Detection of insulins in postmortem tissues: an optimized workflow based on immunopurification and LC-MS/HRMS...

Diabetes is a worldwide disease in perpetual expansion. Type 1 and sometimes type 2 diabetic patients require daily human insulin (HI) or analog administration. Easy access to insulins for insulin-treated diabetics, their relatives, and medical professionals can enable abuse for suicidal or homicidal purpose. However, demonstrating insulin overdose in postmortem blood is challenging. Tissue analyses are contributive, as insulins can accumulate before death or undergo only limited degradation. The present study describes an assay for HI and synthetic analogs (lispro, aspart, glulisine, detemir and degludec, glargine and its main metabolite (M1)) in liver, kidney, muscle, and injection site samples. It is based on a 5-step sample preparation (reduction of tissue sample size, homogenization, extraction, concentration, and immunopurification) associated with liquid chromatography coupled to high-resolution mass spectrometry (LC-MS/HRMS). Selectivity and limit of detection (LOD) for all target analogs were assessed in the above matrices. LOD was determined at 25 ng/g for HI and for analogs except detemir and degludec, where LOD was 50 ng/g in kidney and injection site samples and 80 ng/g in the liver and muscle. The method was applied to13 forensic cases in which insulin use was suspected.