Protamine Sulfate
»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 100USP Heparin Units.
Packaging and storage—
Preserve in tight containers,in a refrigerator.
Loss on drying á731ñ—
Dry it at 105
for 3hours:it loses not more than 5%of its weight.
for 3hours:it loses not more than 5%of its weight.
Ultraviolet absorbance
—
The difference in absorbance of a 1.0%solution in water between 260nm and 280nm against a water blank,is not greater than 0.1(see Spectrophotometry and Light-Scattering á851ñ).
Sulfate—
Dissolve about 150mg,accurately weighed,in 75mLof water,add 5mLof 3Nhydrochloric acid,heat to boiling,and while maintaining at the boiling point,slowly add 10mLof barium chloride TS.Cover the vessel,and allow the mixture to stand on a steam bath for 1hour.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 á461ñ).Not less than 22.5%and not more than 25.5%of N,calculated on the dried basis,is found.
Assay—
Assay preparation—
Dissolve a suitable quantity of Protamine Sulfate,accurately weighed,in Water for Injection to obtain a solution having a concentration of 1mg per mL,calculated on the dried basis.
Heparin preparation—
On the day of the assay,prepare a solution of USP Heparin Sodium RSin saline TSto give a final concentration of 115USP Heparin Units per mL.
Calcium-thromboplastin solution—
Dissolve in calcium chloride solution (1in 50)a quantity of thromboplastin that is sufficient,as determined by preliminary trial if necessary,to produce clotting in about 35seconds in a mixture consisting of equal volumes of plasma and a mixture of 4volumes of saline TSand 1volume of the prepared calcium-thromboplastin solution.
Procedure—
Into each of 10meticulously cleansed,13-×100-mm test tubes pipet 2.5mLof Plasma.Place the tubes in a water bath at 37±0.2,and to each of nine of them add 0.5mLof Assay preparation.Into the tenth tube,to provide the control,pipet 2mLof saline TSand 0.5mLof 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 TSto make 4.5mL.Taking the tubes in random order,add 0.5mLof Calcium-thromboplastin solution,and note the clotting time in each tube in the same manner as for the control tube.
,and to each of nine of them add 0.5mLof Assay preparation.Into the tenth tube,to provide the control,pipet 2mLof saline TSand 0.5mLof 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 TSto make 4.5mL.Taking the tubes in random order,add 0.5mLof 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/WU,
in which NSis the number of USP Heparin Units,and WUis 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 2seconds longer than that in the control tube.
Auxiliary Information—
Staff Liaison:Radhakrishna S Tirumalai,Scientist
Expert Committee:(BBP)Blood and Blood Products
USP28–NF23Page 1667
Pharmacopeial Forum:Volume No.28(6)Page 1862
Phone Number:1-301-816-8339