A: The retention time of the insulin peak in the chromatogram of the Assay preparationcorresponds to the retention time of the appropriate species in the chromatogram of the Identification preparation,as obtained in the Assay.[NOTE—It may be necessary to inject a mixture of Assay preparationand Identification preparation.]

B: Proceed as directed for Identificationtest BunderInsulin Human,except to use 1mg of USP Insulin Reference Standard of the appropriate species to prepare the Standard digest solution,to use 1mg of Insulin to prepare the Test digest solution,and to obtain a resolution,R,between digest fragments IIand IIIof not less than 1.9:meets the requirements.

Solvent— Dissolve 28.4g of anhydrous sodium sulfate in 1000mLof water.Pipet 2.7mLof phosphoric acid into this solution,adjust,if necessary,with ethanolamine to a pHof 2.3,and mix.

Solution A— Prepare a filtered and degassed mixture ofSolventand acetonitrile (82:18).

Solution B— Prepare a filtered and degassed mixture of Solventand acetonitrile (50:50).

Mobile phase— Use variable mixtures of Solution Aand Solution Bas directed for Chromatographic system.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

System suitability solution— Proceed as directed in theAssay.

Standard solution A— Dissolve an accurately weighed quantity of USP Insulin RSof the appropriate species in 0.01Nhydrochloric acid to obtain a solution having a known concentration of about 3.75mg per mL.

Standard solution B— Pipet 1mLof Standard solution Ainto a 10-mLvolumetric flask,dilute with 0.01Nhydrochloric acid to volume,and mix.

Standard solution C— Pipet 1mLof Standard solution Binto a 10-mLvolumetric flask,dilute with 0.01Nhydrochloric acid to volume,and mix.[NOTE—These three Standard solutionsmay be stored at room temperature for up to 12hours and in a refrigerator for up to 48hours.]

Test solution— Transfer about 7.5mg of Insulin to a suitable capped vial,and add 2.0mLof 0.01Nhydrochloric acid.Cap the vial,and shake gently to dissolve.Store this solution for not more than 2hours at room temperature or for not more than 12hours in a refrigerator.

Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph is equipped with a 214-nm detector and a 4.6-mm ×25-cm column that contains packing L1.The column temperature is maintained at 40,and the flow rate is about 1mLper minute.The chromatograph is programmed as follows. Adjust theMobile phasecomposition and the duration of the isocratic elution to obtain a retention time of about 31minutes for insulin,with the A-21desamido insulin eluting just prior to the start of the gradient elution phase.Chromatograph Standard solutions A,B,and C,record the chromatograms,and measure the peak responses as directed for Procedure:calculate the factor X1by the formula: in which rBand rAare the areas of the peak responses obtained for Standard solution Band Standard solution A,respectively.The value of X1is between 0.91and 1.09.Calculate the factor X2by the formula: in which rCand rAare the areas of the peak responses obtained for Standard solution Cand Standard solution A,respectively.The value of X2is between 0.7and 1.3.Chromatograph the System suitability solution,and record the peak responses as directed for Procedure:the resolution,R,between insulin and A-21desamido insulin is not less than 2.0;and the tailing factor for the insulin peak is not more than 1.8.

Procedure— Inject a volume (about 20µL)of the Test solutioninto the chromatograph,record the chromatogram,and measure the areas of the responses for the main insulin peak,the A-21desamido insulin peak,and the peaks from any other impurities.Calculate the percentage of insulin,%I,in the portion of Insulin taken by the formula: in which rIis the peak response for insulin,and rsis the sum of the responses for all of the peaks.Calculate the percentage of A-21desamido insulin,%D,in the portion of Insulin taken by the formula: in which rDis the peak response for A-21desamido insulin,and rsis the sum of the responses for all of the peaks.Calculate the percentage of other insulin related compounds in the portion of Insulin taken by the formula: Not more than 10.0%of A-21desamido insulin is found,and not more than 5.0%of other insulin related compounds is found.For Insulin derived from a single species,measure the responses of any peaks corresponding to beef or pork insulin,and calculate their concentration as a percentage of rs:the amount of cross-contamination is not more than 1.0%.

Arginine solution— Prepare a solution of L-arginine in water containing 1mg per mL.

Mobile phase— Prepare a filtered and degassed mixture ofArginine solution,acetonitrile,and glacial acetic acid (65:20:15).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Resolution solution— Dissolve 4mg of Insulin containing more than 0.4%high molecular weight proteins in 1mLof 0.01Nhydrochloric acid.Store this solution in a refrigerator,and use within 7days.[NOTE—Insulin containing the indicated percentage of high molecular weight proteins may be prepared by allowing Insulin to stand at room temperature for about 5days.]

Test solution— Transfer about 4mg of Insulin to a small vial,add 1mLof 0.01Nhydrochloric acid,and mix to dissolve.Store in a refrigerator,and use within 7days.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 276-nm detector and a 7.8-mm ×30-cm column that contains packing L20.The flow rate is about 0.5mLper minute.Chromatograph the Resolution solution,and record the peak responses as directed forProcedure:the retention times are between 13and 17minutes for the polymeric insulin complexes,about 17.5minutes for the covalent insulin dimer,and between 18and 22minutes for the insulin monomer,with salts eluting after the insulin monomer;and the ratio of the height of the covalent insulin dimer peak to the height of the valley between the covalent insulin dimer peak and the insulin monomer peak is not less than 2.0.

Procedure— Inject a volume (about 100µL)of the Test solutioninto the chromatograph,record the chromatogram,and measure the areas of the peak responses,disregarding any peaks having retention times greater than that of the insulin monomer.Calculate the percentage of high molecular weight proteins in the portion of Insulin taken by the formula: in which SrHis the sum of the responses for all peaks having retention times less than that of the insulin monomer,and rMis the peak response of the insulin monomer:not more than 1.0%is found.

Mobile phase— Dissolve 28.4g of anhydrous sodium sulfate in 1000mLof water,pipet 2.7mLof phosphoric acid into the solution,and adjust with ethanolamine to a pHof 2.3if necessary.Prepare a filtered and degassed mixture of this solution and acetonitrile (74:26).The acetonitrile is warmed to a temperature equal to or higher than 20to avoid precipitation.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

System suitability solution— Dissolve about 1.5mg of Insulin in 1.0mLof 0.01Nhydrochloric acid.Allow to stand at room temperature for not less than 3days to obtain a solution containing not less than 5%of A-21desamido insulin.

Identification preparation— Prepare a solution ofUSP Insulin (Pork)RSandUSP Insulin (Beef)RSin 0.01Nhydrochloric acid containing about 0.6mg of each per mL.

Standard preparation— Dissolve an accurately weighed quantity of USP Insulin RSof the appropriate species in 0.01Nhydrochloric acid to obtain a solution having a known concentration of about 1.5mg per mL.

Assay preparation— Transfer about 15mg of Insulin,accurately weighed,to a 10-mLvolumetric flask,and dissolve in and dilute with 0.01Nhydrochloric acid to obtain a solution having a concentration of about 1.5mg per mL.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 214-nm detector and a 4.6-mm ×15-cm column that contains packing L1.The column temperature is maintained at 40,and the flow rate is about 1mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the relative standard deviation for replicate injections is not more than 1.6%.Chromatograph the System suitability solution,and record the peak responses as directed for Procedure:the resolution,R,between insulin and A-21desamido insulin is not less than 2.0;and the tailing factor for the insulin peak is not more than 1.8.

Procedure— Separately inject equal volumes (about 20µL)of the Assay preparation,theIdentification preparation,and the Standard preparationinto the chromatograph,record the chromatograms,and measure the peak responses for insulin and A-21desamido insulin,using the chromatogram of the Identification preparationto identify the insulin peaks.For Insulin derived from a single species,calculate the potency on the undried basis,in USP Insulin Units per mg,of the Insulin in the Assay preparationby the formula: in which CSis the concentration,in USP Insulin Units per mL,of USP Insulin RSin theStandard preparation;CUis the concentration,in mg per mL,of Insulin in the Assay preparation;and SrUand SrSare the sums of the areas of the insulin and A-21desamido insulin peaks obtained from the chromatograms of the Assay preparationand the Standard preparation,respectively.From the value obtained in the test for Loss on drying,calculate the potency on the dried basis.For Insulin derived from a mixture of beef and pork,calculate the total potency as the sum of the potencies of the beef-and pork-derived insulins,determined separately.