A Universal HPLC Determination of Insulin Potency

Abstract

A liquid chromatographic method for determination of insulin potency is presented. It is applicable to bovine, porcine and human insulins, and to both insulin crystals and insulin formulations. The method uses a unique standard preparation (40 U/ml in 0.6 mg/ml EDTA) which is stable enough to permit use for >1 month. Likewise, samples are prepared in a stable form, so that an autoinjector may be used. NPH insulin suspensions are clarified by addition of heparin, lente insulins are clarified by addition of tetrasodium EDTA, and EDTA is added to neutral regular insulins and insulin crystals. Chromatography was done using a previously described 0.2 M pH 2.0 phosphate/26% CH₃CN mobile phase and a reversed phase column. A three-laboratory study of the method found it to have a precision of pm2% for formulation assays. It was verified to be accurate by comparison with the traditional rabbit biopotency assay, over which the liquid chromatographic assay has several significant advantages.