Test solution: 10 mg per mL, in alcohol.

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.

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— [note—Prepare 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: 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.