Separation and Quantitation of Insulins and Related Substances in Bulk Insulin Crystals and in Injectables by Reversed-Phase High Performance Liquid Chromatography and the Effect of Temperature on the Separation

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Abstract
The Food and Drug Administration performs potency assays of insulin products as part of the insulin certification program required by the Code of Federal Regulations. The official method specified in the United States Pharmacopeia (USP) is a bioassay measuring the depression of blood sugar concentrations in rabbits treated with the insulin under test and the official standard. The insulin products resulting from today's technology include highly purified isolates from bovine or porcine pancreas and insulin that is identical in structure to human insulin. A study of the effects of temperature on the separation of the components in insulin injectables led to the development of a reversed-phase high performance liquid chromatographic (HPLC) method that uses a sulfate buffer/acetonitrile mobile phase at 40°C for the separation and quantitation of bovine, porcine, and human insulins and related substances. This HPLC method reduces analysis time to 1/60 of that required for the bioassay and yields more information about purity than the percent nitrogen determination that is one of the USP official procedures. The results of HPLC analyses were compared with those for the bioassay by means of a potency/area conversion factor computed on a species by species basis. Results for the bioassays and the HPLC determinations for 40 lots of bulk crystalline insulin were compared in this study; in general, the HPLC estimates fell within the 95% confidence interval for combined independent bioassays.