## 4 Methods for the Detection of Xylazine and Its Metabolites  
Several analytical methods [113] have been described for the identification of xylazine in animal-derived food, including high-performance liquid chromatography (HPLC) [114, 115, 116], gas chromatography (GC)/mass spectrometry (MS) [78, 117, 118, 119, 120], and LC-MS [121, 122]. [PERSON] et al. (1991) [106] demonstrated the presence of 4-hydroxy-2,6-dimethylaniline, 2,6-dimethylphenylisothiocyanate, and 4-hydroxy xylazine in urine by comparison with chemically prepared standards with GC/MS. The simultaneous analysis of xylazine and some of its metabolites in animal-derived food has also been addressed. [PERSON] et al. (1993) [123] described the use of an HPLC method for the detection of xylazine along with 2,6-xylidine in bovine and swine kidneys, with 25 ppb as the limit of detection (LOD). [PERSON] et al. (2013) [124] reported a sensitive, accurate, and reproducible  
Figure 4: Commonly detected phase I metabolites. LC-MS/MS method for the detection of xylazine along with 2,6-xylidine in animal tissues (liver, meat, kidney, and fat). Due to its increasing prevalence, xylazine is now often studied along with classical opioids and novel synthetic opioids (NSO) for their detection in the blood and urine of humans [125]. The most recent methods described for the detection of xylazine are summarized in Table 2. [PERSON] et al. (2019) [126] reported the first electroanalytical method for xylazine quantification in pharmaceutical, veterinary formulations, and urine, using a glassy carbon electrode, which is simple, sensitive, and accurate for clinical and forensic applications. The LOD was 120 nmol/L. [PERSON] et al. (2022) [75] described, for the first time, a portable electrochemical device for xylazine detection, consisting of an electrochemical paper-based analytical device (ePAD) integrated with a smartphone, which leads to the detection of xylazine by using differential pulse voltammetry. The sensor is low-cost, single-use (disposable), and able to evaluate traces of xylazine in beverage samples, demonstrating excellent accuracy in the detection of xylazine. The LOD was 0.06 \(\upmu\)g/mL. [PERSON] et al. (2022) [127] reported a laser-etched graphene on a polymeric polyetherimide substrate as an electrochemical device for the detection of xylazine in urine and beverage samples and suggested its use in forensic studies as a low-cost, disposable device. [PERSON] et al. (2022) [128] described a monoclonal-antibody-based lateral flow immunoassay and suggested its use for the detection of xylazine in milk, with an LOD of 0.1 ng/mL. Recently, [PERSON] et al. (2024) [129] described the use of a colorimetric assay using molecularly imprinted polymers with high selectivity for the detection of xylazine in alcoholic beverages. The assay required minimal sample pretreatment. A dip-stick-like device was developed and tested for xylazine detection in gin and tonic drinks, yielding positive results in just 20 s, with an LOD of 1.36 mM. [PERSON] et al. (2024) [130] described an enzyme-linked immunosorbent assay for rapid screening of xylazine in oral fluid. The quantification of the drug was as low as 0.1 ng/mL. [PERSON] et al. (2025) [131] reported a portable electrochemical test strip based on a gold nanoparticles/reduced graphene oxide-modified screen-printed carbon electrode which is capable of rapid detection of xylazine in in raw milk samples with a detection limit as low as 0.02 \(\upmu\)g/L. [PERSON] (2025) [132] studied the use of Raman hyperspectral imaging analysis combined with the multivariate curve resolution-alternating least square algorithm for the detection of xylazine in drug mixtures, including acetaminophen, dipyrrone, and mannitol. The method successfully resolved xylazine at concentrations as low as 5%, 10%, and 25%. [PERSON] et al. (2024) [133] described the use of a portable amperometric sensor with anti-fouling properties to detect xylazine in beverage samples and/or from the residue of consumed beverages.