6\QWKHWLF&DQQDELQRLGVWKH$QDO\WLFDO&KDOOHQJHV

For Forensic Use.

Anthony Macherone Macherone, Ph Ph.D D Applications Scientist Agilent Technologies, Inc. Wilmington DE USA Wilmington,

For Forensic Use.

+LVWRULFDO3HUVSHFWLYH • 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 p activity. y • HU prefaced compounds are named for Hebrew University where these compounds were first synthesized and investigated investigated. • CP 47,497 was initially developed by Pfizer for its analgesic effects.

For Forensic Use.

0ROHFXODU6WUXFWXUHV Delta-9-THC

Si O

Si O H

H O

Classical

HU-210/HU-211 JWH-133

Naphthoylindoles

JWH-073 JWH-122 JWH-210 AM-2201 WIN55,212-2

JWH-081 JWH-200 JWH-398 AM-694

Non-Classical Non Classical

CP47,497(C7) CP55,940 HU-308

For Forensic Use.

6\QWKHWLF&DQQDELQRLGV Current trends • “Herbal Incense” • Numerous compounds • “legal” status • Not picked up in standard urine drug test • Many outlets • Many distributors

For Forensic Use.

0HWKRG'HYHORSPHQW • Sample preparation • Sample extraction • Derivatization • Acquisition • Data analysis

For Forensic Use.

6DPSOH3UHSDUDWLRQ • Lack of homogeneity requires some degree g of mixing g to ensure representative samples are tested. • Matrix difficult to homogenize – Mortar and pestle – Hand-held herb grinder – Electrical devices – Sandpaper

For Forensic Use.

6DPSOH([WUDFWLRQ • Multiple function groups require a generalized approach. Compounds p are p phenols,, alcohols,, indoles,, etc. • Acid/base extraction • Methanol digestion • Matrix contains co-extracted materials

For Forensic Use.

'HULYDWL]DWLRQ • Multiple polar function groups require derivatization for trace analysis y • Poor chromatography g p y • Poor response

For Forensic Use.

+86WDQGDUG Underivatized

Derivatized

Improved Chromatography

Improved Spectral Data

For Forensic Use.

64$FTXLVLWLRQ3DUDPHWHUV • Agilent 5973N-turbo mass spectrometer g p 6890,, autoinjector j and tray y • Gas Chromatograph: • DB-1 column (pt# 128-1012) – 12 meter – 0.2 mm i.d. – 0.33 um film thickness • Helium Flow: 2.6 mL/min. • Constant pressure • Ramp: 50 - 340°C. @ 20°/min. • Scan range: 40 – 620 amu

For Forensic Use.

'DWD$QDO\VLV • Numerous issues y • Trace level analysis • Isomeric pairs – JWH-015 and JWH-073 – JWH-019 JWH 019 and d JWH JWH-122 122 • Similar fragmentation • Co-extractant interferences

For Forensic Use.

:K\8VH'HFRQYROXWLRQ5HSRUWLQJ6RIWZDUH" Deconvolution of mass spectra removes/reduces interferences from chromatographically g p y overlapped pp p peaks:

• Better 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.

Page 12 For Forensic Use.

+RZ'RHV'HFRQYROXWLRQ:RUN" ,RQVZLWKWKHVDPHDEXQGDQFHYVWLPHSURILOHDUHJURXSHG ,RQV ZLWK WKH VDPH DEXQGDQFH YV WLPH SURILOH DUH JURXSHG WRJHWKHUWRFUHDWHVSHFWUD³FOHDQHG´RILQWHUIHUHQFHVIURP RYHUODSSHGSHDNV 7KUHH2YHUODSSHG &KURPDWRJUDSKLF3HDNV

'HFRQYROXWLRQ ,QWHUIHUHQFH

7,& ,QWHUIHUHQFH 7LPH

,QWHUIHUHQFH

,QWHUIHUHQFH  ,QWHUIHUHQFH

7DUJHW

7LPH

P]

7LPH

7LPH

7DUJHW

P]

Page 13 For Forensic Use.

([DPSOH3RVLWLYH³+HUEDO,QFHQVH´6DPSOH Abundance TIC: Positive_2.D\data.ms 5800000 5600000 5400000 5200000 5000000 4800000 4600000 4400000 4200000 4000000 3800000 3600000 3400000 3200000 3000000 2800000 2600000 2400000 2200000 2000000 1800000 1600000 1400000 1200000 1000000 800000 600000 400000 200000 0 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

Ti Time--> >

For Forensic Use.

6HYHUDO&RPSRXQGV+DYH6LPLODU7LPHV$QG6SHFWUD 7KLVSHDNPLJKWEH -:+RU -:+ 6SHFWUXPLVGLUW\ VRLWLVQRW GHILQLWLYH -:+

15

Confidentiality Label October 7, 2011 For Forensic Use.

-:+6SHFWUXP/RRNV%HWWHU%XW,V6WLOO'LUW\ -:+ORRNV PRUHOLNHO\ 6SHFWUXPLVGLUW\ VRLWLVQRW GHILQLWLYH -:+

For Forensic Use.

6HDUFK'LUW\6SHFWUXP$JDLQVW/LEUDU\1RW'HILQLWLYH )LUVWKLWLQ1,676HDUFKLV$0ZLWK PDWFKIDFWRU 6HFRQGKLWLV-:+ZLWK PDWFKIDFWRU 7KLUG KLW LV -:+ ZLWK  PDWFKIDFWRU 7KLUGKLWLV-:+ZLWK PDWFK IDFWRU

327

100 50 0 50

144

41 56 76 41

76

116

200

284

232

270

167

256 284

144

310

327

220

100

360 360

385 411 415

232 40 0

80

120 0

160 60

200 00

240 0

488

435 280 80

320 3 0

Scan 3626 (12.746 min) in C:\ NMS Head to Tail MF=721 RMF=739

17

435

360

400 00

440 0

480 80

520 5 0

AM-694

Confidentiality Label October 7, 2011 For Forensic Use.

1RZ8VH'HFRQYROXWLRQ:LWK$0',6

%HVW0DWFKLV-:+ZLWK PDWFKIDFWRU6LQFH57DQGVSHFWUDO PDWFKDUHEHWWHUWKDQIRU-:+WKLVSHDNLV-:+ 327

100 50 89 101 115

0

200

127

144

89 101 116

50

144

167 167 181

127

100

284 270 213 226 241 254 213 226 241 254 270

100

120

140

160

180

200

220

240

260

280

Head to Tail MF=889 RMF=889

50

144

41 56 76 41

76

116

200

284

232

270

167

256 284

144

310

327

220

100 80

120

160

200

240

320

340

435 360 360

385 411 415

232 40

300

JWH-073

327

100

488

435 280

320

Scan 3626 (12.746 min) in C:\ NMS Head to Tail MF=721 RMF=739

18

310

284

200

Component at scan 3626 (12.746

50

298 298

327 80

0

310

360

400

440

480

520

AM-694 Confidentiality Label October 7, 2011 For Forensic Use.

AMDIS sorts out confusion: AM-694 (blue 435 trace) is overlapped with JWH-073 (red trace). White is Whit i deconvoluted Black is raw spectrum

JWH-073 Library spectrum

19

Confidentiality Label October 7, 2011 For Forensic Use.

$JLOHQW6\QWKHWLF&DQQDELQRLG&RPSHQGLXP

20

Confidentiality Label October 7, 2011 For Forensic Use.

6\QWKHWLF&DQQDELQRLG$SSOLFDWLRQV&'  (1 (1 •Synthetic Synthetic Cannabinoid pdf Compendium (5990-7967EN) (5990 7967EN) •Overview presentation on Synthetic Cannabinoids •Agilent 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

*&444 7$1'(00$6663(&7520(75<

22

Confidentiality Label October 7, 2011 For Forensic Use.

*&444$Q$OWHUQDWLYHWR'56 DRS uses a mathematical algorithm to deconvolute complex spectra p into its component p p parts to facilitate interpretation p

GC/QQQ uses dual stage mass spectrometry to physically isolate the target compounds of interest from potential interferences

For Forensic Use.

24

Confidentiality Label October 7, 2011 For Forensic Use.

444*&0606&RQGLWLRQV Multi Mode Inlet, Pulsed Splitless Mode @ 300 °C: Injection Volume 1 ȝL Column: Agilent HP-5MS UI (19091S-433UI) 30 m x 250 ȝm x 0.25 ȝl, Constant Flow (He) 1.2 mL/min Oven Program 80 °C for 0.17 min then 30 °C/min C/min to 300 °C C for 0 0.5 5 min then 5 °C/min to 340 °C for 5 min Run Time 21.003 min Transfer Line 325 °C C EI Mode Source 300 °C Collision Cell He Quench Gas 2.25 mL/min N2 Collision Gas 1.5 mL/min

For Forensic Use.

6507UDQVLWLRQV CompoundName AMͲ694 AMͲ694 CPͲ47Ͳ497ͲC8 CPͲ47Ͳ497ͲC8 HUͲ211 HUͲ211 JWHͲ015 JWHͲ015 JWHͲ018 JWHͲ018 JWHͲ073 JWH 073 JWHͲ073 JWHͲ081 JWHͲ081 JWHͲ122 JWHͲ122 JWHͲ133 JWHͲ133

PrecursorIon 435 435 377 377 530 446 327 310 341 324 327 310 371 354 338 298 312 269

ProductIon 232 220 191 167 446 299 310 268 167 254 167 254 197 269 268 181 269 93

CollisionEnergy 27 13 29 33 13 21 10 23 23 23 23 23 23 31 23 12 12 23

CompoundName JWHͲ200 JWHͲ200 JWHͲ203 JWHͲ203 JWHͲ250 JWHͲ250 JWHͲ251 JWHͲ251 JWHͲ398 JWHͲ398 RCSͲ4 RCS 4 RCSͲ4 RCSͲ8 RCSͲ8 WIN55212Ͳ3/2 WIN55212Ͳ3/2

PrecursorIon 384 100 339 214 335 214 214 144 375 318 321 264 254 254 100 100

ProductIon 100 56 214 144 214 144 144 116 201 189 264 135 158 144 70 56

CollisionEnergy 23 17 3 17 3 17 17 12 23 23 19 17 13 19 13 15

For Forensic Use.

JWH 073 JWH-073

JWH-018

For Forensic Use.

6507,&&KURPDWRJUDPRI63,&(&RPSRXQGV x10 5 1.4

JWH-203

1.35 1.3 1.25 1.2 1.15 1.1 1.05 1 0.95 0.9 0.85 0.8 0.75

S RCS -4 015 JWH-0 JWH--073 AM-694 JWH--018

0.1 0.05

HU-211

JWH-133

0.2 0 0.15

CP-47 7-497 (C8)

0.3 0.25

WIN N55 2123/2

0.4 0.35

JW WH-200

JWH251

05 0.5 0.45

JWH-0 081

0.6 0.55

JWH--122 RCS-8 JWH-398

JWH250

0.7 0.65

0 7

7.5

8

8.5

9

9.5

10

10.5

11

11.5 12 12.5 13 Counts vs. Acquisition Time (min)

13.5

14

14.5

15

15.5

16

16.5

17

17.5

For Forensic Use.

64YV74³7,&6´ Abundance TIC: Positive_2.D\data.ms 5800000 5600000 5400000 5200000 5000000 4800000 4600000 4400000 4200000 4000000 3800000 3600000 3400000 3200000 3000000 2800000 2600000 2400000 2200000 2000000 1800000 1600000 1400000 1200000 1000000 800000 600000 400000 200000

6LQJOH4XDG Complex chromatogram Post-acquisition deconvolution finds target compounds

0 Time-->

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

7DQGHP4XDG Clean chromatogram Post-acquisition deconvolution not required

29

Confidentiality Label October 7, 2011 For Forensic Use.

([DPSOH&RPSRXQG -:+ -:+ QJPO  QJPO ± QJPO  QJPO Qualifying Ion & Tolerance

Respo onses

Quant Ion

For Forensic Use.

Synthetic Cannabinoids: the Analytical Challenges

*&472) *&4 72)

31

Confidentiality Label October 7, 2011 For Forensic Use.

472))XQGDPHQWDOV Key concepts for TOF:

)OLJKWWXEH &RPPRQZLWK72)

1.

Accurate Mass

2 2.

M Mass R Resolving l i P Power ,RQ0LUURU

&ROOLVLRQFHOO &ROOLVLRQ FHOO &RPPRQZLWK444 ,RQRSWLFV &RPPRQZLWK4 444 2FWRSROH

4XDG0DVV)LOWHU4

2FWRSROH 'HWHFWRU

/HQVDQG

5RXJK S 3XPS

7XUER

7XUER

&ROOLVLRQ&HOO

7XUER

'&4XDG

,RQ3XOVHU

7XUER

For Forensic Use.

0DVV$QDO\VLVIRU72) v = d/t E = ½ m v2 = ½ m (d/t)2 m = (2E/d2) t2 Energy (E) and Distance (d) are fixed 7KHPHDVXUHGPDVVLVSURSRUWLRQDOWR WKHIOLJKWWLPHWLPHRIIOLJKW 

Page 33 For Forensic Use.

:KDWGRHV³([DFW0DVV´PHDQ" Element Atomic Number Exact Mass

0.2874 amu

Page 34

+





&





1





2





C6H6Cl6 C10H12N2O6S C9H21O2PS3 C13H21O3PS C15H17N4Cl C12H21N2O4P C11H20N4O3PS C11H21N4O3P C16H20N2O3

287.8600665 288.0416000 288 0441285 288.0441285 288.0949000 288.1141743 288.1238937 288.1256000 288.1351000 288.1473925

Lindane Carbasulam Terbufos Iprobenfos Myclobutanil Diazoxon Epronaz Pirimetaphos Imazamethabenz

Jan.-Apr. 2009 For Forensic Use.

:K\:KHQ72)472) SQ and TQ • Target analysis, 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.

*&472)$WWULEXWHV Resolving Power – More detection selectivity Mass Accuracy – More qualitative information about the compound – Better library search results if database has accurate mass information Higher Sensitivity compared to SQ SIM y Range g Extended Dynamic MS/MS with Accurate Mass and High Resolving Power – High selectivity and information for structural elucidation

For Forensic Use.

0HWKRG DB-1 (12m x 0.2 mm x 0.33um) 1 5 ml/min constant Flow 1.5 Pulsed, split-less injection 100 C-> 340 C @ 25 C/min EI • High resolution mode (HRM)

For Forensic Use.

SSE+8+50 Si O

Si O H

Unit resolution reference spectrum

H O

Mono isotopic m/z = 530.3611 Uncorrected ǻ = -2.93 ppm Resolution (RS) 17,675

For Forensic Use.

SSE-:++50 O

N

Unit resolution reference spectrum

Mono isotopic m/z = 327.1623 Uncorrected ǻ = 0.04 ppm Resolution (RS) 15,492

For Forensic Use.

SSE&3&DQDORJXH +50 Si O

Si O

Unit resolution reference spectrum

Mono isotopic m/z = 462.3349 Uncorrected ǻ = 4.4 ppm Resolution (RS) 14,512

For Forensic Use.

+8([WUDFWLRQRIDQDO\WHIURPKHDY\PDWUL[ 0DVV 5HVROYLQJ 3RZHU  0DVV5HVROYLQJ3RZHU  x10 8 +ESI TIC Scan Frag=170.0V DG_100 ppb_matrix_03.d 2 1.8 1.6

Matrix TIC: background ~107

1.4 1.2 1 0.8 0.6 0.4 0.2 0 x10 3 +ESI EIC(530.3627) Scan Frag=170.0V DG_100 ppb_matrix_03.d 4.5 4.25 4 3.75 3.5

HU-210 EIC ~ 4.5 x 103

3.25 3 2.75 25 2.5 2.25 2 1.75 1.5 1.25 1 0.75 0.5 0.25 0 5.6

41

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 Counts vs. Acquisition Time (min)

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

Confidentiality Label October 7, 2011 For Forensic Use.

0DVV$FFXUDF\+8 7UXH 0DVV 0HDVXUHG0DVV 7UXH0DVV  0HDVXUHG 0DVV   Delta mass accuracy ~ 3ppm No IRM

42

Confidentiality Label October 7, 2011 For Forensic Use.

&RQFOXVLRQV The described SQ-DRS method and associated library is an effective means of confirming g the p presence of synthetic y cannabinoids by GC/MS • DRS greatly facilitates data interpretation particularly when addressing complex l matrices ti such h as h herbal b l bl blends d

GC/QQQ provides an alternative means of facilitating data p in complex p matrices while improving p g the ratio of interpretation signal 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.

$FNQRZOHGJHPHQWV Fran Diamond, Ph.D. & National Medical Services Thomas JJ. Gluodenis, Gluodenis Jr., Jr Ph Ph.D. D & Tim Conjelko Agilent Technologies Bruce Quimby, Quimby Ph.D. Ph D Agilent Technologies Tom Doherty, Ph.D. Agilent Technologies S fi Aronova, Sofia A Ph D A Ph.D. Agilent il t T Technologies h l i Rafael Acosta Agilent Technologies

44

Confidentiality Label October 7, 2011 For Forensic Use.