Decoding the Unseen: A Systematic Review of the Analytical Performance of Nitazene Test Strips for Identifying...

Nitazenes are highly potent synthetic opioids increasingly detected in illicit drug markets and associated with significant public-health and forensic challenges. Rapid identification tools, such as lateral-flow immunoassay (LFA) test strips, are widely used in harm-reduction and forensic settings; however, their analytical performance for detecting nitazene-class opioids remains insufficiently characterized. This systematic review aims to evaluate the analytical performance of nitazene LFA test strips when used on seized drug materials and laboratory-prepared solutions, compared with mass spectrometry (MS) reference methods (liquid chromatography-tandem mass spectrometry (LC-MS/MS), liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS), and gas chromatography-mass spectrometry (GC-MS)). Outcomes included sensitivity, specificity, cross-reactivity, limits of detection, and operational interferences. Adhering to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses - Diagnostic-Test Accuracy (PRISMA-DTA) guidelines and applying Quality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2) framework, five eligible studies were synthesized, including laboratory evaluations and field-paired analyses. Only studies evaluating LFAs on drug materials - not biological samples - were included. Our results showed that two studies provided paired LFA-mass-spectrometry datasets. Under concentrated preparation conditions (10 mg/1 mL), LFAs demonstrated complete agreement with MS. Sensitivity declined at higher dilution (10 mg/5 mL), while specificity remained high. Analytical detectability varied substantially across nitazene analogues, with N-desethyl-metonitazene exhibiting the greatest sensitivity. Matrix effects, caffeine adulteration (~300 µg/mL), solvent concentrations >10% acetonitrile, and elevated temperatures all reduced line intensity or hindered wicking.  In conclusion, Nitazene LFA test strips show potential value as preliminary material-based screening tools in forensic and harm-reduction applications, but should not be interpreted as confirmatory. Their use requires standardized protocols, conservative interpretation rules, and mandatory mass-spectrometric confirmation. Large, independent, multi-site analytical validation studies are needed to establish reliability, optimize field use, and support integration into drug-checking programs.