Colchicine

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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C₂₂H₂₅NO₆   399.44

Acetamide, N-(5,6,7,9-tetrahydro-1,2,3,10-tetramethoxy-9-oxobenzo[a]heptalen-7-yl)-, (S)-;

Colchicine CAS RN®: 64-86-8; UNII: SML2Y3J35T.

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1 DEFINITION

Colchicine is an alkaloid contained in various species of Colchicum and in other genera. It contains NLT 94.0% and NMT 102.0% of colchicine (C₂₂H₂₅NO₆), calculated on the anhydrous, solvent-free basis.

[Caution-Colchicine is extremely poisonous.]

2 IDENTIFICATION

2.1 A. Spectroscopic Identification Tests 〈197〉, Infrared Spectroscopy: 197K

[Note-Disregard any peak occurring at 1735 cm⁻¹.]

3 ASSAY

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3.1 Procedure

[Note-Perform all dilutions in low-actinic glassware.]

Mobile phase: Dilute 45 mL of 0.5 M monobasic potassium phosphate with water to 450 mL. Add 530 mL of methanol, cool to room temperature, and add methanol to bring the volume to 1000 mL. Adjust with 0.5 M phosphoric acid to a pH of 5.5 ± 0.05, and pass through a 0.45-µm membrane filter.

Standard solution: 6 µg/mL of USP Colchicine RS in a mixture of methanol and water (1:1). This solution is stable for 4 months when stored tightly stoppered and in the dark.

Sample solution: 6 µg/mL of Colchicine in a mixture of methanol and water (1:1)

Chromatographic system

• (See Chromatography 〈621〉, System Suitability.)
• Mode: LC
• Detector: UV 254 nm
• Column: 4.6-mm × 25-cm; packing L7
• Flow rate: 1.0 mL/min
• Injection volume: 20 µL

System suitability

• Sample: Standard solution
• Suitability requirements
• Column efficiency: NLT 4500 theoretical plates
• Relative standard deviation: NMT 2%

Analysis

Samples: Standard solution and Sample solution

Measure the responses for all peaks recorded during 1.5 times the retention time for colchicine.

Calculate the percentage of colchicine (C₂₂H₂₅NO₆) in the portion of Colchicine taken:

Result = (r_u/r_s) × (C_s/C_u) × 100

r_u = peak response from the Sample solution

r_s = peak response from the Standard solution

C_s = concentration of USP Colchicine RS in the Standard solution (µg/mL)

C_u = concentration of Colchicine in the Sample solution (µg/mL)

Acceptance criteria: 94.0%-102.0% on the anhydrous, solvent-free basis

4 IMPURITIES

4.1 Limit of Colchiceine

Analysis: To 5 mL of a 10-mg/mL solution of colchicine, add 2 drops of ferric chloride TS.

Acceptance criteria: No definite green color is produced.

4.2 Limit of Ethyl Acetate

Internal standard solution: Dilute 0.5 mL of n-propyl alcohol with water to 100 mL.

Standard solution: Transfer 1.0 mL of ethyl acetate, 0.5 mL of diacetone alcohol, and 0.5 mL of n-propyl alcohol to a 1000-mL volumetric flask and dilute with water to volume. Mix well. This solution contains 0.9 mg/mL of ethyl acetate.

Sample solution: Add 250 mg of Colchicine, accurately weighed, to a 10-mL volumetric flask, and dissolve in about 8.0 mL of water. Add 1.0 mL of Internal standard solution, dilute with water to volume, and mix well.

Chromatographic system

• (See Chromatography (621), System Suitability.)
• Mode: GC
• Detector: Flame ionization
• Column: 0.53 - mm * 30 - m coated with a 1-µm film of phase G16
• Temperatures
• Injection port: 180°
• Detector: 220°
• Column: See Table 1.

• Carrier gas: Helium
• Flow rate: 5.72 mL/min
• Injection volume: 2 µL
• Injection type: Split ratio, 15:1

System suitability

• Sample: Standard solution
• Suitability requirements
• Relative standard deviation: NMT 15.0% for the peak response ratios of ethyl acetate to the n-propyl alcohol peak, and of diacetone alcohol to the n-propyl alcohol peak, in replicate injections

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of ethyl acetate in the portion of Colchicine taken:

Result = (R_u/R_s) × (C_s/C_u) × 100

R_u = peak response ratio of ethyl acetate to n-propyl alcohol from the Sample solution

R_s = peak response ratio of ethyl acetate to n-propyl alcohol from the Standard solution

C_s = concentration of ethyl acetate in the Standard solution (mg/mL)

C_u = concentration of Colchicine in the Sample solution (mg/mL)

Acceptance criteria: NMT 8.0%

4.3 Chromatographic Purity

Procedure: Proceed as directed in the Assay.

Acceptance criteria: NMT 5.0% other than that due to colchicine, eluting within 1.5 times the retention time for colchicine

4.4 Residual Solvents 〈467〉

Meets the requirements, except that the limit of chloroform is 100 ppm

5 SPECIFIC TESTS

5.1 Optical Rotation 〈781S〉, Procedures, Specific Rotation

Sample solution: 10 mg/mL, in alcohol

Acceptance criteria: −240° to −250°, calculated on the anhydrous, solvent-free basis

5.2 Water Determination 〈921〉, Method I

NMT 2.0%

6 ADDITIONAL REQUIREMENTS

6.1 Packaging and Storage

Preserve in tight, light-resistant containers.

6.2 USP Reference Standards 〈11〉

USP Colchicine RS