Insulin Human Injection

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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1 DEFINITION

Insulin Human Injection is an isotonic, sterile solution of Insulin Human in Water for Injection. It has a potency of NLT 95.0% and NMT 105.0% of the potency stated on the label, expressed in USP Insulin Human Units/mL.

2 IDENTIFICATION

A. The retention time of the major peak of Sample solution A or Sample solution B corresponds to that of the Standard solution, as obtained in the Assay.

3 ASSAY

Procedure

Solution A: Dissolve 28.4 g of anhydrous sodium sulfate in 1000 mL of water. Pipet 2.7 mL of phosphoric acid into the solution, and adjust with ethanolamine to a pH of 2.3, if necessary.

Mobile phase: Acetonitrile and Solution A (26:74).

[Note—The acetonitrile is warmed to NLT 20° to avoid precipitation.]

System suitability solution: 1.5 mg/mL of insulin human in 0.01 N hydrochloric acid. Allow to stand at room temperature for NLT 3 days to obtain a solution containing NLT 5% of A-21 desamido insulin human.

Standard solution: 1.5 mg/mL of USP Insulin Human RS in 0.01 N hydrochloric acid

Sample solution A (for Injection labeled as containing 40 USP Insulin Human Units/mL): Add 2.5 µL of 9.6 N hydrochloric acid for each mL of an accurately measured volume of Injection. Allow the suspension, if present, to clarify, and mix.

Sample solution B (for Injection labeled as containing 100 USP Insulin Human Units/mL): Add 2.5 µL of 9.6 N hydrochloric acid for each mL of an accurately measured volume of Injection. Allow the suspension, if present, to clarify, and mix.

[Note—Pooling several package units may be necessary to obtain su￾cient volume of the sample.] Pipet 2 mL of this solution into a 5-mL volumetric ￾ask, dilute with 0.01 N hydrochloric acid to volume, and mix.

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 214 nm

Column: 4.6-mm × 15-cm; packing L1

Column temperature: 40°

Flow rate: 1 mL/min

Injection volume: 20 µL

System suitability

Samples: System suitability solution and Standard solution

Suitability requirements

Resolution: NLT 2.0 between insulin human and A-21 desamido insulin human, System suitability solution

Tailing factor: NMT 1.8 for the insulin human peak, System suitability solution

Relative standard deviation: NMT 1.6%, Standard solution

Analysis

Samples: Standard solution and either Sample solution A or Sample solution B

Measure the peak responses for insulin human and A-21 desamido insulin human. Calculate the potency, in USP Insulin Human Units/mL, of the Injection taken:

Result = (Σrᵤ / Σrₛ) × C × D

Σrᵤ = sum of the peak responses of insulin human and A-21 desamido insulin human from the Sample solution

Σrₛ = sum of the peak responses of insulin human and A-21 desamido insulin human from the Standard solution

C = concentration of USP Insulin Human RS in the Standard solution (USP Insulin Human Units/mL)

D = dilution factor used to prepare the Sample solution

Acceptance criteria: 95.0%–105.0% of the potency stated on the label, expressed in USP Insulin Human Units/mL

4 OTHER COMPONENTS

Zinc Determination 〈591〉: 10–40 µg for every 100 USP Insulin Human Units

5 PRODUCT-RELATED SUBSTANCES AND IMPURITIES

Physicochemical Analytical Procedures for Insulins, Limit of High Molecular Weight Proteins 〈121.1〉

Proceed as directed in Limit of High Molecular Weight Proteins, except prepare the following Sample solution. It meets the requirements.

Sample solution: Quantitatively add 4 µL of 6 N hydrochloric acid to each mL of an accurately measured volume of Injection, and mix.

Acceptance criteria: NMT 1.7%

6 SPECIFIC TESTS

pH 〈791〉: 7.0–7.8

Particulate Matter in Injections 〈788〉: Meets the requirements for small-volume injections

Bacterial Endotoxins Test 〈85〉: NMT 80 USP Endotoxin Units/100 USP Insulin Human Units

Sterility Tests 〈71〉: Meets the requirements when tested as directed in Test for Sterility of the Product to Be Examined, Membrane Filtration

Injections and Implanted Drug Products 〈1〉: Meets the requirements

7 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve and dispense in the unopened, multiple-dose container provided by the manufacturer. Store in a refrigerator, protect from sunlight, and avoid freezing.

Labeling: Label it to indicate that it has been prepared with Insulin Human produced by methods based on recombinant DNA technology or that it is derived by enzymatic modi￾cation of insulin from porcine pancreas. Label it to state that it is to be stored in a refrigerator and that freezing is to be avoided. The label states the potency in USP Insulin Human Units/mL.

USP Reference Standards 〈11〉

USP Insulin Human RS