International Journal of Molecular Sciences Article Ultra-High Performance Liquid Chromatography-High Resolution Mass Spectrometry and High-Sensitivity Gas Chromatography-Mass Spectrometry Screening of Classic Drugs and New Psychoactive Substances and Metabolites in Urine of Consumers Emilia Marchei 1, * , Maria Alias Ferri 2,3 , Marta Torrens 2,3 , Magí Farré 3,4 , Roberta Pacifici 1 , Simona Pichini 1 and Manuela Pellegrini 1 1 National Centre on Addiction and Doping, Istituto Superiore di Sanità, V.Le Regina Elena 299, 00161 Rome, Italy; roberta.pacifici@iss.it (R.P.); simona.pichini@iss.it (S.P.); manuela.pellegrini@iss.it (M.P.) 2 Drug Addiction Program, Institut de Neuropsiquiatria i Addicions, Institut Hopsital del Mar d’Investigacions Mèdiques (INAD-IMIM), Parc de Salut Mar, 08003 Barcelona, Spain; malias@imim.es (M.A.F.); mtorrens@parcdesalutmar.cat (M.T.) 3 Department of Psychiatry and Pharmacology, Therapeutics and Toxicology, Universitat Autònoma de  Barcelona, 08193 Cerdanyola del Vallés, Spain; mfarre.germanstrias@gencat.cat  4 Clinical Pharmacology Unit, Hospital Universitari Germans Trias i Pujol and Institut de Recerca Citation: Marchei, E.; Ferri, M.A.; GermansTrias i Pujol (HUGTiP-IGTP), 08916 Badalona, Spain Torrens, M.; Farré, M.; Pacifici, R.; * Correspondence: emilia.marchei@iss.it; Tel.: +39-06-49903026 Pichini, S.; Pellegrini, M. Ultra-High Performance Liquid Abstract: The use of the new psychoactive substances is continuously growing and the implemen- Chromatography-High Resolution tation of accurate and sensible analysis in biological matrices of users is relevant and fundamen- Mass Spectrometry and tal for clinical and forensic purposes. Two different analytical technologies, high-sensitivity gas High-Sensitivity Gas chromatography-mass spectrometry (GC-MS) and ultra-high-performance liquid chromatography- Chromatography-Mass Spectrometry high-resolution mass spectrometry (UHPLC-HRMS) were used for a screening analysis of classic Screening of Classic Drugs and New drugs and new psychoactive substances and their metabolites in urine of formed heroin addicts Psychoactive Substances and under methadone maintenance therapy. Sample preparation involved a liquid-liquid extraction. The Metabolites in Urine of Consumers. Int. J. Mol. Sci. 2021, 22, 4000. UHPLC-HRMS method included Accucore™ phenyl Hexyl (100 × 2.1 mm, 2.6 µm, Thermo, USA) https://doi.org/10.3390/ijms22084000 column with a gradient mobile phase consisting of mobile phase A (ammonium formate 2 mM in water, 0.1% formic acid) and mobile phase B (ammonium formate 2 mM in methanol/acetonitrile Academic Editor: João Pedro Silva 50:50 (v/v), 0.1% formic acid) and a full-scan data-dependent MS2 (ddMS2) mode for substances identification (mass range 100–1000 m/z). The GC-MS method employed an ultra-Inert Intuvo GC Received: 1 April 2021 column (HP-5MS UI, 30 m, 250 µm i.d, film thickness 0.25 µm; Agilent Technologies, Santa Clara, CA, Accepted: 12 April 2021 USA) and electron-impact (EI) mass spectra were recorded in total ion monitoring mode (scan range Published: 13 April 2021 40–550 m/z). Urine samples from 296 patients with a history of opioid use disorder were examined. Around 80 different psychoactive substances and/or metabolites were identified, being methadone Publisher’s Note: MDPI stays neutral and metabolites the most prevalent ones. The possibility to screen for a huge number of psychotropic with regard to jurisdictional claims in substances can be useful in suspected drug related fatalities or acute intoxication/exposure occurring published maps and institutional affil- in emergency departments and drug addiction services. iations. Keywords: classic drugs of abuse; new psychoactive substances (NPS); novel synthetic opioids (NSO); urine; liquid chromatography; high-resolution mass spectrometry; gas chromatography- mass spectrometry Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and 1. Introduction conditions of the Creative Commons Attribution (CC BY) license (https:// A new psychoactive substance (NPS) is defined as “a new narcotic or psychotropic creativecommons.org/licenses/by/ drug, in pure form or in preparation, that is not controlled by the United Nations drug 4.0/). Int. J. Mol. Sci. 2021, 22, 4000. https://doi.org/10.3390/ijms22084000 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 4000 2 of 21 conventions, but which may pose a public health threat comparable to that posed by substances listed in these conventions” [1]. In Europe, seizures of NPS mainly concern synthetic cannabinoids which together with synthetic cathinones account for more than 70% of NPS seizures [2]. Nevertheless, the more recent and most toxic NPS showed to be the novel synthetic opioids (NSOs). Since 2009, 57 new NSOs have been detected on Europe’s drug market [2]. Several NSOs were originally synthesized by pharmaceutical companies in their research for analgesic drugs as compounds with a similar chemical structure to natural opiates without addictive properties, but their toxicity or abuse potential posed a very high risk of poisoning to consumers. Whereas some of them were then marketed as prescription drugs, some others were eliminated from the licit market and some others were chemically modified to exclusively enter illicit market [3–5]. The chemical variety of NSOs, ranging from several illicit analogs of fentanyl and derivatives to newly synthesized molecules, make their identification extremely difficult and need the investigation of qualified analysts/toxicologists [6]. Since NSOs and particularly fentanyl-related compounds are active in very low doses, due to their potency and many users are unknowingly consuming these as adulterants in products sold as heroin, or as pain killers [7,8], parent drugs and metabolites are present in biological material at extremely low concentrations. One consequence of this is that they may escape detection because routine testing of these drugs is rarely performed and requires dedicated analytical methods with sufficiently high sensitivity and specificity [9]. The 2020 COVID-19 pandemic has transformed daily life and the different intensity of the lockdown across countries showed important consequences on drug users. The legal restrictions modified their ability to access classic illicit drugs (e.g., heroin, cocaine, cannabinoids) and shifted consumptions towards prescription psychoactive drugs, fre- quently available at home or from the use of psychoactive recreational NPS (e.g., synthetic cathinones, synthetic cannabinoids, phenethylamines to narcotic analgesics such as NSOs or to anxiolytics such as new benzodiazepines [10,11]. Nine new uncontrolled NSOs have been reported during 2020 [12]and the global shortage of heroin due to pandemic may have forced regular users to take other substances with similar effects, such as fentanyl analogs and NSOs [13]. In 2018 the JUSTSO project (analysis, dissemination of knowledge, implementation of Justice and special tests of new synthetic opioids), funded by European Commission, intended to evaluate, test profile and feedback into education and prevention, knowledge related to the NSO currently used in Europe, their nature, effects and associated harm [14]. Our main involvement in the project was to develop and validate analytical method- ologies for the screening analysis of NSO and their metabolites, together with all other possible psychoactive drugs in urine samples of drug users collected in different settings (detoxification units, methadone maintenance clinics, drug addiction services, etc.). Targeted/untargeted screening workflows based on gas or liquid chromatography coupled with mass spectrometry or tandem mass spectrometry (GC-MS, LC-MS and LC- MS/MS, respectively) play a central role in the daily activities of analytical laboratories operating in clinical and forensic toxicology. Specifically, urinalysis with multiple analytical technologies can increase the number of licit and illicit drugs band metabolites with differ- ent physicochemical properties that can be determined [15–23]. New pharmacologically active substances, both licit and illicit, are constantly being introduced and this occurrence has increased demand for new MS solutions that go beyond conventional GC-MS and LC- MS/MS. High-resolution mass spectrometry (HRMS) enables determination of the exact molecular formula (<5 ppm mass error) that can be useful for presumptive assignment of unknowns in general toxicology screenings [18]. Few previous studies performed in this field used one or more than one analytical tool for identification of a high number of unreported psychotropic substances in biological matrices of users. Int. J. Mol. Sci. 2021, 22, 4000 3 of 21 Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC- MS/MS) methodology has been applied not only to detect, but also to quantify 87 NPS and 32 classic illicit drugs and their metabolites in hair and nails [16] and 77 among the most abused NPS in blood, urine and oral fluid [17]. These two assays used only one type of instrument, but required the availability of all the pure standards of analytes under investi- gation for their quantification. Others screening methods coupled LC or GC with detection methods as time-of-flight mass spectrometry for analytical determination of NPS in seized samples [19] or in serum of consumers [20]. Moreover, to solve a complex toxicological fatal case due to NPS, several different analytical methodologies, including 1H nuclear magnetic resonance (NMR), GC–MS and UPLC–MS/MS to examine unambiguously seized material and biological fluids [21]. Finally, a combination of last generation GC-MS and UHPLC-HRMS has been recently employed by our investigation group to determine a selection of synthetic cannabinoids in oral fluid of consumers. Specifically, GC-MS has proven useful to identify and quantify parent compounds whereas UHPLC-HRMS also confirmed the presence of their metabolites in oral fluid [22]. Using the same combination of analytical methodologies, we hereby propose a screen- ing method for urinalysis of principal NSOs, classical drugs of abuse and other NPS with main metabolites using a fast sample extraction. 2. Results 2.1. GC-MS and UHPLC-HRMS Methods A simple and selective screening analysis with simultaneous use of high-sensitivity GC-MS and UHPLC-HRMS was applied for the identification of classic drugs of abuse, new psychoactive substances and metabolites in urine of drug addicts. The extraction procedure was tested with above reported fortified urine samples using different solvents. The mixture of chloroform and isopropanol has been found as the best compromise for the extraction of drugs and with acceptable signal-to noise ratio in an analytical screening, optimizing the extraction times and costs. Furthermore, even if the total analysis time was not short (each chromatographic run was completed in 32 min in GC/MS and 15 min for UHPLC-HRMS) the combined use of two instruments allowed to screen with a high percentage of compounds matched several different substances. The characteristic retention times and monitored m/z ions used for the identification of mostly found substances monitored in urine samples are reported in Table 1. Table 1. List of different target compounds, retention times (Rt) and monitored ions (m/z) using for the screening gas chromatography-mass spectrometry (GC/MS) and ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) analysis. Compound Formula GC/MS UHPLC-HRMS Target Fragment Target Rt Rt Fragment m/z ion m/z ions m/z ion [M+H]+ (min) (min) m/z ions (Q) (Q/q) a (∆-error, ppm) b Anticonvulsants 193(0.21) 194.0963 Carbamazepine C15 H12 N2 O 15.9 236 5.83 237.1022(−2.53) 165(1.4) 192.0805 153(0.06) 154.1227 Gabapentin C9 H17 NO2 7.74 171 110(0.13) 2.79 172.1332(−3.48) 137.0961 81(0.05) 95.0860 126(0.05) 154.0863 Levetiracetam C8 H14 N2 O2 7.75 170 98 (0.33) 2.85 171.1128(−3.51) 126.0914 69(0.13) 141(0.05) 142.1227 Pregabalin C8 H17 NO2 6.54 159 103(0.03) 2.61 160.1332(−3.75) 97.1016 84(0.04) 83.0861 Int. J. Mol. Sci. 2021, 22, 4000 4 of 21 Table 1. Cont. Compound Formula GC/MS UHPLC-HRMS Target Fragment Target Rt Rt Fragment m/z ion m/z ions m/z ion [M+H]+ (min) (min) m/z ions (Q) (Q/q) a (∆-error, ppm) b 206(2.61) 264.0532 Topiramate C12 H21 NO8 S 5.12 324 189 (2.61) 5.19 357.1326 * (2.20) 184.0970 127(1.62) 127.0391 Antidepressants 215 (0.33) 233.1332 Amitriptyline C20 H23 N 9.59 277 202 (0.17) 5.98 278.19033 (−1.94) 191.0861 58 (0.02) 105.0700 139 (0.14) 184.0521 Bupropion C13 H18 ClNO 17.53 239 100 (0.02) 4.52 240.1150 (−2.08) 166.0419 44 (0.01) 131.0731 238 (0.33) 262.1026 Citalopram C20 H21 FN2 O 17.27 324 208 (0.37) 5.44 325.1711(−1.59) 234.0712 58 (0.02) 109.0452 268 (0.34) 270.1044 Clomipramine C19 H23 ClN2 17.22 314 85 (0.23) 6.23 315.1623(−1.59) 86.0964 58 (0.11) 58.0651 238 (0.23) 293.1446 Desmethylcitalopram C19 H19 FN2 O 17.42 310 138 (0.56) 5.40 311.1554(−1.60) 262.1025 44 (0.05) 109.0451 235.1229 208 (2.50) Desmethylmirtazapine C16 H17 N3 19.71 251 4.08 252.1495(−2.38) 209.1073 195 (0.08) 195.0918 208(0.39) 209.1076 Mirtazapine C17 H19 N3 19.51 265 195(0.05) 4.24 266.1652(−1.88) 195.0917 167(0.5) 72.0816 278(0.26) 176.0819 Trazodone C19 H22 ClN5 O 30.50 371 205(0.05) 4.95 372.1586(−1.34) 148.0505 176(0.16) 96.0446 Antipsychotics 282(6.34) 242.0633 Levomepromazine C19 H24 N2 OS 19.32 328 100(6.01) 6.12 329.1682(−1.82) 100.1126 58(0.79) 58.0660 239(0.46) 221.1080 Norquetiapine C17 H17 N3 S 20.01 295 227(0.09) 5.26 296.1216(−1.69) 210.0373 210(0.16) 139.2405 242(0.20) 256.0901 Olanzapine C17 H20 N4 S 19.01 312 229(0.25) 3.09 313.1481(−1.92) 213.0480 213(0.33) 84.0814 239(0.09) 279.0949 Quetiapine C21 H25 N3 O2 S 19.38 383 210(0.04) 5.61 384.1740(−1.56) 253.0792 144(0.06) 221.1071 233(0.09) 191.1179 Risperidone C23 H27 FN4 O2 8.1 410 191(2.04) 4.78 411.2191(−1.22) 110.0600 177(1.30) 69.0334 Amphetamines 91(0.04) 119.0857 Amphetamine C9 H13 N 5.40 135 2.84 136.1121(−3.67) 44(0.005) 91.0547 148 (0.11) 119.0858 Ethylamphetamine C11 H17 N 6.98 163 91 (0.02) 3.38 164.1434 (−3.05) 91.0547 72 (0.005) 136(0.03) 163.0753 MDA C10 H13 NO2 6.67 179 77(0.08) 3.24 180.1019(−3.33) 135.0439 44(0.02) 105.0699 Int. J. Mol. Sci. 2021, 22, 4000 5 of 21 Table 1. Cont. Compound Formula GC/MS UHPLC-HRMS Target Fragment Target Rt Rt Fragment m/z ion m/z ions m/z ion [M+H]+ (min) (min) m/z ions (Q) (Q/q) a (∆-error, ppm) b 135(0.10) 163.0753 MDMA C11 H15 NO2 6.88 193 77(0.83) 3.31 194.1176(−2.58) 135.04393 58(0.01) 105.06986 134(0.25) 119.0855 Methamphetamine C10 H15 N 5.80 149 91(0.04) 3.20 150.1277(−3.99) 91.0541 58(0.01) Benzodiazepines 256(1.15) 250.0974 7- C15 H12 ClN3 O 12.60 285 222(6.82) 4.06 286.0742(−1.75) 222.1025 Aminoclonazepam 194(6.82) 194.0831 264(5.00) 227.0978 7- C H FN O 11.33 283 255(1.53) 4.65 284.1194(−1.76) 256.1243 Aminoflunitrazepam 16 14 3 240(5.55) 148.0631 222(1.64) 224.1182 7- C15 H13 N3 O 15.12 251 195(5.55) 3.20 252.1131(−2.38) 146.0714 Aminonitrazepam 110 (5.55) 121.0762 279(0.64) 274.1208 Alprazolam C17 H13 ClN4 13.54 308 245(2.29) 6.38 309.0902(−1.62) 241.0528 204(0.83) 205.0747 288(1.14) 302.0448 Clonazepam C15 H10 ClN3 O3 12.34 315 280(0.73) 6.18 316.0484(−1.58) 241.0521 234(1.14) 214.0415 324 (0.60) 326.0563 Clonazolam C17 H12 ClN5 O2 17.99 353 249 (1.00) 5.65 354.0752 (−1.69) 319.1064 203 (0.82) 283(0.77) 222.1150 Diazepam C16 H13 ClN2 O 17.66 284 256(0.59) 6.83 285.0789(−2.10) 193.0885 221(1.43) 154.0417 313 (2.64) 314.0388 Etizolam C17 H15 ClN4 S 18.01 342 266 (3.22) 6.54 343.0779 (−1.46) 259.0216 137 (4.83) 341 (0.60) 343.0096 Flubromazolam C17 H12 BrFN4 370 222 (0.45) 6.22 371.0302 (−1.62) 292.1105 195 (2.25) 237.0951 312(0.71) 300.0902 Flunitrazepam C16 H12 FN3 O3 22.31 313 285(0.65) 6.25 314.0936(−1.59) 268.1003 266(0.95) 239.0976 297 (0.55) 299.0625 Flualprazolam C17 H12 ClFN4 326 257 (2.75) 327.0806 (−2.14) 292.1124 222 (0.61) 223.0662 280 (0.44) 268.0842 Nitrazepam C15 H11 N3 O3 24.08 281 253 (0.64) 5.96 282.0873(−2.12) 236.0944 206(0.78) 207.0918 242(1.04) 208.0994 Nordiazepam C15 H11 ClN2 O 18.66 270 235(3.61) 6.41 271.0633(−1.84) 165.0214 207(4.87) 140.0261 268(0.06) 241.0525 Oxazepam C15 H11 N2 O2 Cl 16.70 286 239(0.07) 6.11 287.0581(−2.09) 269.0475 205(0.06) 104.0498 273(0.35) 283.0630 Temazepam C16 H13 ClN2 O2 19.93 300 271(0.12) 6.51 301.0738(−1.99) 256.0715 256(0.86) 255.0681 Int. J. Mol. Sci. 2021, 22, 4000 6 of 21 Table 1. Cont. Compound Formula GC/MS UHPLC-HRMS Target Fragment Target Rt Rt Fragment m/z ion m/z ions m/z ion [M+H]+ (min) (min) m/z ions (Q) (Q/q) a (∆-error, ppm) b Cocaine 168(0.27) 168.1019 Benzoylecgonine C16 H19 NO4 15.15 289 124(0.07) 3.84 290.1387(−1.72) 105.0335 105(0.22) 82.0650 196(0.23) 196.1330 Cocaethylene C18 H23 NO4 15.03 317 82(0.11) 4.72 318.1704(−0.31) 82.0657 105(0.35) 105.0341 272(2.00) 182.1175 Cocaine C17 H21 NO4 14.27 303 182(0.24) 4.25 304.1543(−1.97) 82.0657 82(0.17) 105.0337 182(1.63) 182.1177 Ecgonine methyl C10 H17 NO3 7.12 199 94(0.39) 0.6 200.1281(−2.99) 150.0911 ester 82(0.31) 82.0658 Cannabinoids 300(0.74) 313.2161 11-OH-THC C21 H30 O3 15.91 330 299(0.16) 8.15 331.2267(−1.81) 193.1224 41(1.86) 105.0703 246(0.53) 193.1225 Cannabidiol C21 H30 O2 16.42 314 231(0.06) 8.64 315.2319(−1.59) 135.1169 193(0.75) 93.0704 295(0.11) 293.1901 Cannabinol C21 H26 O2 17.30 310 238(0.79) 8.88 311.2006(−1.61) 241.1224 165(2.36) 223.1118 299(0.79) 193.1223 Delta-9- C21 H30 O2 16.90 314 271(1.66) 9.02 315.2319(−1.59) 123.0441 tetrahydrocannabinol 231(1.01) 93.0701 329 (0.70) 327.1953 THC-COOH C21 H28 O4 17.20 344 299(0.41) 8.26 345.2060(−1.74) 299.2004 41(0.40) 193.1223 Fentanyls and NSOs 189(0.08) 188.1435 4-ANPP C19 H24 N2 18.16 280 146(0.07) 5.04 281.2012(−2.13) 134.0965 91(0.24) 105.0703 231(0.03) 188.1434 Acetyl fentanyl C21 H26 N2 O 18.01 322 188(0.08) 4.89 323.2118 (−1.54) 105.0703 146(0.05) 132.0809 172(0.20) 284.0610 AH-7921 C16 H22 Cl2 N2 O 11.22 329 144(0.20) 3.73 329.1182(−1.52) 189.9555 126(0.05) 172.0610 289(0.01) 268.17651 Alfentanil C21 H32 N6 O3 18.47 416 268(0.03) 5.35 417.2609(−1.20) 197.1284 140(0.04) 165.10223 259(0.05) 202.1588 Alpha- C23 H30 N2 O 18.30 350 146(0.20) 5.50 351.2431(−1.42) 119.0856 methylfentanyl 91(0.25) 91.0546 245 (0.02) 204.1384 Beta- C22 H28 N2 O2 17.52 352 189 (0.05) 4.90 353.2224 (−1.42) 186.1276 Hydroxyfenatnyl 146 (0.03) 132.0809 303(0.01) 134.0965 Carfentanil C24 H30 N2 O3 18.72 394 187(0.05) 5.60 395.2329(−1.52) 105.0702 105(0.08) 113.0600 Int. J. Mol. Sci. 2021, 22, 4000 7 of 21 Table 1. Cont. Compound Formula GC/MS UHPLC-HRMS Target Fragment Target Rt Rt Fragment m/z ion m/z ions m/z ion [M+H]+ (min) (min) m/z ions (Q) (Q/q) a (∆-error, ppm) b 207(0.08) 188.1435 Despropionyl para- C19 H23 FN2 18.45 298 164(0.08) 5.33 299.1918(−2.01) 134.0966 fluorofentanyl 136 (0.40) 105.0703 189(2.77) 188.1436 Fentanyl C22 H28 N2 O 18.89 245 146(1.57) 5.38 337.2279 (−0.30) 105.0703 105(4.27) 132.08010 263(0.01) 234.1289 Fluorofentanyl C22 H27 FN2 O 17.05 354 207(0.04) 3.55 355.2180(−1.69) 188.1433 164(0.02) 105.0699 236 (0.40) 250.1077 Isotonitazene C23 H30 N4 O3 17.76 410 107 (0.12) 7.02 411.2391(−1.21) 100.1109 86 (0.01) 72.0809 257(0.01) 181.1011 MT-45 C24 H32 N2 12.01 348 165(0.17) 4.03 349.2638(−1.72) 169.1699 91(0.05) 87.0916 189(0.12) 150.0915 N-methyl C15 H22 N2 O 17.32 246 96(0.08) 4.20 247.1805(−2.02) 98.0969 Norfentanyl 82(0.22) 69.0707 175(0.09) 204.1038 Norfentanyl C14 H20 N2 O 17.90 232 159(0.12) 3.77 233.1649 (−2.14) 150.0914 83(0.05) 84.0814 279(0.01) 188.1434 Ocfentanil C22 H27 FN2 O2 17.34 370 176(0.05) 4.83 371.2129(−1.62) 134.0966 105(0.05) 105.0702 227(0.02) 228.1230 Remifentanil C20 H28 N2 O5 16.81 376 212(0.02) 4.48 377.2071(−1.33) 146.0964 168(0.01) 113.0600 289(0.01) 355.1838 Sufentanil C22 H30 N2 O2 S 18.50 386 140(0.03) 5.97 387.2101(−1.29) 238.1257 93(0.03) 111.0266 179(0.20) 97.0111 Thienyl fentanyl C19 H24 N2 OS 17.99 328 97(0.03) 4.87 329.1682(−1.82) 82.0657 82(0.04) 172(0.05) 284.0596 U-47700 C16 H22 Cl2 N2 O 10.80 329 125(0.02) 3.52 329.1182(−1.52) 172.9579 84(0.01) 81.0699 Opioids and SOs 268(0.92) 268.1327 6- C H NO4 18.83 327 214(2.44) 3.37 328.1543(−1.82) 211.0753 Monoacetylmorphine 19 21 162(4.40) 165.0698 434 (0.33) 396.2165 Buprenorphine C29 H41 NO4 32.0 467 410(0.17) 5.72 468.3108(−1.28) 84.0808 378 (0.04) 55.0544 229(3.33) 243.1012 Codeine C18 H21 NO3 16.94 299 214(5.00) 2.88 300.1594(−1.28) 215.1065 162(3.00) 58.0659 262(2.17) 249.1509 EDDP C20 H23 N 11.96 277 220(3.09) 5.61 278.1903(−1.43) 234.1275 165(3.82) 186.1275 208(0.08) 235.1355 EMDP C19 H21 N 11.60 263 130(0.17) 5.95 264.1747(−1.89) 234.1275 115(0.20) 220.1121 284(7.80) 283.175 Hydrocodone C18 H21 NO3 16.01 299 242(1.50) 3.35 300.1594(−1.99) 133.0860 185(2.44) 89.0602 Int. J. Mol. Sci. 2021, 22, 4000 8 of 21 Table 1. Cont. Compound Formula GC/MS UHPLC-HRMS Target Fragment Target Rt Rt Fragment m/z ion m/z ions m/z ion [M+H]+ (min) (min) m/z ions (Q) (Q/q) a (∆-error, ppm) b 229(3.12) 185.0597 Hydromorphone C17 H19 NO3 16.35 285 214(4.08) 2.49 286.1438(−1.75) 227.0699 200(5.30) 199.0753 178(0.33) 105.0338 Methadone C21 H27 NO 13.41 309 165(0.25) 6.15 310.2165(−1.93) 265.1584 72(0.03) 223.1116 268(6.67) 201.0912 Morphine C17 H19 NO3 17.18 285 215(2.50) 1.91 286.1438(−1.75) 229.0857 162(2.13) 183.0807 242(6.67) 268.13263 Norcodeine C17 H19 NO3 16.84 285 215 (2.00) 2.91 286.1438(−1.74) 215.10689 148 (2.50) 225.09088 201(0.02) 254.1173 Normorphine C16 H17 NO3 16.12 271 150(1.05) 1.23 272.1281(−2.20) 201.0916 148(1.33) 121.0649 216(1.76) 284.1281 Noroxycodone C17 H19 NO4 15.32 301 201(4.14) 3.17 302.1387(−1.65) 227.0941 188(3.63) 187.0754 253(5.93) 270.1122 Noroxymorphone C16 H17 NO4 15.30 287 202(1.63) 1.78 288.1230 (−2.08) 213.0783 174(4.15) 173.0597 258(4.42) 298.1438 Oxycodone C18 H21 NO4 15.83 315 230(1.91) 3.21 316.1543(−1.90) 256.1330 187(7.64) 241.1093 244(9.07) 284.1278 Oxymorphone C17 H19 NO4 16.25 301 216(2.62) 2.24 302.1387(−1.65) 242.1173 203(6.18) 227.0934 188 (2.00) Tramadol C16 H25 NO2 14.41 263 135 (2.00) 4.13 264.1958(−2.27) 58.0659 58(0.13) Synthetic Cannabinoids 284(1.50) 214.1224 JWH 018 C24 H23 NO 8.55 341 214(1.31) 8.74 342.1852(−1.75) 155.0605 127(0.82) 144.0444 284(1.62) 230.1172 JWH 073 C23 H21 NO 6.98 327 200(0.98) 8.58 328.1696(−1.52) 155.0489 127(0.84) 125.0962 270(0.95) JWH 073 155.0490 C23 H21 NO2 11.10 343 144(1.11) 7.32 344.1645(−1.74) N-4-Hydroxybutyl 127.1062 127(0.77) 314(2.00) 214.1223 JWH 081 C25 H25 NO2 11.57 371 214(1.43) 8.92 372.1958(−1.61) 185.0596 185(1.43) 144.0443 300(1.32) 214.1222 JWH 081 C24 H23 NO2 12.44 357 214(1.31) 8.36 358.1802(−1.39) 171.0438 4-Hydroxynaphtyl 171(1.48) 144.0443 314(1.45) 230.1172 JWH 081 N-5- C25 H25 NO3 19.93 387 230(1.50) 7.70 388.1907(−1.55) 185.0596 Hydroxypentyl 185(0.90) 144.0443 338(1.82) 214.1223 JWH 122 C25 H25 NO 9.33 355 298(1.38) 8.91 356.2009(−1.40) 169.0646 214(1.45) 141.0697 Int. J. Mol. Sci. 2021, 22, 4000 9 of 21 Table 1. Cont. Compound Formula GC/MS UHPLC-HRMS Target Fragment Target Rt Rt Fragment m/z ion m/z ions m/z ion [M+H]+ (min) (min) m/z ions (Q) (Q/q) a (∆-error, ppm) b 284(0.66) JWH 122 N-4- 169.0647 C25 H25 NO2 13.26 371 169(0.92) 7.81 372.1958(−1.61) Hydroxypentyl 141.0698 144(0.96) 284(1.57) JWH 122 N-5- 169.0646 C25 H25 NO2 15.87 371 141(1.29) 7.80 372.1958(−1.61) Hydroxypentyl 141.0697 115(1.56) 352(1.71) 214.1223 JWH 210 C26 H27 NO 10.77 369 312(1.64) 9.21 370.2165(−1.62) 183.0804 214(0.90) 144.0443 298(0.64) 183.0804 JWH 210 N-4- C26 H27 NO2 14.56 385 183(0.86) 8.08 386.2115(−1.29) 155.0854 Hydroxypentyl 144(0.90) 144.0443 368(2.75) 230.1172 JWH 210 N-5- C26 H27 NO2 17.79 385 230(3.24) 8.06 386.2115(−1.29) 183.0803 Hydroxypentyl 144(2.20) 155.0853 296(0.98) 214.1223 UR 144 C21 H29 NO 9.94 311 214(0.13) 9.07 312.2322(−1.60) 125.0962 144(0.40) 97.1016 231 (0.33) 230.1172 UR 144 N-5- C21 H29 NO2 10.70 327 230(0.001) 7.85 328.2271(−1.83) 125.0962 Hydroxypentyl 144(0.10) 97.1016 314(0.90) 232.1129 XLR 11 C21 H28 FNO 10.73 329 232 (0.09) 8.64 330.2228(−1.51) 125.0962 144(0.36) 97.1016 330(0.83) 248.1077 XLR 11 N-4- C21 H28 FNO2 11.73 345 248(0.11) 7.57 346.2177(−1.44) 144.0443 Hydroxypentyl 144(0.29) 67.0550 342 (0.20) AM-2201 C24 H22 FNO 10.35 359 284 (1.25) 360.1764 232 (1.30) Synthetic Cathinones 149(0.25) 126.1278 MDPV C16 H21 NO3 8.23 275 126(0.01) 4.35 276.1594 (−2.17) 149.0232 119(0.50) 119(0.33) 174.1277 4-MEC C12 H17 NO 6.43 191 91(0.17) 3.66 192.1383 (−2.60) 159.1040 72(0.03) 119.0857 149(0.10) 204.1018 Butylone C12 H15 NO3 8.72 221 121(0.20) 3.52 222.1125(−2.25) 174.0913 72(0.02) 72.0815 119(0.20) 160.1121 Mephedrone C11 H15 NO 6.45 177 91(0.10) 3.37 178.1226(−3.36) 145.0886 58(0.02) 119.0857 105(0.20) 146.0965 Methcathinone C10 H13 NO 5.98 163 77(0.07) 2.67 164.107(−1.83) 131.0731 58(0.02) 105.0703 149(0.17) 218.1174 Pentylone C13 H17 NO3 8.13 235 121(0.25) 4.16 236.1281(−2.54) 188.1069 86(0.01) 86.0969 Int. J. Mol. Sci. 2021, 22, 4000 10 of 21 Table 1. Cont. Compound Formula GC/MS UHPLC-HRMS Target Fragment Target Rt Rt Fragment m/z ion m/z ions m/z ion [M+H]+ (min) (min) m/z ions (Q) (Q/q) a (∆-error, ppm) b Miscellaneous 109(0.06) 109.0451 4-FA C9 H12 FN 4.81 153 83(0.10) 2.95 154.1027(−3.24) 137.0761 44(0.01) 114.0917 122(0.03) 150.0499 4-MA or PMA C10 H15 NO 9.50 165 78(0.08) 3.27 166.1226(−3.61) 137.0419 44(0.01) 117.0701 121(0.10) 149.0961 PMMA C11 H17 NO 10.33 179 78(0.13) 3.43 180.1383(−2.78) 121.0649 58(0.01) 154 (0.25) 154.0416 m-CPP C10 H13 ClN2 7.39 196 3.97 197.0840(−3.04) 138 (2.00) 119.0730 209(0.07) 207.0574 Ketamine C13 H16 ClNO 8.28 237 179(0.02) 3.77 238.0993(−2.51) 179.0622 125(0.08) 125.0154 a, Q/q ion abundance ratio; b, delta error (ppm); * Sodium adduct; MDA: 3,4-Methylenedioxyamphetamine; MDMA: 3,4- Methylenedioxymethylamphetamine;11-OH-THC: 11-Hydroxy-delta-9- tetrahydrocannabinol; THC-COOH: 11-nor-9-carboxy-delta- 9- tetrahydrocannabinol carboxilc acid; NSOs: novel Synthetic Opioids;4-ANPP: 4-Aminophenyl-1-phenethylpiperidine or De- spropionyl fentanyl; AH 7921: 3,4-dichloro-N-[[1-(dimethylamino)cyclohexyl]methyl]-benzamide;U-47700: trans-3,4-dichloro-N-[2- (dimethylamino)cyclohexyl]-N-methyl-benzamide; MT-45: 1-cyclohexyl-4-(1,2-diphenylethyl)-piperazine, dihydrochloride; SO: Syn- thetic Opioids; EDDP: 2-Ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine; EMDP: 2-ethyl-5-methyl-3,3-diphenylpyrroline; MDPV: 3,4- Methylendioxy Pyrovalerone; 4-MEC: 4-Methylethcathinone;4-FA: 4-Fluoroamphetamine; 4-MA or PMA: 4-Methoxyamphetamine or para-methoxymethylamphetamine; PMMA: para-methoxymethamphetamine; m-CPP: 1-(3-Chlorophenyl)piperazine, SO: synthetic opioids. The results obtained by screening proficiency urine testing samples from UNODC In- ternational Quality Assurance Program and those from in “NPS-LABVEQ” project showed an excellent agreement (98% agreement as screened substances) between substances de- clared and those found in the samples. Since these latter substances were analyzed at a concentration of 1 ng/mL urine with a signal to noise ratio, calculated at the baseline, always higher than 10, we could assume that our methodologies could screen substances present in concentrations equal or above 1 ng/mL. Moreover, from the analysis of blank urine no additional peaks due to endogenous substances, which could have interfered with the detection, were observed. 2.2. Methods Application Drug screening applied to 296 former heroin addicts under methadone maintenance therapy urine disclosed the presence of different psychoactive prescription drugs, classical drugs of abuse, NSO, NPS and their metabolites. The presence of a certain drug and/or metabolites was confirmed only if both methodologies identified the molecules, which occurred in 95% cases. Pharmaceuticals like benzodiazepines, antidepressants, antipsychotics, anticonvul- sants and opioids were detected. Drugs of abuse (opioids, amphetamines, cocaine and cannabinoids), NPS (synthetic cannabinoids, synthetic cathinones), fentanyls, NSO and other drug classes were also found. The frequency of different drug classes found in urine samples using the developed GC-MS and LC–HRMS screening methods is reported in Figure 1. Pharmaceuticals like benzodiazepines, antidepressants, antipsychotics, anticonvul- sants and opioids were detected. Drugs of abuse (opioids, amphetamines, cocaine and cannabinoids), NPS (synthetic cannabinoids, synthetic cathinones), fentanyls, NSO and other drug classes were also found. The frequency of different drug classes found in urine Int. J. Mol. Sci. 2021, 22, 4000 11 of 21 samples using the developed GC-MS and LC–HRMS screening methods is reported in Figure 1. Figure 1. Percentage plot of classic drugs and new psychoactive substances found in 296 urine samples from former heroin Figure 1. Percentage plot of classic drugs and new psychoactive substances found in 296 urine samples from former heroin users at methadone maintenance clinics and drug addiction services. users at methadone maintenance clinics and drug addiction services. The most frequent found substances (about 90%) were methadone and its metabo- The most frequent found substances (about 90%) were methadone and its metabo- lites, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and 2-ethyl-5-methyl- lites, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine 3,3-diphenylpyrroline (EMDP). Urine samples resulted positive(EDDP)alsoandto2-ethyl-5-methyl- benzodiazepines 3,3-diphenylpyrroline (mainly Clonazepam, Diazepam and their metabolites), antipsychoticsto (EMDP). Urine samples resulted positive also benzodiazepines (principally Risperi- (mainly Clonazepam, done, Quetiapine Diazepam and their and their metabolites), metabolites),(Citalopram, antidepressants antipsychotics (principally Mirtazapine and Risperidone, Quetiapine their metabolites, andand Trazadone their its metabolites), antidepressants psychoactive metabolite (Citalopram, Mirtazap- meta-Chlorophenylpiperazine) ine andand their metabolites, Gabapentin. Trazadone Additional findings and its psychoactive included metabolite samples positives meta-Chloro- for cocaine and its phenylpiperazine) and Gabapentin. Additional findings included metabolites BZE and EME, cannabinoids, amphetamine and synthetic cathinone samples positives for methylene- cocaine and its metabolites BZE and EME, cannabinoids, dioxypyrovalerone and synthetic cannabinoids from JWH family. amphetamine and synthetic cathinone methylenedioxypyrovalerone In urine samples in which methadone andwas synthetic cannabinoids not found, screening from JWH analysis family.the revealed In urine samples in which methadone was not found, screening analysis presence of the opiates (buprenorphine, 6-MAM, morphine, codeine, dextromethorphan), revealed the presence of the opiates (buprenorphine, cocaine, cannabinoids and fentanyl and6-MAM, analogs. morphine, codeine, dextromethorphan), cocaine, cannabinoids and fentanyl and analogs. 2.3. Fentanyl, Fentanyl Analogs and Novel Synthetic Opioids 2.3. Fentanyl, Fentanyl Toxicological Analogsanalysis screening and Novel Synthetic revealed the Opioids presence of fentanyl and analogs and/or metabolites in 23 (7.8%) Toxicological out ofanalysis screening 296 screened urinethe revealed samples. No other presence NSOs were of fentanyl and found. analogs and/orInmetabolites 4 out of 23 in samples, theout 23 (7.8%) substances matched of 296 screened while urine in other samples. Nocases, other parent drug NSOs were was identified by one method and metabolite by the other, or similar compounds were found. determined. In 4 out of 23 samples, the substances matched while in other cases, parent drug was Chromatogram identified by one methodsin GC-MS and UHPLC-HRMS and metabolite by the other, of or 2 positive similar fentanyl samples compounds are were deter- shown in Figures 2 and 3, respectively, and screening results on fentanyl positive samples mined. were reported in Table 2. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 12 of 21 Int. J. Mol. Sci. 2021, 22, 4000 Chromatogram sin GC-MS and UHPLC-HRMS of 2 positive fentanyl samples 12 are of 21 shown in Figures 2 and 3, respectively, and screening results on fentanyl positive samples were reported in Table 2. Figure 2. Representative selected ion monitoring GC-MS chromatograms of: urine samples positive to Fentanyl (A,B) and Figure 2. Representative selected ion monitoring GC-MS chromatograms of: urine samples positive to Fentanyl (A and mass spectrometry or tandem mass spectrometry (MS/MS) full scan mass spectrum used for substance identification (C). B) and mass spectrometry or tandem mass spectrometry (MS/MS) full scan mass spectrum used for substance identifica- tion (C). Int.Int. J. Mol. Sci.Sci. J. Mol. 2021, 22,22, 2021, 4000 x FOR PEER REVIEW 13 of1321of 21 Figure 3. Representative extracted-ion UHPLC-HRMS chromatograms of: (A) urine sample positive to Fentanyl and Norfentanyl (B) urine sample positive to Fentanyl, Norfentanyl and β-hydroxyfentanyl and MS/MS full scan mass spectrum used for substances identification (C). Int. J. Mol. Sci. 2021, 22, 4000 14 of 21 Table 2. Comparison of GC/MS and UHPLC-HRMS fentanyl and/or its metabolites and analogs urine sample screening and confirmation results. Detected Compound Detected Compound Sample Code (GC/MS) UHPLC-HRMS Fentanyl MI-1029 ND Norfentanyl MI-1077 Fluorfentanyl ND MI-1078 N-(3-ethylindole) Norfentanyl ND Fentanyl MI-1079 Fluorfentanyl Beta-Hydroxyfentanyl Norfentanyl BS-2003 Fentanyl Fentanyl MI-3009 Fluoro acetyl Fentanyl ND MI-5016 Fluoro Valeryl fentanyl ND Fentanyl US-010 Fentanyl Norfentanyl US-017 Fentanyl Norfentanyl Beta-hydroxyfentanyl US-039 Fentanyl Fentanyl Norfentanyl US-059 Fluorfentanyl ND US-060 ND Norfentanyl US-065 Fluorfentanyl ND US-077 Fluoro Valeryl fentanyl ND US-083 Fluoro Valeryl fentanyl ND US-095 Fluoro Valeryl fentanyl ND US-109 Fluoro Valeryl fentanyl ND Acetyl-methylfentanyl US-139 ND 2’-fluoro ortho-Fluorofentanyl US-142 Thiofentanyl ND Fentanyl US-144 Fentanyl Norfentanyl Beta-Hydroxyfentanyl US-145 Fluorfentanyl ND US-148 Fentanyl Norfentanyl US-155 Thiofentanyl Norfentanyl ND: not detected. 2.4. Other NPS The NPS, other than NSOs, detected in the 296 analyzed samples by both methodolo- gies belonged to the class of synthetic cathinones (4.4%) and to that of synthetic cannabi- noids (1.3%) (Table 3). Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 15 of 21 Int. J. Mol. Sci. 2021, 22, 4000 15 of 21 Table 3. New psychoactive substances(NPS) found in urine samples under investigation. NPS3.Classes Table Substances New psychoactive substances(NPS) found in urine samples (n) under investigation. MDPV (2) NPS Classes Substances (n) 4-cloro N butylcathinone (1) synthetic cathinones MDPV (2) 4-Methyl-PV8 (6) 4-cloro N butylcathinone (1) synthetic cathinones Fenethylline (4) 4-Methyl-PV8 (6) JWH-122 (1) (4) Fenethylline JWH-032 (1)(1) JWH-122 synthetic cannabinoids JWH-200 JWH-032(1)(1) synthetic cannabinoids JWH-200 UR-144 (1) (1) UR-144 (1) 4-Methyl-PV8: 2-(pyr- n = number of positive samples; MDPV: 3,4-Methylendioxy Pyrovalerone; rolidin-1-yl)-1-(p-tolyl)heptan-1-one. n = number of positive samples; MDPV: 3,4-Methylendioxy Pyrovalerone; 4-Methyl-PV8: 2-(pyrrolidin-1-yl)-1-(p-tolyl)heptan-1-one. The Theanalysis analysisbybyUHPLC-HRMS UHPLC-HRMSmethod methodofofreal realsample sampleobtained obtainedfrom fromthe thesubject subjectthat that results positive to UR-144 showed two peaks with different retention time but similar results positive to UR-144 showed two peaks with different retention time but similar mass mass spectrum spectrum (Figure (Figure 4). 4). FigureFigure 4. Full 4. Fullspectra mass mass spectra of UR-144-pyr of UR-144-pyr and UR-144. and UR-144. 3.3.Discussion Discussion Methadone Methadoneisisfrequently frequentlyprescribed prescribedfor forthe themaintenance maintenancetherapy therapyof ofopioid opioidaddiction addiction detoxification. detoxification.Patients needing Patients needing treatment treatment with thisthis with andand other medications other medicationsoften havehave often co- occurring medical co-occurring andand medical mental illnesses mental thatthat illnesses require medication require treatment medication treatment[24].[24]. Untargeted Untargetedmassmass spectrometry techniqueshave spectrometry techniques havebecome becomeessential essentialtools toolsfor for toxicolog- toxicological ical analysis analysis [25]. [25]. Thepoor The pooravailability availability of of reference reference standards for many many NPS NPS and and metabolites metabolitespresents presentsa large challenge to forensic toxicology laboratories when trying to detect a large challenge to forensic toxicology laboratories when trying to detect and identifyand identify both known both and unknown known and unknownNPS and NPSother xenobiotics. and other What What xenobiotics. toxicologists expectexpect toxicologists both inboth clinical in and forensic clinical analysis and forensic from a from analysis general unknown a general screening unknown procedure screening is the unequivocal procedure is the une- identification quivocal of the xenobiotics identification involvedinvolved of the xenobiotics in intoxication cases, even in intoxication when cases, eventheywhenhave no they evidences have to guidetothe no evidences search. guide the search. In general, the combination of different complementary methods (immunoassays, liquid chromatography and gas chromatography) was shown to be a good approach for Int. J. Mol. Sci. 2021, 22, 4000 16 of 21 screening samples in forensic and clinical toxicology [26]. Currently, the most competent approach for compound identification involves mass spectral library search [27]. We here presented two complementary analytical methods for screening of classic drugs of abuse and new psychoactive substances and metabolites in urine samples. Low resolution GC-MS and high-resolution instruments (UHPLC-HRMS) can both be used to develop efficient screening workflows. It was possible to obtain an identification, based on the obtained mass spectrometry information, of different xenobiotics. The main purpose of this initial screening technique has been to identify samples positive to classical drugs of abuse, NPS and NSOs while simultaneously eliminating negative specimens from any subsequent analytical examination. Once a NPS or a NSO are detected, quantification could be further performed to provide information regarding concentrations found in urine of users and in cases of fatal and non-fatal intoxications. The principal limitation of the presented methodology was the difficulty associated with data processing to get the information from single sample analysis that required quali- fied expertise. Moreover, in some cases it can be extremely difficult to chromatographically separate certain NPS to facilitate identification via mass spectrometry, such as in the case of isomers and isobaric compounds which display the same or significantly related chemical formulae [22,28–32]. In the current method and in agreement with a previous study [29] the isomers JWH- 019 and JWH-122 as well the two metabolites of JWH-122 (JWH 122 N-4-Hydroxypentyl and N-5-Hydroxypentyl) and JWH-210 (JWH 210 N-4-Hydroxypentyl and N-5-Hydroxypentyl) were not distinguishable, since their masses and retention times matched. Otherwise, the isomers UR-144 and UR-144-pyr could be distinguished (Figure 4). Moreover, the opiate family contains a number of isobaric couples that can complicate the correct identification of e.g., morphine versus hydromorphone, or codeine versus hydrocodone. Other potential isobaric/isomeric interfering compounds that we found in our run were amitriptyline ver- sus EDDP, and Tramadol versus O-desmethylvenlafaxine. Nevertheless, in our developed methodology, the above reported substances exhibited different retention times. Isomeric and isobaric substances require gas or liquid chromatographic conditions that enable adequate separation of the compounds prior to MS analysis or include other mass spectrometry data such as m/z, isotope pattern, retention time and fragmentation information [22,30–32]. However, even if the total analysis time was not short, this method could screen several psychoactive substances of different chemical structures in epidemiological studies aimed to disclose the use of compounds with a high risk of toxicity, leading to severe acute intoxications and overdoses. Moreover, High resolution full scan data also provides retrospective analysis for identifying previously unknown drugs of abuse [31]. Indeed, for this particular study, no reference standards were used, but only mass spectrometric libraries and the coupling of both methodologies. As above reported, posi- tivity to a certain substance was only provided when both methodologies, independently run by different operators, matched with the identification of a specific molecule. In agreement with previous studies [15,19,21], the HRMS procedure was shown to be superior to screening by GC-MS, the costs still limit the widespread distribution in routine laboratories. On the other hand, a last generation GC-MS assay highlighted the similar specificity of UHPLC-HRMS and therefore the simultaneous use of the two instruments allowed to demonstrate that a simple and traditional methodology can be used to screen unknown samples this also due to the presence of the latest generation of libraries present in support to toxicologist whose experience allows to identify unknown substances or to exclude false positives. In this concern, analytical methodologies used for the identification of NPS continu- ously emerging in illicit markets should be developed, validated, updated and analytical data should always be shared across different communication platforms to help health professionals involved in clinical and forensic toxicology issue [6,33]. Int. J. Mol. Sci. 2021, 22, 4000 17 of 21 In addition, once substances identification has been accomplished, it can be of interest to confirm and quantify identified substances to expand information on concentration found in biological fluids of consumers and eventually associate obtained data with clinical evidence. In this concern, pure standards of parent compounds and/or metabolites are needed an extensive method validation whatever is the applied methodology (e.g., LC- MS/MS, GC-MS, GC-MS/MS or HRMS) considering the maximum cost-benefit ration for a high throughput laboratory facing with this kind of analyses. 4. Materials and Methods 4.1. Chemicals and Reagents Water, methanol (MeOH) and acetonitrile (ACN) MS grade, chloroform, isopropanol and formic acid analytical grade were purchased by Carlo Erba (Milan, Italy). Ammonium formate, phosphate buffer and N,O-bis-trimethylsilyl-trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) was obtained from Sigma–Aldrich (Milan, Italy). 4.2. Study Design Urine samples collection took place at Consorcio Mar Parc De Salut De Barcelona, Spain and Hospital Universitari Germans Trias i Pujol from March, 2019 through October, 2020. Here, 296 patients with a history of opioid use disorder were enrolled in this study. All individuals were under methadone maintenance therapy (MTT). In this case, 109 pa- tients provided identified urine samples after obtaining a signed informed consent, while 187 accepted to provide an anonymous sample, but no personal information was collected. In order to secure the participants’ privacy, the survey data and collected urine were coded and the local Human Research Ethics Committee of both centers (ref. 2018/2138/I and PI-18-126) approved the study protocol. Prior to analysis aliquots of urine were stored at −20 ◦ C. 4.3. Sample Preparation for Screening Analysis by GC-MS and UHPLC-HRMS A liquid-liquid extraction was performed after diluting 0.5 mL of urine in 1 mL 0.1 M phosphate buffer pH 3.0 and 0.5 mL of the same sample in 0.1 M phosphate buffer pH 10 (the desired pH was eventually adjusted using drops of 1 N HCl or 1N KOH, respectively). The samples were vortex mixed and then the solutions were extracted twice with 1.5 mL chloroform/isopropanol (9:1, v:v). After centrifugation, the organic layer from each buffered sample was divided into two 1.5 mL aliquots and evaporated to dryness at 40 ◦ C under a nitrogen stream. The first dry aliquot was derivatized with a mixture of 25 µL of acetonitrile and 25 µL of N,O-bis-trimethylsilyl-trifluoroacetamide (BSTFA) with 1%trimethylchlorosilane (TMCS) at 70 ◦ C for 30 min. The second dry aliquot was dissolved in 50 µL ethyl acetate. A 1 µL amount of underivatized and derivatized acid and alkaline extracts were injected into the GC-MS system. After the analysis in GC-MS, the underivatized samples were evaporated to dryness under a nitrogen stream and then dissolved in 150 µL of a mixture of mobile phase A (Ammonium formate 2 mM, 0.1% HCOOH) and B (Ammonium formate 2 mM in MeOH/ACN 50/50, 0.1% HCOOH, 1% H2O) (50:50, v/v). 5 µL were injected into UHPLC- HRMS. 4.4. Gas Chromatography-Mass Spectrometry (GC-MS) Instrumentation The GC-MS instrument consisted of an Agilent 7890 A gas chromatograph coupled with 5975 C mass spectrometry detector (Agilent Technologies, PaloAlto, CA, USA). Ultra- Inert GC column Zebron (ZB-Drug-1, 15m × 250 µm i.d, film thickness 0.25 µm; Phe- nomenex, Milan, Italy) was installed. The GC-MS condition for the screening procedure was as follows: splitless injection mode; helium (purity 99%) carrier gas flow 1.2 mL/min; the injection port, ion source, quadrupole and transfer line temperatures were 260, 230, 150 and 320 ◦ C, respectively; Int. J. Mol. Sci. 2021, 22, 4000 18 of 21 column temperature was programmed at 70 ◦ C for 2 min and increased to 190 ◦ C at 30 ◦ C/min and then increased to 290 ◦ C at 5 ◦ C/min for 10 min. Subsequently the programed temperature was increased to 340 ◦ C at 40 ◦ C/min to eliminate impurities from the column. The electron-impact (EI) mass spectra were recorded in total ion monitoring mode (scan range 40–550 m/z). The full scan data files were processed by an Agilent Workstation (Agilent Technolo- gies). The mass spectra international libraries used for peaks identification were NIST Research Library (National Institute of Standards and Technology) 4.5. Ultra-High-Performance Liquid Chromatography-High-Resolution Accurate Masses Spectrometry (UHPLC-HRMS) Instrumentation The UHPLC/ESI Q-Orbitrap system consisted of an Ultimate3000 LC pump and an Ultimate 3000 autosampler coupled with a QExactive Focus mass spectrometer equipped with a heated electrospray ionization (HESI) probe operating in positive ionization mode and the system was controlled by Trace finder 4.0 software (Thermo Fisher Scientific, Bremen, Germany). Separation was performed on an Accucore™ phenyl Hexyl (100 × 2.1 mm, 2.6 µm, Thermo, USA). They were maintained at 40 ◦ C. The flow rate was set at 500 µL/min. Elution was achieved as follow: 99% A for 1 min, linear gradient to 99% B in 10 min, held for 1.5 min. The column re-equilibration was performed with a linear gradient to 99% A in 0.01 min, held for 4.0 min. A heated electrospray ionization (HESI) source in positive/negative ion mode was used for the ionization of compounds. The mass parameters were as follows: ionization voltage was 3.0 kV; sheath gas and auxiliary gas were 35 and 15 arbitrary units, respectively; S-lens RF level 60; vaporizer temperature and capillary temperature were setting both at 320 ◦ C. Nitrogen was used for spray stabilization, for collision induced dissociation experiments in the HCD cell and as the damping gas in the C-trap. The instrument was calibrated in the positive and negative modes every week. Data were acquired in full-scan in data-dependent MS2 (ddMS2) mode. In this mode, both positive and negative high-resolution, full-scan data at resolution of 70 k were collected with a scan range of 100–1000 m/z, then MS2 spectra at a resolution of 17.5 k with an isolation window of 2 m/z were triggered for compounds entered in the inclusion list and expected retention times of the target analytes, with a 1 min time window. The MS and fragmentation data acquired in full scan is processed by Thermo Scientific TraceFinder™ software. This specific software performs a thorough interrogation of the database by making use of the built-in database and mass spectral library of over 1400 com- pounds, retention times, isotope pattern matching, elemental composition determinations to identify and confirm drugs and metabolites in the analyzed samples. Moreover, mz- Cloud Mass Spectral Library was used as mass spectra international library for unknown peak identification (Advanced Mass Spectral Database; www.mzcloud.org, accessed on 1 April 2021). 4.6. Analytical Performance To check the robustness and the reliability of the developed analytical methods, 10 different proficiency urine testing samples from UNODC International Quality Assurance Program (some with no analytes, some with one and some with more substances), whose previous qualitative and quantitative GC–MS results were available, were re-analyzed using the present methods. Moreover, we also tested 10 urine samples fortified with 1 ng/mL 40 different most popular NPS and main metabolites prepared within the framework of an Italian Project (“NPS-LABVEQ” project) founded by Italian antidrug policy department aimed to allow pharmacotoxicological laboratories along the Italian peninsula to identify these substances in biological and non-biological matrices with different NPS [34]. Finally, 20 blank urine Int. J. Mol. Sci. 2021, 22, 4000 19 of 21 samples from laboratory personnel were also tested to check for false positives during the different batches. 5. Conclusions This study presents a comprehensive gas chromatography-mass spectrometry (GC- MS) and liquid chromatography (UHPLC)–high-resolution mass spectrometry (HRMS) general screening procedure for classic drugs and new psychoactive substances in urine of consumers involving an easy, quick and low-cost sample preparation. This screening method based on two different chromatographic and mass spectrometry methodologies can be applied to disclose suspected drugs of abuse related fatalities or acute intoxications occurring in emergency departments and drug addiction services. Author Contributions: “Conceptualization, S.P., M.F., M.T. and R.P.; methodology, E.M., M.P. and M.A.F.; validation, E.M., M.P. and M.A.F.; investigation, S.P., E.M. and M.P. writing—original draft preparation, E.M., M.P.; writing—review and editing, all the authors. All authors have read and agreed to the published version of the manuscript. Funding: Project funded from the European Commission under the Call JUST-2017-AG-DRUG SUPPORTING INITIATIVES IN THE FIELD OF DRUGS POLICY) Topic: JUST-2017-AG-DRUG; Type of action: JUSTDRUGS-AG (GRANT AGREEMENT NUMBER: 806996-JUSTSO).Other funding from Instituto de Salud Carlos III (ISCIII, Fondo de Investigación en Salud (FIS)-Fondo Europeo de Desarrollo Regional (FEDER), Grant Numbers: FIS PI14/00715 and FIS PI17/01962, ISCIII-Red de Trastornos Adictivos RTA Grant Number: RD16/0017/0003 and RD16/0017/0010, AGAUR Gencat Suport Grups de Recerca, Grant Number: 2017 SGR 316 and 2017 SGR 530. The Presidency of the Ministers Council, Department of Antidrug Policies, in Italy. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Human Research Ethics Committees of Parc de Salut MAR (protocol code 2018/8138/I and date of approval 7 February 2019) and the Hospital Universitary Germans Trias i Pujol (protocol code PI-18-126 and date of approval 20 June 2018). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: All data generated or analyzed during this study are included in this published article. Acknowledgments: The authors thank Laura Martucci, Simonetta Di Carlo, Michele Sciotti and Antonella Bacosi for administrative and technical support. Conflicts of Interest: The authors declare no conflict of interest References 1. European Monitoring Centre for Drugs and Drug Addiction. New Psychoactive Substances (NPS). 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