Decoding the Unseen: A Systematic Review of the Analytical Performance of Nitazene Test Strips for Identifying Synthetic Opioids in Seized Drug Materials.

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.