Identification and determination of degradation products of human insulin and its long acting synthetic analogues in urine by means of ESI-LC-MS/MS

The delayed release of the intermediate acting recombinant human insulin formulations (e.g. NPH-insulins) is attributable to the addition of protamine that leads to reversible protamine binding after application in the subcutaneous tissue. In contrast to earlier results, investigations with urine samples obtained from diabetic patients treated with Lantus (LAN) or Levemir (LEV) resulted in no analytical findings of the intact substances in those samples. Thus, further investigations focused on partly degraded metabolic products of insulin occurring in urine. Well known metabolic degradation processes are mainly catalysed by the endogenous enzymes Insulin-Degrading-Enzyme (IDE) and Endosomal-Acidic-Insulinase (EAI), and first experiments started more than 30 years ago. In these studies several cleavage sites at positions A13/14, A14/15, B9/10, 1313/14, 1316/17, B24/25 und B25/26 were identified utilizing in-vitro incubation of 125I-labeled insulin with hepatocytes and subsequent separation by means of liquid-chromatography. In the present study, new cleavage sites of HI, LAN and LEV were identified by LC-MS/MS determination of the respective degradation products in urine samples. Aus dem Text (geändert)