Arginine solution, Mobile phase, System suitability solution, and Chromatographic system— Proceed as directed in the test for Limit of high molecular weight proteins under Insulin.

Test solution— Quantitatively add 4 µL of 6 N hydrochloric acid per mL of an accurately measured volume of Injection, and mix.

Procedure— Proceed as directed for Procedure in the test for Limit of high molecular weight proteins under Insulin. Not more than 2.0% is found.

Mobile phase, Identification preparation, Standard preparation, System suitability solution, and Chromatographic system— Proceed as directed in the Assay under Insulin.

NOTE—The Identification preparation, Standard preparation, and Assay preparation may be stored at room temperature for up to 12 hours or in a refrigerator for up to 48 hours.

Assay preparation 1 (for Injection labeled as containing 40 USP Insulin Units per mL)—Add 2.5 µL of 9.6 N hydrochloric acid per mL of an accurately measured volume of Injection. Allow the suspension, if present, to clarify, and mix.

Assay preparation 2 (for Injection labeled as containing 100 USP Insulin Units per mL)—Add 2.5 µL of 9.6 N hydrochloric acid per mL of an accurately measured volume of Injection. Allow the suspension, if present, to clarify, and mix. [NOTE—Pooling of several package units may be necessary to obtain sufficient volume of the test specimen.] Pipet 2 mL of this solution into a 5-mL volumetric flask, dilute with 0.01 N hydrochloric acid to volume, and mix.

Procedure— Separately inject equal volumes (about 20 µL) of the appropriate Assay preparation, the Identification preparation, and the Standard preparation into the chromatograph, record the chromatograms, and measure the peak responses for insulin and A-21 desamido insulin, using the chromatogram of the Identification preparation to identify the insulin peaks. For Insulin Injection prepared from a single species, calculate the potency, in USP Insulin Units per mL, of the Injection taken by the formula: in which C is the concentration, in USP Insulin Units per mL, of USP Insulin RS in the Standard preparation; D is the dilution factor; and SrU and SrS are the sums of the areas of the insulin and A-21 desamido insulin peaks obtained from the chromatograms of the Assay preparation and the Standard preparation, respectively. For Injection prepared from a mixture of beef and pork insulins, calculate the total potency as the sum of the potencies of both beef and pork insulins, determined as directed above.