technology transition workshop rti international is a trade name of research triangle institute...

Technology Transition Workshop

RTI International is a trade name of Research Triangle Institute

Evaluation AccuTOF DART for Postmortem Toxicology Screening

Peter R. Stout


NIJ Project• Grant No. 2006-DN-BX-K014• Opinions are those of the authors and not

necessarily the U.S. Department of Justice


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


Presentation goals• Equipment set up• Discuss standards run• Urine based testing• Blood/tissue testing• Summary of experience with the instrument• Future work


RTI set up• JEOL TOF• DART• LEAP Technologies CombiPal autosampler• Mass Center software• More recently trying the Schraeder software


AccuTOF DART

SnorkelSnorkel LEAP LEAP AutosamplerAutosampler

Ion Source Ion Source DARTDART


How Autosampler Works


Sample Introduction


AccuTOF- DART Strengths • Minimal sample preparation• Broad range of sensitivity• Rapid analysis• Simultaneous determination

of multi-drug analytes• Sufficient mass accuracy

for formula determination


Reference materials• Examined 112 compounds

– drugs and metabolites

• Methanolic standard materials• Directly introduced


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


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


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


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


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


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


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


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


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


114.06568 227.12207

287.06003115.06819


100 200 300 400 500m/z

0

20


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



100 200 300 400 500m/z

0

20


114.06532 227.12392

228.12840115.06796

Oxazepam (10 µg/mL) with 100 µg/mL creatinine

Creatinine Issues


100 200 300 400 500m/z

0

20


114.06471 227.12042

115.06727 228.12321

Oxazepam (10 µg/mL) with 200 µg/mL creatinine


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


121.06700

278.20765

114.06230310.21327

264.19291163.11918

Methadone=310.216

EDDP=278.190

310.21327

278.20765

-3mmu

+17mmu


Evaluation of Postmortem Blood and Tissue Samples


“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


Cases used

Specimen Cases Analyzed

Blood 28

Liver 11

Kidney 2

Brain 4


Blood method• Acetonitrile added to blood


Blood Method• Vortexed


Blood method• Centrifuged


Blood method• Decanted off supernatant


Blood method• Dry down under N2


Blood method• Reconstitute


SPE method


Human Blood

MS[1];0.892..0.919;-1.0*MS[1];0.697..0.743; / ESI+ / BlankBlood

0

5

10


369.35331

145.05049

370.36761127.0423970.04981

Cholestadiene

Monosaccharide

Monosaccharide

Urea

Plasticizer


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


145.04978 310.21838369.35369

127.0392361.03234311.22723 370.36976180.09375

310.43630

100 ug/mL Methadone in Blank Blood Unextracted


100 200 300 400 500 600m/z

0

20


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


Methadone


100 200 300 400 500m/z

0

100


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


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


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


115.08559

70.05903

116.07321369.37037

Un-extracted


100 200 300 400 500m/z

0

10

20


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


100 200 300 400 500m/z

0

10


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



100 200 300 400 500m/z

0

50


89.10302

132.1003272.07485

147.11139

70.05872203.23126

Postmortem Liver Specimen

Previously reported at 6.6 ug/mL

Un-extracted


100 200 300 400 500m/z

0

50


116.06923 132.10109

166.0860889.10314

72.07511

70.05913 283.27306



Postmortem Liver Specimen


100 200 300 400 500m/z

0

20


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


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


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



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


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/dried/reconstituted in 100 ug/mL ACN

Previously reported at 0.52 g/mL

304.16913


MS[1];4.065..4.084;-1.0*MS[1];3.943..3.965; / ESI+ / PHX720

100 200 300 400 500m/z

0

20


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


83.04527

146.06044

136.07916271.06052183.08659

SPE Extraction

Postmortem Cocaine Case


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


5.016 - BE-d3

min4.5 5 5.5 6 6.5 7 7.50

10000

20000

30000

40000


5.974 - cocaine

min4.5 5 5.5 6 6.5 7 7.5

0

250000

500000

750000

1000000


5.974 - cocaine-d3

min4.5 5 5.5 6 6.5 7 7.5

0

5000

10000


6.579 - cocaethylene

min4.5 5 5.5 6 6.5 7 7.5

0

500000

1000000


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


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


Original case reports

Case 2

mg/L

Methadone 1.1

EDDP Positive

Sertraline 0.14

Desmesthylsertraline 0.76

Doxepin 0.74

Desmethyldoxipin 3.78


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


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


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


Conclusions • At least minimal drug extraction appears

necessary to achieve best sensitivity

• Extensive (solid phase extraction), does not necessarily improve things


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


Conclusions• Strong potential for the instrument

• Issues for implementation:– Sensitivity

– Interferences

– Software (Mass Center)• Mass calibration • Stability• Retrieval of spectra


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


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

technology transition workshop rti international is a trade name of research triangle institute...

Documents

stout slide

tissue slide

formula determination

schraeder software slide

department of justice

instrument future work

accutof dart system

postmortem urine blood