contain some riboflavin. They are, however, poor sources of -carotene and -tocopherol.