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

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

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

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

Procedure (see Chromatography 621)—Apply separately 5-µL portions of the Probenecid test solution and the Probenecid standard solution, a 7-µL portion of the Colchicine test solution, and a 3.5-µL portion of the Colchicine standard solution to 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 UV light: the RF value of the principal spot in the chromatogram obtained from the Probenecid test solution corresponds to that obtained from the Probenecid standard solution. The RF value of the principal spot in the chromatogram obtained from the Colchicine test solution corresponds to that obtained from the Colchicine standard solution.

Medium: pH 6.8 phosphate buffer (see under Buffer Solutions in the section Reagents, Indicators, and Solutions); 900 mL.

Apparatus 2: 50 rpm.

Time: 30 minutes.

Procedure for probenecid— Determine the amount of C13H19NO4S dissolved from UV absorbances at the wavelength of maximum absorbance at about 244 nm of filtered portions of the solution under test, suitably diluted with 0.1 N sodium hydroxide, if necessary, in comparison with a Standard solution having a known concentration of USP Probenecid RS.

Procedure for colchicine— Extract a filtered 200-mL portion of the solution under test with two 25-mL portions of chloroform, collecting the chloroform extracts in a suitable flask. Evaporate the combined extracts to a small volume, and transfer to a 10-mL volumetric flask. Rinse the flask with small portions of chloroform, and add the rinsings to the 10-mL volumetric flask. Dilute with chloroform to volume, and mix. Determine the amount of C22H25NO6 dissolved from absorbances, at the wavelength of maximum absorbance at about 350 nm, 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 C22H25NO6 and C13H19NO4S are dissolved in 30 minutes.

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

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

Procedure— Concomitantly determine the absorbances of the Assay preparation and the Standard preparation at the wavelength of maximum absorbance at about 244 nm, with a suitable spectrophotometer, using 0.1 N sodium hydroxide as the blank. Calculate the quantity, in mg, of C13H19NO4S in the portion of Tablets taken by the formula: in which C is the concentration, in µg per mL, of USP Probenecid RS in the Standard preparation, and AU and AS are the absorbances of the Assay preparation and the Standard preparation, respectively.

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

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

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

Procedure— Concomitantly determine the absorbances of the Assay preparation and the Standard preparation at the wavelength of maximum absorbance at about 350 nm, with a suitable spectrophotometer, using Alcoholic sodium carbonate solution as the blank. Calculate the quantity, in mg, of C22H25NO6 in the portion of Tablets taken by the formula: in which C is the concentration, in µg per mL, of USP Colchicine RS in the Standard preparation, and AU and AS are the absorbances of the Assay preparation and the Standard preparation, respectively.