á121ñINSULIN ASSAYS
The most prominent manifestation of insulin activity,an abrupt decrease in blood glucose,was the basis for biologic assay from the time of the first clinical use of insulin.The procedure,although relatively cumbersome,has the great merit of accurately reflecting the effect on the diabetic patient.The advent of practical yet sophisticated physicochemical methods (e.g.,liquid chromatography)to measure insulin potency quantitatively has resulted in a more accurate and precise compendial test for insulin and insulin products.However,the bioidentity of insulin and insulin products cannot be assessed by these methods.Thus,a qualitative test in rabbits is included in this chapter,and its use is called for in the appropriate monographs.
The Rabbit Blood Sugar Method—Quantitativeis used to determine the potency of Insulin Reference Standards,for the validation of the stability of new insulin preparations,and to determine the specific activities of insulin analogs.

Rabbit Blood Sugar Method—Quantitative
Standard Stock Solution— Dissolve either a suitable quantity of accurately weighed USP Insulin RSor a vial of lyophilized USP Insulin RSof the appropriate species,in sufficient water containing 0.1%to 0.25%(w/v)of either phenol or cresol,1.4%to 1.8%(w/v)of glycerin,and sufficient hydrochloric acid to make a Standard Stock Solutioncontaining 40USP Insulin Units per mLand having a pHbetween 2.5and 3.5,unless otherwise directed in the individual monograph.Store in a cold place,protected from freezing,and use within 6months.
Standard Solutions— Dilute portions of the Standard Stock Solutionto make two solutions,one to contain 1.0USP Insulin Unit per mL(Standard Solution 1),and the other to contain 2.0USP Insulin Units per mL(Standard Solution 2).Use as a diluent a solution containing 0.1%to 0.25%(w/v)of either cresol or phenol,1.4%to 1.8%(w/v)of glycerin,and sufficient hydrochloric acid to produce a pHbetween 2.5and 3.5,unless otherwise directed in the individual monograph.
Assay Solutions— Dilute two volumes of the preparation under test with Standard Stock Solutionto obtain a solution containing 1.0USP Insulin Unit per mL(Assay Solution 1),and a second solution containing 2.0USP Insulin Units per mL(Assay Solution 2).In the case of neutral insulin injection,adjust to a pHof 2.5to 3.5prior to making the dilutions.
Doses of the Solutions To Be Injected— Select on the basis of trial or experience the dose of the dilutions to be injected,the volume of which usually will be between 0.30mLand 0.50mL.For each animal the volume of the Standard Solutionis the same as that of the Assay Solution.
Preparation of Animal— Select suitable,healthy rabbits each weighing not less than 1.8kg.Keep the rabbits in the laboratory for not less than 1week before use in the assay,maintaining them on an adequate uniform diet,with water available at all times.
Procedure— Divide the rabbits into four equal groups of preferably not less than six rabbits each.On the preceding day,approximately 20hours before the assay,provide each rabbit with an amount of food that will be consumed within 6hours.Follow the same feeding schedule before each test day.During the assay,withhold all food until after the final blood specimen is taken.Handle the rabbits with care in order to avoid undue excitement,and inject subcutaneously the doses indicated in the following design,the Second Injection being made on the day after the First Injection or not more than 1week later.
Blood Samples— At 1hour and 2½hours after the time of injection obtain from each rabbit a suitable blood specimen from a marginal ear vein.
Dextrose Determination— Determine the dextrose content of the blood specimens by a suitable procedure that is adapted to automated analysis (see Diagram for Biological Potency of Glucagon by the Rat Hepatocyte Methodunder Automated Methods of Analysis á16ñ).The following procedure may be used.
Anticoagulant Solution— Dissolve 1g of edetate sodium and 200mg of sodium fluoride in 1Lof water,and mix.
Dextrose Standard Preparations— Transfer known concentrations of USP Dextrose RSto suitable vessels,and dilute quantitatively and stepwise with Anticoagulant Solution(1:9)to obtain a range of Dextrose Standard Solutionscontaining between 20and 100mg per 100mLhaving known concentrations similar to the concentrations in the rabbit blood samples.
Test Preparations— Pipet into separate,suitable vessels 0.1mLof each Blood Sampleand 0.9mLof Anticoagulant Solution.
Procedure— Subject the Test Preparationsto dialysis across a semipermeable membrane for a sufficient time so that the dextrose passes through the membrane into a saline TSsolution containing glucose oxidase,horseradish peroxidase,3-methyl-2-benzothiazolinone hydrazone hydrochloride TS,and N,N-dimethylaniline.The absorbances of the Test Preparationsare determined at 600nm in a recording colorimeter.The absorbances of the Dextrose Standard Preparationsare similarly determined at the start and the end of each run.
Group First Injection Second Injection
1 Standard solution 2 Assay solution 1
2 Standard solution 1 Assay solution 2
3 Assay solution 2 Standard solution 1
4 Assay solution 1 Standard solution 2
Calculation— Calculate the response of each rabbit to each injection from the sum of the two blood-sugar values,and subtract its response to Standard Solution 1from that to Standard Solution 2,disregarding the chronological order in which the responses were observed,to obtain the individual differences,y,shown in the accompanying table.
When the data for one or more rabbits are missing in an assay,allow for differences in the sizes of the groups by suitable means (see Replacement of Missing Values under Design and Analysis of Biological Assays á111ñ).
When the number of rabbits,f,carried through the assay is the same in each group,total the y's in each group and compute Ta=–T1+T2+T3–T4and Tb=T1+T2+T3+T4.The logarithm of the relative potency of the test dilutions isM¢=0.301Ta/Tb.The potency of the injection in USP Units per mLequals the antilog (log R+M¢),where R=vS/vU,in which vSis the number of USP Units per mLof the Standard dilution and vUis the number of mLof injection per mLof the Assay dilution.
Determine the confidence interval of the log-relative potencyM¢(see Confidence Intervals for Independent Assaysunder Design and Analysis of Biological Assays á111ñ).If the confidence interval is more than 0.082,which corresponds at P=0.95to confidence limits of about ±10%of the computed potency,repeat the assay until the combined data of the two or more assays,redetermined as described in Combination of Independent Assays under Design and Analysis of Biological Assays á111ñ,meet this acceptable limit.
Group Differences Individual Response (y) Total Response (T)
1 Standard solution 2–Assay solution 1 y1 T1
2 Assay solution 2–Standard solution 1 y2 T2
3 Assay solution 2–Standard solution 1 y3 T3
4 Standard solution 2–Assay solution 1 y4 T4

Bioidentity Test
Proceed as directed for Rabbit Blood Sugar Method—Quantitativewith the following modifications:
Procedure— Divide the rabbits into 4equal groups of 2rabbits each.
Calculation— Proceed as directed for Calculationunder Rabbit Blood Sugar Method—Quantitative,but do not determine the confidence interval of the log-relative potency,M¢.
Interpretation— If the potency value obtained is not less than 15USP Units per mg,the Bioidentity Testrequirement is met.If the potency value is less than 15USP Units per mg,repeat the test using 8more rabbits.If the average potency of the 2sets of tests is not less than 15USP Units per mg,the requirement of the test is met.