Isotonitazene Quantitation and Metabolite Discovery in Authentic Forensic Casework.

Area of Science:

• Forensic Toxicology
• Medicolegal Death Investigation
• Pharmacology

Background:

• The synthetic opioid landscape is evolving with novel substances like isotonitazene emerging.
• Isotonitazene, a benzimidazole-class synthetic opioid, is potent but its prevalence in toxicity cases was previously unquantified.
• Non-fentanyl synthetic opioids pose a growing challenge in forensic casework.

Purpose of the Study:

• To confirm and quantitate isotonitazene in biological samples (blood, urine, vitreous fluid).
• To determine the in vivo metabolic profile of isotonitazene in drug users.
• To assess the prevalence of isotonitazene in medicolegal death investigations.

Main Methods:

• Quantitative analysis of isotonitazene using liquid chromatography tandem mass spectrometry (LC-MS/MS).
• Metabolite identification using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS).
• Standard addition method for accurate quantification in biological matrices.

Main Results:

• Isotonitazene was confirmed in 18 cases, with 9 cases testing positive for no other opioids.
• Average blood concentration was 2.2 ± 2.1 ng/mL; average urine concentration was 2.4 ± 1.4 ng/mL.
• Four isotonitazene metabolites were identified, including N- and O-dealkylation products and 5-amino-isotonitazene.

Conclusions:

• Isotonitazene prevalence is increasing in the US, necessitating its inclusion in routine forensic toxicology testing.
• Toxicologists and medical examiners must be aware of emerging synthetic opioids like isotonitazene.
• Public health messaging should address the dangers of potent novel synthetic opioids.