## 2 Materials and Methods
### Biochemical Assays
Vitamins were assayed using AOAC [29] methods. Vitamins in the various samples were determined by colorimetry. Optical density was measured using a Jenway Model 6300 colorimeter. Calibration curves were obtained from the preparation of a range of solutions of the corresponding vitamin molecule. The samples were prepared as follows:  
Retinol (A): One gram of the sample was placed into a 250 mL flask. After adding 5 mL of pyrogallol solution, 35 mL of ethanol, and 10 mL of potassium hydroxide solution, the mixture was heated for 30 min at 70-80 \({}^{\circ}\)C under a reflux condenser, then allowed to cool under a stream of water. After cooling, 40 mL of distilled water and 100 mL of petroleum ether were added. Extraction was performed by stirring for 3 min. The mixture was then left to settle, and the upper phase was transferred to a separating funnel. The ethereal phase was washed to neutrality with three 50 mL portions of water and filtered through filter paper. A 5 mL sample of the ethereal phase was transferred into a 50 mL flask and diluted with petroleum ether. The retinol concentration of this solution was determined by measuring its optical density at 325 nm. Thiamine (B1): The thiamine content of the samples was determined by adding 50 mL of 0.1 N sulfuric acid to one gram of each sample in a 100 mL volumetric flask. The mixture was heated in a water bath at 100 \({}^{\circ}\)C for 30 min, with frequent stirring. Five milliliters of 2.5 N sodium acetate solution was added to the contents, and the mixture was left to cool. After cooling, the flask was capped and placed in a water bath at 45-50 \({}^{\circ}\)C for 2 h. The resulting solution was made up to 100 mL with distilled water and filtered through filter paper. A 10 mL volume of the filtrate was transferred and mixed with 5 mL of potassium chloride solution. Absorbance was measured at a wavelength of 285 nm. Riboflavin (B2): One gram of each sample was weighed into a 250 mL volumetric flask. To this, 5 mL of 0.1 N sulfuric acid and 5 mL of dichloroethane were added, followed by 90 mL of distilled water. The mixture was stirred and heated on a sand bath for 30 min to extract the riboflavin. Afterward, the mixture was cooled and made up to 250 mL with distilled water, then filtered through filter paper. A 2 mL volume of the filtrate was transferred into another 250 mL volumetric flask and topped up with distilled water. The riboflavin concentration of the solution was determined by measuring its absorbance at 460 nm. Niacin (B3): Five grams of the sample were extracted with 50 mL of distilled water. Extraction was performed by repeated stirring for 30 min. The mixture was then left to settle, and the upper phase was recovered and filtered. This operation was repeated three times with the same amount of distilled water (100 mL). Five milliliters of the combined filtrates were transferred into a 100 mL volumetric flask and topped up with distilled water. The absorbance of the resulting colored solution, measured at a wavelength of 385 nm, was used to determine the nicotinic acid content of the sample. Tocopherol (E): One gram of the sample was weighed and placed in a 250 mL flat-bottomed flask. A solution of 10 mL of ethanol and 20 mL of 1 N sulfuric acid was added. The flask was wrapped in aluminum foil and heated under reflux for 45 min. The resulting solution was cooled for 5 min, followed by the addition of 50 mL of distilled water and transferred to an aluminum foil-covered separating funnel. The unsaponifiable matter in the mixture was extracted five times with 50 mL of dimethyl ether each time. The combined extract was washed with 1 N sulfuric acid solution and dried over anhydrous sodium sulfate. The evaporated extract was immediately dissolved in 15 mL of ethanol, 1 mL of concentrated sulfuric acid, and 1 mL of concentrated nitric acid. The resulting solution was placed in a water bath at 90 \({}^{\circ}\)C for 30 min. After cooling, the tocopherol content of the extract was measured by ultraviolet absorption at 470 nm.