Hyperspectral Raman imaging with multivariate curve resolution-alternating least square (MCR-ALS) analysis for xylazine-containing drug mixtures.

Xylazine, increasingly implicated in illicit opioid overdose deaths, poses a significant public health threat due to its synergistic effects with fentanyl and resistance to naloxone reversal. Despite its rising prevalence, xylazine is not classified as a controlled substance, leading to its exclusion from routine forensic screening. This study introduces a novel analytical method combining Raman hyperspectral imaging with Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS) to detect xylazine in drug mixtures containing common excipients such as acetaminophen, dipyrone, and mannitol. Utilizing only non-negativity constraints, MCR-ALS successfully resolved the Raman spectrum of xylazine at levels as low as 5 % without reference spectra. The method demonstrated robust performance, with percent variance explained (R²) values of 99.60 %, 99.80 %, and 99.91 % for the drug mixtures containing 25 %, 10 %, and 5 % xylazine, respectively.