According to the United States Pharmacopeia and the National Formulary, ”LIMIT OF HIGH MOLECULAR WEIGHT PROTEINS" for GENERAL CHAPTERS "<121.1> PHYSICOCHEMICAL ANALYTICAL PROCEDURES FOR INSULINS" should be carried out with a column filled with L20 packing material. The target analytes included in this method are insulin human, various insulin analogues, and animal derived insulins. Below lists the monographs using this method (at 2023 June). The analysis of insulin bovine using The PROTEIN KW-802.5, a column filled with L20 packing material, confirmed the requirements were met.

System suitability requirementsRetention times (Resolution solution):
  Polymeric insulin complexes: 13 - 17 min
  Covalent insulin dimer: about 17.5 min
  Insulin monomer: 18 - 22 min
  Salts: Eluting after the insulin monomer
Peak-to-valley ratio* (Resolution solution): ≥ 2.0

*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.

Sample: 100 µL
(Resolution solution) 4.0 mg/mL of insulin bovine containing dimer in 0.01 N HCl aq. (prepared according to USP-NF method)

1. 1.High molecular weight proteins
2. 2.Insulin dimer
3. 3. Insulin monomer