A: Its gamma-ray spectrum is identical to that of a specimen of 58Co that exhibits major photopeaks at 0.511 MeV (annihilation radiation) and 0.811 MeV.

B: The retention time of the major peak in the radiochromatogram of the Test solution corresponds to that in the chromatogram of the Standard solution, as obtained in the test for Radiochemical purity.

Procedure for content uniformity— Determine the instrument response of each of 10 Capsules by measurement in a suitable counting assembly and under identical geometric conditions. Calculate the average radioactivity per Capsule. The radioactivities of none of the Capsules differ by more than 10% from the average. The relative standard deviation is less than 3.5%.

Mobile phase— Prepare a solution of 10.0 g of dibasic sodium phosphate in 1000 mL of water, and adjust with phosphoric acid to a pH of 3.5. Prepare a mixture of the solution so obtained and methanol (73.5:26.5), mix, and degas. Use within 2 days. Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard solution— Transfer about 10 mg of USP Cyanocobalamin RS, accurately weighed, to a 100-mL volumetric flask, dilute with Mobile phase to volume, and mix. Transfer 2.0 mL of the solution so obtained to a 100-mL volumetric flask, dilute with Mobile phase to volume, and mix.

Test solution— Dissolve the contents of one Capsule in 1 mL of water, allow to stand for about 10 minutes, and centrifuge. Use the supernatant.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 361-nm detector, a gamma detector adjusted for 58Co, and a 4.6-mm × 25-cm stainless steel column that contains 5-µm packing L7. The flow rate is about 1 mL per minute. Chromatograph the Standard solution, and record the peak responses as directed for Procedure.

Procedure— Inject about 100 µL of the Standard solution into the chromatograph, record the chromatogram for 30 minutes, and note the retention time of the cyanocobalamin peak. Inject 100 µL of the Test solution into the chromatograph, record the chromatogram for three times the retention time of cyanocobalamin, and measure the peak areas using the gamma detector. Calculate the percentage of cyanocobalamin present as cyanocobalamin 58Co in the portion of Capsules taken by the formula: in which rU is the peak area for cyanocobalamin 58Co obtained from the Test solution; and rs is the sum of all the peak areas in the radiochromatogram obtained from the Test solution: not less than 90% of the total radioactivity is found as cyanocobalamin 58Co.