Probenecid standard solution— Prepare a solution of USP Probenecid RSin chloroform having a concentration of about 1mg per mL.

Colchicine standard solution— Prepare a solution of USP Colchicine RSin chloroform having a concentration of about 1mg per mL.

Probenecid test solution— Using a portion of finely powdered Tablets,prepare a filtered solution in chloroform having a concentration of about 1mg of probenecid per mL.

Colchicine test solution— Transfer a quantity of finely powdered Tablets,equivalent to about 0.5mg of colchicine,to a container,add 15mLof water,mix,and filter,collecting the filtrate.Extract the filtrate with 25mLof chloroform,and evaporate the chloroform extract to a volume of about 1mL.

Procedure (see Chromatography á621ñ)—Apply separately 5-µLportions of the Probenecid test solutionand the Probenecid standard solution,a 7-µLportion of the Colchicine test solution,and a 3.5-µLportion of the Colchicine standard solutionto a thin-layer chromatographic plate,coated with a 0.25-mm layer of chromatographic silica gel mixture.Allow the spots to dry,and develop the chromatogram in a solvent system consisting of a mixture of methanol and ammonium hydroxide (100:1.5)until the solvent front has moved about three-fourths of the length of the plate.Remove the plate from the developing chamber,allow the solvent to evaporate,and view the plate under short-wavelength UVlight:the RFvalue of the principal spot in the chromatogram obtained from the Probenecid test solutioncorresponds to that obtained from the Probenecid standard solution.The RFvalue of the principal spot in the chromatogram obtained from the Colchicine test solutioncorresponds to that obtained from the Colchicine standard solution.

Medium: pH6.8phosphate buffer (see under Buffer Solutionsin the section Reagents,Indicators,and Solutions);900mL.

Apparatus 2: 50rpm.

Time: 30minutes.

Procedure for probenecid— Determine the amount of C13H19NO4Sdissolved from UVabsorbances at the wavelength of maximum absorbance at about 244nm of filtered portions of the solution under test,suitably diluted with 0.1Nsodium hydroxide,if necessary,in comparison with a Standard solution having a known concentration of USP Probenecid RS.

Procedure for colchicine— Extract a filtered 200-mLportion of the solution under test with two 25-mLportions of chloroform,collecting the chloroform extracts in a suitable flask.Evaporate the combined extracts to a small volume,and transfer to a 10-mLvolumetric flask.Rinse the flask with small portions of chloroform,and add the rinsings to the 10-mLvolumetric flask.Dilute with chloroform to volume,and mix.Determine the amount of C22H25NO6dissolved from absorbances,at the wavelength of maximum absorbance at about 350nm,of this solution,using chloroform as the blank,in comparison with a Standard solution in chloroform having a known concentration of USP Colchicine RS.

Tolerances— Not less than 80%(Q)of the labeled amounts of C22H25NO6and C13H19NO4Sare dissolved in 30minutes.

Standard preparation— Dissolve an accurately weighed quantity of USP Probenecid RSon 0.1Nsodium hydroxide,and dilute quantitatively and stepwise with 0.1Nsodium hydroxide to obtain a solution having a known concentration of about 10µg per mL.

Assay preparation— Weigh and finely powder not less than 20Tablets.Weigh accurately a portion of the powder,equivalent to about 250mg of probenecid,and transfer to a 250-mLvolumetric flask.Add 0.1Nsodium hydroxide to volume,and mix.Filter a portion of the solution,discarding the first 20mLof the filtrate,pipet 2mLof the filtrate into a 200-mLvolumetric flask,dilute with 0.1Nsodium hydroxide to volume,and mix.

Procedure— Concomitantly determine the absorbances of the Assay preparationand the Standard preparationat the wavelength of maximum absorbance at about 244nm,with a suitable spectrophotometer,using 0.1Nsodium hydroxide as the blank.Calculate the quantity,in mg,of C13H19NO4Sin the portion of Tablets taken by the formula: in which Cis the concentration,in µg per mL,of USP Probenecid RSin the Standard preparation,and AUand ASare the absorbances of the Assay preparationand the Standard preparation,respectively.

Alcoholic sodium carbonate solution— Dissolve 5.0g of anhydrous sodium carbonate in 900mLof water,add 100mLof isopropyl alcohol,and mix.

Standard preparation— Dissolve an accurately weighed quantity of USP Colchicine RSin Alcoholic sodium carbonate solution,and dilute quantitatively and stepwise with Alcoholic sodium carbonate solutionto obtain a solution having a known concentration of about 10µg per mL.

Assay preparation— Weigh and finely powder not less than 20Probenecid and Colchicine Tablets.Weigh accurately a portion of the powder,equivalent to about 1mg of colchicine,and transfer to a 100-mLvolumetric flask.Add 75mLof Alcoholic sodium carbonate solution,shake for 30minutes,dilute with Alcoholic sodium carbonate solution to volume,mix,and filter,discarding the first 20mLof the filtrate.

Procedure— Concomitantly determine the absorbances of the Assay preparationand the Standard preparationat the wavelength of maximum absorbance at about 350nm,with a suitable spectrophotometer,using Alcoholic sodium carbonate solutionas the blank.Calculate the quantity,in mg,of C22H25NO6in the portion of Tablets taken by the formula: in which Cis the concentration,in µg per mL,of USP Colchicine RSin the Standard preparation,and AUand ASare the absorbances of the Assay preparationand the Standard preparation,respectively.