Identification of New Amphetamine-Related Designer Drugs...

Forensic Toxicology Use Only

Identification of New

Amphetamine-Related Designer Drugs

Using Exactive High Resolution

Accurate Mass Spectrometry

2011 Joint SOFT-TIAFT MeetingSan Francisco, California, USA

Thermo Scientific Workshop 28 September 2011

Laboratory of Environmental Hygiene and Forensic Toxicology

Department of Prevention

Azienda ULSS 12 Veneziana, Mestre (Venezia), Italy

Giampietro Frison


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


� 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

The flood of new designer drugs


New A-R drugs encountered at LIAFT

Mephedrone

MDPV

4-MEC 4-FA

Cl-Ph-PipMethylone

Propylone

propyl


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

? ? ? ? ?


Analytical strategy to obtain the structuralcharacterization of new A-R drugs

GC-MSunderiv. drugs

� Poor chromatography

� Non-specific EI MS

GC-MS + derwith 3ClEtClfor

�Improved GC properties

�Distinctive MS behav.

�Highly inform. EI MS

UHPLC-HRMS(Orbitrap)

� Accurate mass measurements

at 100.000 resolv. power

� Study of MH+ collision-

induced product ions

� Comparison of exp. and calc.

MH+ isotopic clusters

� Study of isotopic fine

structure of M+1, M+2, M+3

clusters


GC/MS - Mephedrone

4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

6.31

GC/MSAgilent 7890 II - 5975Full Scan EI (40-450 u)Inj. 1 µl, 250C, split / splitless (1 min)Carrier gas (He), 1 ml/min

Agilent HP-5MS Ultra Inert column30 m x 0.25 mm x 0.25 µm50C (0.5 min), 200C a 30C/min, 300C (5 min) a 10C/min Interf. 280C, EMV + 300 V

Rel. Ab.

Time

Methanolic solution (b.p. 64.5 °C)

Mephedrone

Rel. Ab.

Time

Mephedrone(4-Methyl-Meth-Cathinone)

Rel. Ab.

Time


GC/MS - Mephedrone

GC/MSAgilent 7890 II - 5975Full Scan EI (40-450 u)Inj. 1 µl, 250C, split / splitless (1 min)Carrier gas (He), 1 ml/min


60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

58

91119

162

56 65

Rel. Ab.

m/z

MW 177


Mephedrone deriv. with 3ClEtClfor

MW 177

+ Cl CO O CH2 CCl3

CH3

C O CH2 CCl3

OMW 351

50 µL 2,2,2 - 3ClEtClfor : Ethyl acetate 3 : 7

80 °C, 15 min, to dryness

Residue reconst. with 50 µL Ethyl acetate


GC/MS of Mephedrone - 3ClEtClfor

GC/MS (same instrument, same conditions)Agilent 7890 II - 5975Full Scan EI (40-450 u)Inj. 1 µl, 250C, splitless (1 min)Carrier gas (He), 1 ml/min


6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

11.08

Mephedrone – 3ClEtClfor

Rel. Ab.

Time


GC/MS of Mephedrone - 3ClEtClfor

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380

232

58

11991

160

204316318

351353355

234

236

65

131133135

Rel. Ab.

m/z

CH3

C O CH2 C

O

Cl

Cl

Cl

+ .CH3

C O CH2 C

O

Cl

Cl

Cl

+ .351

353355

316

318

131

133

135

204232

234236

119

91


�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 15N from M+1 C isotope, and 34S from M+2 C isotope

z

φ

r

z

φ

r

zm

k

/=ω

U-HPLC/HR-Orbitrap-MS and A-R drugs

Why choosing the Orbitrap technology


U-HPLC/HR-Orbitrap-MS and A-R drugs

LC: U-HPLC Thermo Scientific Accela 1250Injection: 10 µLColumn: Thermo Scientific Hypersil Gold

50 x 2.1 mm x 1.9 mcmPhase A: H2O, 0,05% HCOOH, 5 mM HCOONH4,pH 5 Phase B: ACN, 0,05% HCOOHFlow: 400 µL/minGradient: 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

Mass detect.: Thermo Scientific “Exactive” Orbitrap HRMSSource: HESI-II Ion MaxSpray volt.: 2,5 KVSheath gas: N2 set to 45 a. u.Aux. gas: N2 set to 5 a. u.Cap.Temp.: 290 °CHESI Temp.: 260°CPolarity PosMass range: 50-800 uMass resol.: 25.000 (HCD on, 25 eV) or 100.000 (HCD off),

No Lock Mass

U-HPLC

HR-Orbitrap-MS

Thermo Scientific Exactive HCD MS

Analytical Conditions


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



1.High chromatographic resolution of analytes

RT: 2.36 - 6.07 SM: 3B

2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0

Time (min)

0

50

100

0

50

100

0

50

100

0

50

100

0

50

100

0

50

100

RT: 3.42AA: 1367658

RT: 3.79AA: 5728285

RT: 3.70AA: 891805

RT: 3.95AA: 5218248

RT: 4.33AA: 7126059 4.60

4.764.11 5.03 5.795.622.47 2.94 3.18 3.85

RT: 4.60AA: 63514174.33

4.764.11 5.03 5.795.622.47 2.94 3.18 3.85

NL: 4.01E5

m/z= 136.1110-136.1124 F: FTMS {1,1} + p ESI Full ms [100.00-800.00] MS ICIS MIX amf-amfsimili-jwh-metilon-propilon

NL: 1.62E6


NL: 2.77E5


NL: 1.61E6


NL: 2.37E6


NL: 2.37E6


RT: 2.48 - 6.03 SM: 3B

2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0

Time (min)

0

50

100

0

50

100

0

50

100

0

50

100

0

50

100

0

50

100

RT: 4.28AA: 3172486

RT: 5.18AA: 4319203

RT: 4.73AA: 7724451

RT: 3.81AA: 1744924

RT: 4.94AA: 559381

RT: 3.30AA: 8608603

NL: 1.09E6


NL: 1.55E6


NL: 2.77E6


NL: 5.02E5


NL: 1.98E5


NL: 1.91E6


“Classic” amphetamines New A-R drugs

Amphetamine

Methamphetamine

MDA

MDMA

MDEA

MBDB

Mephedrone

MDPV

4-MEC

4-FA

Cl-Ph-Pip

Methylone

Propylone


-1,04192,1381192,1383191,1310C12H17NOPropylone

-0,48208,0968208,0967207,0895C11H13NO3Methylone

-1,01197,0838197,0840196,0767C10H13ClN2Cl-Ph-piperazine

-1,30154,1025154,1027153,0954C9H12FN4F-amphetamine

-1,04192,1381192,1383191,1310C12H17NO4-MEC

-1,81276,1589276,1594275,1521C16H21NO3MDPV

-0,56178,1225178,1226177,1154C11H15NOMephedrone

-1,44208,1329208,1332207,1259C12H17NO2MBDB

-1,44208,1329208,1332207,1259C12H17NO2MDEA

-1,03194,1174194,1176193,1103C11H15NO2MDMA

-1,11180,1017180,1019179,0946C10H13NO2MDA

-0,67150,1276150,1277149,1204C10H15NMethamphetamine

-0,73136,1120136,1121135,1048C9H13NAmphetamine

ppmAccurate

massExact mass

∆m at 100KMH+MH+

Exact

mass

Elemental

compositionSubstance

100.000resolving power


2.Accurate mass measurements of MH+ ionic species

Mass

accuracy

< 2 ppm(no lock mass)

∆m = (Accurate mass – Exact mass) / Exact mass x 106


U-HPLC / HR-Orbitrap-MS analytical strategy3.Study of MH+ product ions from MS/MS experiments

MDEA and MBDB: both C12H17NO2, MH+ exact mass 208,1332RT: 3.38 - 5.60

3.5 4.0 4.5 5.0 5.5

Time (min)

0

20

40

60

80

100

Re

lati

ve

Ab

un

da

nc

e

0

20

40

60

80

100

Re

lati

ve

Ab

un

da

nc

e

RT: 4.32AA: 7174432

4.60

4.754.12 4.86 5.04 5.234.02 5.313.83 5.523.653.40

RT: 4.60AA: 6373588

4.32

4.754.12 4.86 5.04 5.234.02 5.313.83 5.523.653.40

NL: 2.93E6


NL: 2.93E6


50 100 150 200

m/z

0

20

40

60

80

100

0

20

40

60

80

100

Re

lati

ve

Ab

un

da

nc

e

208.1329

163.0751

198.1862149.1176105.0699

214.0884

105.0700

135.0441

163.0754

72.0811 208.1332

86.0967

151.0754122.0363 177.1127

NL: 2.63E6

MIX amf-amfsimili-jwh-metilon-propilon#701-704 RT: 4.32-4.33 AV: 2 SB: 6 4.23-4.25 , 4.41-4.44 T: FTMS {1,1} + p ESI Full ms [100.00-800.00]

NL: 2.73E5

MIX amf-amfsimili-jwh-metilon-propilon#702 RT: 4.32 AV: 1 SB: 5 4.23-4.25 , 4.41-4.44 F: FTMS {1,2} + p ESI Full ms2 [email protected] [50.00-800.00]

RT: 3.38 - 5.60

3.5 4.0 4.5 5.0 5.5

Time (min)

0

20

40

60

80

100

Re

lati

ve

Ab

un

da

nc

e

0

20

40

60

80

100

Re

lati

ve

Ab

un

da

nc

e

RT: 4.32AA: 7174432

4.60

4.754.12 4.86 5.04 5.234.02 5.393.833.653.40

RT: 4.60AA: 6373588

4.32

4.754.12 4.86 5.04 5.234.02 5.393.833.653.40

NL: 2.93E6


NL: 2.93E6


50 100 150 200

m/z

0

20

40

60

80

100

0

20

40

60

80

100

Re

lati

ve

Ab

un

da

nc

e

208.1329

177.0908135.0439

198.1897

108.0811

135.0441

72.0811 119.0857 208.1333147.0805 177.0911

NL: 2.24E6

MIX amf-amfsimili-jwh-metilon-propilon#749 RT: 4.61 AV: 1 SB: 8 4.50-4.54 , 4.68-4.72 T: FTMS {1,1} + p ESI Full ms [100.00-800.00]

NL: 6.78E5


MDEA

MBDB

HN

O

O

135,0441

72,0811

163,0754

105,0700133,0648

HN

O

O

135,0441

72,0811

177,0908



3.Study of MH+ product ions from MS/MS experiments4-MEC and Propylone: both C12H17NO, MH+ exact mass 192,1383

RT: 3.94 - 5.55

4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4

Time (min)

0

20

40

60

80

100

Re

lati

ve

Ab

un

da

nc

e

0

20

40

60

80

100

Re

lati

ve

Ab

un

da

nc

e RT: 4.57AA: 6547794

4.73

4.94 5.17 5.23 5.394.29 4.353.99 4.22

RT: 4.73AA: 7730758

4.57

4.94 5.17 5.23 5.394.29 4.353.99 4.22

NL: 3.47E6


NL: 3.47E6


50 100 150 200

m/z

0

20

40

60

80

100

0

20

40

60

80

100

Re

lati

ve

Ab

un

da

nc

e

192.1380

174.1275147.0802119.0852100.1121

145.0885

119.0856

159.1043

131.0730

91.0544192.138372.0811 105.0700

137.0597

NL: 2.67E6


NL: 2.29E5


RT: 3.94 - 5.55

4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4

Time (min)

0

20

40

60

80

100

Re

lati

ve

Ab

un

da

nc

e

0

20

40

60

80

100

Re

lati

ve

Ab

un

da

nc

e RT: 4.57AA: 6547794

4.73

4.94 5.17 5.23 5.394.29 4.353.99 4.22

RT: 4.73AA: 7730758

4.57

4.94 5.17 5.23 5.394.29 4.353.99 4.22

NL: 3.47E6


NL: 3.47E6


50 100 150 200

m/z

0

20

40

60

80

100

0

20

40

60

80

100

Re

lati

ve

Ab

un

da

nc

e

192.1380

174.1275132.0807105.0333

132.0808

91.0544

144.0808117.0574 174.1278

105.0336

192.138386.0967 159.1043

NL: 3.43E6


NL: 2.14E5


4-MEC

Propylone

OHN

105,0336

86,0967

119,0856

72,0811

145,0885 132,0808

propyl

O

HN


94.9331.972071Sulphur32S

0.7632.97145833S

4.2933.96786734S

0.0235.96708136S

75.7834.968853Chlorine35Cl

24.2236.96590337Cl

50.6978.918338Bromine79Br

80.916291

17.999160

16.999132

15.994915

15.000109

14.003074

14.003242

13.003355

12.000000

3.016049

2.014102

1.007825

Mass of atom

49.3181Br

0.20518O

0.03817O

99.757Oxygen16O

0.36815N

99.632Nitrogen14N

*14C

1.0713C

98.93Carbon12C

*Tritium3H

0.115Deuterium2H

99.9885Hydrogen1H

% AbundanceNameSymbol

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)De Laeter JR, Bohlke JK, De Bievre P, Hidaka H, Peiser HS, Rosman KJR, Taylor PDP: Atomic weights of the elements: Review

2000 - (IUPAC technical report). Pure and Applied Chemistry 2003, 75(6):683-800.



4.Comparison of measured and calc. MH+ isotopic clusters

190 191 192 193 194 195 196

m/z

0

10

20

30

40

50

60

70

80

90

100

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

192,1381

193,1415

194,1448 195,1480

192,1383

193,1416

194,1450 195,1484

190 191 192 193 194 195 196

m/z

0

10

20

30

40

50

190 191 192 193 194 195 196

m/z

0

10

20

30

40

50

60

70

80

90

100

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

192,1381

193,1415

194,1448 195,1480

192,1383

193,1416

194,1450 195,1484

MH+measuredisotopic cluster

MH+ calculatedisotopic cluster

272 274 276 278 280 282 284

m/z

0

10

20

30

40

50

60

70

80

90

100

0

10

20

30

40

50

60

70

80

90

100

Rela

tive

Abu

nd

ance

276,1590

277,1624

278,1657

276,1594

277,1628

278,1661

272 274 276 278 280 282 284

m/z

0

10

20

30

40

50

272 274 276 278 280 282 284

m/z

0

10

20

30

40

50

60

70

80

90

100

0

10

20

30

40

50

60

70

80

90

100

Rela

tive

Abu

nd

ance

276,1590

277,1624

278,1657

276,1594

277,1628

278,1661

OHN

4-MEC



O

ON

O

MDPV



4.Comparison of measured and calc. MH+ isotopic clusters

175 176 177 178 179 180 181 182 183

m/z

0

10

20

30

40

50

60

70

80

90

100

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

178,1225

179,1258

180,1291

178,1226

179,1260

180,1294

175 176 177 178 179 180 181 182 183

m/z

0

10

20

30

40

50

60

70

80

90

100

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

178,1225

179,1258

180,1291

178,1226

179,1260

180,1294

194 195 196 197 198 199 200 201 202 203

m/z

0

10

20

30

40

50

60

70

80

90

100

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

197,0838

199,0809

198,0872

200,0842195,0684 201,0873 203,1383196,1177

197,0840

199,0811

198,0874

200,0844201,0878 203,0945



Cl-PhPip



MephedroneN

O H

Cl N NH


U-HPLC / HR-Orbitrap-MS analytical strategy4.Comparison of measured and calc. MH+ isotopic clusters

Relative Isotopic Abundance (RIA)

M+1 / M+0 (13C /12C)

RIA error (%)

RIAmeas – RIAcalc/ RIAcalc x 100

194 195 196 197 198 199 200 201 202 203

m/z

0

10

20

30

40

50

60

70

80

90

100

0

10

20

30

40

50

60

70

80

90

100

Re

lati

ve

Ab

un

da

nc

e

197,0838

199,0809

198,0872

200,0842195,0684 201,0873 203,1383196,1177

197,0840

199,0811

198,0874

200,0844201,0878 203,0945

M+0 (MH+)

M+1

M+2

M+3

M+0

M+1

M+2

M+3

Calculated

Measured

- 0,02Propylone

- 1,76Methylone

- 1,72Cl-Ph-piperazine

- 0,864F-amphetamine

- 3,384-MEC

7,17MDPV

0,02Mephedrone

RIA error (%)Substance

Forensic Toxicology Use Only100

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

197,0838

199,0809

198,0872

200,0842195,0684 201,0873 203,1383196,1177

197,0840



5. Isotopic Fine Structure (IFS) of ion clusters

M+0 (MH+)

M+1M+2

M+3


M+0 of Mephedrone (monoisotopic)

U-HPLC / HR-Orbitrap-MS analytical strategy5. IFS of (M+1), (M+2), (M+3) isotopic peaks vs. (M+0) peaks

178.06 178.08 178.10 178.12 178.14 178.16 178.18 178.20

m/z

0

10

20

30

40

50

60

70

80

90

100

0

10

20

30

40

50

60

70

80

90

100

Rela

tive A

bundance

178.1225

178.1182 178.1278 178.1421178.1054 178.1533178.0831 178.1661178.0743178.0608 178.1780 178.1932

178.1226

NL:7.77E6

20110330amfsimili-mix1-1000_100k#293 RT: 4.35 AV: 1 SB: 8 4.18-4.23 , 4.45-4.50 T: FTMS {1,1} + p ESI Full ms [100.00-800.00]

NL:8.81E5

C 11 H15 NO +H: C 11 H16 N1 O1

pa Chrg 1

C11H16NO

Measured at100.000 RP

Calculated

178,1225

178,1226


M+1 of Mephedrone isotopic cluster (12%M+0)

179,05 179,10 179,15 179,20

m/z

0

10

20

30

40

50

60

70

80

90

100

0

10

20

30

40

50

60

70

80

90

100

Rela

tive A

bundance

179,1259

179,1193179,1322

179,1491179,0645 179,1708 179,1973

179,1260

179,1197

NL:5,78E5

20110330AMFSIMILI-MIX1-1000_100K#290-294 RT: 4,30-4,36 AV: 5 SB: 31 3,97-4,14 , 4,53-4,79 T: FTMS {1;1} + p ESI Full ms [100,00-800,00]

NL:1,05E5

C 11 H15 NO +H: C 11 H16 N1 O1

pa Chrg 1

179,1259

179,1193179,1322

179,1260

179,1197 179,1323


Calculated

C11H1615NO

C1013CH16NO

C11H152HNO




M+2 of Mephedrone isotopic cluster (0.5%M+0)

180,10 180,12 180,14 180,16

m/z

0

10

20

30

40

50

60

70

80

90

100

0

10

20

30

40

50

60

70

80

90

100

Relative A

bundance

180,1292

180,1224180,1583

180,1319

180,1032

180,1294

180,1269

180,1230180,1323

NL:2,88E4


NL:5,67E3

C 11 H15 NO +H: C 11 H16 N1 O1

pa Chrg 1

180,1292

180,1224

180,1319

180,1266

180,1294

180,1230180,1323

180,1269

C913C2H16NO

C11H16N18O

C1013CH16

15NO C1013CH15

2HNO


Calculated



M+3 of Mephedrone isotopic cluster (0.01%M+0)

181,08 181,10 181,12 181,14

m/z

0

10

20

30

40

50

60

70

80

90

100

0

10

20

30

40

50

60

70

80

90

100

Relative A

bundance

181,1302

181,1325

181,1206

181,1302

181,1327

181,1264181,1356181,1239

181,1385

NL:5,83E2


NL:2,15E2

C 11 H15 NO +H: C 11 H16 N1 O1

pa Chrg 1

181,1302

181,1325

181,1302

181,1327

C1013CH16N

18O

C813C3H16NO Measured at

100.000 RP

Calculated


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

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

1 0 0

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

1 0 0

Re

lative

Abu

nda

nce

2 0 8 ,0 9 6 1

2 0 8 ,1 0 3 7

2 0 8 ,0 8 9 5

2 0 8 ,0 7 3 4 2 0 8 ,1 3 5 82 0 8 ,0 3 6 3 2 0 8 ,1 5 9 0 2 0 8 ,1 8 8 3

2 0 8 ,0 9 6 8

N L :4 ,2 4 E 6

2 0 1 1 0 8 2 5 m e ti lo n e -re p 5 7 _ 1 -1 0 0 _ 1 0 0 k -a # 2 0 8 R T : 2 ,9 5 A V : 1 S B : 1 6 2 ,7 7 -2 ,8 8 , 3 ,1 7 -3 ,2 5 T : F T M S {1 ;1 } + p E S I F u ll m s [1 0 0 ,0 0 -8 0 0 ,0 0 ]

N L :8 ,7 7 E 5

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

M+0 of Methylone (monoisotopic)



Calculated

C11H14NO3



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

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

1 0 0

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

1 0 0

Rela

tive A

bun

da

nce

2 0 9 ,0 9 9 4

2 0 9 ,0 9 3 1 2 0 9 ,1 0 4 12 0 9 ,1 1 8 52 0 9 ,0 6 6 7 2 0 9 ,1 3 0 6 2 0 9 ,1 5 0 9

2 0 9 ,1 0 0 2

2 0 9 ,0 9 3 9 2 0 9 ,1 0 3 1

N L :4 ,8 9 E 5


N L :1 ,0 4 E 5

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

C11H1415NO3

C1013CH14NO3

C11H132HNO3

M+1 of Methylone isotopic cluster (12%M+0)


Calculated


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

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

1 0 0

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

1 0 0

Re

lative

Abu

nda

nce

2 1 0 ,1 0 2 7

2 1 0 ,1 0 0 3

2 1 0 ,1 0 6 02 1 0 ,0 9 5 92 1 0 ,0 7 0 8 2 1 0 ,1 3 1 32 1 0 ,1 2 2 92 1 0 ,0 9 3 1 2 1 0 ,1 1 4 52 1 0 ,0 8 3 3

2 1 0 ,1 0 3 5

2 1 0 ,0 9 7 22 1 0 ,1 0 6 5

N L :2 ,6 8 E 4


N L :5 ,6 4 E 3

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

C913C2H14NO3

C11H14NO218O

C1013CH14

15NO3C10

13CH132HNO3

M+2 of Methylone isotopic cluster (0.5%M+0)



Calculated


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

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

1 0 0

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

1 0 0

Rela

tive A

bun

da

nce

2 1 1 ,1 0 3 9

2 1 1 ,1 0 7 3

2 1 1 ,1 2 9 82 1 1 ,1 1 7 32 1 1 ,0 9 9 32 1 1 ,0 7 9 82 1 1 ,0 7 0 2 2 1 1 ,0 8 7 9 2 1 1 ,1 3 4 3

2 1 1 ,1 0 4 4

2 1 1 ,1 0 6 9

2 1 1 ,1 0 0 6 2 1 1 ,1 0 9 8

N L :2 ,9 9 E 3


N L :6 ,4 3 E 2

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

C1013CH14NO2

18O C813C3H14NO3

M+3 of Methylone isotopic cluster (0.01%M+0)



Calculated


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

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

1 0 0

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

1 0 0

Re

lative

Abu

nda

nce

1 9 7 ,0 8 3 5

1 9 7 ,0 8 9 6

1 9 7 ,0 7 6 6 1 9 7 ,1 0 9 1 1 9 7 ,1 3 5 11 9 7 ,0 4 5 91 9 7 ,0 2 2 8 1 9 7 ,1 9 1 71 9 7 ,1 7 3 1

1 9 7 ,0 8 4 0

N L :7 ,2 2 E 6

2 0 1 1 0 8 2 5 a m fs im i li -m i x1 -2 0 0 _ 1 0 0 k -a # 3 4 5 -3 4 7 R T : 4 ,9 1 -4 ,9 3 A V : 3 S B : 1 4 4 ,7 6 -4 ,8 6 , 4 ,9 9 -5 ,0 6 T : F T M S {1 ;1 } + p E S I F u ll m s [1 0 0 ,0 0 -8 0 0 ,0 0 ]

N L :6 ,7 4 E 5

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

C10H14ClN2

M+0 of Cl-Phenyl-Piperazine (monoisotopic)



Calculated



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

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

1 0 0

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

1 0 0

Re

lative

Abu

nda

nce

1 9 8 ,0 8 6 9

1 9 8 ,0 8 0 5

1 9 8 ,0 8 9 8

1 9 8 ,0 9 4 1 1 9 8 ,1 0 3 4 1 9 8 ,1 1 1 91 9 8 ,0 6 5 0 1 9 8 ,0 7 7 3

1 9 8 ,0 8 7 4

1 9 8 ,0 8 1 0

1 9 8 ,0 9 0 3

N L :7 ,6 2 E 5

2 0 1 1 0 8 2 5 a m fs im i li -m i x1 -2 0 0 _ 1 0 0 k -a # 3 4 5 -3 4 7 R T : 4 ,9 1 -4 ,9 3 A V : 3 S B : 1 4 4 ,7 6 -4 ,8 6 , 4 ,9 9 -5 ,0 6 T : F T M S {1 ;1 } + p E S I F u ll m s [1 0 0 ,0 0 -8 0 0 ,0 0 ]

N L :7 ,2 9 E 4

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

C10H14Cl15NN

C913CH14ClN2

C10H132HClN2

M+1 of Cl-Phenyl-Piperazine isotopic cluster (11%M+0)


Calculated


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

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

1 0 0

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

1 0 0

Rela

tive A

bun

da

nce

1 9 9 ,0 8 0 5

1 9 9 ,0 9 0 01 9 9 ,0 7 1 8 1 9 9 ,1 7 9 01 9 9 ,0 3 7 81 9 9 ,0 0 4 9 1 9 9 ,1 1 8 4 1 9 9 ,1 5 8 1

1 9 9 ,0 8 1 1

1 9 9 ,0 9 0 7

N L :3 ,0 0 E 6

2 0 1 1 0 8 2 5 a m fs im i li -m i x1 -2 0 0 _ 1 0 0 k -a # 3 4 6 R T : 4 ,9 2 A V : 1 S B : 6 4 ,4 6 -4 ,5 1 , 4 ,6 5 -4 ,6 6 T : F T M S {1 ;1 } + p E S I F u ll m s [1 0 0 ,0 0 -8 0 0 ,0 0 ]

N L :2 ,1 6 E 5

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

C10H1437ClN2

C813C2H14ClN2



Calculated



2 0 0 ,0 6 2 0 0 ,0 8 2 0 0 ,1 0 2 0 0 ,1 2

m /z

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

1 0 0

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

1 0 0

Re

lative

Abu

nda

nce

2 0 0 ,0 8 3 8

2 0 0 ,0 7 7 6

2 0 0 ,0 8 7 1

2 0 0 ,0 7 3 82 0 0 ,0 6 4 4 2 0 0 ,1 1 5 32 0 0 ,0 4 6 4 2 0 0 ,0 9 9 1

2 0 0 ,0 8 4 4

2 0 0 ,0 7 8 1

2 0 0 ,0 8 7 3 2 0 0 ,0 9 7 0

N L :3 ,4 2 E 5

2 0 1 1 0 8 2 5 a m fs im i li -m i x1 -2 0 0 _ 1 0 0 k -a # 3 4 6 R T : 4 ,9 2 A V : 1 S B : 6 4 ,4 6 -4 ,5 1 , 4 ,6 5 -4 ,6 6 T : F T M S {1 ;1 } + p E S I F u ll m s [1 0 0 ,0 0 -8 0 0 ,0 0 ]

N L :2 ,3 3 E 4

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

C10H132HN2

37ClC10H14N

15N37Cl

C913CH14

37ClN2




Calculated


Conclusions1. 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

Identification of New Amphetamine-Related Designer Drugs...

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