VITAMIN B12 (CYANOCOBALAMIN)

BY NEELOFAR TABASSUM

INTRODUCTION
1. cyanocobalamin is last member of vitamin B complex. It is a water soluble vitamin.

2.

It occurs as dark red crystals or as a crystalline powder. The crystals rapidly absorb moisture to the extent of 10% to 12% at 50% relative humidity.
It is soluble in water and alcohol but insoluble in acetone, chloroform and ether.

3.

SOURCES :
Vitamin B12 has been found only in animals; the chief source is liver, although it is also present in milk, meat, eggs, fish, oysters and clams. Under certain dietary conditions, vitamin B12 may be synthesized by

the intestinal microorganisms.

In general, cyanocobalamin is not present in plant foods except in Spirulina, blue-green algae. Gastric juice contained a factor (intrinsic factor),help in absorption of cyanocobalamin.

Human requirements:
The recommended daily allowance of vitamin B12 is 2 to 4 g for children. 5 g for men and women. Pregnant and lactating mothers require 8 g and 6g daily.

DEFICIENCY OF VITAMIN B12

A nutritional deficiency of this vitamin is usually not observed in most cases of deficiency stem is from failure to absorb the vitamin. The typical deficiency disease, adult pernicious anemia ( anemia caused by failure of erythrocyte formation), is characterized by

R.B.Cs. becoming abnormally large and fewer in number (13

million per cubic millimeter instead of the normal 45 million).

SYMPTOMS: patient weakens, loses weight, demyelinization of

the large nerve fibers of the spinal cord, ands to death.

STRUCTURE OF VITAMIN B12

Mol wt:1500 Molecular formula:

Vitamin B12 is neutral and contains a cyano group; the

cobalt is trivalent and has a coordination number of six. cyano group may be replaced by a variety of ions to give modifications such as chlorocobalamin and hydroxycobalamin.

Acid hydrolysis of cyanocobalamin produces one equivalent of 5,6-dimethylbenzimidazole.

ASSAY METHODS:
cyanocobalamin is assayed by following methods Biological method : Microbial assay method Chemical method : Spectrophotometric method Coulter current distribution method Colorimetric dicyanide method Colorimetric cyanide method Radioactive tracer method(RIA).

SPECTOPHOTOMETRIC METHOD
Spectrophotometric assay is not specific. Red pigments and pseudo vitamin B12 in general show similar UV absorption. The method is useful in determining the conc. of crystalline vitamin B12 or its solution in the absence of interfering substances. Vit B12 exhibits absorption maxima within 1m at 278 and 361m and within 2m at 550m. The absorbance's of the solution prepared for assay are determined at the above maxima in a 1cm quartz cell using water as blank. The ratio A361(m)\A550(m) is between 2.83 and 3.45.

PROCEDURE
4 g of undried cyanocobalamin is accurately weighed on microbalance ,dissolved in sufficient water to make exactly 100ml,mixed well.

The absorbance of the solution is determined at 361m with a suitable spectrophotometer, by using water as blank. %cyanocobalamin=(10000 A/207W)100/(100-C)
A=Absorbance W=wt of sample(mg) C=%loss on drying A1%1cm =207.

CYANIDE COLORIMETRIC METHOD

The colorimetric cyanide assay can be used to determine vitamin B12 or total cobalamin in pharmaceutical product. The method is based upon the quantitative liberation of cyano group of vitamin B12 by illumination with visible light and the subsequent isolation and calorimetric determination of the liberated hydrogen cyanide. This procedure is applicable in the range of 2 to 20 mcg of vitamin B12.

DICYANIDE COLORIMETRIC METHOD

The colorimetric dicyanide method is applicable to the measurement of total cobalamins in pharmaceutical products, fermentation broths and concentrates. This method requires a sample of approximately 200mcg,and based on the difference between the visible spectrum of the dicyanide complex formed in solutions containing excesses of cyanide ions. Pigments and pseudovitamin B12 form dicyanide complexes and consequently the method is non specific.

REFRENCES:
PHARMCEUTICAL ANALYSIS -

TAKERU HIGUCHI
BIOCHEMISTRY- SUNJAY JAIN,J.L

JAIN.

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