Testing for xylazine at Forensic Fluids Laboratories began in the middle of February 2023, and analyses were conducted beginning in March to capture the first full month of testing. Figure 1 shows the overall trends we observed through the first full year of xylazine analysis as a percentage of presumptive positive samples (i.e. samples that screened positive for at least one drug-of-abuse and confirmed positive for at least xylazine). Additionally, we plotted the three-month moving average as a broader statistic of the trends in xylazine positivity rate over this time frame. Using the three-month average trendline, xylazine prevalence peaked at 3.76% by June, dipped to its low in September at 3.14%, and eventually settled at 3.46% by March 2024. On average over the entire year, xylazine was found in 3.41% (1,903) of the 55,691 analyzed samples over 13 months.
Figure 1 Xylazine Positivity Rate
Figure 1. Xylazine total positivity rate (TPR) per month from March 2023 through March 2024. The three-month (quarter-year) moving average is also shown.
Table 1
Drug Classes Found in Xylazine-Positive Samples
| N (% of class) | % of Xylazine Positives |
Fentanyl | 1837 | 96.5 |
Fentanyl | 1837 (100) | 96.5 |
Stimulants | 1218 | 64.0 |
Cocaine | 819 (67.2) | 43.0 |
Methamphetamine | 633 (52.0) | 33.3 |
Amphetamine | 648 (53.2) | 34.1 |
MDMA | 1 (< 0.1) | < 0.1 |
Non-Fentanyl Opiates (NFO) | 836 | 43.9 |
Methadone | 437 (52.3) | 23.0 |
Morphine | 325 (38.9) | 17.1 |
6-monoacetylmorphine | 283 (33.9) | 14.9 |
Buprenorphine | 113 (13.4) | 5.9 |
Tramadol | 62 (7.4) | 3.3 |
Codeine | 24 (2.9) | 1.3 |
Oxycodone | 22 (2.6) | 1.2 |
Hydrocodone | 14 (1.7) | 0.7 |
Cannabinoids | 565 | 29.7 |
Delta-9 THC | 565 (100) | 29.7 |
Table 1 details the drug classes found in xylazine-positive samples. Fentanyl was the most prevalent drug detected with xylazine (96.5%). Given its strong propensity to be found with xylazine compared to the other opiates, we chose to categorize it separately; our findings fall in line with reports that xylazine is most commonly used in combination with fentanyl.[12, 33, 34] Stimulants were also common in xylazine-positive samples (64.0%), followed by non-fentanyl opiates (NFO, 43.9%) and delta-9 THC (29.7%). Individual drugs within these drug classes were also noted in the table.
Table 1 only captures whether a particular drug was observed in a xylazine-positive sample and does not capture any information on how xylazine may have been used in specific combinations with these drugs (i.e. polydrug use). To better understand these trends with xylazine, we looked at the combinations of xylazine with these various drugs based on drug class. Table 2 shows the frequency of xylazine positives in combination with at least one member of another drug class (fentanyl, stimulants, NFO, or cannabinoids).
Table 2
Drug Class Combinations in Xylazine Positive Samples
| N | % of Xylazine Positives |
Single Drug Class Combination | 262 | 13.8 |
Fentanyl | 224 | 11.8 |
Stimulant, NFO, Cannabinoids, or reported alonea | 38 | 2.0 |
Polydrug Class Combinations | 1641 | 86.2 |
Fentanyl + Stimulant | 492 | 25.9 |
Fentanyl + NFO | 215 | 11.3 |
Fentanyl + Cannabinoids | 119 | 6.3 |
Fentanyl + Stimulant + NFO | 367 | 19.3 |
Fentanyl + Stimulant + Cannabinoids | 180 | 9.5 |
Fentanyl + NFO + Cannabinoids | 110 | 5.8 |
Fentanyl + Stimulant + NFO + Cannabinoids | 130 | 6.8 |
Polydrug class combination without Fentanyla | 28 | 1.5 |
a Samples were reported without fentanyl, however trace levels of fentanyl were often present in these samples (data not shown) |
Single drug class combinations with xylazine comprised of only 13.8% of all xylazine positives that were analyzed. As expected, most of this subset of xylazine-positive samples were found with fentanyl (11.8%). The remaining 86.2% of xylazine samples contained multiple drug classes, which are broken down by each grouping. Not surprisingly, fentanyl remained a staple drug within this group. The greatest polydrug-class combinations were fentanyl with stimulants (25.8%) and fentanyl with stimulants and non-fentanyl opiates (19.3%) representing about one-quarter and one-fifth of all xylazine positives respectively. This data indicates not only that xylazine is predominantly found with fentanyl, but also that users of xylazine are most frequently polydrug users. Lastly, while a small contingent of samples was reported without any fentanyl present (3.5% total), examination of the raw LC-MS-MS data often revealed trace levels of fentanyl present (above the limit of detection but below the cutoff value, data not shown), further reinforcing the notion that xylazine is predominantly being used with illicit fentanyl.
Considering that fentanyl was the most prevalent drug detected in xylazine-positive samples, we chose to examine the trends in xylazine positivity among fentanyl-positive samples in our dataset. Figure 2 displays the xylazine positivity rate within samples that confirmed positive for fentanyl over time.
Figure 2 Fentanyl Positive Samples that Contained Xylazine
Figure 2. Percent of fentanyl-positive samples that contained xylazine per month from March 2023 through March 2024. The three-month (quarter-year) moving average is also shown.
The proportion of fentanyl samples that contained xylazine steadily increased between March 2023 and March 2024. As of March 2024, nearly one-third (30.9%, three-month average) of fentanyl-positive samples analyzed at Forensic Fluids Laboratories contained xylazine, up from about one-fifth (21.7%, three-month average) in May 2023. Based on this data, it is evident that xylazine is actively infiltrating the illicit fentanyl market within the regions we monitor.
Lastly, we investigated geospatial trends in xylazine use and spread in Michigan, Ohio, and Indiana. Figures 3, 4, and 5 show the xylazine total positivity rate for samples from counties in Michigan, Ohio and Indiana that were analyzed 1) through May 2023 (three months) and 2) through March 2024 (thirteen months). Xylazine-positive counties are listed along with the percent xylazine-positive samples, number of xylazine-positive samples, and total number of analyzed samples. Xylazine negative counties list the total number of samples analyzed. Counties where we did not collect any eligible samples are demarcated.
Figure 3 Xylazine Positives in Michigan
Figure 3. Heatmap of xylazine prevalence over the first three months and through the first year (13 months) of testing for xylazine of Michigan. The county name, percent and number of xylazine positives are shown for counties that had xylazine positives within the set time period. The total number of samples that screened positive for a drug of abuse is shown in parenthesis for all counties.
Not surprisingly, while the state-level prevalence of xylazine did not change substantially, the county level trends demonstrate significant shifts in xylazine use, as well as the spread of xylazine across the states mapped. In Michigan, the initial xylazine footprint was primarily observed in the southern part of the state. Counties such as Wayne, Saginaw, Montcalm, and Clinton appeared to have had an earlier adoption of xylazine within their respective drug user populations compared to other counties (based on percentages). By the end of our study period, however, an appreciable decline was observed in Saginaw and Clinton counties (> 6% decrease), whereas a more modest decline was observed in Wayne (2.4% decrease) and Montcalm (1.4% decrease). In contrast, Kalamazoo, Jackson, Muskegon, Midland, and Macomb counties saw a slight increase (> 1%) in xylazine prevalence. Likewise, 19 counties demonstrated at least one new xylazine positive after May, potentially indicative of the introduction or increase in use of this drug into user populations over this period. Within this set of emergent counties, Monroe and St. Clair counties both observed prominent xylazine prevalence by the end of the study period (9.1% and 7.9%, respectively). Interestingly, the upper peninsula of Michigan did not have any xylazine detected through March 2024.
Figure 4 Xylazine Positives in Ohio
Figure 4. Heatmap of xylazine prevalence over the first three months and through the first year (13 months) of testing for xylazine of Ohio. The county name, percent and number of xylazine positives are shown for counties that had xylazine positives within the set time period. The total number of samples that screened positive for a drug of abuse is shown in parenthesis for all counties.
In Ohio, xylazine was generally prevalent across the state through May but continued to spread into new counties (25 since May 2023) by the end of the year. The southwest corner of the state appeared to be a particular hot spot for xylazine use. Counties such as Butler, Hamilton, Clermont, Brown, Greene, and Clark demonstrated strong xylazine prevalence through both May and March (> 11% for all counties across both time points). Other counties experiencing appreciable increases in xylazine presence (> 3%) include Highland, Montgomery, Van Wert, Mahoning, and Belmont. In contrast, counties such as Lake, Logan, Athens, Delaware, Franklin, and Wyandot had appreciable decreases (> 3%) in xylazine prevalence over this time frame.
Figure 5 Xylazine Positives in Indiana
Figure 5. Heatmap of xylazine prevalence over the first three months and through the first year (13 months) of testing for xylazine of Indiana. The county name, percent and number of xylazine positives are shown for counties that had xylazine positives within the set time period. The total number of samples that screened positive for a drug of abuse is shown in parenthesis for all counties.
For Indiana, our data is limited in comparison to Michigan and Ohio. Nonetheless, the detection of xylazine in multiple counties by the end of March 2024 illustrates a similar trend to Michigan and Ohio regarding the spread of xylazine across the state over this time. Additionally, Jay, Randolph and Wayne counties, which border alongside xylazine prevalent counties in Ohio, demonstrate the most xylazine prevalent counties in Indiana (in our dataset) by the end of our study period. This observation is potentially indicative of dissemination of xylazine across state borders.
We identified xylazine in other states as well. West Virginia (66), New York (63), Wisconsin (57), Illinois (17), Florida (3), Georgia (1), Missouri (1), and Kentucky (1) all had at least one xylazine-positive sample submitted to our lab from March 2023 through March 2024. The number of samples or geographical spread of samples was not significant in these states to show maps as was done for Michigan, Ohio, and Indiana. An additional file is provided to show the full tally of xylazine positives by state [See Additional file 1].