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

Soluble insulin test solution— Maintain the temperature at 25 ± 1 throughout the procedure. Transfer 5.0 mL of the Injection to a centrifuge tube. Add 20 µL of 1 N sodium hydroxide, and adjust with 0.05 N hydrochloric acid or 0.05 N sodium hydroxide to a pH of 8.20 ± 0.02 if the total zinc concentration is approximately 20 µg per mL or adjust to a pH of 8.35 ± 0.02 if the total zinc concentration is approximately 30 µg per mL. Record the volume, in µL, of acid or base needed to adjust the pH. Mix, and allow to stand for 1 hour. Centrifuge, transfer the supernatant to another centrifuge tube, and repeat the centrifugation. Transfer 2 mL of the supernatant to another tube, add 5 µL of 9.6 N hydrochloric acid, and mix.

Total insulin test solution— Transfer 2 mL of Injection to a suitable vessel, add 5 µL of 9.6 N hydrochloric acid, and allow the suspension to clarify. Dilute the resulting solution with 0.01 N hydrochloric acid to the same theoretical concentration of insulin as the Soluble insulin test solution (e.g., if the Injection is labeled to contain 20% soluble insulin, the dilution factor is 100/20 = 5).

Procedure— Separately inject equal volumes (about 20 µL) of the Soluble insulin test solution and Total insulin test solution into the chromatograph, record the chromatograms, and measure the peak responses for insulin and A-21 desamido insulin. Calculate the amount of soluble insulin human as a percent of the total insulin content of the Injection by the formula: in which D is the dilution factor for the Total insulin test solution; VA is the number of µL added to adjust the pH of the Soluble insulin test solution; and rS and rT are the responses of the Soluble insulin test solution and the Total insulin test solution, respectively. The percent of soluble insulin human is in the range L ± 5, where L is the percent of soluble insulin human stated on the product label.

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

0.1 M Tris buffer solution— Dissolve 3.54 ± 0.01 g of Tris(hydroxymethyl)aminomethane hydrochloride and 3.34 ± 0.01 g of Tris(hydroxymethyl)aminomethane in 500 mL of water. The pH of the 0.1 M Tris buffer solution must be between 8.15 and 8.35. If the pH is outside of this range, discard the solution and prepare fresh; do not adjust the pH.

Soluble insulin test solution— Dilute a suitable volume of Injection with 0.1 M Tris buffer solution to obtain a solution containing about 6 USP Insulin Human Units of soluble insulin per mL (e.g., 2 mL of 70/30 Human Insulin Isophane Suspension and Human Insulin Injection containing 100 USP Insulin Human Units per mL would be diluted with 8 mL of 0.1 M Tris buffer solution to obtain a filtrate that contains 6 USP Insulin Human Units of soluble insulin per mL). Immerse the container in a water bath at 25 ± 1 for 30 ± 2 minutes. Immediately pass this solution through a 0.2-µm filter using a disposable syringe. Transfer 2 parts of the filtrate to a suitable vessel, and add 1 part 0.2 N hydrochloric acid (e.g., the dilution factor for the Soluble insulin test solution that contains 30% soluble insulin is 5 × 3/2 = 7.5).

Total insulin test solution— For each mL of Injection, add 3.0 µL of 9.6 N hydrochloric acid, mix, and allow the suspension to clarify. Dilute the resulting solution with 0.01 N hydrochloric acid to 4 USP Insulin Human Units per mL (e.g., if the product is labeled to contain a total of 100 USP Insulin Human Units per mL, the dilution factor is 25.

Procedure— Separately inject equal volumes (about 50 µL) of the Soluble insulin test solution and the Total insulin test solution, record the chromatograms, and measure the peak responses for insulin and A-21 desamido insulin. Calculate the amount of soluble insulin human as a percent of the total human insulin content of the Injection by the formula: in which DS and DT are the dilution factors for the Soluble insulin test solution and Total insulin test solution, respectively; and rS and rT are the human insulin peak responses obtained from the Soluble insulin test solution and the Total insulin test solution, respectively. The percent of soluble insulin human is in the range L ± 5, where L is the percent of soluble insulin human stated on the product label.