Vitamin contents

To determine the vitamin A content, the AOAC [18] method with some modifications was used. A quantity of 1 g of each sample was weighed and added into a 100 mL flask fitted with a reflux condenser. Then, 20 mL of alcoholic sulphuric acid and absolute alcohol (10 mL) were added to the flask and wrapped with aluminum foil. The mixture was refluxed for 45 min and cooled. Following that, 5 mL of water was added into each flask and transferred to a separating funnel. Diethyl ether was used to extract non-saponified materials. The mixed ether extract was then washed to remove the acid and dried over anhydrous sodium sulphate. The extract was evaporated at low temperatures while protected from sunlight, with final traces of solvent removed in a stream of nitrogen and residues dissolved immediately in 10 mL isopropanol. The extinction of freshly prepared extract in isopropanol was measured at 325 nm against a solvent blank (T1) using a UV-spectrophotometer (Jenway 6305, Bibby Scientific Ltd, U.K.). Thereafter, samples were exposed to U.V. radiation until the extinction no longer decreased with time, and absorbance spectrophotometrically measured (T2). In the same way, the corresponding standard vitamin A solution was determined (for solvent blank = ST1; after exposure to radiation until extinction = ST2) using Eq 2.

To determine the vitamin B₁ content, the AOAC [18] method with some modifications was used. Accurately, 2 g of each sample was weighed, then 0.5 mL of 4-amino phenol together with 5 mL of NH₄OH (0.1 M), thereafter thoroughly mixed, followed by room temperature incubation for 5 min. Next, 10 mL of chloroform was added, which allowed for the observation of two layers. The absorbance of chloroform layer was measured at 430 nm against blank using a UV-spectrophotometer (Jenway 6305, Bibby Scientific Ltd, U.K.). Finally, the Vitamin B₁ content was calculated using Eq 3 below.

To establish the vitamin B₂ content, the AOAC [18] method was used with some modifications. The samples were weighed (2 g) and added to a calibrated test tube. In each test tube, 2 mL glacial acetic acid, 2 mL hydrochloric acid (1 M), 2 mL hydrogen peroxide, 2 mL potassium permanganate (15% w/v), and 2 mL phosphate buffer (pH 6.8) were added. The resulting mixture was mixed thoroughly before measuring its absorbance at 444 nm against blank using a UV-spectrophotometer (Jenway 6305, Bibby Scientific Ltd, U.K.). The content of vitamin B₂ was calculated with the formula in Eq 4.

To determine the niacin content, the AOAC [18] method with some modifications was used. Samples (5 g) were autoclave digested with 1 N sulfuric acid with pH adjusted to 4.5 with 10 N sodium hydroxide, subsequently followed by filtration. Cyanogen bromide was complexed with the supernatants containing extracted niacin to form a purple color. Absorbance of solution was read at 470 nm against blank using a UV-spectrophotometer (Jenway 6305, Bibby Scientific Ltd, U.K.). The concentration of niacin was extrapolated from a standard curve.

To determine the vitamin C content, the AOAC [18] method with some modifications was employed. This involved the titration using diphenol indo 2, 6 –dichlorophenol (DPIP), which required sample (0.2 g) added to 4 mL of buffer solution (containing 1 g/L oxalic acid and 4 g/L sodium acetate anhydrous). The resultant solution was titrated against DPIP (295 mg/L) and sodium bicarbonate (100 mg/L). Vitamin C was calculated from Eq (5) below:

where: M = mass of ascorbic acid titrimetric equivalent to 0.001 M DPIP solution (mg)

100 is the dilution ratio of the sample taken. The second 100 is the scaling factor for conversion per 100 g of raw material, 10 is the titrate volume; V = titrant volume (0.00 1 M DPIP solution) ml; and B = weight of the sample extract used.

To determine the vitamin E content, the method described by Achikanu et al. [19] with some modifications was used. The sample (1 g) was macerated in 20 mL of n-hexane. Thereafter, the mixture was centrifuged for 10 min at 1500 rpm (4000 rpm, Abman, Canada), filtered, and treated with ethanol and alcoholic potassium hydroxide (0.5 N). Subsequently, 2 mL of the filtrate was evaporated to dryness in a boiling water bath (Gallenkamp, England). The residue was mixed with ethanol, ferric chloride (0.2%), and a-a 1-dipyridyl (0.5%). The absorbance of resultant solution was read at 520 nm against the blank using a UV-spectrophotometer (Jenway 6305, Bibby Scientific Ltd, U.K.), after which the concentration of vitamin E was calculated from Eq (6) below: