Hydroxocobalamin

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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C₆₂H₈₉CoN₁₃O₁₅P 1346.36

Cobinamide, dihydroxide, dihydrogen phosphate (ester), mono(inner salt), 3′-ester with 5,6-dimethyl-1-α-d-ribofuranosyl-1H-benzimidazole;

Cobinamide dihydroxide dihydrogen phosphate (ester), mono(inner salt), 3′-ester with 5,6-dimethyl-1-α-d-ribofuranosylbenzimidazole

CAS RN^®: 13422-51-0; UNII: Q40X8H422O.

1 DEFINITION

Hydroxocobalamin contains NLT 95% and NMT 102.0% of hydroxocobalamin (C₆₂H₈₉CoN₁₃O₁₅P), calculated on the anhydrous and solvent-free basis.

2 IDENTIFICATION

Change to read:

A. SPECTROSCOPIC IDENTIFICATION TESTS (197), Infrared Spectroscopy: 197K (CN 1-MAY-2020)

B. COBALT

Sample: 1 mg of Hydroxocobalamin

Analysis: Fuse the Sample 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 boil until dissolved. Add 1 drop of phenolphthalein TS, and add 2 N sodium hydroxide dropwise until a pink color appears. Add 0.5 g of sodium acetate, 0.5 mL of 1 N acetic acid, and 0.5 mL of a 10-mg/mL solution of nitroso R salt. 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 or orange-red color persists after boiling with the addition of hydrochloric acid.

C. The retention time of the major peak of the Sample solution corresponds to that of the Standard solution as obtained in the Assay.

3 ASSAY

3.1 PROCEDURE

Buffer solution: 15.6 g/L of sodium dihydrogen phosphate in water. Adjust with phosphoric acid (1 in 100) to a pH of 3.0.

Mobile phase: See Table 1.

Table 1

Diluent: Methanol, Buffer solution, and water (8:10:82)

Standard solution: 0.1 mg/mL of USP Hydroxocobalamin Chloride RS in Diluent. Mix to homogenize.

Sample solution: 0.1 mg/mL of Hydroxocobalamin in Diluent. Mix to homogenize.

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 351 nm

Column: 4.6-mm × 10-cm and 4.6-mm × 10-cm placed in series; packing L11

Column temperature: 30°

Flow rate: 2.0 mL/min

Injection volume: 20 µL

System suitability

Sample: Standard solution

[Note—The retention time of hydroxocobalamin is about 16 min.]

Suitability requirements

Tailing factor: NMT 2.5

Relative standard deviation: NMT 1.0% from replicate injections

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of hydroxocobalamin (C₆₂H₈₉CoN₁₃O₁₅P) in the portion of Hydroxocobalamin taken:

Result = (r_U/r_S) × (C_S/ C_U) × (M_U/M_S) × 100

r_U = peak response from the Sample solution

r_S = peak response from the Standard solution

Cₛ = concentration of USP Hydroxocobalamin Chloride RS in the Standard solution (mg/mL)

C_U = concentration of Hydroxocobalamin in the Sample solution (mg/mL)

M_U = molecular weight of hydroxocobalamin, 1346.4

M_S = molecular weight of hydroxocobalamin chloride, 1382.8

Acceptance criteria: 95%–102.0% on the anhydrous and solvent-free basis

4 IMPURITIES

4.1 RELATED COMPOUNDS

Buffer solution, Mobile phase, Diluent, and Chromatographic system: Proceed as directed in Assay.

System suitability solution: 0.75 mg/mL of USP Hydroxocobalamin Chloride RS in Diluent

Solution for quantitation: 7.5 µg/mL of USP Hydroxocobalamin Chloride RS in Diluent, diluted from the System suitability solution

Solution for the disregard limit: 0.75 µg/mL of USP Hydroxocobalamin Chloride RS in Diluent, diluted from the Solution for quantitation

Sample solution: 0.75 mg/mL of Hydroxocobalamin in Diluent

System suitability

Sample: System suitability solution

Suitability requirement

Peak-to-valley ratio:² NLT 2.0 between hydroxocobalamin and its closest impurity

Analysis

Sample: Sample solution

Measure the peak responses of the Sample solution. Disregard the peaks with a response smaller than 0.7 times that of the peak of the Solution for the disregard limit.

Calculate the percentage of each impurity in the portion of Hydroxocobalamin taken:

Result = (r_U/r_S) × (C_S/C_U) × (1/F) × (M_r1/M_r2) × 100

r_U = peak response of each individual impurity from the Sample solution

r_S = peak response from the Solution for quantitation

C_S = concentration of USP Hydroxocobalamin Chloride RS in the Solution for quantitation

C_U = concentration of Hydroxocobalamin in the Sample solution

F = response factor, 0.8 for cyanocobalamin and 1.0 for any other impurity

M_r1 = molecular weight of hydroxocobalamin, 1346.4

M_r2 = molecular weight of hydroxocobalamin chloride, 1382.8

Acceptance criteria: See Table 2.

Table 2

4.2 RESIDUAL SOLVENTS (467)

Acceptance criteria

Acetone: NMT 0.5%.

[Note-For Acceptance criteria for any other residual solvents, see the chapter.]

5 SPECIFIC TESTS

PH (791)

Sample solution: 20 mg/mL of solution

Acceptance criteria: 8.0-10.0

WATER DETERMINATION, Method la(921): 14.0%~18.0%

6 ADDITIONAL REQUIREMENTS

PACKAGING AND STORAGE: Preserve in tight, light-resistant containers, and store in a cool, dry place. Do not freeze.

USP REFERENCE STANDARDS (11)

USP Hydroxocobalamin Chloride RS

¹ Chromolith columns (Merck) are based on a reversed phase employing monolithic C-bonded silica with bimodal pore structure (macropore 2 µm; mesopore 13 nm). Example: Merck Chromolith Performance RP-18 endcapped, 4.6-mm × 10-cm, product #1021290001.

²

Peak-to-valley ratio:

p/v = H_p/H_v

p/v = peak-to-valley ratio

H_p = height above the extrapolated baseline of the minor peak

H_v = height above the extrapolated baseline at the lowest point of the curve separating the minor and major peaks

(See Chromatography 〈621〉, Peak-to-Valley Ratio.) NLT 2.0 between hydroxocobalamin and its closest impurity