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

Transcript

1 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

2 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

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

4 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

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

6 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)

7 GC/MS - Mephedrone Rel. Ab. 58 MW 177 GC/MS Agilent 789 II 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

8 Mephedrone deriv. with 3ClEtClfor + Cl CO O CH 2 CCl 3 MW 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, ) G. Frison et al. Rapid Commun. Mass Spectrom. 211, 25,

9 GC/MS of Mephedrone - 3ClEtClfor Rel. Ab CH 3 C O CH O Cl C Cl Cl

10 High resolution/high accuracy MS Mass resolution = m 1 / m 1 m 2 Low resol. MS High resol. MS 1 H = 1 1 H = C = C = O = O = N = N = Ions at 28 Exact mass CO N CH 2 N C 2 H

11 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)!

12 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

13 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

14 1. High chromatographic resolution of analytes HR ion chromatograms of 25 AR-drugs RT: SM: 13G RT: Fluoro-Amphetamine MH + 154,13 RT: SM: 13G 4-MEC MH + 192,1387 RT: 7.14 Pentedrone MH + 192, 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, 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, Time (min) Time (min)

15 1. High chromatographic resolution of analytes HR ion chromatograms of 25 AR-drugs RT: SM: 13G 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: ,4,5-TMo-Amphetamine MH + 226,1442 RT: 7.68 RT: 8.36 RT: 8.11 RT: Acetoxy-DMT MH + 247,1446 Methoxethamine MH + 248,1649 MDPA (IS) MH + 222,1493 RT: SM: 13G RT: 7.51 RT: 7.83 MDPV MH + 276,1597 MDPA (IS) MH + 222,1493 RT: 8.36 RT: 8.11 RT: C-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: RT: Time (min) Time (min)

16 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,

17 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!

18 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, , ,13 1,94 5-APB C 11 H 13 NO 175, , ,27 BenzylPiperazine C 11 H 16 N 2 176, , ,139 2,25 Mephedrone (4-MMC) C 11 H 15 NO 177, , ,123 2,24 3-MMC C 11 H 15 NO 177, , ,123 2,24 Buphedrone C 11 H 15 NO 177, , ,123 2,24 Dimethyltryptamine (DMT) C 12 H 16 N 2 188, , ,1391 2,64 Methyl-Benzyl-Piperazine C 12 H 18 N 2 19, , ,1547 2,9 4-MEC C 12 H 17 NO 191, , ,1387 2,8 Pentedrone C 12 H 17 NO 191, , ,1387 2,8 Chloro-Phenyl-Piperazine C 1 H 13 ClN 2 196, ,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, , ,1493 1,9 m = (Accurate mass Exact mass) / Exact mass x 1 6

19 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, , ,174 2,3 Butylone C 12 H 15 NO 3 221, , ,113 2,25 2,4,5-Trimethoxyamphetamine C 12 H 19 NO 3 225, , ,1442 1,76 Trifluoromethylphenylpiperazine C 11 H 13 F 3 N 2 23, , ,118 1,73 -PVP C 15 H 21 NO 231, , ,17 1,72 4-AcO-DMT C 14 H 18 N 2 O 2 246, , ,1446 2,2 Methoxethamine C 15 H 21 NO 2 247, , ,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, , ,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, , ,722 1,17 m = (Accurate mass Exact mass) / Exact mass x 1 6

20 3. Study of MH+ product ions (MS/MS) 4-MEC and Pentedrone: both C 12 H 17 NO, MH + exact mass 192,1383 RT: MEC RT: 4.57 AA: RT: 4.73 AA: Time (min) 3.47E6 = F: FTMS {1,1} + p ESI Full ms [1.-8.] MS ICIS MIX amf-amfsimili-jwh-metilonpropilon 3.47E6 = F: FTMS {1,1} + p ESI Full ms [1.-8.] MS ICIS MIX amf-amfsimili-jwh-metilonpropilon RT: RT: 4.57 AA: RT: 4.73 AA: Pentedrone Time (min) 3.47E6 = F: FTMS {1,1} + p ESI Full ms [1.-8.] MS ICIS MIX amf-amfsimili-jwh-metilonpropilon 3.47E6 = F: FTMS {1,1} + p ESI Full ms [1.-8.] MS ICIS MIX amf-amfsimili-jwh-metilonpropilon Full scan MS/MS E6 MIX amf-amfsimili-jwh-metilon-propilon#743 RT: 4.57 AV: 1 SB: , 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: , F: FTMS {1,2} + p ESI Full ms2 1.@hcd25.145,885 [5.-8.] 119, Full scan MS/MS E6 MIX amf-amfsimili-jwh-metilon-propilon#769 RT: 4.73 AV: 1 SB: , T: FTMS {1,1} + p ESI Full ms [1.-8.] 86, , E5 MIX amf-amfsimili-jwh-metilon-propilon#768 RT: 4.73 AV: 1 SB: , F: FTMS {1,2} + p ESI Full ms2 1.@hcd25. [5.-8.] 15, ,88 144,88

21 3. Study of MH+ product ions (MS/MS) Buphedrone, 3-MMC, 4-MMC: all C 11 H 15 NO, MH + exact mass 178, Buphedrone MH + 178,123 RT: 5.72 RT: MMC & 4-MMC MH + 178,123 RT: 7.43 R elative Abundance Buphedrone (in source CID, 4 ev) MMC (in source CID, 4 ev) MMC (in source CID, 4 ev)

22 LC/HR-MS & GC/MS (after 3ClEtClfor deriv.) Buphedrone, 3-MMC, 4-MMC deriv. with 2,2,2-trichloroethyl chloroformate O N O O Cl Cl Cl O Cl Cl N O Cl O Buphedrone - 3ClEtClfor MMC - 3ClEtClfor O N O O Cl Cl Cl 4-MMC - 3ClEtClfor

23 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 H Deuterium H Tritium * 12 C Carbon C C * 14 N Nitrogen N O Oxygen O O S Sulphur S S S Cl Chlorine Cl Br Bromine Br 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.

24 4. MH + isotopic patterns M+ (MH + ) Experimental Time (min) Full Scan Cl-Phenyl- Piperazine MH + pattern C M+1 M M+3 37 Cl or 81 Br M+ M+1 M M+3 Calculated

25 4. MH + isotopic patterns Comparison of measured and calculated MH + isotopic patterns O H N 4-MEC O O O N MDPV 1 192, , MH + measured isotopic pattern MH + measured isotopic pattern , , , , , , , MH + calculated isotopic pattern MH + calculated isotopic pattern , , , , ,

26 4. MH + isotopic patterns Comparison of measured and calculated MH + isotopic patterns O H Cl N NH Cl-PhPip N Mephedrone 1 197, , MH + measured isotopic pattern 199, MH + measured isotopic pattern , , , ,84 2,842 21,873 23, , , , MH + calculated isotopic pattern MH + calculated isotopic pattern , ,874 2,844 21,878 23, ,126 18,

27 4. MH + isotopic patterns RIA error Comparison of experimental and calculated MH + isotopic patterns M+ (MH + ) M+1 M+ M+1 M M+2 Experimental M M+3 Calculated RIA Relative Isotopic Abundance M+1 ab / M+ ab ( 13 C / 12 C) RIA error (%) RIA meas RIA calc RIA calc x 1

28 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

29 4. MH + isotopic patterns RIA error Comparison of experimental and calculated MH + isotopic patterns M+ (MH + ) M+1 M+ M+1 M M+2 Experimental M M+3 Calculated RIA Relative Isotopic Abundance M+2 ab / M+ ab (Br or Cl / 12 C) RIA error (%) RIA meas RIA calc RIA calc x 1

30 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

31 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 RP M+1: Complete separation of 15 N and 13 C peaks M+1 198, ,89 M+2 M+3 1. RP M+2: Complete separation of 18 O, 34 S, 37 Cl C 2 peaks 195, ,1177 2,842 21,873 23,

32 5. Fine Structure of MH + isotopic patterns M+1, M+2, M+3 isotopic peaks vs. M+ peaks M+ of Mephedrone (monoisotopic) M+ (MH + ) M+1 M C 11 H 16 NO 178, M , E amfsimili-mix1-1_1k#293 RT: 4.35 AV: 1 SB: , 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 Calculated

33 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+) M+ 1 (MH + ) M M M+3 C 1 13 CH 16 NO C 11 H NO , , , , , , , , , , ,126 C 11 H 152 HNO 5,78E AMFSIMILI-MIX1-1_1K# 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 , , ,5 179,1 179,15 179,2

34 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+) C 13 9 C 2 H 16 NO C 11 H 16 N 18 O C 13 1 CH NO M+ (MH + ) M M M , , , , ,132 18, , , , , ,1319 C 1 13 CH 152 HNO 18,1583 2,88E AMFSIMILI-MIX1-1_1K# 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 ,123 18,123 18, , ,1 18,12 18,14 18,16

35 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+) M+ (MH + ) 2 1 C 1 13 CH 16 N 18 O 181, , , ,1325 C 8 13 C 3 H 16 NO 5,83E AMFSIMILI-MIX1-1_1K# 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 M M M , , ,1327 2,15E2 C 11 H 15 NO +H: C 11 H 16 N 1 O 1 pa Chrg Calculated , , , , ,8 181,1 181,12 181,14

36 5. Fine Structure of MH + isotopic patterns M+1, M+2, M+3 isotopic peaks vs. M+ peaks M+ of Cl-Phenyl-Piperazine (monoisotopic) ,835 7,22E amfsimili-mix1-2_1ka# 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, ] M+ (MH + ) M M C 1 H 14 ClN 2 197, , , , , , , , ,84 M Measured at 1. RP 6,74E5 C 1 H 13 ClN 2 +H: C 1 H 14 Cl 1 N 2 pa Chrg Calculated , 197,5 197,1 197,15 197,2

37 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+) C 9 13 CH 14 ClN 2 198,869 7,62E amfsimili-mix1-2_1ka# 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, ] M+ 1 (MH + ) 198,85 198,65 198, , M M M+3 C 1 H 14 Cl 15 NN ,898 C 1 H 132 HClN 2 198, , ,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 ,81 198,93 198,6 198,7 198,8 198,9 198,1 198,11

38 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+) M+ (MH + ) M M C 1 H ClN 2 199,85 C 8 13 C 2 H 14 ClN 2 199,49 199, , ,9 199, , , ,811 M ,E amfsimili-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 Calculated ,97 199, 199,5 199,1 199,15 199,2

39 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+) C 13 9 CH 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 M M ,42E amfsimili-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 ,781 2,873 2,97 2,6 2,8 2,1 2,12

40 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+) M+ (MH + ) M M+1 M+3 8 M+4 7 C 8 13 C 2 H ClN ,872 21,921 21,132 21,86 21,24 21,46 21, , , ,878 2,E amfsimili-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 Calculated ,814 21,751 21,974 21, 21,5 21,1 21,15

41 UHPLC/HRMS Blood sample RT: 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: RT: 4.21 AA: RT: 3.73 AA: RT: 5.18 AA: RT: 4.96 AA: RT: 4.71 AA: Ketamine Norketamine Dehydronorketamine Hydroxyketamine Hydroxynorketamine Time (min) 5.5E7 = F: FTMS {1,1} + p ESI Full ms [1.-8.] MS ICIS cds5211san9487ketmdpa_inj5uL 1.96E7 = F: FTMS {1,1} + p ESI Full ms [1.-8.] MS ICIS cds5211san9487ketmdpa_inj5uL 5.83E5 = F: FTMS {1,1} + p ESI Full ms [1.-8.] MS ICIS cds5211san9487ketmdpa_inj5uL 4.31E6 = F: FTMS {1,1} + p ESI Full ms [1.-8.] MS ICIS cds5211san9487ketmdpa_inj5uL 3.73E5 = F: FTMS {1,1} + p ESI Full ms [1.-8.] MS ICIS cds5211san9487ketmdpa_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

42 5. Fine Structure of MH + isotopic patterns M+1, M+2, M+3 isotopic peaks vs. M+ peaks Ketamine: M+ (monoisotopic) M+ (MH + ) M M C 13 H 17 ClNO M E prv4783cap8626_ket_sp e_1k#372 RT: 5.41 AV: 1 SB: , 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 Calculated

43 5. Fine Structure of MH + isotopic patterns M+1, M+2, M+3 isotopic peaks vs. M+ peaks Ketamine: M+1 (13% of M+) M+ (MH + ) M M M C CH 17 ClNO C 13 H 17 Cl 15 NO C 13 H 162 HClNO E prv4783cap8626_ket_sp e_1k#372 RT: 5.41 AV: 1 SB: , 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 Calculated

44 5. Fine Structure of MH + isotopic patterns M+1, M+2, M+3 isotopic peaks vs. M+ peaks Ketamine: M+2 (32% of M+) M+ (MH + ) M M M C 13 H ClNO C 13 H 17 ClN 18 O C C 2 H 17 ClNO E prv4783cap8626_ket_sp e_1k#372 RT: 5.41 AV: 1 SB: , 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 Calculated

45 5. Fine Structure of MH + isotopic patterns M+1, M+2, M+3 isotopic peaks vs. M+ peaks Ketamine: M+3 (5% of M+) M+ 3 (MH + ) M M M C CH ClNO C 13 H Cl 15 NO C 13 H 162 H 37 ClNO E prv4783cap8626_ket_sp e_1k#372 RT: 5.41 AV: 1 SB: , 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 Calculated

46 3-MMC in pubic hair Pubic hair sample from a regular user of 3-MMC UHPLC/HRMS analysis Identification of 3-MMC RT: MS/MS product ions of 3-MMC MH + RT: 5.39 AA: RT: 4.96 AA: RT: 5.38 MA: RT: 5.38 AA: RT: 5.38 AA: RT: 5.38 AA: RT: 5.2 AA: RT: 6.21 AA: RT: 6.59 AA: RT: 6.59 AA: RT: 6.57 AA: Time (min) Full Scan MS/MS 3-MMC E7 = F: FTMS {1,1} + p ESI Full ms [1.-8.] MS ICIS 21311prv7417pli-pubici E3 = F: FTMS {1,2} + p ESI Full ms2 1.@hcd25. [5.-8.] MS 21311prv7417pli-pubici E5 = F: FTMS {1,2} + p ESI Full ms2 1.@hcd25. [5.-8.] MS ICIS 21311prv7417pli-pubici E6 = F: FTMS {1,2} + p ESI Full ms2 1.@hcd25. [5.-8.] MS ICIS 21311prv7417pli-pubici E5 = F: FTMS {1,2} + p ESI Full ms2 1.@hcd25. [5.-8.] MS ICIS 21311prv7417pli-pubici E prv7417pli-pubici13613#623 RT: 5.39 AV: 1 SB: , T: FTMS {1,1} + p ESI Full ms [1.-8.] 1.3E prv7417pli-pubici13613#622 RT: 5.38 AV: 1 SB: , F: FTMS {1,2} + p ESI Full ms2 1.@hcd25. [5.-8.]

47 3-MMC metabolites in pubic hair RT: MMC 1.9E7 = 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) MMC metabolites 3-Methylephedrine 3-Methylnorephedrine Time (min) E6 = F: FTMS {1,1} + p ESI Full ms [1.-8.] MS 21311prv7417pli-pubici E5 = F: FTMS {1,1} + p ESI Full ms [1.-8.] MS 21311prv7417pli-pubici E prv7417pli-pubici13613#623 3-MMC MH + RT: 5.39 AV: 1 SB: , T: FTMS {1,1} + p ESI Full ms [1.-8.] E prv7417pli-pubici13613#595 RT: 5.15 AV: 1 SB: , 3-Methylephedrine MH T: FTMS {1,1} + p ESI Full ms [1.-8.] Methylnorephedrine MH E prv7417pli-pubici13613#533 RT: 4.61 AV: 1 SB: T: FTMS {1,1} + p ESI Full ms [1.-8.]

48 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

49 The Forensic Toxicology People at LIATF Thank you very much for your kind attention!