Development and Validation of a Stability-indicating HPLC Method for Determination of Insulin Detemir and Preservatives in their Injection Products

Abstract

Insulin detemir (ID) is a long-acting form of insulin that is characterized by the covalent attachment of a lipophilic tail of myristate, and commercially available as Levemir®. No satisfactory simple isocratic stability-indicating HPLC method has been reported for its quality control. A novel simple and isocratic reversed-phase HPLC method was developed and validated for the simultaneous determination of ID along with its dosage form additives in the available commercial preparations. The method employed C4 column (5μm, 250 × 4.6 mm), a mobile phase consisting of 50 mM phosphate buffer pH 2.7, acetonitrile, triethylamine (62:37:1) and 0.02 g/ml sodium sulfate, that was delivered isocraticaly at a flow rate of 1.5 ml/min, and detection performed at 214nm. The method was properly validated and was shown linear over the range 80–120% of the assay concentration for ID, phenol, and m-cresol. The method was also selective, specific, precise, and accurate. Furthermore, the validated method was applied to separate the major degradation products in those preparations. Forced degradation studies in different pH values, which are the first to be reported for ID, showed that the degradation products were baseline separated from ID itself and/or other formulation additives. Thus, the method has been demonstrated to be a stability-indicating assay.