synthetic cannabinoids the analytical challenges webex · – 0.2 mm i.d. – 0.33 um film ... 81 w...

Anthony Macherone Ph DAnthony Macherone, Ph.DApplications ScientistAgilent Technologies, Inc.Wilmington DE USAWilmington, DE USA

For Forensic Use.

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

• BetterBetter identificationidentification andand confirmationconfirmation ofof analytesanalytes inin highhigh mmatrixatrix ssamplesamples

• Reduces data review time, especially for large forensic screening methods

• Reduces errors in dirty samples

• Identification based on matching entire spectrum cleaned of interferences against library. Much more reliable than target/qualifier ratio method.

Page 12

For Forensic Use.

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.

Confidentiality LabelOctober 7, 201115

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


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


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


For Forensic Use.


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)

21

For Forensic Use.

Synthetic Cannabinoids: the Analytical Challenges


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.


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


For Forensic Use.

Quant Ion Qualifying Ion & Toleranceon

ses

Res

po

For Forensic Use.

Synthetic Cannabinoids: the Analytical Challenges


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


Mono isotopic m/z = 327.1623Uncorrected = 0.04 ppmResolution (RS) 15,492

For Forensic Use.

Si

O

O

Si


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


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


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


For Forensic Use.

synthetic cannabinoids the analytical challenges webex · – 0.2 mm i.d. – 0.33 um film ... 81 w...

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