Multitechnique Approach for Xylazine Detection: Chemical Spot Tests, Immunoassays, and Scanning Electron Microscopy

The infiltration of xylazine into the illicit drug supply, particularly as a fentanyl adulterant, has raised significant safety concerns due to its severe toxicological effects and the absence of an effective antidote for targeted therapeutic intervention. Despite the growing implications of xylazine in overdose cases, presumptive testing methods for its detection remain underdeveloped in forensic and public health settings. This study addresses the urgent need for reliable screening tools by evaluating the applicability of chemical spot tests, microcrystalline tests, commercially available xylazine immunoassay test strips, and other field-deployable drug test kits. A comprehensive panel of chemical spot test reagents was tested, and cobalt thiocyanate, Eosin Y (pH 7.0), and Liebermann, Mandelin, Mecke, Marquis, Scott, and Young reagents produced distinct and rapid color changes with xylazine. In parallel, different microcrystalline test reagents were evaluated, and the resulting microcrystal morphology was assessed by using light microscopy and scanning electron microscopy (SEM). Gold chloride reagent yielded immediate, reproducible, and well-defined microcrystals across different xylazine formulations, establishing it as the most effective reagent for microcrystalline testing. All xylazine test strips yielded clear positive test results when tested with xylazine. In contrast, many commercial and on-site screening tools displayed cross-reactivity or false-positive results with xylazine, raising concerns about its specificity and reliability. These findings highlight the need to develop and validate xylazine-specific presumptive screening tests to facilitate forensic investigations, support harm reduction efforts, and enable informed, safer decision-making by people who use drugs (PWUDs).