Penicillamine Capsules

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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1 DEFINITION

Penicillamine Capsules contain NLT 90.0% and NMT 110.0% of the labeled amount of penicillamine (C₅H₁₁NO₂S).

2 IDENTIFICATION

A. Thin-Layer Chromatography

Standard solution: 100 mg of USP Penicillamine RS in 10 mL of methanol. Add 2 drops of 3 N hydrochloric acid and mix.

Sample solution: Transfer a portion of Capsule contents, containing nominally about 100 mg of penicillamine, to a 10-mL volumetric flask, and dilute with methanol to volume. Add 2 drops of 3 N hydrochloric acid, mix, and filter. Use the filtrate.

Chromatographic system

(See Chromatography 〈621〉, General Procedures, Thin-Layer Chromatography.)

Mode: TLC

Adsorbent: 0.25-mm layer of chromatographic silica gel mixture, heated at 105° for 30 min, and allowed to cool before use

Application volume: 10 μL

Developing solvent system: Butyl alcohol, glacial acetic acid, and water (8:2:2)

Spray reagent: 3-mg/mL solution of ninhydrin in dehydrated alcohol

Analysis

Samples: Standard solution and Sample solution

Separately apply the Sample solution and the Standard solution to the plate. Develop the chromatogram in the Developing solvent system until the solvent front has moved three-fourths 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 Spray reagent, heat it at 105° for 10 min, allow it to cool, and examine it.

Acceptance criteria: The R values, colors, and intensities of the principal spots from the Sample solution correspond to those from the Standard solution.

B. The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay.

3 ASSAY

Procedure

Mobile phase: 6.9 g/L of monobasic sodium phosphate and 0.2 g/L of sodium 1-hexanesulfonate in water. Adjust with phosphoric acid to a pH of 3.0 ± 0.1.

Diluent: 1.0 g/L of edetate disodium in water

System suitability solution: 1 mg/mL of USP Penicillamine RS and 0.1 mg/mL of USP Penicillamine Disulfide RS in Diluent

Standard solution: 1.25 mg/mL of USP Penicillamine RS in Diluent

Sample solution: Nominally equivalent to 1.25 mg/mL of penicillamine in Diluent prepared as follows. Transfer the contents of Capsules (NLT 10) to a suitable volumetric flask. Add the empty Capsule shells to the flask, and add sufficient Diluent to the flask to fill it to three-fourths of its capacity. Shake for 1 min, and allow the mixture to stand for 90 min. Dilute with Diluent to volume. Pass a portion of this solution through a suitable filter of 1-μm or finer pore size, and use the clear filtrate.

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 210 nm

Column: 3.9-mm × 30-cm; 10-μm packing L1

Flow rate: 1.6 mL/min

Injection volume: 20 μL

System suitability

Samples: System suitability solution and Standard solution

[Note - The relative retention times for penicillamine and penicillamine disulfide are 0.7 and 1.0, respectively.]

Suitability requirements

Resolution: NLT 3.0 between penicillamine and penicillamine disulfide, System suitability solution

Relative standard deviation: NMT 1.0%, Standard solution

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of penicillamine (C₅H₁₁NO₂S) in the portion of Capsules taken:

Result = (r_U/r_S) × (C_S/C_U) × 100

r_U = peak response of penicillamine from the Sample solution

r_S = peak response of penicillamine from the Standard solution

C_S = concentration of USP Penicillamine RS in the Standard solution (mg/mL)

C_U = nominal concentration of penicillamine in the Sample solution (mg/mL)

Acceptance criteria: 90.0%–110.0%

4 PERFORMANCE TESTS

Change to read:

Dissolution 〈711〉

4.1 Test 1

Medium: 0.1 N hydrochloric acid; 900 mL

Apparatus 1: 100 rpm

Time: 30 min

Procedure for a pooled sample

Dilute hydrochloric acid: Dilute 37 mL of hydrochloric acid with water to 1 L.

Dilute sulfuric acid: Dilute 1 mL of sulfuric acid with water to 50 mL.

Ammonium sulfamate reagent: 2.5 mg/mL of ammonium sulfamate in Dilute hydrochloric acid N-(1-Naphthyl)ethylenediamine dihydrochloride reagent: 1 mg/mL of N-(1-naphthyl)ethylenediamine dihydrochloride in Dilute hydrochloric acid

Sulfanilamide - mercuric chloride reagent: 1 mg/mL of sulfanilamide and 1 mg/mL of mercuric chloride in Dilute hydrochloric acid

Sodium nitrite reagent: 2 mg/mL of sodium nitrite in Dilute sulfuric acid. Prepare fresh.

Standard solution: 250 μg/mL of USP Penicillamine RS in 0.1 N hydrochloric acid

Sample solution: Withdraw a portion of the solution under test, containing nominally about 278 μg of penicillamine, and pass through a suitable filter.

Blank: Volume of 0.1 N hydrochloric acid equivalent to a volume of the Sample solution

Instrumental conditions

Mode: UV-Vis

Analytical wavelength: 540 nm

Cell: 1 cm

Analysis: Pipet the Sample solution into a 100-mL volumetric flask. Into a similar flask, transfer the reagent Blank, and into a third 100-mL volumetric flask, pipet 1 mL of Standard solution. Treat each flask as follows. Add by pipet 3 mL of Sodium nitrite reagent, and mix by swirling occasionally. After 5 min, add 10 mL of Ammonium sulfamate reagent, swirl, and allow to stand for an additional 5 min. Add 5mL of Sulfanilamide–mercuric chloride reagent, swirl, and immediately add 10 mL of N-(1-Naphthyl)ethylenediamine dihydrochloride reagent. Dilute with water to volume and mix. Determine the absorbances of both solutions against the Blank.

Calculate the percentage of the labeled amount of penicillamine (C₅H₁₁NO₂S) dissolved:

Result = (A_U/A_S) × C_S × V × (1/L) × 100

A_U = absorbance of the Sample solution

A_S = absorbance of the Standard solution

C_S = concentration of USP Penicillamine RS in the Standard solution (mg/mL)

V = volume of the Medium, 900 mL

L = label claim (mg/Capsule)

Tolerances: NLT 80% (Q) of the labeled amount of penicillamine (C₅H₁₁NO₂S) is dissolved.

Procedure for a unit sample

Buffer solution: 50 mM solution of monobasic potassium phosphate buffer at a pH of 3.0

Mobile phase: Methanol and Buffer solution (3:97)

Sample solution: Proceed as directed in Dissolution 〈711〉, Procedure. After 30 min, withdraw 10 mL of solution from each vessel, and immediately pass each aliquot through a polyvinylidene difluoride filter of 0.45-μm pore size. Discard the first 2 mL of filtered solution, and chromatograph the remaining filtrate.

System suitability solution: USP Penicillamine RS at a concentration similar to the Sample solution and 0.002 mg/mL of USP Penicillamine Disulfide RS in 0.1 N hydrochloric acid

Standard solution: USP Penicillamine RS in 0.1 N hydrochloric acid at a concentration similar to the Sample solution

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 210 nm

Column: 4.6-mm × 15-cm; 5-μm packing L1

Flow rate: 1.0 mL/min

Injection volume: 30 μL

System suitability

Samples: System suitability solution and Standard solution

Suitability requirements

Resolution: NLT 2.0 between penicillamine and penicillamine disulfide, System suitability solution

Tailing factor: NMT 2.0, Standard solution

Relative standard deviation: NMT 2.0%, Standard solution

Analysis

Samples: Sample solution and Standard solution

Calculate the percentage of penicillamine (C₅H₁₁NO₂S) dissolved:

Result = (r_U /r_S ) × C_S × V × (1/L) × 100

r_U = peak response from the Sample solution

r_S = peak response from the Standard solution

C_S = concentration of USP Penicillamine RS in the Standard solution (mg/mL)

V = volume of Medium, 900 mL

L = label claim (mg/Capsule)

Tolerances: NLT 80% (Q) of the labeled amount of penicillamine (C₅H₁₁NO₂S) is dissolved.

4.2 Test 2

If the product complies with this test, the labeling indicates that the product meets USP Dissolution Test 2.

Medium: 0.1 N hydrochloric acid; 500 mL

Apparatus 1: 100 rpm

Time: 30 min

Solution A: Transfer 10 mL of phosphoric acid to a 100-mL volumetric flask, and dilute with water to volume.

Buffer: Dissolve 6.8 g of potassium phosphate, monobasic in 1 L of water. Adjust with Solution A to a pH of 3.0.

Mobile phase: Methanol and Buffer (3:97)

Impurity stock solution: 0.1 mg/mL of USP Penicillamine Disulfide RS in Medium. Sonicate to dissolve.

System suitability solution: 0.5 mg/mL of USP Penicillamine RS and 0.01 mg/mL of USP Penicillamine Disulfide RS prepared as follows.

Transfer an appropriate quantity of USP Penicillamine RS to a suitable volumetric flask. Add 10% of the flask volume of Impurity stock solution. Dilute with Medium to volume.

Standard solution: 0.5 mg/mL of USP Penicillamine RS in Medium. Sonicate to dissolve.

Sample solution: Pass a portion of the solution under test through a suitable filter of 0.45-μm pore size.

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 210 nm

Column: 4.6-mm × 15-cm; 3-μm packing L1

Flow rate: 1 mL/min

Injection volume: 10 μL

Column temperature: 30°

Run time: NLT 3 times the retention time of penicillamine

System suitability

Samples: System suitability solution and Standard solution

[Note - The relative retention times for penicillamine and penicillamine disulfide are about 1.0 and 1.1, respectively.]

Suitability requirements

Resolution: NLT 2.0 between penicillamine and penicillamine disulfide, System suitability solution

Tailing factor: NMT 2.0, Standard solution

Relative standard deviation: NMT 2.0%, Standard solution

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled amount of penicillamine (C₅H₁₁NO₂S) dissolved:

Result = (r_U /r_S) × C_S × V × (1/L) × 100

r_U = peak response of penicillamine from the Sample solution

r_S = peak response of penicillamine from the Standard solution

C_S = concentration of USP Penicillamine RS in the Standard solution (mg/mL)

V = volume of Medium, 500 mL

L = label claim (mg/Capsule)

Tolerances: NLT 80% (Q) of the labeled amount of penicillamine (C₅H₁₁NO₂S) is dissolved. (RB 1-Aug-2023)

Uniformity of Dosage Units 〈905〉: Meet the requirements

5 IMPURITIES

Limit of Penicillamine Disulfide

Mobile phase, Diluent, System suitability solution, Sample solution, and Chromatographic system: Proceed as directed in the Assay.

Standard solution: 0.025 mg/mL of USP Penicillamine Disulfide RS in Diluent

System suitability

Samples: System suitability solution and Standard solution

[Note - The relative retention times for penicillamine and penicillamine disulfide are 0.7 and 1.0, respectively.]

Suitability requirements

Resolution: NLT 3.0 between penicillamine and penicillamine disulfide, System suitability solution

Relative standard deviation: NMT 2.0% for penicillamine disulfide, Standard solution

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of penicillamine disulfide (C₅H₁₁NO₂S) in the portion of Capsules taken:

Result = (r_U/r_S) × (C_S/C_U) × 100

r_U = peak area of penicillamine disulfide from the Sample solution

r_S = peak area of penicillamine disulfide from the Standard solution

C_S = concentration of USP Penicillamine Disulfide RS in the Standard solution (mg/mL)

C_U = nominal concentration of penicillamine in the Sample solution (mg/mL)

Acceptance criteria: NMT 2.0%

6 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in tight containers. Store at controlled room temperature.

Add the following:

Labeling: When more than one dissolution test is given, the labeling states the Dissolution test used only if Test 1 is not used. (RB 1-Aug-2023)

USP Reference Standards 〈11〉

USP Penicillamine RS

USP Penicillamine Disulfide RS

3,3′-Dithiodi-d-valine.

C₁₀H₂₀N₂O₄S₂ 296.40