Post-mortem investigation of chronic use of fentanyl and related analogs by hair testing

Fentanyl and its derivatives, including both pharmaceutical and non-pharmaceutical fentanyls, represent the largest group among synthetic opioids. Fentanyls-related deaths have been reported in recreational use/abuse and therapeutic practice. Furthermore, fatalities from tampering with pharmaceutical products so that they can be smoked, snorted, injected, or taken orally have been reported. In the United States, from 2013 to 2019, the age-adjusted rate of deaths involving synthetic opioids increased by 1,040%. Of them, about 50% was caused by fentanyl or derivatives alone or in association with other drugs. Moreover, in 2021, there were more than 80,000 opioid overdose deaths in the U.S. Most of those deaths were mainly driven by synthetic opioids (primarily fentanyls). In addition, since the mid-2010s, an increasing number of unintentional overdose deaths involving opiates and/or fentanyl showed the presence as adulterant of xylazine, a veterinary drug able to worsen hypotension, central nervous system, and respiratory depression caused by opiates. On the basis of the above evidence, it looks necessary to monitor the diffusion of fentanyl and adulterants among the population to understand their role in the overdose-risk environment and possibly prevent fentanyl overdose fatalities. Hair testing can provide essential information regarding previous intake/exposure to xenobiotics in this frame. The use of hair analysis has gained attention over the years, especially for the retrospective investigation of chronic drug abuse and the unique ability of this matrix to serve as a long-term storage site for xenobiotics. The present study has been carried out in collaboration with the Division of Forensics (Dept of Pathology, University of Alabama at Birmingham (UAB), AL, U.S.) and discusses the forensic toxicological value of the results of fentanyl, fentanyl analogs, and xylazine determination in hair from 250 post-mortem cases with different causes and manners of deaths also to verify the usefulness of extensive hair analysis in forensic pathology and epidemiological field. The analytical procedure of hair testing was developed and validated at the Laboratory of Forensic Medicine of the Department of Diagnostics and Public Health of the University of Verona according to international guidelines for the quantitative analysis in forensic toxicology in terms of interferences (selectivity), linearity, sensitivity (limit of detection [LOD] and limit of quantification [LOQ]), intra- and inter-day precision, accuracy (bias), matrix effect (M.E.) and extraction recovery (RE), carryover, and processed sample stability. In 129 out of 250 cases (51,6%), hair samples tested positive for fentanyl, fentanyl analogs, and adulterants, counting 92 males (71.31%) and 37 females (28.69%), with a mean age of 41.72. The manner of death in the subpopulation with positive hair testing was stated as accident n = 92, homicide n = 14, natural n = 10, suicide n = 7, and undetermined n = 6. Regarding the means of death of the subjects who tested positive on hair, the most represented were drug-opioid (n = 76), gun (n = 19), and drug (n = 13). Fentanyl was detected in almost all cases (125/129 cases), usually with analogs (101/125 cases). The analogs included: norfentanyl (n = 83, 66.4 %), acetylfentanyl (n = 16, 12.8 %), ß-hydroxy fentanyl (n = 42, 33.6 %), despropionyl para-fluorofentanyl (n = 26, 20.8%), 4-ANPP (n = 83, 66.4 %). In 51 out of 125 cases, the adulterant xylazine was identified (n = 51, 40.8 %). The hair fentanyls concentrations reported in the literature, ranging from tens to thousands of picograms per milligram, are similar to those determined in our study, and, to the best of our knowledge, this is the first time that xylazine has been detected in hair. The concentration ratio maps of Jefferson County showed several areas of elevated hair-positive subjects. In addition, the maps showing the concentration ratio of xylazine-hair-positive subjects demonstrate a high incidence in a specific neighborhood of Birmingham. Data from this study confirm the usefulness of systematic hair testing in post-mortem investigation, providing a qualitative representation of drug spread in near real-time in a specific area and evidence consistent with chronic organs and tissue pathologic alterations. In addition, several risk factors for positive fentanyls hair testing, such as age and means of death, have been identified. Lastly, data provided evidence that recurrent consumption/exposure to fentanyl could lead to tolerance in subjects who would, therefore, require higher blood concentrations of fentanyl to cause death. Given the constantly increasing diffusion of new synthetic opioids, particularly in North America, and the related social cost of the emergency, applying hair testing on a large non-pre-selected population confirms the usefulness of hair testing for forensic pathology, public health and epidemiological purposes.