Test solution: 10mg per mL,in alcohol.

Internal standard solution— Dilute 0.5mLof n-propyl alcohol with water to 100.0mL.

Standard solution— Pipet 1mLof ethyl acetate and 0.5mLof n-propyl alcohol into a 1000-mLvolumetric flask,add water to volume,and mix.Each mLof Standard solutioncontains 0.90mg of ethyl acetate.

Test solution— Place about 250mg of Colchicine,accurately weighed,in a 10-mLvolumetric flask,dissolve in about 8mLof water,and add 1.0mLof Internal standard solution.Add water to volume,and mix.

Procedure— Determine appropriate sensitivity settings on a gas chromatograph (see Chromatography á621ñ)fitted with a 4-mm ×1.5-m column packed with 20%(w/v)phase G14on support S1,maintaining the column temperature at 75,using nitrogen as the carrier gas,and using a flame-ionization detector.Inject the Standard solutionand 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 45mLof 0.5Mmonobasic potassium phosphate with water to 450mL.Add about 530mLof methanol,cool to room temperature,and add methanol to bring the volume to 1000mL.Adjust with 0.5Mphosphoric acid to a pHof 5.5±0.05,and pass through a 0.45-µm membrane filter.

Standard preparation— Dissolve an accurately weighed quantity of USP Colchicine RSin 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 6µg per mL.This solution is stable for 4months when stored tightly stoppered and in the dark.

Assay preparation— [NOTE—Prepare immediately before use.]Transfer about 60mg of Colchicine,accurately weighed,to a 500-mLvolumetric flask,dissolve in a mixture of methanol and water (1:1),dilute with the same mixture to volume,and mix.Pipet 5mLof this solution into a 100-mLvolumetric flask,dilute with the same mixture to volume,and mix.

Chromatographic system (see Chromatography á621ñ)—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 1mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the column efficiency is not less than 4500theoretical plates,the retention time for colchicine is between 5.5and 9.5minutes,and the relative standard deviation for replicate injections is not more than 2%.

Procedure— Separately inject equal volumes (about 20µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for all peaks recorded during 1.5times the retention time for colchicine.Calculate the quantity,in mg,of C22H25NO6in the Colchicine taken by the formula: in which Cis the concentration,in µg per mL,of the Standard preparation;and rUand rSare the colchicine peak responses obtained from the Assay preparationand the Standard preparation,respectively.