The author(s) shown below used Federal funding provided by the U.S. Department of Justice to prepare the following resource: Document Title: Statistical and Mass Spectral Tools for the Identification and Characterization of Synthetic Phenethylamines Author(s): Ruth Waddell Smith, A. Daniel Jones, Victoria L. McGuffin Document Number: 252917 Date Received: May 2019 Award Number: 2015-IJ-CX-K008 This resource has not been published by the U.S. Department of Justice. This resource is being made publically available through the Office of Justice Programs’ National Criminal Justice Reference Service. 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. This resource was prepared by the author(s) using Federal funds provided by the U.S. Department of Justice. 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 Purpose of the Project ..................................................................................................................... 1 Project Design and Methods ........................................................................................................... 2 Project Findings .............................................................................................................................. 3 Goal 1. Further develop and validate a method for the statistical comparison of mass spectra . 3 Goal 2. Develop a spectral interpretation scheme for structural class characterization of synthetic phenethylamines based on gas chromatography-mass spectrometry .......................... 6 Goal 3. Develop mass defect filters as a tool for rapid classification of synthetic phenethylamines .......................................................................................................................... 7 Implications for Criminal Justice Policy and Practice in the US .................................................. 10 References ..................................................................................................................................... 11 Appendices .................................................................................................................................... 12 Appendix 1. Compounds included in this study ....................................................................... 12 Appendix 1A. Phenethylamines ................................................................................................ 12 Appendix 1B. Tryptamines ....................................................................................................... 13 Appendix 2: GC-MS instrument and method parameters ......................................................... 14 Appendix 3: Mass spectral data processing methods ................................................................ 17 Appendix 4 Statistical comparison of APB-phenethylamines, 2C-phenethylamines, NBOMe- phenethylamines, and tryptamines ............................................................................................ 18 Appendix 5: Characterization scheme to assign compounds to APB-, 2C-, or NBOMe- phenethylamine subclasses ........................................................................................................ 23 Appendix 6: Characterization examples based on low-resolution mass spectral data .............. 25 Appendix 7: Classification of phenethylamines and tryptamines using principal components analysis and linear discriminant analysis .................................................................................. 29 Appendix 7.1 Feature selection using PCA .............................................................................. 29 Appendix 7.2 Feature selection using an informed chemical approach .................................... 31 Appendix 7.3 Classification models based on linear discriminant analysis ............................. 34 Appendix 8: Characterization examples based on high-resolution mass spectral data ............. 37 Appendix 9: Scholarly products ................................................................................................ 42 A8.1 Publications ...................................................................................................................... 42 A8.2 Presentations ..................................................................................................................... 42 A8.3 Planned future products .................................................................................................... 43 This resource was prepared by the author(s) using Federal funds provided by the U.S. Department of Justice. 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. Purpose of the Project Synthetic designer drugs appeared on the market in 2008 and have continued to pose problems in forensic laboratories since then, primarily in terms of identification and characterization. These compounds are manufactured to maintain the psychoactive effects of a currently controlled substance but with a slight structural change that circumvents current legislation. This results in a variety of compounds that contain the same core structure but which differ in the position or the identity of a single substituent. Current methods of identification rely on gas chromatography-mass spectrometry (GC-MS), typically using electron ionization (EI) and a single quadrupole mass analyzer. While EI affords sufficient fragmentation for structural elucidation, the single quadrupole generates low-resolution, nominal mass data. Thus, definitive identification of structurally similar compounds based on a visual comparison of low-resolution mass spectral data is difficult. Further, with the rapid emergence of new analogs, oftentimes no reference material is immediately available for comparison to aid in the identification. In this work, methods were developed to aid in both the identification and characterization of synthetic designer drugs, initially focusing on three structural subclasses of the synthetic phenethylamines. For identification, a statistical comparison method was refined and applied to demonstrate comparison of two mass spectra with a statistical assessment of the veracity of the identification (Goal 1). For characterization, mass spectral features of the three synthetic phenethylamines subclasses were probed to identify those features that were sufficiently characteristic of the subclass. These features formed the basis of a flow-chart style characterization scheme with the intention that such a scheme could be used to characterize a new analog according to structural subclass (Goal 2). Further, the utility of high-resolution mass spectrometry for designer drug characterization was demonstrated. With the accurate mass data 1 This resource was prepared by the author(s) using Federal funds provided by the U.S. Department of Justice. 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. afforded, the mass defect of an ion can be determined. These mass defects were exploited to increase specificity in the characterization (Goal 3). Finally, project findings will be disseminated as a webinar through the Forensic Technology Center of Excellence, most likely during summer 2018 (Goal 4). Project Design and Methods Synthetic phenethylamine reference standards were purchased from Cayman Chemical (Ann Arbor, MI) throughout this project. These standards included representative compounds from three structural subclasses: the 2,5-dimethoxy (2C-), the aminopropylbenzofuran (APB-), and the 2,5-dimethoxy-N-(2-methoxybenzyl) (NBOMe-) phenethylamines. Particularly for the 2C- and NBOMe-phenethylamines, care was taken to ensure that a range of possible substituents (e.g., halogens, sulfur, and nitro groups) was included. These compounds were primarily used to develop and initially test the developed methods. Additional phenethylamines from other subclasses, as well as cathinones and tryptamines, were also purchased to further test the robustness of the methods developed. All compounds included in this study are shown in Appendix 1. Each standard was analyzed by gas chromatography-mass spectrometry, with both a single quadrupole mass analyzer (GC-QMS) to generate low-resolution data and with a time-of- flight mass analyzer (GC-TOFMS) to generate high-resolution data. Instrument and method parameters are given in Appendix 2. The low-resolution data were used to refine and test the statistical comparison method and to develop the first characterization scheme (Goals 1 and 2), while the high-resolution data were used to define mass defect filters and hence, increase specificity of the characterization scheme (Goal 3). The data processing methods are included in Appendix 3. 2 This resource was prepared by the author(s) using Federal funds provided by the U.S. Department of Justice. 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. Project Findings Goal 1. Further develop and validate a method for the statistical comparison of mass spectra A statistical approach for the comparison of two