Development and validation of an LC-Orbitrap-HRMS method for quantification of human insulin and selected insulin analogues with application on two forensic cases

Insulin, a peptide hormone essential for blood glucose regulation, is typically quantified in clinical practice using immunoassays. While sensitive, these assays lack specificity and are often inadequate for the precise qualification and quantification in forensic practices. To address this limitation, we developed and validated an LC-Orbitrap-HRMS method for the quantification of intact insulin and its analogues in four postmortem matrices: plasma, liver, kidney, and adipose tissue. Method validation demonstrated linearity for human insulin in plasma from 0.1 to 100 ng/mL, with accuracy between 90.1 % and 106.9 %. Notably, in challenging matrices such as adipose tissue, the LLOQ was 1 ng/mL, with accuracy range from 94.1 % to 100.2 %, confirming robust analytical performance. The validated method was applied on two forensic cases involving suspected insulin overdose. Insulin concentrations in subcutaneous adipose tissue from the injection sites were significantly elevated, measuring 743.1 ng/mL and 53.2 ng/mL, respectively. These results demonstrate the utility of the method in providing reliable evidence for the investigation of suspected fatal insulin intoxication.