Heparin Sodium
»Heparin Sodium is the sodium salt of sulfated glycosaminoglycans present as a mixture of heterogeneous molecules varying in molecular weights.It is present in mammalian tissues and is usually obtained from the intestinal mucosa or other suitable tissues of domestic mammals used for food by man.It is purified to retain a combination of activities against different fractions of the blood clotting sequence.It is composed of polymers of alternating derivatives of D-glucosamine (N-sulfated,O-sulfated,or N-acetylated)and uronic acid (L-iduronic acid or D-glucuronic acid)joined by glycosidic linkages.The component activities of the mixture are in ratios corresponding to those shown by the USP Heparin Sodium Reference Standard.Some of these components have the property of prolonging the clotting time of blood.This occurs mainly through the formation of a complex of each component with the plasma proteins antithrombin IIIand heparin cofactor IIto potentiate the inactivation of thrombin.Other coagulation proteases in the clotting sequence,such as activated factor X,are also inhibited.The potency of Heparin Sodium,calculated on the dried basis,is not less than 140USP Heparin Units in each mg,and not less than 90.0percent and not more than 110.0percent of the potency stated on the label.
NOTE—The USP Heparin Unit is defined by the USP Heparin Sodium Reference Standard and can be independent of International Units.The respective units are not equivalent (see General Notices).The Unit for Anti-Factor Xaactivity is defined by the USP Heparin Sodium Reference Standard and is equivalent in potency to that Standard.
Packaging and storage— Preserve in tight containers,and store below 40,preferably at room temperature.
Labeling— Label it to indicate the tissue and the animal species from which it is derived.
Identification— It meets the requirements of the flame test for Sodium á191ñ.
Bacterial endotoxins á85ñ— It contains not more than 0.03USP Endotoxin Unit per USP Heparin Unit.
Add the following:
Sterility á71ñ(where it is labeled as sterile): meets the requirements.USP28
pHá791ñ: between 5.0and 7.5,in a solution (1in 100).
Loss on drying á731ñ— Dry it in vacuum at 60for 3hours:it loses not more than 5.0%of its weight.
Residue on ignition á281ñ: between 28.0%and 41.0%.
Protein— To 1mLof a solution (1in 100)add 5drops of trichloroacetic acid solution (1in 5):no precipitate or turbidity forms.
Change to read:
Anti-factor Xaactivity—
pH8.4Buffer— Dissolve amounts of tris(hydroxymethyl)aminomethane,edetic acid,and sodium chloride in water containing 0.1%of polyethylene glycol 6000to obtain a solution having concentrations of 0.050M,0.0075M,and 0.175M,respectively.Adjust,if necessary,with hydrochloric acid or sodium hydroxide solution to a pHof 8.4.
Antithrombin IIIsolution— Reconstitute an accurately weighed quantity of antithrombin III(see Reagent Specificationsunder Reagents,Indicators,and Solutions)in pH8.4Bufferto obtain a solution having a concentration of 1.0Antithrombin III Unit per mL.
Factor Xasolution— Reconstitute an accurately weighed quantity of bovine factor Xa(see Factor Xain Reagent Specificationsunder Reagents,Indicators,and Solutions)in pH8.4Bufferto obtain a solution that gives an absorbance value between 0.65and 1.25at 405nm when assayed as described below but using 30µLof pH8.4Bufferinstead of 30µLof the Standard solutionsor the Test solutions.
Note—Factor Xasolutioncontains about 3nanokatalytic units per mL,but can vary depending upon the manufacturer of factor Xaor the substrate used.
Chromogenic substrate solution— Prepare a solution of a suitable chromogenic substrate for amidolytic test (see Reagent Specificationsunder Reagents,Indicators,and Solutions)specific for factor Xain water to obtain a concentration of about 1mM.
Stopping solution— Prepare a 20%(v/v)solution of acetic acid in water.
Standard solutions— Dilute an accurately measured volume of USP Heparin Sodium RSwith pH8.4Bufferto obtain at least 5(out of 7below)solutions having known activities of about 0.375,0.3125,0.25,0.188,0.125,0.0625,and 0.0313USP Heparin Unit per mL.
Test solutions— Dissolve or dilute an accurately measured quantity of Heparin Sodium in pH8.4Buffer,and dilute with the same buffer to obtain solutions having activities approximately equal to those of the Standard solutions.
Procedure— [Note—Perform the test with each Standard solutionand Test solutionin duplicate.]To each of a series of suitable plastic tubes placed in a water bath set at 37,transfer 120µLof pH8.4Buffer.Then separately transfer 30µLof the different dilutions of the Standard solutionsor the Test solutions to the tubes.Add 150µLof Antithrombin IIIsolution,prewarmed at 37for 15minutes,to each tube,mix,and incubate for 2minutes.Add 300µLof Factor Xasolution,prewarmed at 37for 15minutes,to each tube,mix,and incubate for 2minutes.Add 300µLof Chromogenic substrate solution,prewarmed at 37for 15minutes,to each tube,mix,and incubate for exactly 2minutes.Add 150µLof Stopping solutionto each tube,and mix.Prepare a blank for zeroing the spectrophotometer by adding the reagents in reverse order,starting with the Stopping solutionand ending with the addition of 150µLof pH8.4Buffer,and excluding the Standard solutionsor the Test solutions.Record the absorbance at 405nm against the blank.
Calculations— Plot the log of the absorbance values of the Standard solutionsand the Test solutionsversus heparin concentrations in USP Units.Construct separate straight lines of best fit using least-squares linear regression analyses for the Standard solutionsand the Test solutions,and determine the slope for each regression line.Calculate the potency of Heparin Sodium by the formula:
P(ST/SS),
in which Pis the potency of USP Heparin Sodium RS;and STand SSare the slopes of the lines from the Test solutionsand the Standard solutions,respectively.Express the Anti-factor Xapotency of the Test solution as a percentage of the heparin concentration determined in the Assay.Calculate the percentage of anti-factor Xaactivity against anticoagulant activity by the formula:
100(anti-factor Xapotency /anticoagulant potency).
Not less than 80%and not more than 120%is found.
Nitrogen content,Method Iá461ñ: between 1.3%and 2.5%,calculated on the dried basis,the procedure for Nitrates and Nitrates Absentbeing used.
Assay—
Standard preparation— Determine by preliminary trial,if necessary,approximately the minimum quantity of USP Heparin Sodium RSwhich,when added in 0.8mLof saline TS,maintains fluidity in 1mLof prepared plasma for 1hour after the addition of 0.2mLof calcium chloride solution (1in 100).This quantity is usually between 1and 3USP Heparin Units.On the day of the assay prepare a Standard preparationsuch that it contains,in each 0.8mLof saline TS,the above-determined quantity of the Reference Standard.
Assay preparation— Dissolve about 25mg of Heparin Sodium,accurately weighed,in sufficient saline TSto give a concentration of 1mg per mL,and dilute quantitatively to a concentration estimated to correspond to that of the Standard preparation.
Preparation of plasma— Collect blood from sheep directly into a vessel containing 8%sodium citrate solution in the proportion of one volume to each 19volumes of blood to be collected.Mix immediately by gentle agitation and inversion of the vessel.Promptly centrifuge the blood,and pool the separated plasma.To a 1-mLportion of the pooled plasma in a clean test tube add 0.2mLof calcium chloride solution (1in 100),and mix.Consider the plasma suitable for use if a solid clot forms within 5minutes.To store plasma for future use,subdivide the pooled lot into portions not exceeding 100mLin volume,and store in the frozen state,preventing even partial thawing prior to use.For use in the assay,thaw the frozen plasma in a water bath at a temperature not exceeding 37.Remove particulate matter by straining the thawed plasma through a coarse filter.
Procedure— To meticulously clean 13-mm ×100-mm test tubes add graded amounts of the Standard preparation,selecting the amounts so that the largest does not exceed 0.8mLand so that they correspond roughly to a geometric series in which each step is approximately 5%greater than the next lower.To each tube so prepared add sufficient saline TSto make the total volume 0.8mL.Add 1.0mLof prepared plasma to each tube.Then add 0.2mLof calcium chloride solution (1in 100),note the time,immediately insert a suitable stopper in each tube,and mix the contents by inverting three times in such a way that the entire inner surface of the tube is wet.
In the same manner set up a series using the Assay preparation,completing the entire process of preparing and mixing the tubes of both the Standard preparationand the Assay preparationwithin 20minutes after the addition of the prepared plasma.One hour,accurately timed,after the addition of the calcium chloride,determine the extent of clotting in each tube,recognizing three grades (0.25,0.50,and 0.75)between zero and full clotting (1.0).If the series does not contain 2tubes graded more than 0.5and 2tubes graded less than 0.5,repeat the assay,using appropriately modified Standardand Assay preparation.
Calculation— Convert to logarithms the volumes of Standard preparationused in the successive 5or 6tubes that bracket a grade of clotting of 0.5,including at least 2tubes with a larger and 2tubes with a smaller grade than 0.5.Number and list the tubes serially,and tabulate for each the grade of clotting observed in each tube.From the log-volumes,x,and separately from their corresponding grades of clotting,y,compute the paired averages xiand yiof Tubes 1,2,and 3,of Tubes 2,3,and 4,of Tubes 3,4,and 5,and,where the series consists of 6tubes,of Tubes 4,5,and 6,respectively.If for one of these paired averages the average grade,yi,is exactly 0.50,the corresponding xiis the median log-volume of the Standard preparation,xS.Otherwise,interpolate xSfrom the paired values of yi,xiand yi +1,xi +1that fall immediately below and above grade 0.5as
xS=xi+(yi-0.5)(xi +1-xi)/(yi-yi +1).
From the paired data on the tubes of the Assay preparation,compute similarly its median log-volume xU.
The log potency of the Assay preparationis
M=xS-xU+log R,
where R=vS/vUis the ratio of the USP Heparin Units (vS)per mLof the Standard preparationto the mg (vU)of Heparin Sodium per mLof the Assay preparation.
Repeat the assay independently,and average the two or more values of Mto obtain bar(M).If the second determination of Mdiffers by more than 0.05from the first determination,continue the assay until the log confidence interval computed as directed under Confidence Intervals for Individual Assaysin Design and Analysis of Biological Assays á111ñdoes not exceed 0.20.The potency of Heparin Sodium in USP Heparin Units per mg is P*=antilog bar(M).
Auxiliary Information— Staff Liaison:Radhakrishna S Tirumalai,Scientist
Expert Committee:(BBP)Blood and Blood Products
USP28–NF23Page 941
Pharmacopeial Forum:Volume No.29(6)Page 1898
Phone Number:1-301-816-8339