Protamine Sulfate
(proe' ta meen sul' fate).
DEFINITION
Protamine Sulfate is a purified mixture of simple protein principles obtained from the sperm or testes of suitable species of fish, which has the property of neutralizing heparin. Each mg of Protamine Sulfate, calculated on the dried basis, neutralizes NLT 100 USP Heparin Units.
ASSAY
•  Procedure
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 the 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 Assay in triplicate for a total of 18 determinations.
Calculate the number of USP Heparin Units in the volume of Titrant added at the endpoint per mg of Protamine Sulfate. Calculate the USP Heparin Units neutralized per mg of Protamine Sulfate taken:
Result = (VT × CT)/(VS × CS)
 VT = = volume of Titrant added (mL) CT = = concentration of Titrant (USP Heparin Units/mL) VS = = volume of the Sample solution (mL) CS = = concentration of Protamine Sulfate (mg/mL)
Calculate the potency of the Protamine Sulfate as the average of the 18 values. Calculate the 3 standard deviations for the results obtained with each of the Sample solutions. Calculate the 3 standard deviations for the results obtained with each of the 3 independent assays. The Assay is valid if each of the 6 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.
OTHER COMPONENTS
Acceptance criteria: 22.5%–25.5% of N on the dried basis
SPECIFIC TESTS
Dry a sample at 105 for 3 h: it loses NMT 5% of its weight.
•  Ultraviolet Absorbance
Sample solution: 1.0% solution of Protamine Sulfate in water
Spectrometric conditions
Mode: UV
Wavelength range: 260–280 nm
Blank: Water
Acceptance criteria: The difference in absorbance of the Sample solution at 260–280 nm against the Blank is NMT 0.1.
•  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 at 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