Advancing Insulin Analysis in Varied Matrices for the Investigation of Suspected Hypoglycaemic Death

Diabetes mellitus is a collection of glucose control disorders resulting from deficiency in insulin production/action. In 2021, 6.7 million diabetes-related deaths were reported worldwide. Since many of these deaths were due to chronic diabetes-related conditions, this figure is likely an underestimate. This study sought to improve the investigation of dysglycaemia-related deaths, particularly post-mortem analysis of recombinant human insulin and insulin analogues. It was hypothesised that a move away from traditional matrices and methods must be undertaken, along with standardisation of data interpretation provided to Coroners.

To achieve this, an end-to-end evaluation of the analytical and reporting process for post-mortem insulin was undertaken.

Roche Cobas® and Siemens Immulite® immunoassays were compared for insulin analysis. These demonstrated acceptable recoveries of endogenous insulin (106.5% and 88.3%, respectively), but poor performance for exogenous insulin compounds. Comparison of assays, e.g., Levemir® 2.0% and 12.5% (P = < 0.0001), respectively, and comparison of insulin compounds on a single assay, e.g., NovoRapid® and Levemir® (Cobas®), 11.8% and 2.0% (P = 0.0007), respectively, was poor.

A novel high-resolution accurate-mass liquid chromatography–mass spectrometry (HRAM LC–MS) vitreous humour insulin method was developed. Qualitative performance was demonstrated for five insulin compounds, with variability from expected mass/charge of ≤ 4.89 parts per million for all mass/charge groups detected.

Quantitative performance for NovoRapid® demonstrated linearity up to 2000 pmol/L (R2 = 0.9987), and a lower limit of quantification of 47.2 pmol/L. In one post-mortem case, two insulin compounds, Humalog® and NovoRapid®, were detected. It is unusual for these to be prescribed in combination, suggesting deliberate administration of excess insulin.

To advance the investigation of dysglycaemia-related deaths, migration away from the use of immunoassays is clearly indicated, given the poor recovery of exogenous insulin compounds. The importance of HRAM LC–MS vitreous humour insulin analysis was highlighted by the detection of a previously-unreported potential insulin overdose. The outcomes from this study would collectively improve the investigation of dysglycaemia-related deaths, enhancing the service provided to families of the deceased.