Method development for the quantification of nine nitazene analogs and brorphine in Dried Blood Spots utilizing liquid chromatography – tandem mass spectrometry
Open accessAbstract
The detection of nitazenes in biological fluids is increasingly needed as they are repeatedly reported in intoxication and overdose cases. A simple method for the quantification of low levels of nine nitazene analogs and brorphine in Dried Blood Spots (DBS) was developed and validated. 10 μL of spiked whole blood is deposited on a Capitainer®B card and allowed to dry. The spot is punched out, and extracted with 500 μL methanol:acetonitrile (3:1 v/v) added with 1.5 μL of fentanyl-D5 as the internal standard. After stirring, sonication, and centrifugation of the vial, the solvent is dried under nitrogen, the extract is reconstituted in 30 μL methanol, and 1 μL is injected into a UHPLC-MS/MS instrument. The method validation showed linear calibration in the 1-50 ng/mL range, LOD values ranging between 0.3 ng/mL (isotonitazene) and 0.5 ng/mL (brorphine), average CV% and bias% within 15 % and 10 % for all compounds, respectively. The matrix effect due to blood and filter paper components was within 85-115 % while recovery was between 15-20 %. Stability tests against time and temperature showed no significant variations for storage periods up to 28 days. Room temperature proved to represent the best samples storage conditions. UHPLC-MS/MS proved capable to reliably identify all target analytes at low concentration even in small specimen volumes, as those obtained from DBS cards, which in turn confirmed to be effective and sustainable micro-sampling devices. This procedure improves the efficiency of toxicological testing and provides an innovative approach for the identification of the nitazene class of illicit compounds.
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Liquid chromatography-mass spectrometry-based techniques, such as liquid chromatography-triple-quadrupole mass spectrometry and liquid chromatography-time-of-flight mass spectrometry, have become the preferred approaches for screening and quantification in forensic and toxicological laboratories.
“Liquid chromatography–mass spectrometry-based techniques, such as liquid chromatography–triple-quadrupole mass spectrometry and liquid chromatography–time-of-flight mass spectrometry, have become the preferred approaches for screening and quantification in forensic and toxicological laboratories.”
From Nitazenes: The Emergence of a Potent Synthetic Opioid Threat (2025) · cites this paper as [54]
These advanced LC-MS systems can achieve the low limits of detection and quantification required for nitazenes.
“These advanced systems can achieve the low limits of detection and quantification required.”
From Nitazenes: The Emergence of a Potent Synthetic Opioid Threat (2025) · cites this paper as [54]
High-resolution mass spectrometry is particularly valuable for non-targeted screening and structural elucidation of new nitazene analogues.
“High-resolution mass spectrometry is particularly valuable for non-targeted screening and structural elucidation of new analogues [52,53,54,55,56].”
From Nitazenes: The Emergence of a Potent Synthetic Opioid Threat (2025) · cites this paper as [54]
Forensic laboratories are adapting to analyse nitazenes in alternative matrices such as hair for retrospective exposure assessment, dried blood spots for micro-sampling in clinical settings, and wastewater for early warning and epidemiological monitoring.
“Forensic laboratories are also adapting to analyse nitazenes in alternative matrices such as hair [62] for retrospective exposure assessment, dried blood spots [54,63] for micro-sampling in clinical settings, and wastewater for early warning and epidemiological monitoring [50,64].”
From Nitazenes: The Emergence of a Potent Synthetic Opioid Threat (2025) · cites this paper as [54]
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- Authors
- Ververi, C., Galletto, M., Massano, M., Alladio, E., Vincenti, M., & Salomone, A.
- Affiliations
- University of Turin (IT)
- Corresponding author
- Ververi, Christina
- ORCID
- Venue
- Journal of Pharmaceutical and Biomedical Analysis
- Publisher
- Elsevier BV
- Published
- 2024-04
- Volume
- 241
- Pages
- 115975
- ISSN
- 0731-7085 (ISSN-L)
- OpenAlex
- W4390811391
- Cited by
- 34
- References
- 40
- Citations by year
- 2026: 16 · 2025: 13 · 2024: 5
- FWCI
- 8.03
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Journal of Pharmaceutical and Biomedical Analysis (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Institutional Research Information System University of Turin (University of Turin) (submittedVersion, other-oa)
- Funders
- Dipartimenti di Eccellenza (2023-2027, D13 C22003520001)
- Topics
- Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Toxicology › Forensic Toxicology and Drug Analysis
- Physical Sciences › Chemistry › Analytical Chemistry › Analytical Methods in Pharmaceuticals
- Life Sciences › Immunology and Microbiology › Immunology › Biosimilars and Bioanalytical Methods
- Keywords
- nitazenes, dried blood spots, isotonitazene, fentanyl analogs, illicit drug detection, toxicological testing, UPLC-MS/MS, method validation, matrix effects, sample storage stability, microsampling
- MeSH terms
- Methanol, Chromatography, High Pressure Liquid, Chromatography, Liquid, Imidazoles, Piperidines, Reproducibility of Results, Tandem Mass Spectrometry, Dried Blood Spot Testing
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