# Laboratory Methods

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# Materials and methods
## a.	Sample preparation
+ i.	Drug samples from the community were received as part of a mail-in drug checking program. Samples were sent into the laboratory in 4.0-mL vials containing the sample dissolved in 1.5mL acetonitrile.  Samples were evaluated based upon participant-provided information of the expected substance. If necessary, the sample was diluted, or an appropriate extraction was performed. Samples were provided as either powder (approximately 10 mg), reside swabbed from the inside of an empty bag, a fraction (approximately ¼) of a tablet, or a used cotton. <br>
+ ii.	Approximately 500μL of the extract was filtered into a 2.0-mL autosampler vial.<br>
+ iii.	Derivatization (if necessary) - Silylation using Bis(trimethylsilyl)trifluoroacetamide with 1% Trimethylchlorosilane (BSTFA w/ 1% TMCS Sigma Aldrich) was utilized. The solvent was evaporated using a Thermo Scientific Savant SPD131DDA SpeedVac Concentrator. Equal parts of ethyl acetate and BSTFA (approximately 150μL) were added to the sample vial and heated at 70°C for 30 minutes. The samples were ready for injection on the GCMS.<br><br>
## b.	Analytical method
+ i.	Samples were analyzed with a ThermoScientific Exactive GC with an electron ionization (EI) source. Compounds in the drug samples were identified qualitatively using a Thermo Scientific Q Exactive GC Orbitrap GC-MS System with a TriPlus RSH Autosampler. Gas chromatography separation was performed using a Thermo TraceGOLD TG-5SilMS column (30 m x 0.25 mm × 0.25 μm). One microliter injections were carried out in split mode using a 20:1 ratio mode with Helium as a carrier gas (constant flow 1.5mL/min). The GC oven temperature programming started at 100 °C and was ramped to 300 °C at a rate of 30 °C/min, with a 9-min hold time. The total run time was 17 minutes. The inlet temperature was 280°C, while the ion source was 230 °C and the MS transfer line was set at 280 °C. The AGC target was set to 1×106.   A 2.3-minute filament delay was used. Samples were acquired in positive mode in full SCAN mode with a range of m/z 40–500 and a resolution of 60,000.<br>
+ ii.	Xcalibur Qual Browser Version 4.5.445.18 (ThermoFisher, Breman, Germany) was used to analyze the data. Compound identification was performed using mass spectral libraries for fragmentation pattern analysis: SWGDRUG MS Library Version 3.10 (Scientific Working Group for the Analysis of Seized Drugs), Cayman Chemical (Ann Arbor, MI) and NIST 20 (National Institute of Standards and Technology, 2020 Version). If necessary, the retention time of compounds was compared analytical reference standards. Standards were purchased from Cayman Chemical Company and Cerilliant Corporation (Round Rock, TX).<br>

# Attribution
For all publications resulting from the use of instrumentation in the MS Core Laboratory, please include in the publication acknowledgements: 
“We thank the University of North Carolina’s Department of Chemistry Mass Spectrometry Core Laboratory, Brandie Ehrmann and Diane Weatherspoon for their assistance with mass spectrometry analysis.”<br><br>

# Funding
Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number R35GM118055 for purchase of mass spectrometry equipment. Sample collection infrastructure was supported by the Foundation for Opioid Response Efforts and the North Carolina General Assembly through the UNC Collaboratory.