A: The retention time of the major peak in the chromatogram of the Assay preparation corresponds to that in the chromatogram of the Standard preparation, as obtained in the Assay.

B: Determine the peptide fragments, using the following peptide mapping procedure.

Solvent— Proceed as directed in the Assay.

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

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

Mobile phase— Use variable mixtures of Solution A and Solution B as directed for Chromatographic system. Make adjustments if necessary (see System Suitability under Chromatography 621).

System suitability solution— Dissolve an accurately weighed quantity of Insulin Lispro in 0.01 N hydrochloric acid to obtain a solution containing about 3.5 mg per mL. Allow to stand at room temperature to obtain a solution containing between 0.8% and 11% A-21 desamido insulin lispro.

Test solution— Dissolve about 3.5 mg of Insulin Lispro in 1.0 mL of 0.01 N hydrochloric acid. Store this solution for not more than 56 hours in a refrigerator.

Chromatographic system (see Chromatography 621)— 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 1 mL per minute. The chromatograph is programmed as follows. Adjust the Mobile phase composition and duration of the isocratic elution to obtain a retention time of about 41 minutes for insulin lispro, with A-21 desamido insulin lispro eluting near the start of the linear gradient phase. Chromatograph the System suitability solution, and record the peak responses as directed for Procedure: the resolution, R, between insulin lispro and A-21 desamido insulin lispro is not less than 2.5; and the tailing factor for the insulin lispro peak is not more than 2.0.

Procedure— Proceed as directed for Procedure in the test for Related compounds under Insulin: not more than 1.00% of A-21 desamido insulin lispro is found; not more than 0.50% of any other individual insulin lispro related compound is found; and not more than 2.00% of total impurities, excluding A-21 desamido insulin lispro, is found.

Solvent— Dissolve 28.4 g of anhydrous sodium sulfate in 1000 mL of water, mix, and adjust with phosphoric acid to a pH of 2.3.

Mobile phase— Mix 745 mL of Solvent and 255 mL of acetonitrile. Make adjustments if necessary (see System Suitability under Chromatography 621).

System suitability solution— Dissolve an accurately weighed quantity of Insulin Lispro in 0.01 N hydrochloric acid to obtain a solution having a concentration of about 1 mg per mL. Allow to stand at room temperature to obtain a solution containing between 0.8% and 11% A-21 desamido insulin lispro.

Standard preparation— Dissolve an accurately weighed quantity of USP Insulin Lispro RS in 0.01 N hydrochloric acid to obtain a solution having a known concentration of about 0.7 mg per mL.

Assay preparation— Dissolve an accurately weighed portion of Insulin Lispro in 0.01 N hydrochloric acid to obtain a solution having a concentration of about 0.8 mg per mL.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 214-nm detector and a 4.6-mm × 10-cm column that contains packing L1. The column temperature is maintained at 40, and the flow rate is about 0.8 mL per minute. Adjust the Mobile phase to provide a retention time of about 24 minutes for the main insulin lispro peak. Chromatograph three replicate injections of the System suitability solution, and record the peak responses as directed for Procedure: the resolution, R, between insulin lispro and A-21 desamido insulin lispro is not less than 3.0; the tailing factor for the insulin lispro peak is not more than 1.5; and the relative standard deviation for replicate injections is not more than 1.1%.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the areas for the major peaks. Calculate the potency, in USP Insulin Lispro Units per mg, on the as-is basis by the formula: in which CS is the concentration, in USP Insulin Lispro Units per mL, of USP Insulin Lispro RS in the Standard preparation; CU is the concentration, in mg per mL, of Insulin Lispro in the Assay preparation; and rU and rS are the insulin lispro peak areas obtained from the Assay preparation and the Standard preparation, respectively. From the value obtained in the test for Loss on drying, calculate the potency on the dried basis.