A: Infrared Absorption á197Mñ(50mg in 300mg).

B: Dissolve 10mg in 5mLof water,and add 1drop of 5Nsodium hydroxide and 20mg of ninhydrin:a blue or violet-blue color is produced immediately.

C: Dissolve 20mg in 4mLof water,add 2mLof phosphotungstic acid solution (1in 10),and heat nearly to boiling:a deep blue color is produced immediately.

Test solution: 50mg per mL,in 1.0Nsodium hydroxide.

pH2.5Buffer— Dissolve 100g of monobasic potassium phosphate in water,add 0.2mLof hydrochloric acid,dilute with water to 1000mL,and mix.Adjust,if necessary,with phosphoric acid or with 10Npotassium hydroxide to a pHof 2.5.

Standard preparation— Prepare as directed for Penicillin Gin Table 2under Antibiotics—Microbial Assays á81ñ,except to prepare a final stock solution containing 100Penicillin G Units per mLand six test dilutions ranging from 0.005Penicillin G Unit per mLto 0.2Penicillin G Unit per mL,and to use a median dose of the Standard of 0.050Penicillin G Unit per mL.

Test preparation— Dissolve 1.0g in water to make 18.0mL,transfer 9.0mLof this solution to a separator,add 20mLof amyl acetate and 1mLof pH2.5Buffer,and shake.Allow the layers to separate,and draw off the aqueous layer into a second separator,retaining the amyl acetate extract in the first separator.Check the pHof the aqueous layer,and if it is greater than 3.0adjust it with hydrochloric acid to a pHof 2.5,and extract with 20mLof amyl acetate.Discard the aqueous layer,and add the amyl acetate extract to the first separator.Wash the combined amyl acetate extracts with 10mLof diluted pH2.5Buffer(1in 10),and discard the aqueous layer.Extract the amyl acetate with 10.0mLof Buffer No.1(see Phosphate Buffers and Other Solutionsin the section Media and Diluentsunder Antibiotics—Microbial Assays á81ñ).Use a portion of the buffer extract as Test solution A.To a 5-mLportion of the extract add 0.1mLof penicillinase solution,and incubate at 36to 37.5for 60minutes (Test solution B).

Preparation of inoculum— Prepare as directed under Antibiotics—Microbial Assays á81ñ,using Micrococcus luteus(ATCC9341)as the test organism,and an inoculum that gives clear sharp zones of inhibition 17mm to 21mm in diameter with the median dose level of the Standard.

Procedure— Proceed as directed for the Cylinder-Plate Methodunder Antibiotics—Microbial Assays á81ñ,using 10mLof Medium 1for the base layer and 4mLof inoculated Medium 4for the seed layer,and incubating the plates at 29to 31,except on each test plate to fill 2cylinders with Test solution A,2cylinders with Test solution B,and 2cylinders with the median dose of the Standard.If Test solution Ayields no zone of inhibition,the test is negative for penicillin.If Test solution Ayields a zone of inhibition and Test solution Bdoes not,penicillin is present.Determine its level from the standard curve:not more than 0.01Penicillin G Unit is found in each mLof Test solution A(0.2Penicillin G Unit per g).

NOTE—Mercuric dithizonate is light-sensitive.Perform this test in subdued light.

Dithizone stock solution— Dissolve 40mg of dithizone in 1000mLof chloroform.

Dithizone titrant— Dilute 30.0mLof Dithizone stock solutionwith chloroform to 100.0mL.This solution contains approximately 12mg of dithizone per liter.

Standard solution— Transfer 135.4mg of mercuric chloride to a 100-mLvolumetric flask,add 0.25Nsulfuric acid to volume,and mix.This solution contains the equivalent of 100mg of Hg in 100mL.

Diluted standard solution— Pipet 2mLof Standard solutioninto a 100-mLvolumetric flask,add 0.25Nsulfuric acid to volume,and mix.Each mLof this solution contains the equivalent of 20µg of Hg.

Standardization— Pipet 1mLof Diluted standard solutioninto a 250-mLseparator,and add 100mLof 0.25Nsulfuric acid,90mLof water,and 10mLof hydroxylamine hydrochloride solution (1in 5).Then add 1mLof edetate disodium solution (1in 50),1mLof glacial acetic acid,and 5mLof chloroform,shake for 1minute,allow to separate,and discard the chloroform layer.To the solution add Dithizone titrant,in portions of 0.3mLto 0.5mL,from a 10-mLburet.After each addition,shake the mixture 20times,and allow the chloroform layer to separate and discard it.Continue until an addition of Dithizone titrantremains green after the shaking.Calculate the quantity,in µg,of mercury equivalent to 1mLof Dithizone titrantby dividing 20by the number of mLof Dithizone titrantadded.

Procedure— Transfer 500mg of Penicillamine to a 650-mL Kjeldahl flask containing a few glass beads,incline the flask at an angle of about 45,and add 2.5mLof nitric acid through a small funnel placed in the mouth of the flask.Allow the mixture to stand at room temperature until nitrous oxide fumes are evolved and vigorous reaction subsides (5to 30minutes).Add 2.5mLof sulfuric acid through the funnel,and heat,gently at first and then to the production of fumes of sulfur trioxide,then cool.Cautiously add 2.5mLof nitric acid,again heat to the production of sulfur trioxide fumes,and cool.Repeat the treatment with nitric acid and heat,then cool,and cautiously add 50mLof water,rinsing the funnel and collecting the rinsings in the flask.Remove the funnel,boil the solution down to approximately half its volume (about 25mL),and cool to room temperature.Transfer to a 250-mLseparator with the aid of water,and add water to make about 50mL.Add 1mLof edetate disodium solution (1in 50)and 1mLof glacial acetic acid,and extract with small portions of chloroform until the last chloroform extract remains colorless.Discard the chloroform extract,and add 50mLof 0.25Nsulfuric acid,90mLof water,and 10mLof hydroxylamine hydrochloride solution (1in 5).Add Dithizone titrant,in portions of 0.3mLto 0.5mL,from a 10-mLburet.After each addition,shake the mixture 20times,and allow the chloroform layer to separate and discard it.Continue until an addition of Dithizone titrantremains green after the shaking.Calculate the amount of mercury present:the limit is 10µg (0.002%).

Diluent ,Mobile phase,and Resolution solution—Prepare as directed in the Assay.

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 Assay.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 Penicillamine taken by the formula: in which Cis the concentration,in mg per mL,of USP Penicillamine Disulfide RSin the Standard preparation,and rUand rSare the penicillamine disulfide peak responses obtained from the Test preparationand the Standard preparation,respectively:not more than 1.0%of penicillamine disulfide is found.

Diluent— Dissolve 1.0g of edetate disodium in water to make 1000mLof solution.

Mobile phase— Dissolve 6.9g of monobasic sodium phosphate and 0.20g of sodium 1-hexanesulfonate in water to make 1000mLof solution.Adjust with phosphoric acid to a pHof 3.0±0.1,and filter through a suitable filter of 1µm or finer porosity.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Resolution solution— Prepare a solution in Diluentcontaining about 1mg of USP Penicillamine RSand 0.1mg of USP Penicillamine Disulfide RSper mL.

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

Assay preparation— Transfer about 125mg of Penicillamine,accurately weighed,to a 100-mLvolumetric flask,dissolve in and dilute with Diluentto volume,and mix.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 210-nm detector and a 3.9-mm ×30-cm column containing packing L1.The flow rate is about 1.6mLper minute.Chromatograph the Resolution solution,and record the responses as directed for Procedure:the relative retention times are about 0.7for penicillamine and 1.0for penicillamine disulfide,and the resolution,R,between the penicillamine peak and the penicillamine disulfide peak is not less than 3.0.Chromatograph the Standard preparation,and record the responses as directed for Procedure:the relative standard deviation for replicate injections is not more than 1.0%.

Procedure— [NOTE—Use peak areas where peak responses are indicated.]Separately inject equal volumes (about 20µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of penicillamine (C5H11NO2S)in the portion of Penicillamine 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.