Xylazine, a veterinary sedative, has been found as an adulterant of heroin in street drugs in Puerto Rico. It was found in combination with free morphine and 6-acetylmorphine, codeine, cocaine and benzoylecgonine in postmortem cases at the Puerto Rico Institute of Forensic Sciences (PRIFS). Xylazine is not approved for human use because it has been proven harmful. Currently, three separate analyses are required to determine all the aforementioned drugs at the PRIFS's toxicology laboratory. To reduce analysis time consumption, sample volume, run time, sample preparation and cost, a high-throughput ultra-high-pressure liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous quantification of xylazine, free morphine, 6-acetylmorphine, codeine, cocaine and benzoylecgonine in 0.25 mL postmortem blood by protein precipitation, fulfilling confirmation criteria with three transitions for each compound with acceptable relative ion intensities. Linearity was established between 10-1,000 ng/mL. Total run time was 2.5 min. Limit of detection was 1 ng/mL for cocaine and xylazine, 2 ng/mL for 6-acetylmorphine and 10 ng/mL for free morphine, codeine and benzoylecgonine. The intra-day and inter-day precision and accuracy was less than 15.6%. Process efficiencies ranged from 35.9 to 123.4% and recoveries from 59.9 to 110.1%. The developed method was successfully applied to casework.

The use of xylazine as a drug of abuse has emerged worldwide in the last 7 years, including Puerto Rico. Clinical findings reported that xylazine users present greater physiological deterioration, than heroin users. The aim of this study was to assess the xylazine toxicity on endothelial cells, as this is one of the first tissues impact upon administration. Human umbilical vein endothelial cells in culture were treated with xylazine, cocaine, 6-monoacetylmorphine (heroin metabolite) and its combinations, at concentrations of 0.10 µM to 400 µM, for periods of 24, 48 and 72 hours. IC50 were calculated and the Annexin V assay implemented to determine the cell death mechanism. Results indicated IC50 values at 24 hours as follow: xylazine 62 µM, cocaine 210 µM, 6-monoacetylmorphine 300 µM. When these drugs were combined the IC50 value was 57 µM. Annexin V results indicated cell death by an apoptosis mechanism in cells treated with xylazine or in combination. Results demonstrated that xylazine use inhibits the endothelial cell proliferation, at lower concentrations than cocaine and 6-monoacetylmorphine. These findings contribute to the understanding of the toxicity mechanisms induced by xylazine on endothelial cells.

Human xylazine (XYL) abuse among addicts has received great interest due to its potential toxic effects upon addicts and the need to understand the mechanism of action associated with the potential health effects. XYL is an alpha-2 agonist restricted to veterinarian applications, without human medical applications. Our previous work demonstrated that XYL and its combination with cocaine (COC) and/or 6-monoacetylmorphine (6-MAM) induce cell death through an apoptotic mechanism. The aim of this study was to determine the effect of xylazine on the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) as well as DNA damage on endothelial cell. Human umbilical vein endothelial cells (HUVEC) were treated with XYL (60 μM), COC (160 μM), 6-MAM (160 μM), camptothecin (positive control, 50 μM), XYL/COC (50 μM), XYL/6-MAM (50 μM), and XYL/COC/6-MAM (40 μM) for a period of 24 hours. Generation of intracellular ROS, RNS, and DNA fragmentation were analyzed using a fluorometric assay. Results reveal that XYL and 6-MAM increase levels of ROS; no induction of RNS production was observed. The combination of these drugs shows significant increase in DNA fragmentation in G2/M phase, while XYL, COC, and 6-MAM, without combination, present higher DNA fragmentation in G0/G1 phase. These findings support that these drugs and their combination alter important biochemical events aligned with an apoptotic mechanism of action in HUVEC.