Protamine Sulfate

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

Protamine sulfate is a purified mixture of peptides obtained from the sperm or testes of Salmonidae, which has the property of neutralizing heparin. Each mg of Protamine Sulfate, calculated on the dried basis, neutralizes NLT 100 USP Heparin Units. It contains NLT 90.0% and NMT 110.0% of protamine sulfate, calculated on the dried basis.

2 IDENTIFICATION

A. The retention times of the four major peaks of the Sample solution correspond to those of the Standard solution, as obtained in the Assay.

B. Identification Tests—General 〈191〉, Sulfate: Meets the requirements

C. Bioidentity

Sample solution A: 0.15 mg/mL of Protamine Sulfate in water

Sample solution B: Dilute 2.0 mL of Sample solution A with water to 3.0 mL.

Sample solution C: Dilute 1.0 mL of Sample solution A with water to 3.0 mL.

Titrant: USP Heparin Sodium for Assays RS in water (about 80–120 USP Heparin Units/mL)

Analysis:

[Note—Titrate each Sample solution in duplicate.] Transfer a volume of each Sample solution to the analytical cell of a suitable colorimeter, and set the apparatus for measurement at a suitable wavelength (none is critical) in the visible range. Add Titrant in small volumes until there is a sharp increase in the absorbance, and note the volume of Titrant added. Perform the entire Analysis in triplicate for a total of 18 determinations.

Calculate the number of USP Heparin Units per mg of Protamine Sulfate in the volume of Titrant added at the endpoint.

Calculate the USP Heparin Units neutralized per mg of Protamine Sulfate taken:

Result = (V_T × C_T)/(V_S × C_U)

V_T = volume of Titrant added (mL)

C_T = concentration of Titrant (USP Heparin Units/mL)

V_S = volume of each Sample solution (mL)

C_U = concentration of Protamine Sulfate in each Sample solution (mg/mL)

Calculate the potency of the Protamine Sulfate as the average of the 18 values. Calculate the three standard deviations for the results obtained with each Sample solution. Calculate the three standard deviations for the results obtained with each of the three independent assays. The test is valid if each of the six standard deviations is NMT 5% of the average result.

Acceptance criteria: Each mg of Protamine Sulfate neutralizes NLT 100 USP Heparin Units on the dried basis.

3 ASSAY

3.1 Procedure

Solution A: 0.1 M monobasic sodium phosphate. Adjust with phosphoric acid to a pH of 1.8. Pass through a membrane filter of 0.45-µm pore size, and degas before use.

Solution B: Solution A and acetonitrile (93.5:6.5)

Mobile phase: See Table 1.

Table 1

[Note—Initial gradient composition may be adjusted as appropriate to obtain sufficient resolution. The end of the gradient can be increased to re-equilibrate the column for the next injection.]

Standard solution: 0.5 mg/mL of USP Protamine Sulfate RS in 0.01 M hydrochloric acid

Sample solution: 0.5 mg/mL of Protamine Sulfate in 0.01 M hydrochloric acid

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 214 nm

Column: 4.6-mm × 25-cm; 5-µm packing L1

Column temperature: 55°

Flow rate: 1 mL/min

Injection volume: 100 µL

System suitability

Sample: Standard solution

Suitability requirements

Retention time: The chromatogram of the Standard solution must show four major peaks (in increasing elution order: protamine peptides 1, 2, 3, and 4) with protamine peptide 4 eluting no later than 15 min.

[Note—See the reference chromatogram provided with the USP Protamine Sulfate RS certificate.]

Resolution: NLT 2.0 between protamine peptides 1 and 2, calculated by the tangent method

Relative standard deviation: NMT 2.0% for the total integrated areas of at least six replicate injections, using vertical drop-down integration

Analysis

Samples: Standard solution and Sample solution

Separately inject equal volumes of the Standard solution (at least six injections) and the Sample solution, record the chromatograms for approximately 30 min, and measure the responses for all the peaks using a full scale comparable to the height of the largest peak and using vertical drop-down integration.

Calculate the percentage of protamine sulfate in the portion of Protamine Sulfate taken:

Result = = Σ[(r_U/r_S) × (C_S/C_U)] × 100

r_U = peak response from the Sample solution

r_S = peak response from the Standard solution

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

C_U = concentration of Protamine Sulfate in the Sample solution (mg/mL)

Acceptance criteria: 90.0%–110.0% on the dried basis

4 IMPURITIES

Residual Solvents 〈467〉: Meets the requirements

Chromatographic Purity

Solution A, Solution B, Mobile phase, Standard solution, Sample solution, Chromatographic system, and System suitability: Proceed as directed in the Assay.

Analysis

Sample: Sample solution

Proceed as directed in the Assay.

Calculate the percentage purity of Protamine Sulfate from the four protamine peptide peaks:

Result = [(r_U1 + r_U2 + r_U3 + r_U4)/r_T] × 100

r_U1 = peak response of protamine peptide 1 from the Sample solution

r_U2 = peak response of protamine peptide 2 from the Sample solution

r_U3 = peak response of protamine peptide 3 from the Sample solution

r_U4 = peak response of protamine peptide 4 from the Sample solution

r_T = sum of all the peak responses from the Sample solution

Acceptance criteria: NLT 92%

Change to read:

Iron 〈241〉, Procedures, Procedure 1 (CN 1-Jun-2023): NMT 10 ppm

Methylmercury: NMT 10 ppm, using a validated analytical procedure.

[Note—Methylmercury determination is not necessary when the content for total mercury is less than the limit for methylmercury.]

5 SPECIFIC TESTS

5.1 Bacterial Endotoxins Test 〈85〉: NMT 7.0 USP Endotoxin Units/mg

5.2 Loss on Drying 〈731〉

Analysis: Dry at 105° for 3 h.

Acceptance criteria: NMT 5%

5.3 Ultraviolet Absorbance

Sample solution: 1.0% Solution of Protamine Sulfate in water

Instrumental conditions

Mode: UV

Wavelength range: 260–280 nm

Blank: Water

Acceptance criteria: The difference between the absorbance of the Sample solution at 260–280 nm and the Blank is NMT 0.1.

5.4 Sulfate

Sample: 150 mg

Analysis: Dissolve the Sample in 75 mL of water, add 5 mL of 3 N hydrochloric acid, heat to boiling, and while maintaining the boiling point, slowly add 10 mL of barium chloride TS. Cover the vessel, and allow the mixture to stand on a steam bath for 1 h. Filter, wash the precipitate with several portions of hot water, dry, and ignite to constant weight. The weight of the barium sulfate, multiplied by 0.4117, represents the weight of sulfate in the portion of Protamine Sulfate taken.

Acceptance criteria: 16%–22% on the dried basis

6 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in tight and tamper-proof containers. Store at controlled room temperature or at 2°–8°.

USP Reference Standards 〈11〉

USP Endotoxin RS

USP Heparin Sodium for Assays RS

USP Protamine Sulfate RS