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RTI International is a trade name of Research Triangle Institute
Evaluation AccuTOF DART for Postmortem Toxicology Screening
Peter R. Stout
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NIJ Project• Grant No. 2006-DN-BX-K014• Opinions are those of the authors and not
necessarily the U.S. Department of Justice
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Objective• Evaluate the Jeol Ltd. AccuTOF DART system
as a novel application for use in postmortem toxicology laboratories– Qualitative
– Screening methodology
• Specifically postmortem– Urine
– Blood and tissue
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Presentation goals• Equipment set up• Discuss standards run• Urine based testing• Blood/tissue testing• Summary of experience with the instrument• Future work
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RTI set up• JEOL TOF• DART• LEAP Technologies CombiPal autosampler• Mass Center software• More recently trying the Schraeder software
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AccuTOF DART
SnorkelSnorkel LEAP LEAP AutosamplerAutosampler
Ion Source Ion Source DARTDART
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How Autosampler Works
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Sample Introduction
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AccuTOF- DART Strengths • Minimal sample preparation• Broad range of sensitivity• Rapid analysis• Simultaneous determination
of multi-drug analytes• Sufficient mass accuracy
for formula determination
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Reference materials• Examined 112 compounds
– drugs and metabolites
• Methanolic standard materials• Directly introduced
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IsomersDrug M+H
Pentobarbital/Amobarbital 227.138
Codeine/Hydrocodone 300.159
2-Oxo-3-hydroxy LSD/3-Oxo-3-hydroxy LAMPA 356.196
Morphine/Hydromorphone/Norcodeine 286.143
Benzoylecgonine/Norcocaine 290.138
PMA/Ephedrine 166.122
Cocaine/Scopolamine 304.154
Talbutal/Butalbutal 225.123
Phentermine/methamphetamine 150.120
9-THC/6-THC 315.224
Imiprimine/Despropyl Fentanyl 281.193
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Near Isomers
Near isomers M+H
Phentermine/triethanolamine 150.120/150.105
Methamphetamine/triethanolamine 150.120/150.105
Amitriptyline/venlafaxine 278.183/278.204
Tramadol/nortriptyline 264.188/264.167
Ranatidine/Clomipramine 315.141/315.154
Diphenhydramine/Tripelennamine 255.162/255.173
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Urine Method• Drug standards diluted in blank human urine• Glass probe dipped in sample• Analyzed with DART in positive mode• DART temperature @ 300°C• Mass calibrated using polyethylene glycol• Orifice 1 voltage set at 20V• Archived previously confirmed postmortem
samples analyzed
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Urine Components
MS[1];0.410..0.429;-1.0*MS[1];0.502..0.518; / ESI+ / Urea
40 60 80 100 120 140 160 180 200 220m/z
Intensity (8400)
121.07725114.07152 227.13577
76.07708189.14361
122.08170141.06898 180.07311
61.04613
Urea+HUrea+H Creatinine+HCreatinine+H CreatinineCreatinineDimer +HDimer +H
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Cocaine 100 µg/mL M + H = 304.1548
MS[1];0.922..0.951;-1.0*MS[1];0.841..0.852; / ESI+ / SpikedStds
100 200 300 400 500m/z
0
2000
4000
Intensity (4588)
304.15305
114.06555227.12712
121.07086305.16568189.14444
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Methamphetamine 100 µg/mL M+H = 150.1278
MS[1];5.383..5.416;-1.0*MS[1];5.536..5.562; / ESI+ / Drugs_Urine2
100 200 300 400m/z
0
8000
Intensity (9628)
121.06932114.06251227.12679
150.12732
189.1361776.06356
228.13489
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MS[1];5.542..5.551;-1.0*MS[1];5.778..5.802; / ESI+ / Drugs_Urine3
100 200 300 400 500m/z
0
50
Intensity (66467)x103
121.06372
114.05774 227.12192 343.05224
Triazolam 100 µg/mL M + H = 343.0508
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LOD for Drug Analytes in Urine (µg/mL)Drugs LOD Drug LOD
Amitriptyline 1.5 Heroin 25
Amphetamine 3.1 Hydrocodone 3.1
Benzoylecgonine 12.5 Methadone 3.1
Caffeine 15 Methamphetamine 6.2
Cocaethylene 12.5 Morphine 3.1
Cocaine 1.2 Nicotine 1.5
Codeine 12.5 Oxycodone 3.1
Dextromethorphan 3.1 Oxazepam (25)
Diphenhydramine 3.1 PCP 0.78
Diazepam 3.1 Propoxyphene 1.5
Ecgonine Methyl Ester 1.5 9-THC 3.1
Fentanyl 0.31 Trazodone 50
Fluoxetine 12.5 Triazolam 3.1
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MS[1];0.747..0.781;-1.0*MS[1];0.843..0.872; / ESI+ / Oxazepam_creatinine
100 200 300 400 500m/z
10
20
Intensity (24930)x103
114.06568 227.12207
287.06003115.06819
MS[1];0.267..0.284;-1.0*MS[1];0.357..0.374; / ESI+ / Oxazepam_creatinine
100 200 300 400 500m/z
0
20
Intensity (26260)x103
287.05609
269.04555289.05561
Creatinine Issues
Oxazepam (10 µg/mL) Oxazepam (10 µg/mL) in DI water in DI water
(M+H 287.0578)(M+H 287.0578)
Oxazepam (10 µg/mL) Oxazepam (10 µg/mL) with 10 µg/mL creatininewith 10 µg/mL creatinine
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MS[1];0.964..0.977;-1.0*MS[1];1.060..1.071; / ESI+ / Oxazepam_creatinine
100 200 300 400 500m/z
0
20
Intensity (30740)x103
114.06532 227.12392
228.12840115.06796
Oxazepam (10 µg/mL) with 100 µg/mL creatinine
Creatinine Issues
MS[1];1.271..1.282;-1.0*MS[1];1.213..1.219; / ESI+ / Oxazepam_creatinine
100 200 300 400 500m/z
0
20
Intensity (32070)x103
114.06471 227.12042
115.06727 228.12321
Oxazepam (10 µg/mL) with 200 µg/mL creatinine
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Archived Case Example
MS[1];0.644..0.653;-1.0*MS[1];0.560..0.573; / ESI+ / Specimen3
100 200 300 400 500 600m/z
0
20
Intensity (35188)x103
121.06700
278.20765
114.06230310.21327
264.19291163.11918
Methadone=310.216
EDDP=278.190
310.21327
278.20765
-3mmu
+17mmu
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Evaluation of Postmortem Blood and Tissue Samples
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“Minimal” Blood/Tissue Method
• 2 mL of ACN added to 1 mL of sample • Mixture was shaken and vortexed • Spun in centrifuge at 2500 rpm for 5 minutes • ~100uL of ACN saved for DART analyses • Remainder poured off and dried down at 45°C • Reconstituted in 100 uL of ACN and analyzed
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Cases used
Specimen Cases Analyzed
Blood 28
Liver 11
Kidney 2
Brain 4
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Blood method• Acetonitrile added to blood
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Blood Method• Vortexed
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Blood method• Centrifuged
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Blood method• Decanted off supernatant
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Blood method• Dry down under N2
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Blood method• Reconstitute
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SPE method
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Human Blood
MS[1];0.892..0.919;-1.0*MS[1];0.697..0.743; / ESI+ / BlankBlood
0
5
10
Intensity (11364)x103
369.35331
145.05049
370.36761127.0423970.04981
Cholestadiene
Monosaccharide
Monosaccharide
Urea
Plasticizer
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Methadone
MS[1];0.901..0.919;-1.0*MS[1];1.034..1.046; / ESI+ / MethadoneBloodStd
100 200 300 400 500 600m/z
0
10
Intensity (14841)x103
145.04978 310.21838369.35369
127.0392361.03234311.22723 370.36976180.09375
310.43630
100 ug/mL Methadone in Blank Blood Unextracted
MS[1];1.558..1.586;-1.0*MS[1];1.475..1.499; / ESI+ / MethadoneBloodStd
100 200 300 400 500 600m/z
0
20
Intensity (33857)x103
310.21614
311.22056103.03683 391.28790
100 ug/mL Methadone in Blank Blood Extracted in 1:3 ACN
310.21838 M + H =310.216
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Methadone
MS[1];2.218..2.235;-1.0*MS[1];2.127..2.144; / ESI+ / MethadoneBloodStd
100 200 300 400 500m/z
0
100
Intensity (119380)x103
310.21528
311.2189283.05517 123.07828391.28396
100 ug/mL Methadone in Blank Blood Extracted in 1:3 ACN/dried/reconstituted in 100 ug/mL
MS[1];2.022..2.060;-1.0*MS[1];2.159..2.172; / ESI+ / MethadoneBloodStd1_0.1
100 200 300 400 500m/z
0
20
Intensity (29040)x103
123.07901
83.05484
124.0867082.04631 369.35311 531.40165
81.03864 391.29149153.07949 310. 215
1 ug/mL Methadone in Blank Blood Extracted in 1:3 ACN/dried/reconstituted in 100 ug/mL ACN
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Postmortem Aorta Blood Specimen
MS[1];0.801..0.819;-1.0*MS[1];0.719..0.734; / ESI+ / MethadonePMSpec
100 200 300 400 500m/z
0
20
Intensity (35821)x103
115.08559
70.05903
116.07321369.37037
Un-extracted
MS[1];1.472..1.500;-1.0*MS[1];1.365..1.382; / ESI+ / MethadonePMSpec
100 200 300 400 500m/z
0
10
20
Intensity (24315)x103
116.06926
90.05135
369.3662172.07556 166.09047
Extracted in 1:3 ACN
Previously reported at 2.8 g/mL
Nondetected
Technology Transition WorkshopPostmortem Aorta Blood Specimen
MS[1];2.140..2.167;-1.0*MS[1];1.884..1.905; / ESI+ / MethadonePMSpec
100 200 300 400 500m/z
0
10
Intensity (13490)x103
83.05599 369.35466
123.06486
310.2196161.03354
124.08542 370.36700283.27234
311.22838
Extracted in 1:3 ACN/dried/reconstituted in 100 ug/mL ACN
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MS[1];2.833..2.853;-1.0*MS[1];3.212..3.237; / ESI+ / MethadonePMSpec
100 200 300 400 500m/z
0
50
Intensity (79400)x103
89.10302
132.1003272.07485
147.11139
70.05872203.23126
Postmortem Liver Specimen
Previously reported at 6.6 ug/mL
Un-extracted
MS[1];3.516..3.554;-1.0*MS[1];3.702..3.737; / ESI+ / MethadonePMSpec
100 200 300 400 500m/z
0
50
Intensity (64335)x103
116.06923 132.10109
166.0860889.10314
72.07511
70.05913 283.27306
Extracted in 1:3 ACN
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Postmortem Liver Specimen
MS[1];4.184..4.241;-1.0*MS[1];3.998..4.044; / ESI+ / MethadonePMSpec
100 200 300 400 500m/z
0
20
Intensity (33340)x103
116.0691889.10344
131.1169272.07504
310.21660
166.08548
305.24859 311.2225370.05897 192.14268
Extracted in 1:3 ACN/dried/reconstituted in 100 ug/mL
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Cocaine
MS[1];1.635..1.655;-1.0*MS[1];1.742..1.760; / ESI+ / CocaineBloodStd1.0
0
5000
Intensity (8087)
85.02309 369.34847
103.03658
145.05039
579.50032
370.38104 607.5356761.03199163.06130 304.15982
579.86476
1 ug/mL Cocaine in blank blood extracted in 1:3 ACN
MS[1];2.285..2.311;-1.0*MS[1];2.454..2.480; / ESI+ / CocaineBloodStd1.0
100 200 300 400 500 600m/z
0
5000
Intensity (7913)
369.33659 392.27727
83.06060
304.15470
1 ug/mL Cocaine in Blank Blood Extracted in 1:3 ACN/dried/reconstituted in 100 ug/mL ACN
M+H=304.154
304.15962
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Cocaine
0.1 ug/mL Cocaine in Blank Blood Extracted in 1:3 ACN/dried/reconstituted in 100 ug/mL ACN
MS[1];1.531..1.556;-1.0*MS[1];1.369..1.410; / ESI+ / CocaineBloodSt0.1
0
5000
Intensity (7590)
369.33584 392.27919123.05165121.06803
338.33018
61.02662
304.15352 513.33852393.29659
132.10483
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MS[1];2.134..2.171;-1.0*MS[1];2.044..2.057; / ESI+ / MethadonePMSpec
250 260 270 280 290 300 310 320m/z
0
1000
2000
Intensity (2405)
310.21957283.27227281.25948 305.25414
311.22795
304.16913263.23950257.26864 279.26405 299.30686
Postmortem Aorta Blood Specimen
MS[1];1.470..1.496;-1.0*MS[1];1.364..1.382; / ESI+ / MethadonePMSpec
100 200 300 400 500 600m/z
0
5000
Intensity (7110)
132.1013890.05135
369.36621
72.07556 166.09047
304.16921
Extracted in 1:3 ACN
Extracted in 1:3 ACN/dried/reconstituted in 100 ug/mL ACN
Previously reported at 0.52 g/mL
304.16913
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MS[1];4.065..4.084;-1.0*MS[1];3.943..3.965; / ESI+ / PHX720
100 200 300 400 500m/z
0
20
Intensity (29414)x103
123.08085
83.05810124.08767
369.3348882.05064
152.0710561.03487370.34608338.33046
? BE=290.138Propoxyphene=340.226Norpropoxyphene=326.211
•All drugs not found•Could not see without concentrating
100 200 300 400 500m/z
0
10
Intensity (10955)x103
83.04527
146.06044
136.07916271.06052183.08659
SPE Extraction
Postmortem Cocaine Case
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Postmortem case by LC/MS and SPE
min4.5 5 5.5 6 6.5 7 7.5
0
500000
1000000
1500000
MSD1 290, EIC=289.7:290.7 (C:\DATA\LCMSD_1\DATA\AAFS_RTI_SQLF\RTI_COCMETH0009.D) ES-API, Pos, SIM, Frag: 125 (TT)
5.019 - BE
5.500
min4.5 5 5.5 6 6.5 7 7.5
0
100000
200000
300000
MSD1 293, EIC=292.7:293.7 (C:\DATA\LCMSD_1\DATA\AAFS_RTI_SQLF\RTI_COCMETH0009.D) ES-API, Pos, SIM, Frag: 125 (TT)
5.016 - BE-d3
min4.5 5 5.5 6 6.5 7 7.50
10000
20000
30000
40000
MSD1 304, EIC=303.7:304.7 (C:\DATA\LCMSD_1\DATA\AAFS_RTI_SQLF\RTI_COCMETH0009.D) ES-API, Pos, SIM, Frag: 125 (TT)
5.974 - cocaine
min4.5 5 5.5 6 6.5 7 7.5
0
250000
500000
750000
1000000
MSD1 307, EIC=306.7:307.7 (C:\DATA\LCMSD_1\DATA\AAFS_RTI_SQLF\RTI_COCMETH0009.D) ES-API, Pos, SIM, Frag: 125 (TT)
5.974 - cocaine-d3
min4.5 5 5.5 6 6.5 7 7.5
0
5000
10000
MSD1 318, EIC=317.7:318.7 (C:\DATA\LCMSD_1\DATA\AAFS_RTI_SQLF\RTI_COCMETH0009.D) ES-API, Pos, SIM, Frag: 125 (TT)
6.579 - cocaethylene
min4.5 5 5.5 6 6.5 7 7.5
0
500000
1000000
MSD1 321, EIC=320.7:321.7 (C:\DATA\LCMSD_1\DATA\AAFS_RTI_SQLF\RTI_COCMETH0009.D) ES-API, Pos, SIM, Frag: 125 (TT)
6.578 - cocaethylene-d3
BE
BE-d3
cocaine
cocaine-d3
cocaethylene
cocaethylene-d3
BE 1253 ng/mL, Cocaine 8.8 ng/mL, CE 2.7 ng/mL
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Original case reports
Case 1
mg/L
Cocaine 0.03
EME 0.22
BE 1.76
Oxycodone 0.08
Propoxyphene 0.45
Norpropoxyphene 0.91
Alprazolam Positive
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Original case reports
Case 2
mg/L
Methadone 1.1
EDDP Positive
Sertraline 0.14
Desmesthylsertraline 0.76
Doxepin 0.74
Desmethyldoxipin 3.78
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Comparison to “Traditional” MS platforms
Case 1 Case 2
Platform COC BE CE Methadone
Original Quant 30ng/ml 1,760ng/ml ND 1,100 ng/ml
LC/MS 8.8 1253 2.7 1156
LC/Trap* Detected Detected Detected Detected
LC/TOF 12.5 1632 6.4 754**
LC/QTOF 26.1 1539 10.4 898
LC/QQQ 1.1 1448 0.1 1134
DART-TOF ND ND ND
* No quantitation attempted on the trap** Source was saturated, resulting in poor quantitation
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Table of Drugs Analyzed in PM Specimens
Acetaminophen
Alprazolam
Amphetamine
Atropine
Benzoylecgonine
Caffeine
Carbamazepine
Carisoprodol
Chlorpheniramine
Citalopram
Clonazepam
Cocaine
Codeine
Cotinine
Cyclobenzaprine
Desmethylcitalopram
Desmethyldoxepin
Dextromethorphan
Diazepam
Diphenhydramine
EME
Fluoxetine
Fluvoxamine
Gabapentin
Hydrocodone
Ibuprophen
Levorphanol
MDMA
Methamp
Methadone
Methylphenidate
Meprobamate
Metoprolol
Mirtazapine
Morphine
Nicotine
Nortriptyline
Olanzapine
Oxcarbazepine
Oxycodone
Oxymorphone
Paroxetine
PCP
Promethazine
Propoxyphene
Quetiapine
Temazepam
THC
Topiramate
Trazadone
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Conclusions• Reasonable sensitivity for some drugs• Greater sensitivity for some drugs over others• Creatinine appears to interfere with ionization
of some drugs• Requirement for some sample preparation to
mitigate interference from matrix• Auto sampler helps with more consistent
sample introduction
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Conclusions • At least minimal drug extraction appears
necessary to achieve best sensitivity
• Extensive (solid phase extraction), does not necessarily improve things
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Conclusions• Drug detected in spiked blood have better
sensitivity than in postmortem specimens at the same or greater concentrations
• Not ideal for detecting therapeutic drug levels– Caveat the post mortem samples used are
old and compound degradationmay have occurred
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Conclusions• Strong potential for the instrument
• Issues for implementation:– Sensitivity
– Interferences
– Software (Mass Center)• Mass calibration • Stability• Retrieval of spectra
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Conclusions• Sensitivity
– GIST/Vapur addition may improve sensitivity
• Software – Schraeder software addresses many issues
• Mass calibration • Retrieval of spectra
– Still acquire using Mass Center• Potential stability issues• Be proactive about disk management
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Acknowledgement
• Jeri Ropero-Miller• Nichole Bynum• National Institutes of Justice
– Award #2006-DN-BX-K014
• LEAP Technologies• Maricopa County, Washington State Police
and OCME-NC• SPEWare