Untargeted metabolomics reveals a reproducible signature associated with fatal insulin intoxication

Abstract Fatal insulin intoxication remains difficult to diagnose because insulin rapidly degrades after death, limiting the reliability of direct biochemical measurements. We hypothesised that insulin excess induces systemic metabolic alterations that persist beyond insulin degradation and can be captured using postmortem metabolomics. High-resolution mass spectrometry (HRMS)-based metabolomics was applied to a national cohort (2017–2024), including 51 fatal insulin intoxications and non-insulin deaths for comparison. Orthogonal partial least squares-discriminant analysis (OPLS-DA) models were trained on cases collected between 2017 and 2022. Performance was evaluated using two temporally distinct test sets (2023–2024), a matched validation cohort ( n = 59) and a heterogeneous cohort ( n = 154), with 14 insulin cases included. An insulin-associated metabolomic signature comprising 91 features demonstrated reproducible discrimination. In the matched cohort, insulin intoxication classification achieved 100% sensitivity and 73% specificity. In the heterogeneous cohort, 100% sensitivity was maintained with 72% specificity. No insulin false negatives were observed in either test set. Significant alterations were observed across acylcarnitines, fatty acids/lipids, and purine/nucleoside metabolites. Fatal insulin intoxication is associated with a reproducible metabolomic signature. While not suitable as a confirmatory diagnostic approach, the high sensitivity and absence of false negatives suggests potential utility in supporting exclusion of insulin intoxication in death investigations.