Slide 1: Synthetic Cannabinoids the Analytical Challenges
6\QWKHWLF&DQQDELQRLGVWKH$QDO\WLFDO&KDOOHQJHV For Forensic Use. Anthony Macherone Macherone, Ph Ph.D D Applications Scientist Agilent Technologies, Inc. Wilmington DE USA Wilmington, For Forensic Use.
Slide 2: Molecular Structures
0ROHFXODU6WUXFWXUHV Delta-9-THC Si O Si O H H O Classical Naphthoylindoles Non-Classical Non Classical HU-210/HU-211 JWH-073 JWH-081 CP47,497(C7) JWH-133 JWH-122 JWH-200 CP55,940 JWH-210 JWH-398 HU-308 AM-2201 AM-694 WIN55,212-2 For Forensic Use.
Slide 3: Synthetic Cannabinoids: Current trends
6\QWKHWLF&DQQDELQRLGV Current trends • “Herbal Incense” • Numerous compounds • “legal” status • Not picked up in standard urine drug test • Many outlets • Many distributors For Forensic Use.
Slide 4: Method Development
0HWKRG'HYHORSPHQW • Sample preparation • Sample extraction • Derivatization • Acquisition • Data analysis For Forensic Use.
Slide 5: Sample Preparation
6DPSOH3UHSDUDWLRQ • Lack of homogeneity requires some degree g of mixing g to ensure representative samples are tested. • Matrix difficult to homogenize – Mortar and pestle – Hand-held herb grinder – Electrical devices – Sandpaper For Forensic Use.
Slide 6: Sample Extraction
6DPSOH([WUDFWLRQ • Multiple function groups require a generalized approach. Compounds p are pphenols,, alcohols,, indoles,, etc. • Acid/base extraction • Methanol digestion • Matrix contains co-extracted materials For Forensic Use.
Slide 7: Derivatization
'HULYDWL]DWLRQ • Multiple polar function groups require derivatization for trace analysis y • Poor chromatography g p y • Poor response For Forensic Use.
Slide 8: HU Standard
+86WDQGDUG Underivatized Derivatized Improved Chromatography Improved Spectral Data For Forensic Use.
Slide 9: SQ Acquisition Parameters
64$FTXLVLWLRQ3DUDPHWHUV • Agilent 5973N-turbo mass spectrometer g p 6890,, autoinjector • Gas Chromatograph: j and tray 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. • Constant pressure • Ramp: 50 - 340°C. @ 20°/min. • Scan range: 40 – 62
Slide 10: Data Analysis
'DWD$QDO\VLV • Numerous issues y • Trace level analysis • Isomeric pairs – JWH-015 and JWH-073 – JWH-019 JWH 019 and d JWH JWH-122 122 • Similar fragmentation • Co-extractant interferences For Forensic Use.
Slide 11: Why Use Deconvolution Reporting Software?
:K\8VH'HFRQYROXWLRQ5HSRUWLQJ6RIWZDUH" Deconvolution of mass spectra removes/reduces interferences from chromatographically g p y overlapped pp p peaks: • Better identification and confirmation of analytes in high matrix samples • Reduces data review time, especially for large forensic screening meth
Slide 12: How Does Deconvolution Work?
+RZ'RHV'HFRQYROXWLRQ:RUN" ,RQVZLWKWKHVDPHDEXQGDQFHYVWLPHSURILOHDUHJURXSHG ,RQV ZLWK WKH VDPH DEXQGDQFH YV WLPH SURILOH DUH JURXSHG WRJHWKHUWRFUHDWHVSHFWUD³FOHDQHG´RILQWHUIHUHQFHVIURP RYHUODSSHGSHDNV 7KUHH2YHUODSSHG 'HFRQYROXWLRQ &KURPDWRJUDSKLF3HDNV ,QWHUIHUHQFH 7,& ,QWHUIHUHQFH ,QWHUIHUHQFH 7LPH P]
Slide 13: Example Positive Herbal Incense Sample
([DPSOH3RVLWLYH³+HUEDO,QFHQVH´6DPSOH Abundance TIC: Positive_2.D\data.ms 5800000 5600000 5400000 5200000 5000000 4800000 4600000 4400000 4200000 4000000 3800000 3600000 3400000 3200000 3000000 2800000 2600000 2400000 2200000 2000000 1800000 1600000 1400000 1200000 1000000 800000 600000 400000 200000
Slide 14: Several Compounds Have Similar Times And Spectra
6HYHUDO&RPSRXQGV+DYH6LPLODU7LPHV$QG6SHFWUD 7KLVSHDNPLJKWEH -:+RU -:+ 6SHFWUXPLVGLUW\ VRLWLVQRW GHILQLWLYH -:+ Confidentiality Label 15 October 7, 2011 For Forensic Use.
Slide 15: JWH Spectrum Looks Better But Is Still Dirty
-:+6SHFWUXP/RRNV%HWWHU%XW,V6WLOO'LUW\ -:+ORRNV PRUHOLNHO\ 6SHFWUXPLVGLUW\ VRLWLVQRW GHILQLWLYH -:+ For Forensic Use.
Slide 16: Search Dirty Spectrum Against Library Not Definitive
6HDUFK'LUW\6SHFWUXP$JDLQVW/LEUDU\1RW'HILQLWLYH )LUVWKLWLQ1,676HDUFKLV$0ZLWK PDWFKIDFWRU 6HFRQGKLWLV-:+ZLWK PDWFKIDFWRU 7KLUG KLW LV -:+ ZLWK PDWFKIDFWRU 7KLUGKLWLV-:+ZLWK PDWFK IDFWRU 327 100 435 232 284 50 200 310 144 270 360 41 56 76 167 385 411 488 0 256 284 327 415 76 116 360 50 41 144 220 100 2
Slide 17: Now Use Deconvolution With AMDIS
1RZ8VH'HFRQYROXWLRQ:LWK$0',6 %HVW0DWFKLV-:+ZLWK PDWFKIDFWRU6LQFH57DQGVSHFWUDO PDWFKDUHEHWWHUWKDQIRU-:+WKLVSHDNLV-:+ 327 100 284 50 200 310 127 144 270 89 101 115 167 213 226 241 254 298 0 89 101 116 167 181 213 226 298 50 144 241 254 270 310 127 200 284 100 327 80 100 120 140 160 180 200 220 240 260
Slide 18: AMDIS sorts out confusion
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 Confidentiality Label 19 October 7, 2011 For Forensic Use.
Slide 19: Agilent Synthetic Cannabinoid Compendium
$JLOHQW6\QWKHWLF&DQQDELQRLG&RPSHQGLXP Confidentiality Label 20 October 7, 2011 For Forensic Use.
Slide 20: Synthetic Cannabinoid Applications CD
6\QWKHWLF&DQQDELQRLG$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) 21 For Forensic Use.
Slide 21: Synthetic Cannabinoids: the Analytical Challenges
Synthetic Cannabinoids: the Analytical Challenges *&444 7$1'(00$6663(&7520(75< Confidentiality Label 22 October 7, 2011 For Forensic Use.
Slide 22: GC/QQQ An Alternative to DRS
*&444$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.
Slide 23: For Forensic Use.
Confidentiality Label 24 October 7, 2011 For Forensic Use.
Slide 24: QQQ GC/MS/MS Conditions
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 m
Slide 25: SRM Transitions
6507UDQVLWLRQV CompoundName PrecursorIon ProductIon CollisionEnergy CompoundName PrecursorIon ProductIon CollisionEnergy AMͲ694 435 232 27 JWHͲ200 384 100 23 AMͲ694 435 220 13 JWHͲ200 100 56 17 CPͲ47Ͳ497ͲC8 377 191 29 JWHͲ203 339 214 3 CPͲ47Ͳ497ͲC8 377 167 33 JWHͲ203 214 144 17 HUͲ211 530 446 13 JWH
Slide 26: JWH-073 JWH-018
JWH 073 JWH-073 JWH-018 For Forensic Use.
Slide 27: SRM TIC Chromatogram of SPICE Compounds
6507,&&KURPDWRJUDPRI63,&(&RPSRXQGV x10 5 1.4 1.35 JWH-203 1.3 1.25 1.2 1.15 1.1 1.05 1 0.95 0.9 0.85 0.8 0.75 0.7 JWH- 0.65 250 0.6 0.55 JWH- 05 0.5 251 JWH-398 0.45 RCS-8 0.4 N55 212- 0.35 WH-200 0.3 7-497 JWH--073 JWH--018 0.25 JWH--122 015 081 AM-694 WIN JW 0.2 0 JWH-- RCSS JWH-0 JWH-0 HU-211 CP-
Slide 28: SQ vs TQ TICs
64YV74³7,&6´ Abundance TIC: Positive_2.D\data.ms 5800000 5600000 5400000 6LQJOH4XDG 5200000 5000000 4800000 Complex chromatogram 4600000 4400000 Post-acquisition deconvolution finds 4200000 4000000 3800000 target compounds 3600000 3400000 3200000 3000000 2800000 2600000 2400000 2200000 2000000 18000
Slide 29: Example Compound
([DPSOH&RPSRXQG -:+ -:+ QJPO QJPO ± QJPO QJPO Quant Ion Qualifying Ion & Tolerance onses Respo For Forensic Use.
Slide 30: Synthetic Cannabinoids: GC/Q-TOF
Synthetic Cannabinoids: the Analytical Challenges *&472) *&4 72) Confidentiality Label 31 October 7, 2011 For Forensic Use.
Slide 31: TOF Fundamentals
472))XQGDPHQWDOV Key concepts for TOF: )OLJKWWXEH 1. Accurate Mass &RPPRQZLWK72) 2 2. M Mass R Resolving l i P Power ,RQ0LUURU &ROOLVLRQFHOO &ROOLVLRQ FHOO &RPPRQZLWK444 ,RQRSWLFV &RPPRQZLWK4 444 4XDG0DVV)LOWHU 4 2FWRSROH 2FWRSROH 'HWHFWRU ,RQ3XOVHU /HQVDQG &ROOLVLRQ&HOO '&4XDG 5RXJK 7XUER 7XUER 7XU
Slide 32: Mass Analysis for TOF
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 WKHIOLJKWWLPH WLPHRIIOLJKW Page 33 For Forensic Use.
Slide 33: What does Exact Mass mean?
:KDWGRHV³([DFW0DVV´PHDQ" Element Atomic Number Exact Mass + & 1 2 C6H6Cl6 287.8600665 Lindane C10H12N2O6S 288.0416000 Carbasulam C9H21O2PS3 288 0441285 288.0441285 Terbufos C13H21O3PS 288.0949000 Iprobenfos 0.2874 amu C15H17N4Cl 288.1141743 Myclobutanil C12H21N2O4P 288.1238937 Diazoxon C11H20N4O3PS
Slide 34: Why / When TOF/Q-TOF
: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.
Slide 35: GC/Q-TOF Attributes
*&472)$WWULEXWHV Resolving Power – More detection selectivity Mass Accuracy – More qualitative information about the compound – Better library search results if database has accurate mass information Higher Sensitivity compared to SQ SIM y Extended Dynamic Range g MS/MS with Accurate Mass and High R
Slide 36: Method
0HWKRG DB-1 (12m x 0.2 mm x 0.33um) 1 5 ml/min constant Flow 1.5 Pulsed, split-less injection 100 C-> 340 C @ 25 C/min EI • High resolution mode (HRM) For Forensic Use.
Slide 37: HU-210 HRM
SSE+8+50 Si O Si O H H Unit resolution reference spectrum O Mono isotopic m/z = 530.3611 Uncorrected ǻ = -2.93 ppm Resolution (RS) 17,675 For Forensic Use.
Slide 38: JWH-018 HRM
SSE-:++50 O N Unit resolution reference spectrum Mono isotopic m/z = 327.1623 Uncorrected ǻ = 0.04 ppm Resolution (RS) 15,492 For Forensic Use.
Slide 39: CP-47,497 HRM
SSE&3 &DQDORJXH +50 Si O Si O Unit resolution reference spectrum Mono isotopic m/z = 462.3349 Uncorrected ǻ = 4.4 ppm Resolution (RS) 14,512 For Forensic Use.
Slide 40: HU-210 Extraction of analyte from heavy matrix
+8([WUDFWLRQRIDQDO\WHIURPKHDY\PDWUL[ 0DVV 5HVROYLQJ 3RZHU 0DVV5HVROYLQJ3RZHU x10 8 +ESI TIC Scan Frag=170.0V DG_100 ppb_matrix_03.d 2 1.8 1.6 1.4 Matrix TIC: background ~107 1.2 1 0.8 0.6 0.4 0.2 0 x10 3 +ESI EIC(530.3627) Scan Frag=170.0V DG_100 ppb_matrix_03.d 4.5 4.25 4 3.75 3.5 3.25 HU-210 EIC ~
Slide 41: Mass Accuracy HU-210
0DVV$FFXUDF\+8 7UXH 0DVV 0HDVXUHG0DVV 7UXH0DVV 0HDVXUHG 0DVV Delta mass accuracy ~ 3ppm No IRM Confidentiality Label 42 October 7, 2011 For Forensic Use.
Slide 42: Conclusions
&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 suchh as h herbal b l bl blends d GC/QQQ provides
Slide 43: Acknowledgements
$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 TTechno