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Technology Transition Workshop
Evaluation AccuTOF DART for Postmortem
Toxicology Screening
Peter R. Stout
RTI International is a trade name of Research Triangle Institute
Technology Transition Workshop
NIJ Project
• Grant No. 2006-DN-BX-K014
• Opinions are those of the authors and not
necessarily the U.S. Department of Justice
Technology Transition Workshop
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
Snorkel
Ion Source
DART
LEAP
Autosampler
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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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Isomers
Drug
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
D9-THC/D6-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
Intensity (8400)
114.07152
121.07725
227.13577
5000
76.07708
189.14361
122.08170
61.04613
141.06898
180.07311
0
40
60
80
100
120
140
160
180
200
m/z
Urea+H
Creatinine+H
Creatinine
Dimer +H
220
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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
Intensity (4588)
304.15305
4000
114.06555
227.12712
2000
121.07086
305.16568
189.14444
0
100
200
300
400
m/z
500
600
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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
Intensity (9628)
8000
114.06251
121.06932
227.12679
150.12732
189.13617
76.06356
228.13489
0
100
200
300
400
m/z
500
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Triazolam 100 µg/mL
M + H = 343.0508
MS[1];5.542..5.551;-1.0*MS[1];5.778..5.802; / ESI+ / Drugs_Urine3
3
x10
Intensity (66467)
121.06372
50
114.05774
227.12192
343.05224
0
100
200
300
400
m/z
500
600
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LOD for Drug Analytes in Urine (µg/mL)
Drugs
Amitriptyline
Amphetamine
Benzoylecgonine
Caffeine
Cocaethylene
Cocaine
Codeine
Dextromethorphan
Diphenhydramine
Diazepam
Ecgonine Methyl Ester
Fentanyl
Fluoxetine
LOD
1.5
3.1
12.5
15
12.5
1.2
12.5
3.1
3.1
3.1
1.5
0.31
12.5
Drug
Heroin
Hydrocodone
Methadone
Methamphetamine
Morphine
Nicotine
Oxycodone
Oxazepam
PCP
Propoxyphene
D9-THC
Trazodone
Triazolam
LOD
25
3.1
3.1
6.2
3.1
1.5
3.1
(25)
0.78
1.5
3.1
50
3.1
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Creatinine Issues
MS[1];0.267..0.284;-1.0*MS[1];0.357..0.374; / ESI+ / Oxazepam_creatinine
3
x10
Intensity (26260)
287.05609
20
269.04555
289.05561
Oxazepam (10 µg/mL)
in DI water
(M+H 287.0578)
0
100
200
300
400
500
600
m/z
MS[1];0.747..0.781;-1.0*MS[1];0.843..0.872; / ESI+ / Oxazepam_creatinine
3
x10
Intensity (24930)
114.06568
227.12207
Oxazepam (10 µg/mL)
with 10 µg/mL creatinine
20
10
287.06003
115.06819
100
200
300
400
m/z
500
600
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Creatinine Issues
MS[1];0.964..0.977;-1.0*MS[1];1.060..1.071; / ESI+ / Oxazepam_creatinine
3
x10
Intensity (30740)
114.06532
227.12392
115.06796
228.12840
Oxazepam (10 µg/mL) with
100 µg/mL creatinine
20
0
100
200
300
400
500
600
m/z
MS[1];1.271..1.282;-1.0*MS[1];1.213..1.219; / ESI+ / Oxazepam_creatinine
3
x10
Intensity (32070)
114.06471
227.12042
115.06727
228.12321
Oxazepam (10 µg/mL) with
200 µg/mL creatinine
20
0
100
200
300
400
m/z
500
600
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Archived Case Example
MS[1];0.644..0.653;-1.0*MS[1];0.560..0.573; / ESI+ / Specimen3
3
x10
Intensity (35188)
121.06700
EDDP=278.190
+17mmu
278.20765
278.20765
20
310.21327
310.21327
114.06230
163.11918
264.19291
0
100
200
300
Methadone=310.216
-3mmu
400
m/z
500
600
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Evaluation of Postmortem
Blood and Tissue Samples
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“Minimal” Blood/Tissue Method
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•
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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
Blood
Liver
Kidney
Brain
Cases Analyzed
28
11
2
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
3
x10
Intensity (11364)
Cholestadiene
369.35331
10
Monosaccharide
145.05049
5
Urea
Monosaccharide
70.04981
0
127.04239
370.36761
Plasticizer
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Methadone
100 ug/mL Methadone in Blank Blood Unextracted
MS[1];0.901..0.919;-1.0*MS[1];1.034..1.046; / ESI+ / MethadoneBloodStd
3
x10
Intensity (14841)
310.21838
310.21838
145.04978
M + H =310.216
369.35369
10
61.03234 127.03923
180.09375
310.43630
311.22723
370.36976
0
100
200
300
400
500
600
700
m/z
100 ug/mL Methadone in Blank Blood Extracted in 1:3 ACN
MS[1];1.558..1.586;-1.0*MS[1];1.475..1.499; / ESI+ / MethadoneBloodStd
3
x10
Intensity (33857)
310.21614
20
311.22056
103.03683
391.28790
0
100
200
300
400
m/z
500
600
700
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Methadone
100 ug/mL Methadone in Blank Blood Extracted in 1:3
ACN/dried/reconstituted in 100 ug/mL
MS[1];2.218..2.235;-1.0*MS[1];2.127..2.144; / ESI+ / MethadoneBloodStd
3
x10
Intensity (119380)
310.21528
100
83.05517
311.21892
123.07828
391.28396
0
100
200
300
400
500
600
700
600
700
m/z
1 ug/mL Methadone in Blank Blood Extracted in 1:3
ACN/dried/reconstituted in 100 ug/mL ACN
MS[1];2.022..2.060;-1.0*MS[1];2.159..2.172; / ESI+ / MethadoneBloodStd1_0.1
3
x10
Intensity (29040)
123.07901
20
83.05484
124.08670
82.04631
369.35311
531.40165
310. 215
153.07949
81.03864
391.29149
0
100
200
300
400
m/z
500
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Postmortem Aorta Blood
Specimen
Un-extracted
MS[1];0.801..0.819;-1.0*MS[1];0.719..0.734; / ESI+ / MethadonePMSpec
3
x10
Intensity (35821)
115.08559
Nondetected
70.05903
Previously reported at
2.8 mg/mL
20
116.07321
369.37037
0
100
200
300
400
500
600
700
500
600
700
m/z
Extracted in 1:3 ACN
MS[1];1.472..1.500;-1.0*MS[1];1.365..1.382; / ESI+ / MethadonePMSpec
3
x10
Intensity (24315)
116.06926
20
90.05135
10
369.36621
166.09047
72.07556
0
100
200
300
400
m/z
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Postmortem Aorta Blood
Specimen
Extracted in 1:3 ACN/dried/reconstituted in 100 ug/mL ACN
MS[1];2.140..2.167;-1.0*MS[1];1.884..1.905; / ESI+ / MethadonePMSpec
3
x10
Intensity (13490)
83.05599
369.35466
123.06486
10
310.21961
61.03354
124.08542
283.27234
370.36700
311.22838
0
100
200
300
400
m/z
500
600
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Postmortem Liver Specimen
Un-extracted
MS[1];2.833..2.853;-1.0*MS[1];3.212..3.237; / ESI+ / MethadonePMSpec
3
x10
Intensity (79400)
Previously reported at
6.6 ug/mL
89.10302
50
132.10032
72.07485
147.11139
203.23126
70.05872
0
100
200
300
400
500
600
500
600
m/z
Extracted in 1:3 ACN
MS[1];3.516..3.554;-1.0*MS[1];3.702..3.737; / ESI+ / MethadonePMSpec
3
x10
Intensity (64335)
116.06923
132.10109
50
89.10314
166.08608
72.07511
283.27306
70.05913
0
100
200
300
400
m/z
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Postmortem Liver Specimen
Extracted in 1:3 ACN/dried/reconstituted in 100 ug/mL
MS[1];4.184..4.241;-1.0*MS[1];3.998..4.044; / ESI+ / MethadonePMSpec
3
x10
Intensity (33340)
116.06918
89.10344
20
131.11692
310.21660
72.07504
166.08548
192.14268
70.05897
305.24859
311.22253
0
100
200
300
400
m/z
500
600
700
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Cocaine
1 ug/mL Cocaine in blank blood extracted in 1:3 ACN
MS[1];1.635..1.655;-1.0*MS[1];1.742..1.760; / ESI+ / CocaineBloodStd1.0
Intensity (8087)
85.02309
5000
M+H=304.154
369.34847
103.03658
145.05039
61.03199
163.06130
304.15962
304.15982
579.50032
370.38104
607.53567
579.86476
0
1 ug/mL Cocaine in Blank Blood Extracted in 1:3
ACN/dried/reconstituted in 100 ug/mL ACN
MS[1];2.285..2.311;-1.0*MS[1];2.454..2.480; / ESI+ / CocaineBloodStd1.0
Intensity (7913)
369.33659
392.27727
83.06060
5000
304.15470
0
100
200
300
400
m/z
500
600
700
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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
Intensity (7590)
123.05165
121.06803
369.33584
392.27919
338.33018
5000
61.02662
304.15352
132.10483
0
393.29659
513.33852
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Postmortem Aorta Blood
Specimen
Extracted in 1:3 ACN
MS[1];1.470..1.496;-1.0*MS[1];1.364..1.382; / ESI+ / MethadonePMSpec
Previously reported at
0.52 mg/mL
Intensity (7110)
90.05135
132.10138
369.36621
5000
166.09047
72.07556
304.16921
0
100
200
300
400
500
600
700
m/z
Extracted in 1:3 ACN/dried/reconstituted in 100 ug/mL ACN
MS[1];2.134..2.171;-1.0*MS[1];2.044..2.057; / ESI+ / MethadonePMSpec
Intensity (2405)
283.27227
2000
310.21957
281.25948
1000
305.25414
311.22795
304.16913
304.16913
263.23950
257.26864
279.26405
338.35709
299.30686
0
250
260
270
280
290
300
m/z
310
320
330
340
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Postmortem Cocaine Case
MS[1];4.065..4.084;-1.0*MS[1];3.943..3.965; / ESI+ / PHX720
3
x10
Intensity (29414)
•All drugs not found
•Could not see without concentrating
123.08085
83.05810
20
124.08767
82.05064
61.03487
369.33488
152.07105
338.33046
370.34608
0
100
200
300
400
500
600
m/z
? BE=290.138
MS[1];1.489..1.522;-1.0*MS[1];1.358..1.415; / ESI+ / AAFSExtractionNorpropoxyphene=326.211 Propoxyphene=340.226
3
x10
Intensity (10955)
10
83.04527
SPE Extraction
146.06044
136.07916
183.08659
271.06052
0
100
200
300
400
m/z
500
600
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Postmortem case by LC/MS and SPE
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
1500000
BE
1000000
500000
5.500
0
4.5
5
5.5
MSD1 293, EIC=292.7:293.7 (C:\DATA\LCMSD_1\DATA\AAFS_RTI_SQLF\RTI_COCMETH0009.D)
6
ES-API, Pos, SIM, Frag: 125 (TT)
6.5
7
7.5
m in
6
ES-API, Pos, SIM, Frag: 125 (TT)
6.5
7
7.5
m in
6.5
7
7.5
m in
6.5
7
7.5
m in
7.5
m in
7.5
m in
5.016 - BE-d3
300000
200000
100000
BE-d3
0
4.5
5
5.5
MSD1 304, EIC=303.7:304.7 (C:\DATA\LCMSD_1\DATA\AAFS_RTI_SQLF\RTI_COCMETH0009.D)
5.974 - cocaine
40000
30000
cocaine
20000
10000
0
4.5
5
5.5
MSD1 307, EIC=306.7:307.7 (C:\DATA\LCMSD_1\DATA\AAFS_RTI_SQLF\RTI_COCMETH0009.D)
6
ES-API, Pos, SIM, Frag: 125 (TT)
5.974 - cocaine-d3
1000000
750000
500000
250000
0
cocaine-d3
4.5
5
5.5
MSD1 318, EIC=317.7:318.7 (C:\DATA\LCMSD_1\DATA\AAFS_RTI_SQLF\RTI_COCMETH0009.D)
6
ES-API, Pos, SIM, Frag: 125 (TT)
6.579 - cocaethylene
10000
cocaethylene
5000
0
4.5
5
5.5
MSD1 321, EIC=320.7:321.7 (C:\DATA\LCMSD_1\DATA\AAFS_RTI_SQLF\RTI_COCMETH0009.D)
6
ES-API, Pos, SIM, Frag: 125 (TT)
6.5
7
6.578 - cocaethylene-d3
1000000
cocaethylened3
500000
0
4.5
5
5.5
6
6.5
BE 1253 ng/mL, Cocaine 8.8 ng/mL, CE 2.7 ng/mL
7
Technology Transition Workshop
Original case reports
Case 1
Cocaine
EME
BE
Oxycodone
Propoxyphene
Norpropoxyphene
Alprazolam
mg/L
0.03
0.22
1.76
0.08
0.45
0.91
Positive
Technology Transition Workshop
Original case reports
Case 2
Methadone
EDDP
Sertraline
Desmesthylsertraline
Doxepin
Desmethyldoxipin
mg/L
1.1
Positive
0.14
0.76
0.74
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
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
LC/MS
* No quantitation attempted on the trap
** Source was saturated, resulting in poor quantitation
Technology Transition Workshop
Table of Drugs Analyzed in PM Specimens
a
a
Acetaminophen
Cyclobenzaprine
Methamp
Alprazolam
Desmethylcitalopram
Methadone
a
a
a
a
a
a a
a a
a
a
a
PCP
Promethazine
Amphetamine
Desmethyldoxepin
Methylphenidate
Propoxyphene
Atropine
Dextromethorphan
Meprobamate
Quetiapine
Metoprolol
Temazepam
a
a
a
Benzoylecgonine
Diazepam
Caffeine
Diphenhydramine
Mirtazapine
Carbamazepine
EME
Morphine
Carisoprodol
Fluoxetine
Chlorpheniramine
Fluvoxamine
Citalopram
Gabapentin
a
Nicotine
Nortriptyline
Olanzapine
Clonazepam
Hydrocodone
Cocaine
Ibuprophen
Oxycodone
Levorphanol
Oxymorphone
MDMA
Paroxetine
Codeine
Cotinine
Oxcarbazepine
THC
Topiramate
Trazadone
Technology Transition Workshop
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
Technology Transition Workshop
Conclusions
• At least minimal drug extraction appears
necessary to achieve best sensitivity
• Extensive (solid phase extraction), does not
necessarily improve things
Technology Transition Workshop
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 degradation
may have occurred
Technology Transition Workshop
Conclusions
• Strong potential for the instrument
• Issues for implementation:
– Sensitivity
– Interferences
– Software (Mass Center)
• Mass calibration
• Stability
• Retrieval of spectra
Technology Transition Workshop
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
Technology Transition Workshop
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