(kol' chi seen).
Acetamide, N-(5,6,7,9-tetrahydro-1,2,3,10-tetramethoxy-9-oxobenzo[a]heptalen-7-yl)-, (S)-.
» Colchicine is an alkaloid contained in various species of Colchicum and in other genera. It contains not less than 94.0 percent and not more than 101.0 percent of C22H25NO6, calculated on the anhydrous, solvent-free basis.
[CautionColchicine is extremely poisonous. ]
Packaging and storage Preserve in tight, light-resistant containers.
USP Reference standards 11
USP Colchicine RS
Identification, Infrared Absorption 197K [noteDisregard any peak occurring at 1735 cm1. ]
Specific rotation 781S: between 240 and 250, calculated on the anhydrous, solvent-free basis.
Test solution: 10 mg per mL, in alcohol.
Water, Method I 921: not more than 2.0%.
Limit of colchiceine To 5 mL of a solution (1 in 100) add 2 drops of ferric chloride TS: no definite green color is produced.
Limit of ethyl acetate
Internal standard solution Dilute 0.5 mL of n-propyl alcohol with water to 100.0 mL.
Standard solution Pipet 1 mL of ethyl acetate and 0.5 mL of n-propyl alcohol into a 1000-mL volumetric flask, add water to volume, and mix. Each mL of Standard solution contains 0.90 mg of ethyl acetate.
Test solution Place about 250 mg of Colchicine, accurately weighed, in a 10-mL volumetric flask, dissolve in about 8 mL of water, and add 1.0 mL of Internal standard solution. Add water to volume, and mix.
Procedure Determine appropriate sensitivity settings on a gas chromatograph (see Chromatography 621) fitted with a 4-mm × 1.5-m column packed with 20% (w/v) phase G14 on support S1, maintaining the column temperature at 75, using nitrogen as the carrier gas, and using a flame-ionization detector. Inject the Standard solution and the Test solution, determine the peak height for ethyl acetate relative to the peak height for n-propyl alcohol, and calculate the percentage, by weight, of ethyl acetate in the portion of Colchicine taken: not more than 8.0% is found.
Chromatographic purity The sum of the responses of any peaks other than that due to colchicine, eluting within 1.5 times the retention time for colchicine, is not more than 5.0% of the sum of all responses, obtained as directed in the Assay.
Residual solvents 467: meets the requirements, except that the limit of chloroform is 100 ppm.
Assay [notePerform all dilutions in low-actinic glassware. ]
Mobile phase Dilute 45 mL of 0.5 M monobasic potassium phosphate with water to 450 mL. Add about 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 preparation Dissolve an accurately weighed quantity of USP Colchicine RS in a mixture of methanol and water (1:1), and dilute quantitatively and stepwise with the same mixture to obtain a solution having a known concentration of about 6 µg per mL. This solution is stable for 4 months when stored tightly stoppered and in the dark.
Assay preparation [notePrepare immediately before use. ] Transfer about 60 mg of Colchicine, accurately weighed, to a 500-mL volumetric flask, dissolve in a mixture of methanol and water (1:1), dilute with the same mixture to volume, and mix. Pipet 5 mL of this solution into a 100-mL volumetric flask, dilute with the same mixture to volume, and mix.
Chromatographic system (see Chromatography 621)The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm × 25-cm column that contains packing L7. The flow rate is about 1 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the column efficiency is not less than 4500 theoretical plates, the retention time for colchicine is between 5.5 and 9.5 minutes, and the relative standard deviation for replicate injections is not more than 2%.
Procedure Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for all peaks recorded during 1.5 times the retention time for colchicine. Calculate the quantity, in mg, of C22H25NO6 in the Colchicine taken by the formula:
10C(rU / rS)in which C is the concentration, in µg per mL, of the Standard preparation; and rU and rS are the colchicine peak responses obtained from the Assay preparation and the Standard preparation, respectively.
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USP35NF30 Page 2763Pharmacopeial Forum: Volume No. 33(3) Page 396