Cyanocobalamin

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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1 DEFINITION

Cyanocobalamin contains NLT 96.0% and NMT 102.0% of cyanocobalamin (C₆₃H₈₈CoN₁₄O₁₄P), calculated on the dried basis. 

2 IDENTIFICATION

2.1 A. SPECTROSCOPIC IDENTIFICATION TESTS (197), Ultraviolet-Visible Spectroscopy: 197U

Wavelength range: 200-700 nm

Sample solution: Prepare as directed in the Assay.

Acceptance criteria: The absorption spectrum exhibits maxima at 278 ± 1, 361 ± 1, and 550 ± 2 nm. The absorbance ratio A₃₆₁/A₂₇₈ Is 1.70-1.90, and the absorbance ratio A₃₆₁/A₂₇₈ is 3.15-3.40.

2.2 B.

Sample solution: Fuse 1 mg of Cyanocobalamin with 50 mg of potassium pyrosulfate in a porcelain crucible. Cool, break up the mass with a glass rod, add 3 mL of water, and dissolve by boiling.

Analysis: Add 1 drop of phenolphthalein TS, and add sodium hydroxide solution (100 mg/mL), dropwise, until just pink. Add 500 mg of sodium acetate, 0.5 mL of 1 N acetic acid, and 0.5 mL of nitroso R salt solution (2 mg/mL). Add 0.5 mL of hydrochloric acid, and boil for 1 min.

Acceptance criteria: A red or orange-red color appears immediately after the addition of nitroso R salt. The red color persists after boiling with the addition of hydrochloric acid.

2.3 C. HPLC

Mobile phase and Chromatographic system: Proceed as directed in the test for Related Compounds.

Standard solution: 50 µg/mL of Cyanocobalamin from USP Cyanocobalamin (Crystalline) RS in Mobile phase. Use within 1 h.

Sample solution: 50 µg/mL of Cyanocobalamin in Mobile phase. Use within 1 h.

Acceptance criteria: The retention time of the major peak of the Sample solution corresponds to that of the Standard solution.

3 ASSAY

3.1 PROCEDURE

Sample solution: 30 µg/mL of Cyanocobalamin in water

Instrumental conditions

(See Ultraviolet-Visible Spectroscopy (857).)

Mode: UV

Analytical wavelength: 361 nm

Cell: 1 cm

Blank: Water

Analysis

Sample: Sample solution

Calculate the percentage of cyanocobalamin (C₆₃H₈₈CoN₁₄O₁₄P) in the portion of Cyanocobalamin taken:

Result = A_u/(A_s x C_u)

Au = absorbance of the Sample solution

As = specific absorbance (E^1%) of cyanocobalamin at 361 nm (100 mL . g^-1 . cm^-1), 207

C_u = concentration of Cyanocobalamin in the Sample solution (g/mL)

Acceptance criteria: 96.0%-102.0% on the dried basis

4 IMPURITIES

Change to read:

4.1 RELATED COMPOUNDS

Solution A: 10 g/L of disodium hydrogen phosphate, dodecahydrate (USP 1-Dec-2023) in water

Mobile phase: Mixture of methanol and Solution A (26.5: 73.5). Adjust with phosphoric acid to a pH of 3.5.

System suitability solution: Dissolve 25 mg of Cyanocobalamin in 10 mL of water, warming if necessary. Allow to cool, add 5 mL of a 1.0-g/L solution of tosylchloramide sodium and 0.5 mL of 0.05 M hydrochloric acid, and then dilute with water to 25 mL. Shake and allow to stand for 5 min. Dilute 1.0 mL of this solution with Mobile phase to 10 mL, and inject immediately.

Quantitative limit solution: 1 µg/mL of Cyanocobalamin in Mobile phase. Use within 1 h.

Sample solution: 1 mg/mL of Cyanocobalamin in Mobile phase. Use within 1 h.

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

Detector: UV 361 nm

Column: 4.6-mm × 25-cm; 5-µm packing L7

Column temperature: 35°

Flow rate : 0.8 mL/min

Injection volume: 20 µL

System suitability

Samples: System suitability solution and Quantitative limit solution

[NOTE-The System suitability solution should exhibit two major peaks, cyanocobalamin and 78,8ẞ-lactocyanocobalamin. The relative retention times for the two peaks are 1.0 and 1.2, respectively.] FICIAL

Suitability requirements

Resolution : NLT 2.5 between cyanocobalamin and 78,8ẞ-lactocyanocobalamin, System suitability solution

Signal-to-noise ratio: NLT 5.0 for the major peak, Quantitative limit solution

Analysis

Sample: Sample solution

[NOTE-The run time should be at least three times the retention time of the cyanocobalamin peak.]

Identify the impurities listed in Table 1, and measure the peak responses.

Calculate the percentage of individual impurities in the portion of Cyanocobalamin taken:

Result = (r_U/r_T) x 100

r_U = peak response of each impurity from the Sample solution

r_T = sum of all the peak responses from the Sample solution 

Acceptance criteria: See Table 1. [NOTE-Disregard any peak less than 0.1%.]

Table 1

5 SPECIFIC TESTS

5.1 LOSS ON DRYING (731)

Sample: 25 mg

Analysis: Dry the Sample in a suitable vacuum drying apparatus at 105° and at a pressure of NMT 5 mm of mercury for 2 h.

Acceptance criteria: NMT 12.0%

6 ADDITIONAL REQUIREMENTS

6.1 PACKAGING AND STORAGE:

Preserve in tight, light-resistant containers, and store at controlled room temperature.

6.2 USP REFERENCE STANDARDS (11)

USP Cyanocobalamin (Crystalline) RS