Procedure
[NOTEPerform the following test in subdued light.
] Transfer about 40 mg of Hydroxocobalamin, accurately weighed, to a 25-mL volumetric flask, dissolve in carbon dioxide-free water, dilute with carbon dioxide-free water to volume, and mix. Transfer 1.0-mL portions of this solution to each of two glass-stoppered test tubes. To one of the tubes, designated
B, add 3.0 mL of
pH 4.0 Buffer, and mix. To the other tube, designated
U, add 3.0 mL of
pH 9.3 Buffer, and mix. Determine the absorbance of solution
U, in a 1-cm cell, at the wavelength of maximum absorbance at about 550 nm, with a suitable spectrophotometer, using solution
B as the blank. Calculate the percentage of pH-dependent cobalamins, as hydroxocobalamin, by the formula:
(100,000A) / (19.66W),
in which
A is the absorbance of solution
U, and
W is the weight, in mg, of Hydroxocobalamin taken: the content, calculated on the dried basis, is between 95.0% and 102.0%.