Vitamin B12 (cyanocobalamine)
Cyanocobalamine is a cobalt co-ordinated complex with composition C16O4H86–92N14O13PC0.
Cyanocobalamine occurs as dark red crystals or as a crystalline powder red pigments and pseudo vitamin B12 in general show similar UV absorption. The method is useful in determineermination of the concentration of crystalline vitamin B12 or its solutions in the absence of interfering substances. It exhibits maxima at 278nm and 361nm. The absorbance of solution of cyanocobalamine prepared for the assay are determineermined at the above maxima in a 1cm quartz cell with a suitable spectrophotometer by using water as a blank.
Counter current distribution (CCD) method:
By this procedure, one can determine cyanocobalamine in a mixture with cobalamine, pigments and pseudocobalamine by means of pre treatment with cyanide. The total cobalamine content can be determine by conversion of cobalamine to cyanocobalamine. This method is useful in determine of purity of crystalline cyanocobalamine, the concentration of B12 in infection and the assay of oral solids and crude cobalamine concentrates.
Procedure: Sample extraction.
Sample not more than 0.5µg but at least 50µg of vitamin B12 is weighed and put in a 250ml flask + 50ml water+ 5ml of 10%KCn + 5ml of 2% NaNO2 and boiled for 5mins. 1ml of HCHO is added and filtered through filter paper and the residue is washed with suffering water to make upto 100ml.
Cresol butanol extraction :
To 50ml clear filtrate, add 5ml of a 1:1 cresol solution mixture. Shaken -> the lower part of mixture (cresol + CCl4) is drawn by 10ml syringe-> To the above ->add 4ml of 5NH2SO4 (crude pseudo cobalamine and pigments are removed in this step) -> To above, 74ml of Na2CO3+ 2%KCn solution and centrifuge. The top aqua layer is drawn by means of a syringe -> To washed cresol, add CCl4 solution + 20ml CHCL3 + 5ml butyl alcohol + 5mlwater shaken and centrifuged. The aqua layer is drawn into 25ml flask and solution is made upto 25ml with water.
Benzyl alcohol+ water are shaken, they are allowed to settle, and they are centrifuged before use to remove entrained liquid. The two liquids are referred to as the benzyl phase and the aqua phase respectively. The above extraction phase taken is treated with the benzyl phase and aqua phase. The aqua solutions are extracted and diluted to 10ml with water and vitamin B12 in the phases is determined spectrophotometrically or colourimetrically.
Cyanide colourimetric method:
Used to determine cyanocobalamine or total cobalamine in pharmaceutical products. The method is based on the Quantitative liberation of the cyano group of vitamin B12 by illumination with visible light and subsequent isolation and colourimetric determine of the liberated vitamin B12.
Di-cyanide colorimetric method:
This method which requires a sample of approximate 200µg is based on difference between the visible spectrum of vitamin B12 and the spectrum of di-cyanide complex formed in solutions containing excess cyanide ions.
Radioactive tracer method:
For the determination of vitamin B12 content of complex mixtures the use of a tracer form of the vitamin containing radioactive cobalt is used. It is used for determine of cyanocobalamin alone or to the determination of cyanocobalamin + those of vitamin B12 analogy which are readily convertible to cyanocobalamin by treatment with cyanide ion. The principle of the method depends upon the use of certain extractive and other procedures, designed to remove impurities which would interfere, and upon computing the percentage recovery of the vitamin from the percentage recovery of its radioactive compound. The method of measurement is by its absorption at 361nm.