2.4. Determination of vitamin C concentration

Vitamin C (ascorbic acid) concentration was determined by the method described by Angirekula et al. [16]. Determination was made after devitalisation with 2, 4 dinitrophenylhydrazine.

Principle: Ascorbic acid is oxidized by copper to form dihydroascorbic acid and diketoguloric acid. These products are treated with 2, 4 DNP to form a derivative of bis 2,4 dinitrophenylhydrazine. This compound in conc. H₂SO₄ undergoes a rearrangement to form a product with an absorption band that is measured at 520 nm. This reaction is seen in the presence of thiourea to provide an unduly reducing reaction which helps to produce an inter substance from non-ascorbic acid chromogens.

Reagents

TCA (trichloroacetic Acid) 5% and 10% in distilled water

2,4 dinitrophenylhydrazine/thiourea/copper (DTC) solution (add 0.4 g thiourea, 0.05 g CuSO4 5H₂O and 30 g 2, 4 dinitrophenylhydrazine and bring to a total volume of 100 ml with 9 NH₂SO₄.

A 65% H₂SO₄

Procedure: Serum (1 ml) of and 1 ml of ice cold 10% TCA were mixed well and centrifuged for 20 min at 3500 rpm. Then, 0.5 ml of supernatant and 1 ml of DTC were taken and incubated for 3 h at 37 °C. To convert this into an inter arrange product, 0.75 ml of ice cold 65% H₂SO₄ was added. Then mixed well and cooled. The final product was read at 520 nm.