Identification of New Amphetamine-Related Designer Drugs Using Exactive High Resolution Accurate Mass Spectrometry Giampietro Frison

Home , Indane, MBDB, MDAT, MDMAI

2011 Joint SOFT-TIAFT Meeting San Francisco, California, USA Thermo Scientific Workshop 28 September 2011 Identification of New Amphetamine-Related Designer Drugs Using Exactive High Resolution Accurate Mass Spectrometry Giampietro Frison

Laboratory of Environmental Hygiene and Forensic Toxicology Department of Prevention Azienda ULSS 12 Veneziana, Mestre (Venezia), Italy

Forensic Toxicology Use Only Forensic toxicology at the LIATF

Seized drugs

• Determination of active ingredients, adulterants, diluents in seized material for Police and Court

Biological samples

• Determination of alcohol, psychoactive drugs, other drugs of toxicological interest in biofluids

• Driving under the influence of alcohol and drugs

• Post-mortem toxicology

• Drug-facilitated crimes

• Other forensic toxicology applications

Forensic Toxicology Use Only The flood of new designer drugs

Indane analogues of Ecstasy’s (MDAI, MDMAI)

Tetraline analogues of Ecstasy’s (2-AT, MDAT)

Tryptamines

Cannabinomimetics (JWH-18, JWH-073, JWH-200, JWH-250,CP-47,497, ... )

Beta-keto-amphetamines (designer cathinones)

Piperazines

Forensic Toxicology Use Only New A-R drugs encountered at LIAFT

Mephedrone 4-MEC 4-FA

MDPV Methylone Cl-Ph-Pip

propyl

Propylone Forensic Toxicology Use Only The flood of new designer drugs ? ? ?? ?

The LIAFT experience with new designer drugs Increasing number of police seizures and/or intoxication cases Need to promptly obtain their structural characterization , in spite of poor availability of reference (p/m) standards

Forensic Toxicology Use Only Analytical strategy to obtain the structural characterization of new A-R drugs

GC-MS GC-MS + der UHPLC-HRMS underiv. drugs with 3ClEtClfor (Orbitrap)

Poor chromatography Improved GC properties Accurate mass measurements at 100.000 resolv. power Non-specific EI MS Distinctive MS behav. Study of MH + collision- Highly inform. EI MS induced product ions

Comparison of exp. and calc. MH + isotopic clusters

Study of isotopic fine structure of M+1, M+2, M+3 clusters Forensic Toxicology Use Only GC/MS - Mephedrone

Rel. Ab. 6.31

Mephedrone (4-Methyl-Meth-Cathinone)

Methanolic solution (b.p. 64.5 °C)

4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 Time GC/MS Agilent 7890 II - 5975 Agilent HP-5MS Ultra Inert column Full Scan EI (40-450 u) 30 m x 0.25 mm x 0.25 µm Inj. 1 µl, 250C, split / splitless (1 min) 50C (0.5 min), 200C a 30C/min, 300C (5 min) a 10C/min Carrier gas (He), 1 ml/min Interf. 280C, EMV + 300 V

Forensic Toxicology Use Only GC/MS - Mephedrone

Rel. Ab. 58

MW 177

56 65 91 119 162 0 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 m/z GC/MS Agilent 7890 II - 5975 Agilent HP-5MS Ultra Inert column Full Scan EI (40-450 u) 30 m x 0.25 mm x 0.25 µm Inj. 1 µl, 250C, split / splitless (1 min) 50C (0.5 min), 200C a 30C/min, 300C (5 min) a 10C/min Carrier gas (He), 1 ml/min Interf. 280C, EMV + 300 V Forensic Toxicology Use Only Mephedrone deriv. with 3ClEtClfor

+ Cl CO O CH 2 CCl 3

50 µL 2,2,2 - 3ClEtClfor : Ethyl acetate 3 : 7 MW 177 80 °C, 15 min, to dryness

Residue reconst. with 50 µL Ethyl acetate

CH 3

C O CH 2 CCl 3 O MW 351

Forensic Toxicology Use Only GC/MS of Mephedrone - 3ClEtClfor

Rel. Ab. 11.08

Mephedrone – 3ClEtClfor

6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 Time GC/MS (same instrument, same conditions) Agilent 7890 II - 5975 Agilent HP-5MS Ultra Inert column Full Scan EI (40-450 u) 30 m x 0.25 mm x 0.25 µm Inj. 1 µl, 250C, splitless (1 min) 50C (0.5 min), 200C a 30C/min, 300C (5 min) a 10C/min Carrier gas (He), 1 ml/min Interf. 280C, EMV + 300 V Forensic Toxicology Use Only GC/MS of Mephedrone - 3ClEtClfor

. 91 + CH 3 Cl 351 353 Cl 355 C O CH 2 C Cl O 131 232 204 133 316 119 234 135 318 236 Rel. Ab. 232 234

58 91 119 131 236 133 65 135 351 160 316 204 353 318 355

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 m/z Forensic Toxicology Use Only U-HPLC/HR-Orbitrap-MS and A-R drugs Why choosing the Orbitrap technology

r z k ω = m / z φ

Non-targeting approach (Full Scan) and “all ions” MS/MS

Retrospective detection of analytes without new MS runs

High resolving power: 100.000 at m/z 200

High mass accuracy: < 5ppm (e.c.), < 2ppm (i.c.)

High mass accur. for M+0 and M+1, M+2, ....ions: EC assign. with high confidence

100.000 RP: full separation of 15 N from M+1 C isotope, and 34 S from M+2 C isotope Forensic Toxicology Use Only U-HPLC/HR-Orbitrap-MS and A-R drugs

U-HPLC Thermo Scientific LC: U-HPLC Thermo Scientific Accela 1250 Exactive HCD MS Injection: 10 µL Column: Thermo Scientific Hypersil Gold 50 x 2.1 mm x 1.9 mcm

Phase A: H2O, 0,05% HCOOH, 5 mM HCOONH 4,pH 5 Phase B: ACN, 0,05% HCOOH Analytical Conditions Flow: 400 µL/min Gradient: 2% B, to 30% B in 5 min, to 98% B in 5 min, maint. 2 min, to 2% B in 4 min, maint. 2 min. Temp.: Column 40 °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: N2 set to 45 a. u. Aux. gas: N2 set to 5 a. u. Cap.Temp.: 290 °C HESI Temp.: 260 °C Polarity Pos Mass range: 50-800 u Mass resol.: 25.000 (HCD on, 25 eV) or 100.000 (HCD off), No Lock Mass Forensic Toxicology Use Only 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 clusters

5. Examination of the isotopic fine structure (IFS) of the (M+1), (M+2), (M+3) isotopic peaks relative to the monoisotopic (M+0 ) peaks Forensic Toxicology Use Only U-HPLC / HR-Orbitrap-MS analytical strategy

1. High chromatographic resolution of analytes

“Classic” amphetamines New A-R drugs RT: 2.36 - 6.07 SM: 3B RT: 2.48 - 6.03 SM: 3B RT: 3.42 NL: 4.01E5 RT: 4.28 NL: 1.09E6 AA: 1367658 m/z= 136.1110-136.1124 F: FTMS AA: 3172486 m/z= 178.1217-178.1235 F: FTMS 100 100 {1,1} + p ESI Full ms [100.00-800.00] {1,1} + p ESI Full ms [100.00-800.00] Amphetamine MS ICIS MIX Mephedrone MS ICIS MIX 50 amf-amfsimili-jwh-metilon-propilon 50 amf-amfsimili-jwh-metilon-propilon

0 0 RT: 3.79 NL: 1.62E6 RT: 5.18 NL: 1.55E6 AA: 5728285 m/z= 150.1265-150.1281 F: FTMS AA: 4319203 m/z= 276.1580-276.1608 F: FTMS 100 100 {1,1} + p ESI Full ms [100.00-800.00] {1,1} + p ESI Full ms [100.00-800.00] MS ICIS MIX MS ICIS MIX 50 Methamphetamine amf-amfsimili-jwh-metilon-propilon 50 MDPV amf-amfsimili-jwh-metilon-propilon

0 0 RT: 3.70 NL: 2.77E5 RT: 4.73 NL: 2.77E6 AA: 891805 m/z= 180.1005-180.1023 F: FTMS AA: 7724451 m/z= 192.1373-192.1393 F: FTMS 100 100 {1,1} + p ESI Full ms [100.00-800.00] {1,1} + p ESI Full ms [100.00-800.00] MS ICIS MIX MS ICIS MIX amf-amfsimili-jwh-metilon-propilon 4-MEC Propylone amf-amfsimili-jwh-metilon-propilon 50 MDA 50

0 0 RT: 3.95 NL: 1.61E6 RT: 3.81 NL: 5.02E5 AA: 5218248 m/z= 194.1161-194.1181 F: FTMS AA: 1744924 m/z= 154.1019-154.1035 F: FTMS 100 100 {1,1} + p ESI Full ms [100.00-800.00] {1,1} + p ESI Full ms [100.00-800.00] MS ICIS MIX 4-FA MS ICIS MIX 50 MDMA amf-amfsimili-jwh-metilon-propilon 50 amf-amfsimili-jwh-metilon-propilon

0 0 RT: 4.33 NL: 2.37E6 RT: 4.94 NL: 1.98E5 AA: 7126059 4.60 m/z= 208.1317-208.1337 F: FTMS AA: 559381 m/z= 197.0830-197.0850 F: FTMS 100 100 {1,1} + p ESI Full ms [100.00-800.00] {1,1} + p ESI Full ms [100.00-800.00] MS ICIS MIX MS ICIS MIX 50 MDEA amf-amfsimili-jwh-metilon-propilon 50 Cl-Ph-Pip amf-amfsimili-jwh-metilon-propilon

2.47 2.94 3.18 3.85 4.114.76 5.03 5.62 5.79 0 0 RT: 4.60 NL: 2.37E6 RT: 3.30 NL: 1.91E6 4.33 AA: 6351417 m/z= 208.1317-208.1337 F: FTMS AA: 8608603 m/z= 208.0956-208.0976 F: FTMS 100 100 {1,1} + p ESI Full ms [100.00-800.00] {1,1} + p ESI Full ms [100.00-800.00] MBDB MS ICIS MIX MS ICIS MIX 50 amf-amfsimili-jwh-metilon-propilon 50 Methylone amf-amfsimili-jwh-metilon-propilon

2.47 2.94 3.18 3.85 4.114.76 5.03 5.62 5.79 0 0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 Time (min) Time (min) Forensic Toxicology Use Only U-HPLC / HR-Orbitrap-MS analytical strategy

2. Accurate mass measurements of MH + ionic species

MH + MH + ∆m at 100K 100.000 Elemental Exact resolving power Substance composition mass Accurate Exact mass ppm mass

Amphetamine C9H13 N 135,1048 136,1121 136,1120 -0,73

Methamphetamine C10 H15 N 149,1204 150,1277 150,1276 -0,67

MDA C10 H13 NO 2 179,0946 180,1019 180,1017 -1,11

MDMA C H NO 193,1103 194,1176 194,1174 -1,03 11 15 2 Mass MDEA C12 H17 NO 2 207,1259 208,1332 208,1329 -1,44 accuracy MBDB C12 H17 NO 2 207,1259 208,1332 208,1329 -1,44

Methylone C H NO 207,0895 208,0967 208,0968 -0,48 11 13 3 < 2 ppm

Mephedrone C11 H15 NO 177,1154 178,1226 178,1225 -0,56 (no lock mass)

MDPV C16 H21 NO 3 275,1521 276,1594 276,1589 -1,81

4-MEC C12 H17 NO 191,1310 192,1383 192,1381 -1,04

Propylone C12 H17 NO 191,1310 192,1383 192,1381 -1,04

4F-amphetamine C9H12 FN 153,0954 154,1027 154,1025 -1,30

Cl-Ph-piperazine C10 H13 ClN 2 196,0767 197,0840 197,0838 -1,01

∆m = (Accurate mass – Exact mass) / Exact mass x 10 6 Forensic Toxicology Use Only U-HPLC / HR-Orbitrap-MS analytical strategy 3. Study of MH + product ions from MS/MS experiments + MDEA and MBDB: both C 12 H17 NO 2, MH exact mass 208,1332

RT: 3.38 - 5.60 RT: 3.38 - 5.60 NL: 2.93E6 NL: 2.93E6 RT: 4.32 4.60 m/z= 208.1317-208.1337 F: RT: 4.32 4.60 m/z= 208.1317-208.1337 F: 100 AA: 7174432 FTMS {1,1} + p ESI Full ms 100 AA: 7174432 FTMS {1,1} + p ESI Full ms [100.00-800.00] MS ICIS MIX [100.00-800.00] MS ICIS MIX 80 amf-amfsimili-jwh-metilon- 80 amf-amfsimili-jwh-metilon- propilon propilon 60 60

40 MDEA 40

20 20 Relative Abundance Relative Abundance 3.40 3.65 3.83 4.02 4.124.75 5.04 4.86 5.23 5.31 5.52 3.40 3.65 3.83 4.02 4.124.75 4.865.04 5.23 5.39 0 0 RT: 4.60 NL: 2.93E6 RT: 4.60 NL: 2.93E6 AA: 6373588 m/z= 208.1317-208.1337 F: AA: 6373588 m/z= 208.1317-208.1337 F: 100 4.32 FTMS {1,1} + p ESI Full ms 100 4.32 FTMS {1,1} + p ESI Full ms [100.00-800.00] MS ICIS MIX [100.00-800.00] MS ICIS MIX 80 amf-amfsimili-jwh-metilon- 80 amf-amfsimili-jwh-metilon- propilon propilon 60 60 MBDB 40 40

20 20 Relative Abundance Relative Abundance 3.40 3.65 3.83 4.02 4.124.75 5.04 4.86 5.23 5.31 5.52 3.40 3.65 3.83 4.02 4.124.75 4.865.04 5.23 5.39 0 0 3.5 4.0 4.5 5.0 5.5 3.5 4.0 4.5 5.0 5.5 Time (min) Time (min)

208.1329 NL: 2.63E6 208.1329 NL: 2.24E6 100 100 MIX MIX amf-amfsimili-jwh-metilon-propilon#701- amf-amfsimili-jwh-metilon-propilon#749 80 704 RT: 4.32-4.33 AV: 2 SB: 6 80 RT: 4.61 AV: 1 SB: 8 4.50-4.54 , 4.23-4.25 , 4.41-4.44 T: FTMS {1,1} + 4.68-4.72 T: FTMS {1,1} + p ESI Full ms p ESI Full ms [100.00-800.00] 60 60 [100.00-800.00]

40 40 72,0811 198.1897 72,0811 20 163.0751 214.0884 20 Relative Abundance Relative Abundance NL: 2.73E5 135.0439 177.0908 105.0699 149.1176 198.1862 MIX 108.0811 NL: 6.78E5 0 0 105.0700 amf-amfsimili-jwh-metilon-propilon#702 135.0441 MIX H 100 RT: 4.32 AV: 1 SB: 5 H 4.23-4.25 , 100 amf-amfsimili-jwh-metilon-propilon#748N 4.41-4.44 F: FTMS {1,2} + p ESI Full ORT: 4.61 AV: 1 SB: 7 4.50-4.54 , ms2 [email protected] [50.00-800.00] 80 135.0441 O 80 4.68-4.72 F: FTMS {1,2} + p ESI Full ms2 [email protected] [50.00-800.00] 60 60 O 163.0754 O 177,0908 40 163,0754 40 86.0967 135,0441 135,0441 20 72.0811 208.1332 20 151.0754 72.0811 119.0857 147.0805 208.1333 122.0363 177.1127 177.0911 0 133,0648 0 50 100 150 200 105,0700 50 100 150 200 m/z m/z Forensic Toxicology Use Only U-HPLC / HR-Orbitrap-MS analytical strategy 3. Study of MH + product ions from MS/MS experiments + 4-MEC and Propylone: both C 12 H17 NO, MH exact mass 192,1383

RT: 3.94 - 5.55 RT: 3.94 - 5.55 NL: 3.47E6 NL: 3.47E6 4.73 m/z= 192.1373-192.1393 F: 4.73 m/z= 192.1373-192.1393 F: 100 100 RT: 4.57 FTMS {1,1} + p ESI Full ms RT: 4.57 FTMS {1,1} + p ESI Full ms AA: 6547794 [100.00-800.00] MS ICIS MIX AA: 6547794 [100.00-800.00] MS ICIS MIX 80 amf-amfsimili-jwh-metilon- 80 amf-amfsimili-jwh-metilon- propilon propilon 60 4-MEC 60 40 40

20 20 Relative Abundance 3.99 4.22 4.29 4.35 4.94 5.17 5.235.39 Relative Abundance 3.99 4.22 4.29 4.35 4.94 5.17 5.235.39 0 0 RT: 4.73 NL: 3.47E6 RT: 4.73 NL: 3.47E6 AA: 7730758 m/z= 192.1373-192.1393 F: AA: 7730758 m/z= 192.1373-192.1393 F: 100 FTMS {1,1} + p ESI Full ms 100 FTMS {1,1} + p ESI Full ms 4.57 [100.00-800.00] MS ICIS MIX 4.57 [100.00-800.00] MS ICIS MIX 80 amf-amfsimili-jwh-metilon- 80 amf-amfsimili-jwh-metilon- propilon propilon 60 60 Propylone

40 40

20 20 Relative Abundance 3.99 4.22 4.29 4.35 4.94 5.17 5.235.39 Relative Abundance 3.99 4.22 4.29 4.35 4.94 5.17 5.235.39 0 0 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 Time (min) Time (min)

192.1380 NL: 2.67E6 192.1380 NL: 3.43E6 100 100 MIX MIX amf-amfsimili-jwh-metilon-propilon#743 amf-amfsimili-jwh-metilon-propilon#769 80 RT: 4.57 AV: 1 SB: 6 4.45-4.49 , 80 RT: 4.73 AV: 1 SB: 5 4.64-4.67 , 4.65-4.67 T: FTMS {1,1} + p ESI Full ms 4.84-4.87 T: FTMS {1,1} + p ESI Full ms 60 [100.00-800.00] 60 [100.00-800.00]

40 72,0811 40 86,0967

20 174.1275 20 Relative Abundance O Relative Abundance 174.1275 O 100.1121 119.0852 147.0802 NL: 2.29E5 105.0333 132.0807 NL: 2.14E5 0 0 145.0885 MIX H 132.0808 MIX H 100 amf-amfsimili-jwh-metilon-propilon#742N 100 amf-amfsimili-jwh-metilon-propilon#768N RT: 4.57 AV: 1 SB: 5 4.45-4.49 , RT: 4.73 AV: 1 SB: 6 4.64-4.67propyl , 4.65-4.67 F: FTMS {1,2} + p ESI Full 91.0544 4.84-4.87 F: FTMS {1,2} + p ESI Full 80 80 ms2 [email protected] [50.00-800.00] ms2 [email protected] [50.00-800.00]

60 131.0730 60 119.0856 145,0885 132,0808 40 40 105.0336 137.0597 159.1043 119,0856 144.0808 105,0336 117.0574 174.1278 20 91.0544 20 192.1383 86.0967 72.0811 105.0700 192.1383 159.1043 0 0 50 100 150 200 50 100 150 200 m/z m/z Forensic Toxicology Use Only Elements, their isotopes, and EC calc.

Data acquired even by Symbol Name Mass of atom % Abundance 1H Hydrogen 1.007825 99.9885 ultrahigh mass 2H Deuterium 2.014102 0.115 3H Tritium 3.016049 * accuracy and mass 12 C Carbon 12.000000 98.93 resolution can be 13 C 13.003355 1.07 14 C 14.003242 * insufficient for 14 N Nitrogen 14.003074 99.632 calculating unique 15 N 15.000109 0.368

elemental 16 O Oxygen 15.994915 99.757 compositions without 17 O 16.999132 0.038 18 O 17.999160 0.205 information about 32 S Sulphur 31.972071 94.93 isotope ratios 33 S 32.971458 0.76 34 S 33.967867 4.29 36 S 35.967081 0.02 Natural occurring 35 Cl Chlorine 34.968853 75.78 elements can be 37 Cl 36.965903 24.22

monoisotopic (F, Na, P, 79 Br Bromine 78.918338 50.69 81 Br 80.916291 49.31 I) or polyisotopic (H, De Laeter JR, Bohlke JK, De Bievre P, Hidaka H, Peiser HS, Rosman KJR, Taylor PDP: Atomic weights of the elements: Review C, N, O, S, Cl, Br) 2000 - (IUPAC technical report). Pure and Applied Chemistry 2003, 75(6): 683-800. Forensic Toxicology Use Only U-HPLC / HR-Orbitrap-MS analytical strategy 4. Comparison of measured and calc. MH + isotopic clusters O O H N O 4-MEC MDPV N O

192,1381 276,1590 100 100

90 90

80 80 70 + 70 + 60 MH measured 60 MH measured 50 isotopic cluster 50 isotopic cluster 40 40 RelativeRelativeAbundance Abundance RelativeRelative Abundance Abundance 30 30

20 20 277,1624 193,1415 10 10 194,1448 195,1480 278,1657 0 0 192,1383 276,1594 100 100

90 90

80 80 70 MH + calculated 70 MH + calculated 60 60 50 isotopic cluster 50 isotopic cluster 40 40

30 30

20 20 277,1628 193,1416 10 10 194,1450 195,1484 278,1661 0 0 190 191 192 193 194 195 196 272 274 276 278 280 282 284 m/z m/z Forensic Toxicology Use Only U-HPLC / HR-Orbitrap-MS analytical strategy 4. Comparison of measured and calc. MH + isotopic clusters O H N Cl N NH Cl-PhPip Mephedrone

178,1225 197,0838 100 100 90 90 80 80 70 70 + + 60 60 MH measured MH measured 50 50 isotopic cluster isotopic cluster 40 40 199,0809 RelativeRelativeAbundance Abundance RelativeAbundance 30 30

20 20 179,1258 198,0872 10 10 200,0842 195,0684 196,1177 201,0873 203,1383 180,1291 0 0 197,0840 178,1226 100 100

90 90

80 80 70 MH + calculated 70 MH + calculated 60 60

50 isotopic cluster 50 isotopic cluster 40 40 199,0811 30 30

20 20 198,0874 179,1260 10 10 200,0844 201,0878 203,0945 180,1294 0 0 194 195 196 197 198 199 200 201 202 203 175 176 177 178 179 180 181 182 183 m/z m/z Forensic Toxicology Use Only U-HPLC / HR-Orbitrap-MS analytical strategy 4. Comparison of measured and calc. MH + isotopic clusters

197,0838 100 Relative Isotopic Abundance (RIA ) 90 M+0 (MH +) 13 12 80 M+1 / M+0 ( C / C) 70 Measured 60 50 M+2 RIA error (%) 40 199,0809 Relative30 Abundance RIA – RIA / RIA x 100 20 M+1 meas calc calc 198,0872 M+3 10 200,0842 195,0684 196,1177 201,0873 203,1383 0 197,0840 100 Substance RIA error (%) 90 M+0 80 Methylone - 1,76 70 Calculated

60 Mephedrone 0,02 50 M+2 MDPV 7,17 40 199,0811 30 4-MEC - 3,38 M+1 20 Propylone - 0,02 198,0874 M+3 10 200,0844 201,0878 203,0945 4F-amphetamine - 0,86 0 194 195 196 197 198 199 200 201 202 203 m/z Cl-Ph-piperazine - 1,72 Forensic Toxicology Use Only U-HPLC / HR-Orbitrap-MS analytical strategy 5. Isotopic Fine Structure (IFS ) of ion clusters

197,0838 + 100 + MH measured M+0 (MH ) isotopic cluster 90

40 199,0809 RelativeAbundance 30 M+2

20 M+1 198,0872 M+3 10 200,0842 195,0684 196,1177 201,0873 203,1383 0 197,0840 100 Forensic Toxicology Use Only U-HPLC / HR-Orbitrap-MS analytical strategy 5. IFS of (M+1), (M+2), (M+3) isotopic peaks vs. (M+0) peaks M+0 of Mephedrone (monoisotopic )

178,1225178.1225 NL: 100 7.77E6 90 20110330amfsimili-mix1- 1000_100k#293 RT: 4.35 AV: 1 80 SB: 8 4.18-4.23 , 4.45-4.50 T: FTMS {1,1} + p ESI Full ms 70 [100.00-800.00]

50 C11 H16 NO 40 Measured at Relative Abundance Relative 30 20 100.000 RP 10 178.0608 178.0743 178.0831 178.1054 178.1182 178.1278 178.1421 178.1533 178.1661 178.1780 178.1932 0 178.1226 100 178,1226 H NO +H: 90 11 15 CNL:11 H 16 N 1 O 1 pa Chrg 1 80 8.81E5 C 70

50 40 Calculated 30

0 178.06 178.08 178.10 178.12 178.14 178.16 178.18 178.20 m/z Forensic Toxicology Use Only U-HPLC / HR-Orbitrap-MS analytical strategy 5. IFS of (M+1), (M+2), (M+3) isotopic peaks vs. (M+0) peaks M+1 of Mephedrone isotopic cluster (12% M+0)

100 179,1259179,1259 NL: 5,78E5 90 20110330AMFSIMILI-MIX1- 80 13 1000_100K#290-294 RT: 4,30-4,36 C10 CH16 NO AV: 5 SB: 31 3,97-4,14 , 4,53-4,79 70 T: FTMS {1;1} + p ESI Full ms 60 [100,00-800,00] 50 C H 2HNO 40 15 11 15 Measured at C11 H16 NO Relative Abundance Relative 30 20 100.000 RP 10 179,1322 179,1193179,1193 0 179,0645 179,1491 179,1708 179,1973 100 179,1260179,1260 90 11 H 15 NO +H: C 11 H 16 N 1 O 1 80 pa Chrg 1 70 60 NL: 50 1,05E5 40 CalculatedC 30 20 10 179,1197179,1197 179,1323 0 179,05 179,10 179,15 179,20 m/z Forensic Toxicology Use Only U-HPLC / HR-Orbitrap-MS analytical strategy 5. IFS of (M+1), (M+2), (M+3) isotopic peaks vs. (M+0) peaks M+2 of Mephedrone isotopic cluster (0.5% M+0)

100 180,1292180,1292 NL: 2,88E4 90 13 180,1319180,1319 C9 C2H16 NO 20110330AMFSIMILI-MIX1- 80 1000_100K#290-294 RT: 4,30-4,36 AV: 5 SB: 31 3,97-4,14 , 4,53-4,79 70 T: FTMS {1;1} + p ESI Full ms 60 18 [100,00-800,00] C11 H16 N O 50 40 Measured at

Relative Abundance Relative 180,1266 13 2 30 13 15 C CH HNO C10 CH16 NO 10 15 20 100.000 RP 10 180,1224 180,1224 180,1583 0 180,1032 100 180,1294180,1294 90 11 H 15 NO +H: C 11 H 16 N 1 O 1 80 pa Chrg 1 70 60 NL: 50 5,67E3 40 CalculatedC 180,1269180,1269 30 20 10 180,1230180,1230 180,1323180,1323 0 180,10 180,12 180,14 180,16 m/z Forensic Toxicology Use Only U-HPLC / HR-Orbitrap-MS analytical strategy 5. IFS of (M+1), (M+2), (M+3) isotopic peaks vs. (M+0) peaks M+3 of Mephedrone isotopic cluster (0.01% M+0)

100 181,1302181,1302 NL: 5,83E2 90 181,1325181,1325 20110330AMFSIMILI-MIX1- 80 1000_100K#290-294 RT: 4,30-4,36 13 18 C10 CH16 N O AV: 5 SB: 31 3,97-4,14 , 4,53-4,79 70 T: FTMS {1;1} + p ESI Full ms 60 [100,00-800,00] 50 13 40 C8 C3H16 NO Measured at

Relative Abundance Relative 30 20 100.000 RP 10 181,1206 0 100 181,1302181,1302 90 181,1327 11 H 15 NO +H: 181,1327 C 11 H 16 N 1 O 1 80 pa Chrg 1 70 60 NL: 50 2,15E2 40 CCalculated 30 20 10 181,1264 181,1239 181,1356 0 181,1385 181,08 181,10 181,12 181,14 m/z Forensic Toxicology Use Only U-HPLC / HR-Orbitrap-MS analytical strategy 5. IFS of (M+1), (M+2), (M+3) isotopic peaks vs. (M+0) peaks M+0 of Methylone (monoisotopic )

208,0961 N L : 1 0 0 4 ,2 4E 6 20110825metilone-rep57_1- 9 0 100_100k-a#208 RT: 2,95 AV: 1 S B : 16 2,77-2,88 , 3,17-3,25 T: 8 0 FTM S {1;1} + p E S I Full m s [100,00-800,00] 7 0 C11 H14 NO 3 6 0

5 0 208,0895 4 0 Measured at

RelativeAbundance 3 0

2 0 100.000 RP

1 0 208,0363 208,0734 208,1037 208,1358 208,1590 208,1883 0 208,0968 N L : 1 0 0 8 ,7 7E 5 9 0 C 1 1 H 1 3 N O 3 + H : C 1 1 H 1 4 N 1 O 3 8 0 p a C h rg 1

7 0

6 0

5 0 4 0 Calculated 3 0

2 0

1 0

0 2 0 8 ,0 0 2 0 8 ,0 5 2 0 8 ,1 0 2 0 8 ,1 5 2 0 8 ,2 0 m /z Forensic Toxicology Use Only U-HPLC / HR-Orbitrap-MS analytical strategy 5. IFS of (M+1), (M+2), (M+3) isotopic peaks vs. (M+0) peaks M+1 of Methylone isotopic cluster (12% M+0)

209,0994 N L : 1 0 0 4 ,8 9E 5 20110825metilone-rep57_1- 9 0 100_100k-a#208 RT: 2,95 AV: 1 13 S B : 16 2,77-2,88 , 3,17-3,25 T: 8 0 C10 CH14 NO 3 FTM S {1;1} + p E S I Full m s [100,00-800,00] 7 0

6 0

5 0 15 2 4 0 C11 H14 NO3 C11 H13 HNO 3 Measured at

RelativeAbundance 3 0

2 0 100.000 RP

1 0 209,0931 209,1041 209,0667 209,1185 209,1306 209,1509 0 209,1002 N L : 1 0 0 1 ,0 4E 5 9 0 C 1 1 H 1 3 N O 3 + H : C 1 1 H 1 4 N 1 O 3 p a C h rg 1 8 0

7 0

6 0

5 0 4 0 Calculated 3 0

2 0

1 0 209,0939 209,1031 0 2 0 9 ,0 6 2 0 9 ,0 8 2 0 9 ,1 0 2 0 9 ,1 2 2 0 9 ,1 4 m /z Forensic Toxicology Use Only U-HPLC / HR-Orbitrap-MS analytical strategy 5. IFS of (M+1), (M+2), (M+3) isotopic peaks vs. (M+0) peaks M+2 of Methylone isotopic cluster (0.5% M+0)

210,1027 N L : 1 0 0 2 ,6 8E 4 20110825metilone-rep57_1- 9 0 100_100k-a#208 RT: 2,95 AV: 1 210,1003 13 S B : 16 2,77-2,88 , 3,17-3,25 T: 8 0 C9 C2H14 NO 3 FTM S {1;1} + p E S I Full m s 18 [100,00-800,00] 7 0 C11 H14 NO 2 O 6 0

5 0 13 2 4 0 13 15 C CH HNO Measured at C10 CH14 NO3 10 13 3 RelativeAbundance 3 0

2 0 100.000 RP

1 0 210,0959 210,1060 210,0708 210,0833 210,0931 210,1145 210,1229 210,1313 0 210,1035 N L : 1 0 0 5 ,6 4E 3 9 0 C 1 1 H 1 3 N O 3 + H : C 1 1 H 1 4 N 1 O 3 8 0 p a C h rg 1

7 0

6 0

5 0 4 0 Calculated 3 0

2 0

1 0 210,0972 210,1065 0 2 1 0 ,0 7 2 1 0 ,0 8 2 1 0 ,0 9 2 1 0 ,1 0 2 1 0 ,1 1 2 1 0 ,1 2 2 1 0 ,1 3 m /z Forensic Toxicology Use Only U-HPLC / HR-Orbitrap-MS analytical strategy 5. IFS of (M+1), (M+2), (M+3) isotopic peaks vs. (M+0) peaks M+3 of Methylone isotopic cluster (0.01% M+0)

211,1039 N L : 1 0 0 2 ,9 9E 3 20110825metilone-rep57_1- 9 0 13 100_100k-a#208 RT: 2,95 AV: 1 13 18 C8 C3H14 NO 3 S B : 16 2,77-2,88 , 3,17-3,25 T: 8 0 C10 CH14 NO 2 O FTM S {1;1} + p E S I Full m s [100,00-800,00] 7 0

6 0

5 0 211,1073 4 0 Measured at

RelativeAbundance 3 0 211,1173 211,1298 211,0993 2 0 211,0702 211,0798 211,0879 100.000 RP 211,1343 1 0

0 211,1044 N L : 1 0 0 6 ,4 3E 2 9 0 C 1 1 H 1 3 N O 3 + H : C 1 1 H 1 4 N 1 O 3 p a C h rg 1 8 0

7 0

6 0

5 0 4 0 Calculated 3 0 211,1069

2 0

1 0 211,1006 211,1098 0 2 1 1 ,0 7 2 1 1 ,0 8 2 1 1 ,0 9 2 1 1 ,1 0 2 1 1 ,1 1 2 1 1 ,1 2 2 1 1 ,1 3 m /z Forensic Toxicology Use Only U-HPLC / HR-Orbitrap-MS analytical strategy 5. IFS of (M+1), (M+2), (M+3) isotopic peaks vs. (M+0) peaks M+0 of Cl-Phenyl-Piperazine (monoisotopic )

197,0835 N L : 1 0 0 7 ,2 2E 6 20110825amfsimili-mix1-200_100k- 9 0 a#345-347 RT: 4,91-4,93 AV: 3 SB: 14 4,76-4,86 , 4,99-5,06 T: FTM S 8 0 {1;1} + p E SI Full m s [100,00-800,00] 7 0 C10 H14 ClN 2 6 0

5 0 197,0896 4 0 Measured at

RelativeAbundance 3 0

2 0 100.000 RP

1 0 197,0228 197,0459 197,0766 197,1091 197,1351 197,1731 197,1917 0 197,0840 N L : 1 0 0 6 ,7 4E 5 9 0 C 1 0 H 1 3 C lN 2 + H : C 1 0 H 1 4 C l 1 N 2 8 0 p a C h rg 1

7 0

6 0

5 0 4 0 Calculated 3 0

2 0

1 0

0 1 9 7 ,0 0 1 9 7 ,0 5 1 9 7 ,1 0 1 9 7 ,1 5 1 9 7 ,2 0 m /z Forensic Toxicology Use Only U-HPLC / HR-Orbitrap-MS analytical strategy 5. IFS of (M+1), (M+2), (M+3) isotopic peaks vs. (M+0) peaks M+1 of Cl-Phenyl-Piperazine isotopic cluster (11% M+0)

198,0869 N L : 1 0 0 7 ,6 2E 5 20110825amfsimili-mix1-200_100k- 9 0 a#345-347 RT: 4,91-4,93 AV: 3 SB: 13 14 4,76-4,86 , 4,99-5,06 T: FTM S 8 0 C9 CH14 ClN 2 {1;1} + p E SI Full m s [100,00-800,00] 7 0

6 0

5 0 C H Cl 15 NN C H 2HClN 4 0 10 14 10 13 2 Measured at 198,0898

RelativeAbundance 3 0

2 0 100.000 RP

1 0 198,0805 198,0650 198,0773 198,0941 198,1034 198,1119 0 198,0874 N L : 1 0 0 7 ,2 9E 4 9 0 C 1 0 H 1 3 C lN 2 + H : C 1 0 H 1 4 C l 1 N 2 8 0 p a C h rg 1

7 0

6 0

5 0 4 0 Calculated 3 0

2 0

1 0 198,0810 198,0903 0 1 9 8 ,0 6 1 9 8 ,0 7 1 9 8 ,0 8 1 9 8 ,0 9 1 9 8 ,1 0 1 9 8 ,1 1 m /z Forensic Toxicology Use Only U-HPLC / HR-Orbitrap-MS analytical strategy 5. IFS of (M+1), (M+2), (M+3) isotopic peaks vs. (M+0) peaks M+2 of Cl-Phenyl-Piperazine isotopic cluster (33% M+0)

199,0805 N L : 1 0 0 3 ,0 0E 6 37 20110825amfsimili-mix1- 9 0 C H Cl N 200_100k-a#346 RT: 4,92 AV: 1 10 14 2 S B : 6 4,46-4,51 , 4,65-4,66 T: 8 0 FTM S {1;1} + p ES I Full m s [100,00-800,00] 7 0

6 0

5 0 4 0 13 Measured at C8 C2H14 ClN 2 RelativeAbundance 3 0

2 0 100.000 RP

1 0 199,0049 199,0378 199,0718 199,0900 199,1184 199,1581 199,1790 0 199,0811 N L : 1 0 0 2 ,1 6E 5 9 0 C 1 0 H 1 3 C lN 2 + H : C 1 0 H 1 4 C l 1 N 2 p a C h rg 1 8 0

7 0

6 0

5 0 4 0 Calculated 3 0

2 0

1 0 199,0907 0 1 9 9 ,0 0 1 9 9 ,0 5 1 9 9 ,1 0 1 9 9 ,1 5 1 9 9 ,2 0 m /z Forensic Toxicology Use Only U-HPLC / HR-Orbitrap-MS analytical strategy 5. IFS of (M+1), (M+2), (M+3) isotopic peaks vs. (M+0) peaks M+3 of Cl-Phenyl-Piperazine isotopic cluster (3% M+0)

200,0838 N L : 1 0 0 3 ,4 2E 5 20110825amfsimili-mix1- 9 0 13 37 200_100k-a#346 RT: 4,92 AV: 1 C9 CH14 Cl N2 S B : 6 4,46-4,51 , 4,65-4,66 T: 8 0 FTM S {1;1} + p ES I Full m s [100,00-800,00] 7 0

6 0 2 37 5 0 C10 H13 HN2 Cl 15 37 4 0 C10 H14 N N Cl Measured at

RelativeAbundance 3 0

2 0 100.000 RP 200,0871 1 0 200,0776 200,0464 200,0644 200,0738 200,0991 200,1153 0 200,0844 N L : 1 0 0 2 ,3 3E 4 9 0 C 1 0 H 1 3 C lN 2 + H : C 1 0 H 1 4 C l 1 N 2 8 0 p a C h rg 1

7 0

6 0

5 0 4 0 Calculated 3 0

2 0

1 0 200,0781 200,0873 200,0970 0 2 0 0 ,0 6 2 0 0 ,0 8 2 0 0 ,1 0 2 0 0 ,1 2 m /z Forensic Toxicology Use Only Conclusions 1. Efficient chromatographic separation of A-R drugs 2. Accurate mass measurements of MH + ionic species with a mass accuracy < 2 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 clusters (RIA error < 10% for all new A-R drugs)

5. Isotopic fine structure of the (M+1), (M+2), (M+3) isotopic peaks completely in accordance with theoretical clusters

Elucidation of elemental composition and structural characteristics of new amphetamine-related designer drugs

Forensic Toxicology Use Only