Capabilities of High Resolution/High Accuracy. Mass Spectrometry in Structural. Characterization of New Psychoactive

New Drugs 214 May 14-15, 214, Rome Capabilities of High Resolution/High Accuracy Mass Spectrometry in Structural Characterization of New Psychoactive Substances with Amphetamine-like Properties Giampietro Frison Laboratory of Environmental Hygiene and Forensic Toxicology (LIATF) Department of Prevention Azienda ULSS 12 Veneziana, Mestre (Venezia), Italy

The LIATF experience with NPS????? Large number of police seizures and some intoxication cases Need to promptly obtain NPS structural characterization, in spite of poor availability of reference (p/m) standards

25 A-R drugs identified at LIAFT Mephedrone (4-MMC) 3-MMC 4-MEC Buphedrone Methylone Butylone Pentedrone -PVP MDPV

25 A-R drugs identified at LIAFT Benzyl-Piperazine Benzyl-Methyl-Piperazine TriF-Methyl-Phenyl- Piperazine Cl-Phenyl-Piperazine 5-APB Methoxethamine DMT 4-AcO-DMT DET

25 A-R drugs identified at LIAFT 2C-E 2C-B bk-2c-b 2,4,5-TMA 4-FA 25I-NBOMe 25B-NBOMe

Analytical strategy to obtain the structural characterization of new A-R drugs GC-MS of underivatized drugs GC-MS after derivatization with 3Cl-ethyl-chloroformate UHPLC-HRMS (High resolution/high accuracy MS)

GC/MS - Mephedrone Rel. Ab. 58 MW 177 GC/MS 56 65 91 119 6 8 1 12 14 16 18 2 22 24 26 28 3 32 34 Agilent 789 II - 5975 Full Scan EI (4-45 u) Inj. 1 l, 25C, split / splitless (1 min) Carrier gas (He), 1 ml/min 162 Agilent HP-5MS Ultra Inert column 3 m x.25 mm x.25 m 5C (.5 min), 2C a 3C/min, 3C (5 min) a 1C/min Interf. 28C, EMV + 3 V

Mephedrone deriv. with 3ClEtClfor + Cl CO O CH 2 CCl 3 MW 177 5 L 2,2,2-3ClEtClfor : Ethyl acetate 3 : 7 8 C, 15 min, to dryness Residue reconst. with 5 L Ethyl acetate CH 3 C O CH2 CCl3 O MW 351 1) G. Frison et al. Rapid Commun. Mass Spectrom. 25, 19, 919 927 2) G. Frison et al. Rapid Commun. Mass Spectrom. 211, 25, 387 39

GC/MS of Mephedrone - 3ClEtClfor Rel. Ab. 91 119 232 234 236 CH 3 C O CH 2 24 232 O 234 131 133 135 Cl C Cl Cl 316 318 +. 351 353 355 58 65 91 119 131 133 135 16 24 316 318 351 353 355 4 6 8 1 12 14 16 18 2 22 24 26 28 3 32 34 36 38 236

High resolution/high accuracy MS Mass resolution = m 1 / m 1 m 2 Low resol. MS High resol. MS 1 H = 1 1 H = 1.7825 12 C = 12 12 C = 12. 16 O = 16 16 O = 15.994915 14 N = 14 14 N = 14.374 Ions at 28 Exact mass CO + 27.994915 N + 2 28.6158 CH 2 N + 28.18723 C 2 H + 4 28.31299

High resolution/high accuracy MS Mass accuracy The ability to measure the exact mass (accurate mass) of a monoisotopic ion m = (Accurate mass Exact mass) Exact mass. 1 6 High resolution High accuracy MS Elemental Composition (EC)!

U-HPLC/HR-Orbitrap-MS k m / High mass resolution: 1. at 2 High mass accuracy: < 5 ppm (ext. cal.) < 2 ppm (int.cal.) z U-HPLC LC: U-HPLC Thermo Scientific Accela 125 Injection: 1 µl Column: Thermo Scientific Hypersil Gold 5 x 2.1 mm x 1.9 mcm Phase A: H 2 O,,5% HCOOH, 5 mm HCOONH 4,pH 5 Phase B: ACN,,5% HCOOH Flow: 4 µl/min Gradient: 2% B, to 5% B in 8 min, to 98% B in 5 min, maint. 2 min, to 2% B in 2 min, maint. 2 min. Temp.: Column 4 C, samples 15 C HR-Orbitrap-MS Mass detect.: Thermo Scientific Exactive Orbitrap HRMS Source: HESI-II Ion Max Spray volt.: 2,5 KV Sheath gas: N 2 set to 45 a. u. Aux. gas: N 2 set to 5 a. u. Cap.Temp.: 29 C HESI Temp.: 26 C Polarity Pos Mass range: 5-8 u Mass resol.: 25. (HCD on, 25 ev) or 1. (HCD off), No Lock Mass

U-HPLC / HR-Orbitrap-MS analytical strategy 1. High chromatographic resolution of analytes 2. Accurate mass measurements of MH + ionic species in full scan conditions 3. Study of MH + collision-induced product ions obtained in MS/MS experiments 4. Comparison of experimental and calculated MH + isotopic patterns 5. Examination of the isotopic fine structure (IFS) of the M+1, M+2, M+3 isotopic peaks relative to the monoisotopic M+ peaks

1. High chromatographic resolution of analytes HR ion chromatograms of 25 AR-drugs RT: 1.97-14.3 1 5 SM: 13G RT: 5.92 5-Fluoro-Amphetamine MH + 154,13 RT: 1.98-14.5 1 5 SM: 13G 4-MEC MH + 192,1387 RT: 7.14 Pentedrone MH + 192,1387 1 5 1 5 1 5 1 5 1 5 1 5 RT: 4.2 Buphedrone MH + 178,123 RT: 5.2 RT: 5.72 RT: 6.45 RT: 6.53 RT: 7.23 RT: 7.43 RT: 7.75 Benzyl-Piperazine MH + 177,139 5-APB MH + 176,174 3-MMC & 4-MMC MH + 178,123 DMT MH + 189,1391 Methyl-Benzyl-Piperazine MH + 191,1547 RT: 8.11 MDPA (IS) MH + 222,1493 1 5 1 5 1 5 1 5 1 5 RT: 5.29 RT: 7.92 RT: 7.98 Methylone MH + 28,973 2C-E MH + 21,1493 DET MH + 217,174 RT: 8.36 RT: 8.7 RT: 8.11 Chloro-Phenyl-Piperazine MH + 197,845 RT: 8.69 MDPA (IS) MH + 222,1493 2 3 4 5 6 7 8 9 1 11 12 13 1 Time (min) 2 3 4 5 6 7 8 9 1 11 12 13 14 Time (min)

1. High chromatographic resolution of analytes HR ion chromatograms of 25 AR-drugs RT: 1.96-14.2 SM: 13G 1 5 1 5 1 5 1 5 1 5 1 5 1 5 RT: 6.4 Butylone MH + 222,113 RT: 7.43 RT: 6.63 TriF-Methyl-Phenyl-Piperazine MH + 231,118 -PVP MH + 232,17 RT: 7.11 2,4,5-TMo-Amphetamine MH + 226,1442 RT: 7.68 RT: 8.36 RT: 8.11 RT: 8.99 4-Acetoxy-DMT MH + 247,1446 Methoxethamine MH + 248,1649 MDPA (IS) MH + 222,1493 RT: 1.94-14.5 1 5 1 5 1 5 1 5 1 5 1 5 SM: 13G RT: 7.51 RT: 7.83 MDPV MH + 276,1597 MDPA (IS) MH + 222,1493 RT: 8.36 RT: 8.11 RT: 8.61 2C-B MH + 26,285 Bk-2C-B MH + 274,78 25B-NBOMe MH + 38,86 25I-NBOMe MH + 428,722 RT: 1.81 RT: 1.33 RT: 11.13 RT: 1.59 2 3 4 5 6 7 8 9 1 11 12 13 1 Time (min) 2 3 4 5 6 7 8 9 1 11 12 13 14 Time (min)

1. High chromatographic resolution of analytes High chromatographic resolution and on-going optimization of analytical conditions help to minimize co-elution and ion suppression Co-elution and ion suppression significantly hinder the ability to make an accurate mass mesurement and, in some cases, become more important than the instrument mass resolution and mass measurement accuracy Croley TR et al. The chromatographic role in high resolution mass spectrometry for nontargeted analysis. J Am. Soc. Mass Spectrom, 212, 23, 1569-1578.

High resolution/high accuracy MS m = (Accurate mass Exact mass) Exact mass. 1 6 The better is the measure of exact mass the lower is the number of EC formulae!

2. MH + accurate mass measurements Acc. mass meas. of MH + ionic species for 25 AR-drugs at 1. RP (no lock mass) Substance Elemental composition Exact mass MH + MH + Δm at 1K Exact mass Accurate mass (ppm) 4-Fluoro-Amphetamine C 9 H 12 FN 153,954 154,127 154,13 1,94 5-APB C 11 H 13 NO 175,997 176,17 176174 2,27 BenzylPiperazine C 11 H 16 N 2 176,1313 177,1386 177,139 2,25 Mephedrone (4-MMC) C 11 H 15 NO 177,1153 178,1226 178,123 2,24 3-MMC C 11 H 15 NO 177,1153 178,1226 178,123 2,24 Buphedrone C 11 H 15 NO 177,1153 178,1226 178,123 2,24 Dimethyltryptamine (DMT) C 12 H 16 N 2 188,1313 189,1386 189,1391 2,64 Methyl-Benzyl-Piperazine C 12 H 18 N 2 19,147 191,1543 191,1547 2,9 4-MEC C 12 H 17 NO 191,131 192,1383 192,1387 2,8 Pentedrone C 12 H 17 NO 191,131 192,1383 192,1387 2,8 Chloro-Phenyl-Piperazine C 1 H 13 ClN 2 196,767 197,84 197,845 2,53 Methylone C 11 H 13 NO 3 27,895 28,968 28,973 2,4 2C-E C 12 H 19 NO 2 29,1415 21,1489 21,1493 1,9 m = (Accurate mass Exact mass) / Exact mass x 1 6

2. MH + accurate mass measurements Acc. mass meas. of MH + ionic species for 25 AR-drugs at 1. RP (no lock mass) Substance Elemental composition Exact mass MH + MH + Δm at 1K Exact mass Accurate mass (ppm) Diethyltryptamine (DET) C 14 H 2 N 2 216,1626 217,1699 217,174 2,3 Butylone C 12 H 15 NO 3 221,152 222,1125 222,113 2,25 2,4,5-Trimethoxyamphetamine C 12 H 19 NO 3 225,1365 226,1438 226,1442 1,76 Trifluoromethylphenylpiperazine C 11 H 13 F 3 N 2 23,131 231,114 231,118 1,73 -PVP C 15 H 21 NO 231,1623 232,1696 232,17 1,72 4-AcO-DMT C 14 H 18 N 2 O 2 246,1368 247,1441 247,1446 2,2 Methoxethamine C 15 H 21 NO 2 247,1572 248,1645 248,1649 1,61 2C-B C 1 H 14 BrNO 2 259,28 26,281 26,285 1,54 Bk-2C-B C 1 H 12 BrNO 3 273,1 274,74 274,78 1,82 MDPV C 16 H 21 NO 3 275,1521 276,1594 276,1597 1,8 25B-NBOMe C 18 H 22 BrNO 3 379,783 38,856 38,86 1,5 25I-NBOMe C 18 H 22 INO 3 427,646 428,719 428,722 1,17 m = (Accurate mass Exact mass) / Exact mass x 1 6

3. Study of MH+ product ions (MS/MS) 4-MEC and Pentedrone: both C 12 H 17 NO, MH + exact mass 192,1383 RT: 3.94-5.55 1 8 6 4 2 1 8 6 4 2 4-MEC 3.99 4.22 4.29 4.35 3.99 4.22 4.29 4.35 4.73 RT: 4.57 AA: 6547794 4.57 RT: 4.73 AA: 773758 4.94 5.17 5.23 5.39 4.94 5.17 5.23 5.39 4. 4.2 4.4 4.6 4.8 5. 5.2 5.4 Time (min) 3.47E6 = 192.1373-192.1393 F: FTMS {1,1} + p ESI Full ms [1.-8.] MS ICIS MIX amf-amfsimili-jwh-metilonpropilon 3.47E6 = 192.1373-192.1393 F: FTMS {1,1} + p ESI Full ms [1.-8.] MS ICIS MIX amf-amfsimili-jwh-metilonpropilon RT: 3.94-5.55 1 8 6 4 2 1 8 6 4 2 3.99 4.22 4.29 4.35 3.99 4.22 4.29 4.35 4.73 RT: 4.57 AA: 6547794 4.57 RT: 4.73 AA: 773758 4.94 5.17 5.23 5.39 Pentedrone 4.94 5.17 5.23 5.39 4. 4.2 4.4 4.6 4.8 5. 5.2 5.4 Time (min) 3.47E6 = 192.1373-192.1393 F: FTMS {1,1} + p ESI Full ms [1.-8.] MS ICIS MIX amf-amfsimili-jwh-metilonpropilon 3.47E6 = 192.1373-192.1393 F: FTMS {1,1} + p ESI Full ms [1.-8.] MS ICIS MIX amf-amfsimili-jwh-metilonpropilon 1 8 6 4 2 1 8 6 4 2 Full scan MS/MS 174.1275 1.1121 119.852 147.82 145.885 91.544 72.811 15.7 131.73 119.856 137.597 159.143 192.138 192.1383 5 1 15 2 2.67E6 MIX amf-amfsimili-jwh-metilon-propilon#743 RT: 4.57 AV: 1 SB: 6 4.45-4.49, 4.65-4.67 T: FTMS {1,1} + p ESI Full ms [1.-8.] 72,811 O H 2.29E5 MIX N amf-amfsimili-jwh-metilon-propilon#742 RT: 4.57 AV: 1 SB: 5 4.45-4.49, 4.65-4.67 F: FTMS {1,2} + p ESI Full ms2 1.@hcd25.145,885 [5.-8.] 119,856 1 8 6 4 2 1 8 6 Full scan MS/MS 91.544 15.333 132.87 132.88 174.1275 192.138 4 15.336 117.574 144.88 174.1278 2 192.1383 86.967 159.143 5 1 15 2 3.43E6 MIX amf-amfsimili-jwh-metilon-propilon#769 RT: 4.73 AV: 1 SB: 5 4.64-4.67, 4.84-4.87 T: FTMS {1,1} + p ESI Full ms [1.-8.] 86,967 159,143 2.14E5 MIX amf-amfsimili-jwh-metilon-propilon#768 RT: 4.73 AV: 1 SB: 6 4.64-4.67, 4.84-4.87 F: FTMS {1,2} + p ESI Full ms2 1.@hcd25. [5.-8.] 15,336 132,88 144,88

3. Study of MH+ product ions (MS/MS) Buphedrone, 3-MMC, 4-MMC: all C 11 H 15 NO, MH + exact mass 178,1226 1 5 Buphedrone MH + 178,123 RT: 5.72 RT: 6.45 3-MMC & 4-MMC MH + 178,123 RT: 7.43 R elative Abundance 1 95 9 85 8 75 7 65 6 55 5 45 4 35 3 25 2 Buphedrone (in source CID, 4 ev) 91.621 131.748 133.856 128.646 117.619 144.816 16.113 15 119.898 162.1192 1 95.566 15.399 158.98 178.1231 5 65.473 72.895 89.466 147.85 13.69 57.658 79.628 115.592 141.9597 149.853 168.818 5 6 7 8 9 1 11 12 13 14 15 16 17 18 1 95 9 85 8 75 7 65 6 55 5 45 4 35 3 25 2 15 1 5 3-MMC (in source CID, 4 ev) 143.741 145.89 16.113 119.898 2 91.621 15 147.85 13.672 1 117.745 13.69 162.1191 178.1232 5 93.776 115.592 158.98 58.737 65.473 78.55 84.9679 128.646 132.825 168.818 182.99 5 6 7 8 9 1 11 12 13 14 15 16 17 18 1 95 9 85 8 75 7 65 6 55 5 45 4 35 3 25 4-MMC (in source CID, 4 ev) 91.621 119.898 143.74 145.89 16.113 147.85 13.673 117.745 13.69 162.1192 93.776 115.592 158.98 178.1232 58.737 65.473 78.55 89.465 128.646 132.825 168.811 5 6 7 8 9 1 11 12 13 14 15 16 17 18

LC/HR-MS & GC/MS (after 3ClEtClfor deriv.) Buphedrone, 3-MMC, 4-MMC deriv. with 2,2,2-trichloroethyl chloroformate 1.698 1.487 O N O O Cl Cl Cl O Cl Cl N O Cl O Buphedrone - 3ClEtClfor 1.894 3-MMC - 3ClEtClfor O N O O Cl Cl Cl 4-MMC - 3ClEtClfor 1.3 1.4 1.5 1.6 1.7 1.8 1.9 11. 11.1

Elements, their isotopes, and EC calc. Data acquired even by ultrahigh mass accuracy and mass resolution can be insufficient for calculating unique elemental compositions without information about isotope ratios Natural occurring elements can be monoisotopic (F, Na, P, I) or polyisotopic (H, C, N, O, S, Cl, Br) Symbol Name Mass of atom % Abundance 1 H Hydrogen 1.7825 99.9885 2 H Deuterium 2.1412.115 3 H Tritium 3.1649 * 12 C Carbon 12. 98.93 13 C 13.3355 1.7 14 C 14.3242 * 14 N Nitrogen 14.374 99.632 15 N 15.19.368 16 O Oxygen 15.994915 99.757 17 O 16.999132.38 18 O 17.99916.25 32 S Sulphur 31.97271 94.93 33 S 32.971458.76 34 S 33.967867 4.29 36 S 35.96781.2 35 Cl Chlorine 34.968853 75.78 37 Cl 36.96593 24.22 79 Br Bromine 78.918338 5.69 81 Br 8.916291 49.31 De Laeter JR, Bohlke JK, De Bievre P, Hidaka H, Peiser HS, Rosman KJR, Taylor PDP: Atomic weights of the elements: Review 2 - (IUPAC technical report). Pure and Applied Chemistry 23, 75(6):683-8.

4. MH + isotopic patterns 1 95 9 85 7.98 1 9 24.778 M+ (MH + ) Experimental 8 75 7 65 6 55 5 45 4 35 3 25 2 15 1 5 1 95 9 85 8 75 7 65 6 55 5 45 4 35 3 25 2 15 1 5.79 1.47 2.12 5.38 2.86 3.63 4.18 6.86 8.44 8.8 1.2 1.92 11.63 12.51.2 13.62 6.8 7.37 14.89 1 2 3 4 5 6 7 8 9 1 11 12 13 14 15 1 Time (min) Full Scan 16.1954 114.964 125.9891 136.223 144.125 Cl-Phenyl- Piperazine MH + pattern 151.363 156.41 167.139 178.1229 187.1233 195.877 197.837 199.8 21.859 28.939 1 11 12 13 14 15 16 17 18 19 2 21 8 7 6 5 4 3 2 1 1 9 8 7 6 5 4 3 13 C M+1 M+2 242.749 M+3 37 Cl or 81 Br 241.811 242.277 238.144 239.1287 24.183 241.1997 243.782 244.2382 245.911 24.786 M+ M+1 M+2 242.756 M+3 Calculated 2 241.819 1 243.79 244.823 245.832 238 239 24 241 242 243 244 245 246

4. MH + isotopic patterns Comparison of measured and calculated MH + isotopic patterns O H N 4-MEC O O O N MDPV 1 192,1381 1 276,159 9 9 8 8 7 6 5 4 3 MH + measured isotopic pattern 7 6 5 4 3 MH + measured isotopic pattern 2 1 1 192,1383 193,1415 194,1448 195,148 2 1 1 277,1624 278,1657 276,1594 9 9 8 7 6 5 MH + calculated isotopic pattern 8 7 6 5 MH + calculated isotopic pattern 4 4 3 3 2 1 193,1416 194,145 195,1484 19 191 192 193 194 195 196 2 1 277,1628 278,1661 272 274 276 278 28 282 284

4. MH + isotopic patterns Comparison of measured and calculated MH + isotopic patterns O H Cl N NH Cl-PhPip N Mephedrone 1 197,838 1 178,1225 9 8 7 6 5 4 3 MH + measured isotopic pattern 199,89 9 8 7 6 5 4 3 MH + measured isotopic pattern 2 1 1 198,872 195,684 196,1177 197,84 2,842 21,873 23,1383 2 1 1 178,1226 179,1258 18,1291 9 9 8 7 6 5 MH + calculated isotopic pattern 8 7 6 5 MH + calculated isotopic pattern 4 3 199,811 4 3 2 1 198,874 2,844 21,878 23,945 194 195 196 197 198 199 2 21 22 23 2 1 179,126 18,1294 175 176 177 178 179 18 181 182 183

4. MH + isotopic patterns RIA error Comparison of experimental and calculated MH + isotopic patterns 1 9 8 7 6 5 4 3 2 1 1 9 8 7 6 5 4 3 24.778 M+ (MH + ) M+1 M+ M+1 M+2 242.749 M+2 Experimental M+3 241.811 242.277 238.144 239.1287 24.183 241.1997 243.782 244.2382 245.911 24.786 242.756 M+3 Calculated 2 241.819 1 243.79 244.823 245.832 238 239 24 241 242 243 244 245 246 RIA Relative Isotopic Abundance M+1 ab / M+ ab ( 13 C / 12 C) RIA error (%) RIA meas RIA calc RIA calc x 1

4. MH + isotopic patterns RIA error Substance RIA error (%) (M+1/M+) 4-Fluoro-Amphetamine,53 5-APB,88 BenzylPiperazine -,14 Mephedrone (4-MMC) 1,56 3-MMC,68 Buphedrone 1,73 Dimethyltryptamine (DMT) -,34 Methyl-Benzyl-Piperazine -1,34 4-MEC 1,5 Pentedrone,55 Chloro-Phenyl-Piperazine -,2 Methylone -,94 2C-E -,28 Substance RIA error (%) (M+1/M+) Diethyltryptamine (DET) -,18 Butylone -,43 2,4,5-Trimethoxyamphetamine -,89 Trifluoromethylphenylpiperazine,7 -PVP -,25 4-AcO-DMT -,27 Methoxethamine,32 2C-B -4,42 Bk-2C-B -3,7 MDPV -,3 25B-NBOMe,62 25I-NBOMe -2,11

4. MH + isotopic patterns RIA error Comparison of experimental and calculated MH + isotopic patterns 1 9 8 7 6 5 4 3 2 1 1 9 8 7 6 5 4 3 24.778 M+ (MH + ) M+1 M+ M+1 M+2 242.749 M+2 Experimental M+3 241.811 242.277 238.144 239.1287 24.183 241.1997 243.782 244.2382 245.911 24.786 242.756 M+3 Calculated 2 241.819 1 243.79 244.823 245.832 238 239 24 241 242 243 244 245 246 RIA Relative Isotopic Abundance M+2 ab / M+ ab (Br or Cl / 12 C) RIA error (%) RIA meas RIA calc RIA calc x 1

4. MH + isotopic patterns RIA error Substance Halogen atom RIA error (%) (M+2/M+) Chloro-Phenyl-Piperazine Cl,4 2C-B Br -,27 bk-2c-b Br 2,22 25B-NBOMe Br 1,23

5. Fine Structure of MH + isotopic patterns Isotopic Fine Structure of M+1, M+2, M+3 peaks vs. M+ peak 1 M+ (MH + ) 197,838 MH + measured isotopic pattern 9 8 7 6 5 4 3 2 1 1. RP M+1: Complete separation of 15 N and 13 C peaks M+1 198,872 199,89 M+2 M+3 1. RP M+2: Complete separation of 18 O, 34 S, 37 Cl... 13 C 2 peaks 195,684 196,1177 2,842 21,873 23,1383 197 84

5. Fine Structure of MH + isotopic patterns M+1, M+2, M+3 isotopic peaks vs. M+ peaks M+ of Mephedrone (monoisotopic) 1 9 8 7 6 5 4 3 2 1 236.673 1 9 8 7 6 5 4 3 238.991 2 1 1 9 8 7 M+ (MH + ) M+1 M+2 24.961 24.622 C 11 H 16 NO 178,1225 178.1225 178.68 178.743 178.831 178.154 178.1182 178.1278 178.1421 178.1533 178.1661 178.178 178.1932 178.1226 M+3 239.124 237.634 238.62 238.134 239.1426 241.994 24.1258 242.128 243.184 244.178 178,1226 7.77E6 21133amfsimili-mix1-1_1k#293 RT: 4.35 AV: 1 SB: 8 4.18-4.23, 4.45-4.5 T: FTMS {1,1} + p ESI Full ms [1.-8.] Measured at 1. RP 8.81E5 C 11 H 15 NO +H: C 11 H 16 N 1 O 1 pa Chrg 1 6 5 4 3 Calculated 2 1 178.6 178.8 178.1 178.12 178.14 178.16 178.18 178.2

5. Fine Structure of MH + isotopic patterns M+1, M+2, M+3 isotopic peaks vs. M+ peaks M+1 of Mephedrone isotopic pattern (12%M+) 1 9 8 7 6 5 4 3 2 1 236.673 238.991 1 9 8 7 6 5 4 3 2 M+ 1 (MH + ) 1 9 8 M+1 7 5 4 M+2 24.961 24.622 M+3 C 1 13 CH 16 NO C 11 H 16 15 NO 239.124 237.634 238.62 238.1346239.1426 241.994 24.1258 242.128 243.184 244.178 179,1193 179,1259 179,1322 179,645 179,1193 179,1322 179,1491 179,178 179,1973 179,126 179,126 C 11 H 152 HNO 5,78E5 21133AMFSIMILI-MIX1-1_1K#29-294 RT: 4,3-4,36 AV: 5 SB: 31 3,97-4,14, 4,53-4,79 T: FTMS {1;1} + p ESI Full ms [1,-8,] Measured at 1. RP 1,5E5 C 11 H 15 NO +H: C 11 H 16 N 1 O 1 pa Chrg 1 Calculated 3 2 1 179,1197 179,1323 179,5 179,1 179,15 179,2

5. Fine Structure of MH + isotopic patterns M+1, M+2, M+3 isotopic peaks vs. M+ peaks M+2 of Mephedrone isotopic pattern (.5%M+) 1 9 8 7 6 5 4 3 2 1 236.673 238.991 1 9 8 7 6 5 4 3 2 1 1 9 8 7 6 5 4 3 C 13 9 C 2 H 16 NO C 11 H 16 N 18 O C 13 1 CH 15 16 NO M+ (MH + ) M+1 239.124 M+2 24.961 24.622 M+3 241.994 244.178 237.634 238.62 238.134 239.1426 24.1258 242.128 243.184 18,1266 18,1224 18,1224 18,1292 18,132 18,1294 18,1269 18,1269 18,1292 18,1319 18,1319 C 1 13 CH 152 HNO 18,1583 2,88E4 21133AMFSIMILI-MIX1-1_1K#29-294 RT: 4,3-4,36 AV: 5 SB: 31 3,97-4,14, 4,53-4,79 T: FTMS {1;1} + p ESI Full ms [1,-8,] Measured at 1. RP 5,67E3 C 11 H 15 NO +H: C 11 H 16 N 1 O 1 pa Chrg 1 Calculated 2 1 18,123 18,123 18,1323 18,1323 18,1 18,12 18,14 18,16

5. Fine Structure of MH + isotopic patterns M+1, M+2, M+3 isotopic peaks vs. M+ peaks M+3 of Mephedrone isotopic pattern (.1%M+) 1 9 8 7 238.991 1 9 8 7 6 5 4 3 M+ (MH + ) 2 1 C 1 13 CH 16 N 18 O 181,126 181,132 181,1325 181,1325 C 8 13 C 3 H 16 NO 5,83E2 21133AMFSIMILI-MIX1-1_1K#29-294 RT: 4,3-4,36 AV: 5 SB: 31 3,97-4,14, 4,53-4,79 T: FTMS {1;1} + p ESI Full ms [1,-8,] Measured at 1. RP 6 5 4 3 2 1 236.673 1 9 M+1 8 7 M+2 24.961 24.622 M+3 239.124 237.634 238.62 238.134 239.1426 241.994 24.1258 242.128 243.184 244.178 181,132 181,1327 181,1327 2,15E2 C 11 H 15 NO +H: C 11 H 16 N 1 O 1 pa Chrg 1 6 5 4 Calculated 3 2 181,1264 1 181,1239 181,1356 181,1385 181,8 181,1 181,12 181,14

5. Fine Structure of MH + isotopic patterns M+1, M+2, M+3 isotopic peaks vs. M+ peaks M+ of Cl-Phenyl-Piperazine (monoisotopic) 1 9 8 197,835 7,22E6 211825amfsimili-mix1-2_1ka#345-347 RT: 4,91-4,93 AV: 3 SB: 14 4,76-4,86, 4,99-5,6 T: FTMS {1 ;1 } + p E S I F ull m s [1, -8, ] 1 9 8 7 6 5 4 3 2 1 236.673 238.991 7 6 5 4 3 M+ (MH + ) 2 1 1 9 M+1 8 7 M+2 24.961 24.622 C 1 H 14 ClN 2 197,896 197,228 197,459 197,766 197,191 197,1351 197,1731 197,1917 197,84 M+3 239.124 237.634 238.62 238.134 239.1426 241.994 24.1258 242.128 243.184 244.178 Measured at 1. RP 6,74E5 C 1 H 13 ClN 2 +H: C 1 H 14 Cl 1 N 2 pa Chrg 1 6 5 4 Calculated 3 2 1 197, 197,5 197,1 197,15 197,2

5. Fine Structure of MH + isotopic patterns M+1, M+2, M+3 isotopic peaks vs. M+ peaks M+1 of Cl-Phenyl-Piperazine isotopic pattern (11%M+) 1 9 8 C 9 13 CH 14 ClN 2 198,869 7,62E5 211825amfsimili-mix1-2_1ka#345-347 RT: 4,91-4,93 AV: 3 SB: 14 4,76-4,86, 4,99-5,6 T: FTMS {1 ;1 } + p E S I F ull m s [1, -8, ] 1 9 8 7 6 5 4 3 2 1 236.673 238.991 7 6 5 4 3 2 M+ 1 (MH + ) 198,85 198,65 198,773 198,874 1 9 8 M+1 7 5 4 M+2 24.961 24.622 M+3 C 1 H 14 Cl 15 NN 239.124 237.634 238.62 238.1346239.1426 241.994 24.1258 242.128 243.184 244.178 198,898 C 1 H 132 HClN 2 198,941 198,134 198,1119 Measured at 1. RP 7,29E4 C 1 H 13 ClN 2 +H: C 1 H 14 Cl 1 N 2 pa Chrg 1 Calculated 3 2 1 198,81 198,93 198,6 198,7 198,8 198,9 198,1 198,11

5. Fine Structure of MH + isotopic patterns M+1, M+2, M+3 isotopic peaks vs. M+ peaks M+2 of Cl-Phenyl-Piperazine isotopic pattern (33%M+) 1 9 8 7 6 5 4 3 2 1 236.673 238.991 1 9 8 7 6 5 4 3 M+ (MH + ) 2 1 1 9 M+1 8 7 M+2 24.961 24.622 C 1 H 14 37 ClN 2 199,85 C 8 13 C 2 H 14 ClN 2 199,49 199,378 199,718 199,9 199,1184 199,1581 199,179 199,811 M+3 239.124 237.634 238.62 238.134 239.1426 241.994 24.1258 242.128 243.184 244.178 3,E6 211825amfsimili-mix1-2_1k-a#346 RT: 4,92 AV: 1 SB: 6 4,46-4,51, 4,65-4,66 T: F TM S {1 ;1 } + p E S I F ull m s [1,-8,] Measured at 1. RP 2,16E5 C 1 H 13 ClN 2 +H: C 1 H 14 Cl 1 N 2 pa Chrg 1 6 5 4 Calculated 3 2 1 199,97 199, 199,5 199,1 199,15 199,2

5. Fine Structure of MH + isotopic patterns M+1, M+2, M+3 isotopic peaks vs. M+ peaks M+3 of Cl-Phenyl-Piperazine isotopic pattern (3%M+) 1 9 8 7 6 5 4 3 2 1 236.673 238.991 1 9 8 7 6 5 4 3 2 1 1 9 8 7 6 5 4 C 13 9 CH 37 14 ClN 2 C 1 H 14 N 15 N 37 Cl 2,838 2,871 C 1 H 132 HN 2 37 Cl 2,776 2,464 2,644 2,738 2,991 2,1153 2,844 M+ (MH + ) M+1 239.124 M+2 24.961 24.622 M+3 241.994 244.178 237.634 238.62 238.134 239.1426 24.1258 242.128 243.184 3,42E5 211825amfsimili-mix1-2_1k-a#346 RT: 4,92 AV: 1 SB: 6 4,46-4,51, 4,65-4,66 T: F TM S {1 ;1 } + p E S I F ull m s [1,-8,] Measured at 1. RP 2,33E4 C 1 H 13 ClN 2 +H: C 1 H 14 Cl 1 N 2 pa Chrg 1 Calculated 3 2 1 2,781 2,873 2,97 2,6 2,8 2,1 2,12

5. Fine Structure of MH + isotopic patterns M+1, M+2, M+3, M+4 isotopic peaks vs. M+ peaks M+4 of Cl-Phenyl-Piperazine isotopic pattern (.2%M+) 1 9 8 7 6 5 4 3 2 1 236.673 238.991 1 9 8 7 6 5 4 3 M+ (MH + ) 2 1 1 M+2 24.961 9 24.622 M+1 M+3 8 M+4 7 C 8 13 C 2 H 14 37 ClN 2 239.124 237.634 238.62 238.134 239.1426 241.994 24.1258 242.128 243.184 244.178 21,872 21,921 21,132 21,86 21,24 21,46 21,1179 21,1625 21,1422 21,878 2,E4 211825amfsimili-mix1-2_1k-a#346 RT: 4,92 AV: 1 SB: 6 4,46-4,51, 4,65-4,66 T: F TM S {1 ;1 } + p E S I F ull m s [1,-8,] Measured at 1. RP 1,13E3 C 1 H 13 ClN 2 +H: C 1 H 14 Cl 1 N 2 pa Chrg 1 6 5 4 3 Calculated 2 1 21,814 21,751 21,974 21, 21,5 21,1 21,15

UHPLC/HRMS Blood sample RT:.91-8.7 1 5 1 5 1 5 1 5 1 5 Cl O Cl O Cl O Cl O HO Cl O HO H N NH 2 NH 2 H N NH 2 RT: 4.14 AA: 1355983 RT: 4.21 AA: 1424771 RT: 3.73 AA: 91726 RT: 5.18 AA: 215424 RT: 4.96 AA: 737233 RT: 4.71 AA: 1859188 Ketamine Norketamine Dehydronorketamine Hydroxyketamine Hydroxynorketamine 1. 1.5 2. 2.5 3. 3.5 4. 4.5 5. 5.5 6. 6.5 7. 7.5 8. Time (min) 5.5E7 = 238.974-238.112 F: FTMS {1,1} + p ESI Full ms [1.-8.] MS ICIS 213429cds5211san9487ketmdpa_inj5uL 1.96E7 = 224.819-224.855 F: FTMS {1,1} + p ESI Full ms [1.-8.] MS ICIS 213429cds5211san9487ketmdpa_inj5uL 5.83E5 = 222.662-222.698 F: FTMS {1,1} + p ESI Full ms [1.-8.] MS ICIS 213429cds5211san9487ketmdpa_inj5uL 4.31E6 = 254.922-254.962 F: FTMS {1,1} + p ESI Full ms [1.-8.] MS ICIS 213429cds5211san9487ketmdpa_inj5uL 3.73E5 = 24.767-24.85 F: FTMS {1,1} + p ESI Full ms [1.-8.] MS ICIS 213429cds5211san9487ketmdpa_inj5uL Ketamine: 23 ng/ml Norketamine: 85 ng/ml Identification of metabolites: Chromatographic behaviour Accurate mass measurements of MH + ionic species Study of MH + collision-induced product ions in MS/MS experiments Comparison of experimental and calculated MH + isotopic clusters

5. Fine Structure of MH + isotopic patterns M+1, M+2, M+3 isotopic peaks vs. M+ peaks Ketamine: M+ (monoisotopic) 1 9 8 7 6 5 4 3 2 1 236.673 238.991 1 9 8 7 6 5 4 3 M+ (MH + ) 2 1 1 9 M+1 239.124 8 7 M+2 24.961 24.622 241.994 244.178 237.634 238.62 238.134 239.1426 24.1258 242.128 243.184 C 13 H 17 ClNO 238.991 237.9888 238.11 238.479 238.7 238.91 238.145 238.134 238.151 238.1748 238.27 238.993 M+3 7.87E6 212117prv4783cap8626_ket_sp e_1k#372 RT: 5.41 AV: 1 SB: 11 5.26-5.32, 5.57-5.64 T: FTMS {1,1} + p ESI Full ms [1.-8.] Measured at 1. RP 6.54E5 C 13 H 16 ClNO +H: C 13 H 17 Cl 1 N 1 O 1 pa Chrg 1 6 5 4 3 Calculated 2 1 237.95 238. 238.5 238.1 238.15 238.2 238.25

5. Fine Structure of MH + isotopic patterns M+1, M+2, M+3 isotopic peaks vs. M+ peaks Ketamine: M+1 (13% of M+) 1 9 8 7 6 5 4 3 2 1 236.673 238.991 1 9 8 7 6 5 4 3 M+ (MH + ) 2 1 1 9 M+1 239.124 8 7 M+2 24.961 24.622 M+3 241.994 244.178 237.634 238.62 238.134 239.1426 24.1258 242.128 243.184 239.124 C 13 12 CH 17 ClNO C 13 H 17 Cl 15 NO C 13 H 162 HClNO 239.959 238.9944 239.688 239.178 239.1426 239.127 1.5E6 212117prv4783cap8626_ket_sp e_1k#372 RT: 5.41 AV: 1 SB: 11 5.26-5.32, 5.57-5.64 T: FTMS {1,1} + p ESI Full ms [1.-8.] Measured at 1. RP 9.19E4 C 13 H 16 ClNO +H: C 13 H 17 Cl 1 N 1 O 1 pa Chrg 1 6 5 4 3 Calculated 2 1 239.964 239.156 238.95 239. 239.5 239.1 239.15 239.2 239.25

5. Fine Structure of MH + isotopic patterns M+1, M+2, M+3 isotopic peaks vs. M+ peaks Ketamine: M+2 (32% of M+) 1 9 8 7 6 5 4 3 2 1 236.673 238.991 1 9 8 7 6 5 4 3 M+ (MH + ) 2 1 1 9 M+1 239.124 8 7 M+2 24.961 24.622 M+3 241.994 244.178 237.634 238.62 238.134 239.1426 24.1258 242.128 243.184 C 13 H 17 37 ClNO 24.961 C 13 H 17 ClN 18 O C 13 11 C 2 H 17 ClNO 239.9961 24.622 24.98 24.161 24.1258 24.1533 24.1794 24.216 24.2282 24.964 2.53E6 212117prv4783cap8626_ket_sp e_1k#372 RT: 5.41 AV: 1 SB: 11 5.26-5.32, 5.57-5.64 T: FTMS {1,1} + p ESI Full ms [1.-8.] Measured at 1. RP 2.9E5 C 13 H 16 ClNO +H: C 13 H 17 Cl 1 N 1 O 1 pa Chrg 1 6 5 4 3 Calculated 2 1 24.16 239.95 24. 24.5 24.1 24.15 24.2 24.25

5. Fine Structure of MH + isotopic patterns M+1, M+2, M+3 isotopic peaks vs. M+ peaks Ketamine: M+3 (5% of M+) 1 9 8 7 6 5 4 3 2 1 236.673 238.991 1 9 8 7 6 5 4 M+ 3 (MH + ) 2 1 1 M+1 9 239.124 8 7 M+2 24.961 24.622 M+3 241.994 244.178 237.634 238.62 238.134 239.1426 24.1258 242.128 243.184 241.994 C 13 12 CH 37 17 ClNO C 13 H 37 17 Cl 15 NO C 13 H 162 H 37 ClNO 241.927 241.165 241.1179 241.1543 241.997 3.41E5 212117prv4783cap8626_ket_sp e_1k#372 RT: 5.41 AV: 1 SB: 11 5.26-5.32, 5.57-5.64 T: FTMS {1,1} + p ESI Full ms [1.-8.] Measured at 1. RP 2.94E4 C 13 H 16 ClNO +H: C 13 H 17 Cl 1 N 1 O 1 pa Chrg 1 6 5 4 3 Calculated 2 1 241.934 241.126 24.95 241. 241.5 241.1 241.15 241.2 241.25

3-MMC in pubic hair Pubic hair sample from a regular user of 3-MMC UHPLC/HRMS analysis Identification of 3-MMC RT: 3.44-7.3 1 1 1 1 1 1 8 6 4 2 1 8 6 4 2 MS/MS product ions of 3-MMC MH + RT: 5.39 AA: 36285666 RT: 4.96 AA: 476377 RT: 5.38 MA: 2882 4.69 4.87 5. 5.67 5.97 6.4 6.71 7.1 RT: 5.38 AA: 1556873 RT: 5.38 AA: 4228799 RT: 5.38 AA: 723617 RT: 5.2 AA: 149153 RT: 6.21 AA: 51932 RT: 6.59 AA: 17244 RT: 6.59 AA: 363488 RT: 6.57 AA: 62459 3.5 4. 4.5 5. 5.5 6. 6.5 7. Time (min) Full Scan 1.744 15.9537 MS/MS 3-MMC 119.852 125.9854 6 7 8 9 1 11 12 13 14 15 16 17 18 1.9E7 = 178.1217-178.1235 F: FTMS {1,1} + p ESI Full ms [1.-8.] MS ICIS 21311prv7417pli-pubici13613 9.23E3 = 178.1217-178.1235 F: FTMS {1,2} + p ESI Full ms2 1.@hcd25. [5.-8.] MS 21311prv7417pli-pubici13613 5.1E5 = 16.119-16.1125 F: FTMS {1,2} + p ESI Full ms2 1.@hcd25. [5.-8.] MS ICIS 21311prv7417pli-pubici13613 1.36E6 = 145.876-145.89 F: FTMS {1,2} + p ESI Full ms2 1.@hcd25. [5.-8.] MS ICIS 21311prv7417pli-pubici13613 2.49E5 = 119.848-119.86 F: FTMS {1,2} + p ESI Full ms2 1.@hcd25. [5.-8.] MS ICIS 21311prv7417pli-pubici13613 176.181 178.122 18.1289 16.1116 144.9811 167.115 145.888 16.1123 119.858 143.861 147.87 91.546 58.655 62.9294 73.288 84.96 99.9873 117.72 13.654 158.968 162.1189 178.123 1.7E7 21311prv7417pli-pubici13613#623 RT: 5.39 AV: 1 SB: 19 3.96-4.11, 5.54-5.69 T: FTMS {1,1} + p ESI Full ms [1.-8.] 1.3E6 21311prv7417pli-pubici13613#622 RT: 5.38 AV: 1 SB: 18 3.99-4.2, 5.53-5.63 F: FTMS {1,2} + p ESI Full ms2 1.@hcd25. [5.-8.]

3-MMC metabolites in pubic hair RT: 3.22-7.8 1 5 5.39 3-MMC 1.9E7 = 178.1217-178.1235 F: FTMS {1,1} + p ESI Full ms [1.-8.] MS 21311prv7417pli-pubici13613 Identification of 3- MMC metabolites in pubic hair: 3-Methylephedrine 3-Methylnorephedrine Distinction between external contamination of hair sample and intake of drug(s) 1 5 1 5 1 5 1 5 1 5 3.44 3.52 3.7 3.8 3.96 4.13 4.27 4.37 4.58 4.82 4.96 5.1 5.27 5.67 5.77 5.94 6.1 6.2 6.39 6.46 6.6 6.83 6.89 5.15 3-MMC 3.71 3.9 4.16 4.23 4.58 4.84 5.42 5.51 5.63 5.8 6.6 6.32 6.39 6.67 6.81 6.98 4.6 4.72 metabolites 3-Methylephedrine 3-Methylnorephedrine 3.3 3.52 3.64 3.87 3.94 4.18 4.28 4.44 4.87 5. 5.27 5.6 5.82 5.92 6.13 6.36 6.48 6.7 6.84 7.5 3.4 3.6 3.8 4. 4.2 4.4 4.6 4.8 5. 5.2 5.4 5.6 5.8 6. 6.2 6.4 6.6 6.8 7. Time (min) 148.1121 164.171 15.424 117.1386 131.856 146.61 151.348 171.1484 178.1226 186.167 193.9739 22.182 1 11 12 13 14 15 16 17 18 19 2 3.58E6 = 18.1374-18.1392 F: FTMS {1,1} + p ESI Full ms [1.-8.] MS 21311prv7417pli-pubici13613 3.19E5 = 166.1218-166.1234 F: FTMS {1,1} + p ESI Full ms [1.-8.] MS 21311prv7417pli-pubici13613 178.122 1.7E7 21311prv7417pli-pubici13613#623 3-MMC MH + RT: 5.39 AV: 1 SB: 8 5.28-5.32, 5.56-5.66 T: FTMS {1,1} + p ESI Full ms [1.-8.] 176.181 15.9537 114.97 119.852 125.9853 139.1216 145.882 16.1116 167.113 18.1288 193.9715 27.1573 18.1383 3.55E6 21311prv7417pli-pubici13613#595 RT: 5.15 AV: 1 SB: 7 4.99-5.6, 3-Methylephedrine MH + 5.26-5.31 T: FTMS {1,1} + p ESI Full ms 162.1278 [1.-8.] 15.696 114.921 125.9868 131.854 144.9851 151.359 158.9643 167.133 171.9933 182.1454 193.9739 199.191 166.1227 3-Methylnorephedrine MH + 2.61E5 21311prv7417pli-pubici13613#533 RT: 4.61 AV: 1 SB: 4 4.48-4.54 T: FTMS {1,1} + p ESI Full ms [1.-8.]

Conclusions 1. Efficient chromatographic separation of A-R drugs 2. Accurate mass measurements of MH + ionic species with a mass accuracy < 2-3 ppm for all A-R drugs 3. Characteristic collision-induced product ions of MH + ions with same ECs 4. Fully superimposable experimental and calculated MH + isotopic patterns (RIA error < 5% for all A-R drugs) 5. Isotopic fine structure of the M+1, M+2, M+3 isotopic peaks completely in accordance with theoretical patterns Elucidation of elemental composition and structural characteristics of new amphetamine-related drugs

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