» 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 not less than 100 USP Heparin Units.
Packaging and storage Preserve in tight containers, in a refrigerator.
Loss on drying 731 Dry it at 105 for 3 hours: it loses not more than 5% of its weight.
Ultraviolet absorbance The difference in absorbance of a 1.0% solution in water between 260 nm and 280 nm against a water blank, is not greater than 0.1 (see Spectrophotometry and Light-Scattering 851).
Sulfate Dissolve about 150 mg, accurately weighed, 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 hour. 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. Not less than 16% and not more than 22%, calculated on the dried basis, is found.
Nitrogen content Determine the nitrogen content as directed under Method II (see Nitrogen Determination 461). Not less than 22.5% and not more than 25.5% of N, calculated on the dried basis, is found.
Assay preparation Dissolve a suitable quantity of Protamine Sulfate, accurately weighed, in Water for Injection to obtain a solution having a concentration of 1 mg per mL, calculated on the dried basis.
Plasma Prepare as directed for Preparation of plasma in the Assay under Heparin Sodium.
Heparin preparation On the day of the assay, prepare a solution of USP Heparin Sodium RS in saline TS to give a final concentration of 115 USP Heparin Units per mL.
Calcium-thromboplastin solution Dissolve in calcium chloride solution (1 in 50) a quantity of thromboplastin that is sufficient, as determined by preliminary trial if necessary, to produce clotting in about 35 seconds in a mixture consisting of equal volumes of plasma and a mixture of 4 volumes of saline TS and 1 volume of the prepared calcium-thromboplastin solution.
Procedure Into each of 10 meticulously cleansed, 13- × 100-mm test tubes pipet 2.5 mL of Plasma. Place the tubes in a water bath at 37 ± 0.2, and to each of nine of them add 0.5 mL of Assay preparation. Into the tenth tube, to provide the control, pipet 2 mL of saline TS and 0.5 mL of Calcium-thromboplastin solution, noting the time, to the nearest second, of adding the latter. While mixing with a wire loop, note the time of the first appearance of fibrin fibers, and record it to the nearest second. The elapsed time is the normal clotting time of the plasma. Pipet into the nine remaining tubes the following volumes, in mL, of Heparin preparation: 0.43, 0.45, 0.47, 0.49, 0.50, 0.51, 0.53, 0.55, and 0.57, respectively. To each tube add saline TS to make 4.5 mL. Taking the tubes in random order, add 0.5 mL of Calcium-thromboplastin solution, and note the clotting time in each tube in the same manner as for the control tube.
Calculation Calculate the number of USP Heparin Units neutralized per mg taken by the formula:
NS / WUin which NS is the number of USP Heparin Units, and WU is the number of mg of Protamine Sulfate in the last tube prior to the first one in which the clotting time is not less than 2 seconds longer than that in the control tube.
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USP32NF27 Page 3434Pharmacopeial Forum: Volume No. 28(6) Page 1862