Validation of a Fully Automated Immunoaffinity Workflow for the Detection and Quantification of Insulin Analogs by LC–MS-MS in Postmortem Vitreous Humor

Abstract

The analysis of biological specimens collected at autopsy for the presence of exogenous insulin(s) is of special interest in select death investigations as they may be suspected in the cause of a death. Technical challenges include the limited stability of insulin, and the forensic requirement of differentiating endogenous insulin from pharmaceutical analogs. A novel method was developed for the detection and quantification of human insulin, Glulisine, Lispro, Aspart, Glargine and Detemir in vitreous fluid. An immunoaffinity extraction procedure is performed followed by separation of the insulin α- and β-chains. Liquid chromatography tandem mass spectrometry analysis of the β-chain allows for the unequivocal identification of each insulin analog. The analytical measurement range for each insulin was 0.5–25 ng/mL. The method was evaluated for accuracy, precision, carryover, interferences and stability. Eight vitreous fluid samples collected from cases where untoward insulin use was suspected were subjected to analysis. Positive results were obtained from three samples, and a detailed case history is provided for one of these cases. Even though insulin instability in postmortem biological fluid remains a challenge, this method allows for a reliable forensic-level analysis in vitreous fluid.

Detection of intact insulin analogues in post‐mortem vitreous humour—Application to forensic toxicology casework

“…An alternative PM matrix for insulin analysis that yields a more definitive interpretation regarding suspected OD cases is the VH. Not subjected to the same degradation processes pertinent in blood (viz., haemolysis and esterification), insulin detections in this matrix have been described for numerous suicide/OD PM cases 6–9 . Insulins can penetrate through the blood‐ocular barrier into the VH as a result of high systemic levels, such as those experienced in a poisoning event.…”

“…Given the concentration‐dependent mode of transport into the VH from the peripheral system, levels >0.5 ng/ml are believed significant and strongly indicative of an overdose event. In reference to insulin intoxications, reported levels in PM VH determined by LC–MS range from <1 ng/ml to approximately 3.6 ng/ml 2,7–9 . With some instances, detection has been achieved up to 8 days PM, indicating some window of opportunity for detection and stability in the VH 4 .…”

“…Linearity was assessed over the calibration range 0.3–40 ng. Stability of these insulins has previously been assessed, 8 and therefore, steps were taken to minimise protein loss through the utilisation of low‐protein binding apparatus.…”

“…The vitreous humour (VH) has been explored as an alternative matrix for PM toxicology and proposed as a promising exhibit for the detection of insulin and synthetic analogues, 2,4,[6][7][8][9] establishing COD in some of these published cases. The VH is anatomically isolated, resistant to microbiological contamination with bacterial degradation and is more stable and less affected by PM changes than blood.…”

Alternative LC-MS/MS Platforms and Data Acquisition Strategies for Proteomic Genotyping of Human Hair Shafts

“…Proteomics has many promising applications in a legal context, with recent advances being made in body fluid identification, drug interactions, sex estimation, and human identification (HID) [1][2][3][4][5][6]. Proteomic genotyping is the analysis of protein sequence variants, termed single amino acid polymorphisms (SAPs), to infer single nucleotide polymorphism (SNP) alleles.…”