A new nitazene did not become fully validated merely because it resembled drugs already on an autopsy panel. Walton and colleagues extended a nine-analyte assay to butonitazene and etodesnitazene using separate standard-addition verification. Butonitazene also required a longer chromatographic program before its peak could be captured properly.
The original scope was a defined list
The original LC–tandem quadrupole MS validation included isotonitazene, protonitazene, metonitazene, etonitazene, clonitazene, flunitazene and three isotonitazene-related metabolites. It assessed calibration, accuracy, precision, interference, recovery, matrix effects, dilution, carryover, detection and quantification limits, processed-sample stability and urine matrix matching.
Butonitazene and etodesnitazene were later additions. Table I explicitly identified them as outside the full validation scope and evaluated through standard addition only. That distinction did not mean their results had no analytical support; it meant the support came from a different set of experiments.
Why did butonitazene require a method change?
The original chromatographic run lasted seven minutes. Butonitazene eluted at 7.63 minutes, beyond that program. The investigators extended the final organic hold from 0.5 to 1.5 minutes so it could be detected and quantified properly.
Etodesnitazene eluted at 2.81 minutes and was added without the same chromatographic change. The different behavior demonstrates why adding a compound name to a target list is not enough. Its retention under the actual separation conditions must be established.
A missing peak under an unsuitable program would not establish that the specimen contained no drug. Conversely, extending a run does not independently establish every quantitative performance characteristic.
What did the separate verification establish?
Both added analytes were assessed with reference standards and standard addition. Their reported verified linear interval was 0.2–50 ng/mL, with detection limits below 0.1 ng/mL. No interference was found from the evaluated matrix, internal standard, analyte or commonly encountered drugs.
For butonitazene, controls prepared at 5 and 10 ng/mL gave 5.9 and 9.8 ng/mL, deviations of +18% and −2%. Etodesnitazene controls at those levels gave 5.2 and 8.9 ng/mL, deviations of +4% and −11%. Recoveries were 88% and 80%, respectively.
These were verification findings under specified conditions, not a complete duplication of the original nine-analyte validation. The original panel’s accuracy, storage, dilution and matrix results cannot simply be assigned to the two new drugs.
What did the authentic postmortem findings show?
The broader project received 171 biological specimens from 114 selected investigations during 2019–2021, including 47 postmortem and 67 driving-under-the-influence cases. Specimens were suspected of containing new synthetic opioids; this was not a prevalence survey of all deaths.
Butonitazene was quantified in three postmortem specimens: unspecified blood at 3.2 ng/mL, serum at 2.4 ng/mL and urine at 10 ng/mL. Those were specimen counts and must not automatically be described as three independent fatal poisonings.
Etodesnitazene was quantified in one central-blood specimen at 30 ng/mL. A separate peripheral-blood finding was qualitative only. The qualitative finding did not supply another exact concentration or a paired central/peripheral ratio.
Screening, quantitation and interpretation had separate limits
Added analytes outside the full panel were screened using LC–quadrupole time-of-flight MS, followed by standard-addition quantitation with LC–tandem quadrupole MS after verification for each drug. That workflow was more evidence than an unconfirmed library suggestion, while retaining the narrower validation scope.
Authentic storage histories ranged from less than a month to about a year. Blood and fluid specimens were generally refrigerated, while bile, liver and brain were frozen before shipment, then prepared and refrigerated. There was no uniform postmortem interval or collection-site history for every case.
Neither the new peak nor its concentration established an administered dose, impairment level or independent cause of death. Preserve the method version, target list, matrix, reporting qualifier and the experiments actually performed.
For the underlying calibration approach and its limits, see standard addition in postmortem specimens.
Primary source
Walton SE, Krotulski AJ, Logan BK. A Forward-Thinking Approach to Addressing the New Synthetic Opioid 2-Benzylbenzimidazole Nitazene Analogs by Liquid Chromatography–Tandem Quadrupole Mass Spectrometry (LC–QQQ-MS). Journal of Analytical Toxicology. 2022;46:221–231. doi:10.1093/jat/bkab117.
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