A: Transfer a portion of finely powdered Tablets,equivalent to about 100mg of penicillamine,to a 10-mLvolumetric flask,dilute with methanol to volume,add 2drops of 3Nhydrochloric acid,mix,and filter.Use the filtrate as the test solution.Prepare a Standard solution by dissolving 100mg of USP Penicillamine RSin 10mLof methanol,adding 2drops of 3Nhydrochloric acid,and mixing.Separately apply 10-µLportions of the test solution and the Standard solution to a suitable thin-layer chromatographic plate (see Chromatography á621ñ)coated with a 0.25-mm layer of chromatographic silica gel mixture,heated at 105for 30minutes,and allowed to cool before use.Allow the spots to dry,and develop the chromatogram in a solvent system consisting of a mixture of butyl alcohol,glacial acetic acid,and water (8:2:2)until the solvent front has moved about three-fourths of the length of the plate.Remove the plate,mark the solvent front,allow the solvent to evaporate,and place the plate in an atmosphere of iodine vapors.After a few minutes,spray the plate with a 1in 300solution of ninhydrin in dehydrated alcohol,heat it at 105for about 10minutes,allow it to cool,and examine it:the RFvalues,colors,and intensities of the principal spots obtained from the test solution correspond to those obtained from the Standard solution.

B: Aportion of powdered Tablets responds to Identificationtest Cunder Penicillamine.

Medium: 0.5%edetate disodium and 0.05%sodium lauryl sulfate solution;900mL.

Apparatus 1: 100rpm.

Time: 60minutes.

Mobile phase— Prepare a filtered and degassed solution of 0.01Mdibasic sodium phosphate and 0.01Mmonobasic potassium phosphate (60:40).If necessary,adjust the solution by the addition of 0.01Mdibasic sodium phosphate or 0.01Mmonobasic potassium phosphate to a pHof 7.0±0.1.

Standard solution— Prepare a solution of USP Penicillamine RSin 0.5%edetate disodium and 0.05%sodium lauryl sulfate solution having an accurately known concentration of about 0.28mg per mL.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 254-nm detector and a 3.9-mm ×30-cm column that contains packing L1.The flow rate is about 1mLper minute.Chromatograph replicate injections of the Standard solution,and record the peak responses as directed for Procedure:the relative standard deviation is not more than 2.0%,and the resolution factor between the solvent peak and penicillamine is not less than 1.5.

Procedure— Separately inject equal volumes (about 80µL)of the Standard solutionand a filtered portion of the solution under test into the chromatograph,record the chromatograms,measure the responses for the major peaks,and calculate the amount of C5H11NO2Sdissolved per Tablet.

Tolerances— Not less than 80%(Q)of the labeled amount of C5H11NO2Sis dissolved in 60minutes.

Diluent— Prepare as directed in the Assay.

Mobile phase,Resolution solution,and Chromatographic system— Proceed as directed in the Assayunder Penicillamine.

Standard preparation— Dissolve an accurately weighed quantity of USP Penicillamine Disulfide RSin Diluentto obtain a solution having a known concentration of about 0.025mg per mL.

Test preparation— Use the Assay preparation.

Chromatographic system— Proceed as directed in the Assayunder Penicillamine.Chromatograph the Standard preparation,and record the penicillamine disulfide peak responses as directed for Procedure:the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— [NOTE—Use peak areas where peak responses are indicated.]Separately inject equal volumes (about 20µL)of the Standard preparationand the Test preparationinto the chromatograph,record the chromatograms,and measure the responses for the penicillamine disulfide peaks.Calculate the percentage of penicillamine disulfide (C10H20N2O4S2)in the portion of Tablets taken by the formula: in which Cis the concentration,in mg per mL,of USP Penicillamine Disulfide RSin the Standard preparation,Lis the quantity,in mg,of penicillamine in each Tablet based on the labeled amount,and rUand rSare the penicillamine disulfide peak responses obtained from the Test preparationand the Standard preparation,respectively:not more than 3.0%of penicillamine disulfide is found.

Diluent— Dissolve 5.0g of edetate disodium in water to make 1000mLof solution.

Mobile phase,Resolution solution,and Chromatographic system— Proceed as directed in the Assayunder Penicillamine.

Standard preparation— Dissolve an accurately weighed quantity of USP Penicillamine RSin Diluentto obtain a solution having a known concentration of about 1.25mg per mL.

Assay preparation— Weigh and finely powder not less than 20Tablets.Transfer an accurately weighed portion of the powder,equivalent to about 250mg of penicillamine,to a 200-mLvolumetric flask,add about 150mLof Diluent,shake for 5minutes,and allow the mixture to stand for 90minutes.Dilute with Diluentto volume,and mix.Filter a portion of this solution through a suitable filter of 1µm or finer porosity,and use the clear filtrate as the Assay preparation.

Procedure— Proceed as directed for Procedurein the Assayunder Penicillamine.Calculate the quantity,in mg,of penicillamine (C5H11NO2S)in the portion of Tablets taken by the formula: in which Cis the concentration,in mg per mL,of USP Penicillamine RSin the Standard preparation,and rUand rSare the penicillamine peak responses obtained from the Assay preparationand the Standard preparation,respectively.