Anthony Macherone Ph DAnthony Macherone, Ph.DApplications ScientistAgilent Technologies, Inc.Wilmington DE USAWilmington, DE USA
For Forensic Use.
For Forensic Use.
• Initially chemicals were synthesized for medicinal research.
• JWH prefaced compounds are named for John W. Huffman at Clemson University for research on the relationship between the structure of drugs and brain receptor activity.p y
• HU prefaced compounds are named for Hebrew University where these compounds were first synthesized and investigatedcompounds were first synthesized and investigated.
• CP 47,497 was initially developed by Pfizer for its analgesic effects.
For Forensic Use.
Delta-9-THC
Si
HO
Si
O
Classical Naphthoylindoles Non-Classical
O
H
Classical
HU-210/HU-211
Naphthoylindoles
JWH-073 JWH-081
Non Classical
CP47,497(C7)JWH-133 JWH-122 JWH-200
JWH-210 JWH-398AM-2201 AM-694WIN55,212-2
CP55,940HU-308
For Forensic Use.
Current trends• “Herbal Incense”• Numerous compounds• Numerous compounds• “legal” status• Not picked up in standardNot picked up in standard
urine drug test• Many outlets• Many distributors
For Forensic Use.
• Sample preparation
• Sample extraction
• Derivatization
• Acquisition
• Data analysis
For Forensic Use.
• Lack of homogeneity requires some degree of mixing to g gensure representative samples are tested.
• Matrix difficult to homogenizeMortar and pestle– Mortar and pestle
– Hand-held herb grinder– Electrical devices– Sandpaper
For Forensic Use.
• Multiple function groups require a generalized approach. Compounds are phenols, alcohols, indoles, etc.p p , , ,
• Acid/base extraction
• Methanol digestion
• Matrix contains co-extracted materials
For Forensic Use.
• Multiple polar function groups require derivatization for trace analysisy
• Poor chromatographyg p y
• Poor response
For Forensic Use.
Underivatized Derivatized
Improved Chromatography
Improved Spectral Data
For Forensic Use.
• Agilent 5973N-turbo mass spectrometer• Gas Chromatograph: 6890, autoinjector and trayg p , j y• DB-1 column (pt# 128-1012)
– 12 meter– 0.2 mm i.d.– 0.33 um film thickness
• Helium Flow: 2.6 mL/min.Helium Flow: 2.6 mL/min.• Constant pressure• Ramp: 50 - 340°C. @ 20°/min.• Scan range: 40 – 620 amu
For Forensic Use.
• Numerous issues• Trace level analysisy• Isomeric pairs
– JWH-015 and JWH-073JWH 019 d JWH 122– JWH-019 and JWH-122
• Similar fragmentation• Co-extractant interferences
For Forensic Use.
Deconvolution of mass spectra removes/reduces interferences from chromatographically overlapped peaks:g p y pp p
• Better identification and confirmation of analytes in high matrix samplesBetter identification and confirmation of analytes in high matrix samples
• Reduces data review time, especially for large screening methods
• Reduces false positives and false negatives in dirty samples
• Identification based on matching entire spectrum cleaned of interferences against library. Much more reliable than target/qualifier ratio method.
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For Forensic Use.
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For Forensic Use.
5200000
5400000
5600000
5800000
Abundance
TIC: Positive_2.D\data.ms
3800000
4000000
4200000
4400000
4600000
4800000
5000000
2400000
2600000
2800000
3000000
3200000
3400000
3600000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00
0
200000
400000
600000
800000
Ti >Time-->
For Forensic Use.
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For Forensic Use.
For Forensic Use.
0
50
100
41 56 76144 167
200 232
270
284
284
310
327
360385 411
415
435
488
40 80 120 160 200 240 280 320 360 400 440 480 520
50
100
41 76 116 144220
232
256 284 327 360415
435
Scan 3626 (12.746 min) in C:\ NMS AM-694Head to Tail MF=721 RMF=739
0 80 0 60 00 0 80 3 0 360 00 0 80 5 0
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For Forensic Use.
100284
327
0
50
50
89
89
101
101
115
116
127
127
144
144
167
167 181
200
200
213
213
226
226
241
241
254
254
270
270
284
284
298
298
310
310
Component at scan 3626 (12.746 JWH-073Head to Tail MF=889 RMF=889
80 100 120 140 160 180 200 220 240 260 280 300 320 340
100127 200 284
327
0
50
100
41 56 76144 167
200 232
270
284310
327
360385 411
435
488
40 80 120 160 200 240 280 320 360 400 440 480 520
50
100
41 76 116 144220
232
256 284 327 360415
435
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Scan 3626 (12.746 min) in C:\ NMS AM-694Head to Tail MF=721 RMF=739
40 80 120 160 200 240 280 320 360 400 440 480 520
For Forensic Use.
AMDIS sorts out confusion:
AM-694 (blue 435 trace) is overlappedoverlapped with JWH-073 (red trace).
Whit iWhite is deconvoluted
Black is raw spectrum
JWH-073 Library spectrum
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For Forensic Use.
Confidentiality LabelOctober 7, 201120
For Forensic Use.
•Synthetic Cannabinoid pdf Compendium (5990-7967EN)Synthetic Cannabinoid pdf Compendium (5990 7967EN)
•Overview presentation on Synthetic Cannabinoids
Agilent ChemStation library files•Agilent ChemStation library files
•DRS Library files
•Read me files (instructions for use)
www.agilent.com/chem/cannabinoidcd
21
For Forensic Use.
Synthetic Cannabinoids: the Analytical Challenges
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For Forensic Use.
DRS uses a mathematical algorithm to deconvolute complex spectra into its component parts to facilitate interpretationp p p p
GC/QQQ uses dual stage mass spectrometry to physicallyGC/QQQ uses dual stage mass spectrometry to physically isolate the target compounds of interest from potential interferences
For Forensic Use.
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For Forensic Use.
Multi Mode Inlet, Pulsed Splitless Mode @ 300 °C: Injection Volume 1 LColumn: Agilent HP-5MS UI (19091S-433UI) 30 m x 250 m x 0.25 l, Constant Flow (He) 1.2 mL/min1.2 mL/minOven Program
80 °C for 0.17 minthen 30 °C/min to 300 °C for 0 5 minthen 30 C/min to 300 C for 0.5 minthen 5 °C/min to 340 °C for 5 min
Run Time 21.003 minTransfer Line 325 °CTransfer Line 325 CEI ModeSource 300 °CCollision CellCollision CellHe Quench Gas 2.25 mL/minN2 Collision Gas 1.5 mL/min
For Forensic Use.
Compound Name Precursor Ion Product Ion Collision Energy Compound Name Precursor Ion Product Ion Collision EnergyAM 694 435 232 27 JWH 200 384 100 23AM 694 435 220 13 JWH 200 100 56 17CP 47 497 C8 377 191 29 JWH 203 339 214 3CP 47 497 C8 377 167 33 JWH 203 214 144 17HU 211 530 446 13 JWH 250 335 214 3HU 211 446 299 21 JWH 250 214 144 17JWH 015 327 310 10 JWH 251 214 144 17JWH 015 310 268 23 JWH 251 144 116 12JWH 018 341 167 23 JWH 398 375 201 23JWH 018 324 254 23 JWH 398 318 189 23JWH 073 327 167 23 RCS 4 321 264 19JWH 073 327 167 23 RCS 4 321 264 19JWH 073 310 254 23 RCS 4 264 135 17JWH 081 371 197 23 RCS 8 254 158 13JWH 081 354 269 31 RCS 8 254 144 19JWH 122 338 268 23 WIN55 212 3/2 100 70 13JWH 122 298 181 12 WIN55 212 3/2 100 56 15JWH 133 312 269 12JWH 133 269 93 23
For Forensic Use.
JWH 073JWH-073
JWH-018
For Forensic Use.
5x10 5x10
1.15
1.2
1.25
1.3
1.35
1.4 JWH-203
0.85
0.9
0.95
1
1.05
1.1
0 5
0.55
0.6
0.65
0.7
0.75
0.8
-
JWH
-25
0
0.2
0.25
0.3
0.35
0.4
0.45
0.5
- 7-49
7
JWH
251
S 015
-073 94 -018
-122
RC
S-8
JWH
-398
081
WH
-200
N55
212
-
0
0.05
0.1
0.15
0
Counts vs. Acquisition Time (min)7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 13.5 14 14.5 15 15.5 16 16.5 17 17.5
JWH
-13
3
CP
-47
(C8)
HU
-211
RC
S-4
JWH
-0JW
H-
AM
-6JW
H-
JWH
-
JWH
-0 JW WIN
3/2
For Forensic Use.
38000004000000420000044000004600000480000050000005200000540000056000005800000
AbundanceTIC: Positive_2.D\data.ms
Complex chromatogramPost-acquisition deconvolution findstarget compounds
12000001400000160000018000002000000220000024000002600000280000030000003200000340000036000003800000 target compounds
2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.000
200000400000600000800000
1000000
Time-->
Clean chromatogramPost-acquisition deconvolution not required
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For Forensic Use.
Quant Ion Qualifying Ion & Toleranceon
ses
Res
po
For Forensic Use.
Synthetic Cannabinoids: the Analytical Challenges
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For Forensic Use.
Key concepts for TOF:
1. Accurate Mass
2 M R l i P2. Mass Resolving Power
For Forensic Use.
v = d/t
E = ½ m v2 = ½ m (d/t)2
m = (2E/d2) t2
Energy (E) and Distance (d) are fixed
Page 33
For Forensic Use.
Element Atomic Number Exact MassElement Atomic Number Exact Mass
C6H6Cl6 287.8600665 LindaneC10H12N2O6S 288.0416000 CarbasulamC9H21O2PS3 288 0441285 TerbufosC9H21O2PS3 288.0441285 TerbufosC13H21O3PS 288.0949000 IprobenfosC15H17N4Cl 288.1141743 MyclobutanilC12H21N2O4P 288.1238937 Diazoxon
0.2874 amu
C11H20N4O3PS 288.1256000 EpronazC11H21N4O3P 288.1351000 PirimetaphosC16H20N2O3 288.1473925 Imazamethabenz
Jan.-Apr. 2009Page 34
For Forensic Use.
SQ and TQ • Target analysis enforcement• Target analysis, enforcement
– However, what else is present?
TOF/Q-TOF• Targeted and non-targeted analysis• Profiling: Origin, manufacturer, adulterants• Structure elucidation / confirmation (Q-TOF)
For Forensic Use.
Resolving Power
– More detection selectivityMass Accuracy
– More qualitative information about the compoundBetter library search results if database has accurate mass information– Better library search results if database has accurate mass information
Higher Sensitivity compared to SQ SIM
Extended Dynamic Rangey g
MS/MS with Accurate Mass and High Resolving Power
– High selectivity and information for structural elucidation
For Forensic Use.
DB-1 (12m x 0.2 mm x 0.33um)
1 5 ml/min constant Flow1.5 ml/min constant Flow
Pulsed, split-less injection
100 C-> 340 C @ 25 C/min
EI• High resolution mode (HRM)
For Forensic Use.
Si
O
H
HO
Si
O
Unit resolution reference spectrum
Mono isotopic m/z = 530.3611Uncorrected = -2.93 ppm
Resolution (RS) 17,675
For Forensic Use.
O
N
Unit resolution reference spectrum
Mono isotopic m/z = 327.1623Uncorrected = 0.04 ppmResolution (RS) 15,492
For Forensic Use.
Si
O
O
Si
Unit resolution reference spectrum
Mono isotopic m/z = 462.3349Uncorrected = 4.4 ppmResolution (RS) 14,512
For Forensic Use.
8x10
1.8
2
+ESI TIC Scan Frag=170.0V DG_100 ppb_matrix_03.d
0.8
1
1.2
1.4
1.6
Matrix TIC: background ~107
0
0.2
0.4
0.6
3x10 +ESI EIC(530.3627) Scan Frag=170.0V DG_100 ppb_matrix_03.d
2 5
2.75
3
3.25
3.5
3.75
4
4.25
4.5
HU-210 EIC ~ 4.5 x 103
0.5
0.75
1
1.25
1.5
1.75
2
2.25
2.5
Confidentiality LabelOctober 7, 201141
0
0.25
Counts vs. Acquisition Time (min)5.6 5.7 5.8 5.9 6 6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9 7 7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8 7.9 8 8.1 8.2 8.3 8.4 8.5 8.6 8.7 8.8 8.9
For Forensic Use.
Delta mass accuracy ~ 3ppmDelta mass accuracy 3ppmNo IRM
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For Forensic Use.
The described SQ-DRS method and associated library is an effective means of confirming the presence of synthetic g p ycannabinoids by GC/MS• DRS greatly facilitates data interpretation particularly when addressing
l t i h h b l bl dcomplex matrices such as herbal blends
GC/QQQ provides an alternative means of facilitating data interpretation in complex matrices while improving the ratio of p p p gsignal to chemical noise
GC/Q-TOF offers the investigator the ability to perform targeted and non-targeted analysis, structure elucidation and confirmation
For Forensic Use.
Fran Diamond, Ph.D. & National Medical Services
Thomas J Gluodenis Jr Ph D & Tim Conjelko AgilentThomas J. Gluodenis, Jr., Ph.D. & Tim Conjelko Agilent Technologies
Bruce Quimby Ph D Agilent TechnologiesBruce Quimby, Ph.D. Agilent Technologies
Tom Doherty, Ph.D. Agilent Technologies
S fi A Ph D A il t T h l iSofia Aronova, Ph.D. Agilent Technologies
Rafael Acosta Agilent Technologies
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For Forensic Use.