The author(s) shown below used Federal funds provided by the U.S. Department of Justice and prepared the following final report: Document Title: Identification and Prevalence Determination of Novel Recreational Drugs and Discovery of Their Metabolites in Blood, Urine and Oral Fluid Author(s): Amanda L.A. Mohr, Melissa Friscia, Barry K. Logan Document No.: 250338 Date Received: October 2016 Award Number: 2013-DN-BX-K018 This report has not been published by the U.S. Department of Justice. To provide better customer service, NCJRS has made this federally funded grant report available electronically. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. Identification and Prevalence Determination of Novel Recreational Drugs and Discovery of Their Metabolites in Blood, Urine, and Oral Fluid Award Number: 2013-DN-BX-K018 Amanda L.A. Mohr, Melissa Friscia, Barry K. Logan This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. Abstract Designer drug products which contain a variety of unregulated psychoactive constituents have become mainstream on the illicit drug market. These compounds, collectively known as novel psychoactive substances (NPS) are generally abused for their stimulatory and euphoric effects. Because of their physical and mind-altering effects, NPS are commonly used at electronic dance music (EDM) festivals to enhance attendees’ experience of the music and the event. Their widespread use at EDM festivals has been well documented and several adverse events and fatalities associated with the ingestion of these emerging recreational drugs have been reported in the United States. The diversity and rapid turnover in the prevalence of any particular NPS at any given point of time has created several challenges for public health officials, law enforcement, and forensic science communities. Over the course of two years, blood, urine and oral fluid samples were collected from EDM festival attendees, in addition to survey data regarding prescription and recreational drug use within the last week, with the aims of discovering emerging NPS, ascertaining their overall prevalence, evaluating the viability of oral fluid as an alternative matrix for drug detection compared to blood, and determining patterns of use and trends, especially for NPS within this population. Rapid changes in the drug market of synthetic compounds frequently cause epidemiological studies to be published long after drugs have cycled through the peak of their popularity with users, and the scope of testing frequently fails to detect, identify or report the most recently available drugs. Additionally, incomplete literature exists regarding the identity of metabolites of many NPS, making laboratories abilities to maintain a current scope difficult and incomplete. To address this issue, in vitro metabolism studies for alpha-pyrrolidinophenone (alpha-PVP), methylone and dimethylone were carried out using human liver microsomes. Metabolites identified using this in vitro process were subsequently compared to metabolites produced in vivo from authentic human drug user samples described below to determine the extent to which each metabolite could be detected in authentic biological specimens of recreational users and which metabolites would serve as the most valuable biological markers of use. Over 2014 and 2015, biological samples were collected from 396 individuals (126 blood samples; 227 urine samples; 122 oral fluid samples screened with the Alere DDS2; and 384 oral fluid samples collected with the Immunalysis Quantisal™ oral fluid collector). In survey questions, seventy-two percent of the participants had reported using a recreational drug or medicinal substance within the last week. Users most commonly reported using marijuana and alcohol, which were followed by “Molly” and cocaine; Of the 396 individuals tested, approximately 75% of the population was positive in at least one biological specimen for drugs and/or alcohol. With respect to NPS and/or 3,4-methylenedioxy-methamphetamine (MDMA), 37% of the positive samples were confirmed in at least one biological matrix for one or more NPS and/or MDMA. In 2014, several samples were confirmed for alpha-PVP (n=17), however in 2015 there was not a single positive case for alpha-PVP. Instead, increasing numbers of subjects were positive for the NPS ethylone, which demonstrates and supports the high rates of turnover NPS. Page 1 of 86 This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. In comparing the three matrices, there was good agreement between the specimens with respect to reporting positive results. The study demonstrated the value of using oral fluid as a specimen for drug detection compared to blood. Finally, through the use of in vitro metabolism studies, metabolic pathways for alpha-PVP and dimethylone were proposed. These metabolites were subsequently identified in authentic blood, urine and oral fluid specimens. The most prevalent metabolite for alpha-PVP was the 5-OH-PVP metabolite and for dimethylone were methylated dimethylone in blood and oral fluid and the hydroxylated dimethylone in urine. In a few cases, the parent drug was not confirmed, however, the presence of unique metabolites could be used in many cases to indicate which parent drug the subject had ingested. Page 2 of 86 This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. Table of Contents Executive Summary ....................................................................................................... 4-19 Introduction ................................................................................................................ 20-23 Methods ....................................................................................................................... 24-36 Human Subjects ....................................................................................................... 24 Sample Collection ............................................................................................... 25-35 Sample Analysis ................................................................................................... 26-29 Blood .......................................................................................................... 26-29 Urine ........................................................................................................... 29-32 Oral Fluid .................................................................................................... 32-35 Metabolite Identification ........................................................................... 35-36 Unknown Analysis ........................................................................................... 36 Results and Discussion ................................................................................................ 36-73 Survey Data ......................................................................................................... 36-37 Blood Samples ..................................................................................................... 38-43 Urine Samples ..................................................................................................... 43-49 Oral Fluid Samples .............................................................................................. 49-60 Metabolite Identification .................................................................................... 60-68 Alpha-PVP ................................................................................................... 60-66 Dimethylone ............................................................................................... 66-71 Unidentified Analytes ........................................................................................ 71-73 Conclusions .................................................................................................................. 74-78 Discussion of Findings ......................................................................................... 74-76 Implications for Policy and Practice .................................................................... 76-78 Implications for Further Research ...................................................................... 79-81 References ................................................................................................................... 81-85 Dissemination of Research Findings ........................................................................... 85-86 Page 3 of 86 This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the