DART Workshop

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DART Workshop Ames, IAAugust 2008
Use of the AccuTOF-DART system for the forensic
analysis of drugs of abuse Bob Steiner Virginia
Department of Forensic Science Central
Laboratory Richmond, VA 804-786-4707
x22347 robert.steiner_at_dfs.virginia.gov
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GLOSSARY OF TERMS

• AccuTOF Accurate mass Time Of Flight mass
  spectrometer
• DART Direct Analysis in Real Time
  atmospheric pressure ion source
• Orifice 1 inlet to the AccuTOF raising
  voltage causes fragmentation of ions via
  collision induced dissociation
• Profile Spectrum multiple data points to
  describe the mass peaks of a spectrum have
  chromatogram appearance
• Centroid to find the center of a mass peak
  collected in profile mode gives resulting
  histogram spectrum as a mass vs. abundance pair
• PEG600 polyethylene glycol polymer with an
  average MW of 600 Da used for internal mass
  calibration
• Internal mass calibration PEG600 spectrum run
  within a data file allows to accurately set the
  mass axis for all spectra in that file

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GLOSSARY OF TERMS

• Function switching method that allows
  changing the orifice 1 voltage rapidly during
  data collection ori 1 voltage changes every
  0.25 sec
• Protonated or deprontonated molecule the
  addition or subtraction of a hydrogen from the
  neutral molecule, depending on polarity of DART
• Millimass unit (mmu) accuracy of TOF spectra
  measured to thousandths of a Dalton acceptable
  spectra are lt 5 mmu from calculated mass
• JEOL-DX file text file of data points in a
  mass spectrum can be easily exported to other
  software for analysis

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GLOSSARY OF TERMS

• SearchFromList library searching program
  searches by empirical formula and matching
  user-library spectra
• Drugs_Neutral_Masses library of empirical
  formulae of drugs
• Drug Prep Library_ori20 library spectra of
  pharmaceuticals at orifice 1 voltage of 20 V
  also libraries at ori30, 60 and 90 V
• Drug Std Library_ori20 library spectra of
  primary standards at orifice 1 voltage of 20 V
  also libraries at ori30, 60 and 90 V
• Mirror spectrum display of unknown (positive)
  and known (negative) spectra in SearchFromList
  program

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DART METHODOLOGY

• How do I use the AccuTOF-DART for drug samples?
• Types of samples
• Best sampling methods
• Function Switching
• Calibration considerations

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HEADSPACE SAMPLING
1. Start acquisition 2. Open container near DART
gas stream 3. Remove container Cant get simpler
than that!!!!!!
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SAMPLING OF SOLIDS
Hold solid material in DART gas stream using
tweezers
Drawbacks 1. Hard to hold 2. MESSY 3.
Non-homogeneous samples
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MORE SOLID SAMPLING DRAWBACKS
Coated tablets!
GAS STREAM IS HOT!!!
4-chloro-2,5-dimethoxyamphetamine
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DRIED ON TUBE METHOD
Deposit sample onto tubes with syringe Can
measure how much sample is deposited Turned out
not so good!
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DRIED ON TUBE METHOD
Loading cap tubes with sample Even 1 uL runs
down tube Inconsistent results obtained
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Injecting sample into gas stream with syringe
Draw up samples from ALS vials Can measure amount
injected Turned out not so good!
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LIQUID SAMPLING
Dissolve sample into suitable solvent Dip
capillary tube in liquid/hold wand in DART gas
stream
Advantages 1. EASY!!! 2. Can control
concentration of sample 3. Homogeneous
sample Preferred method at DFS!!!
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DART sampling by hand is an ART!
Consistency from one person to the next is
difficult to obtain!!!
Buy an AutoDART???
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FUNCTION SWITCHING
In general, ionization of molecules with DART
produces hydride addition or subtraction products
of the molecular ion.
Methyl stearate spectrum showing MH ion at
299.2931 Da.
No diagnostic ions
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Collision-Induced Dissociation

• Definition An ion/neutral species
  interaction wherein the projectile ion is
  dissociated as a result of interaction with a
  target species. This is brought about by
  conversion of part of the translation energy of
  the ion to internal energy in the ion during
  collision.
• High vacuum prevents this in EI
• Basis behind triple stage quadrupole systems
• Commonly done in LCMS systems to promote
  fragmentation
• Performed in the AccuTOF by raising orifice 1
  voltage

http//mass-spec.lsu.edu/msterms/index.php/Collis
ion-Induced_Dissociation
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EFFECT OF RAISING ORIFICE 1 VOLTAGE
Orifice 1 15V
Orifice 1 30V
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EFFECT OF RAISING ORIFICE 1 VOLTAGE
Orifice 1 45V
Orifice 1 60V
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Function Switching allows collection of data at
SEVERAL orifice 1 voltages AT ONCE!
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Noscapine
Orifice 1 20
Function Switching results showing fragmentation
at various orifice 1 voltages
Orifice 1 30
Orifice 1 60
Orifice 1 90
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RUNNING A SAMPLE ON THE AccuTOF-DART
Lock and chk stds
More PEG
PEG
Sample
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Mass calibrations
Calibration at acquisition PEGH Set at tune
(after cleaning) Global mass calibration
Internal mass calibration temp_PEG Within your
data file Fine tunes your calibration
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Run PEG before AND/OR after samples within your
datafile! Intensity matters!!
Peggy the Dimetrodon
PEG
PEG
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GOOD PEG vs BAD PEG
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Where to average your peak for best PEG intensity?
TIC
profile
30V data
centroid
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TIC
Less intensity!
profile
centroid
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Centroiding peaks difficult with low intensity
ions
Interfering peak or noise
tailing due to kinetic energy spread in TOF
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LOCK MASS AND CHECK STANDARDS

• Mix of cocaine, methamphetamine and nefazodone
• MWs span the mass range
• Cocaine is used as drift compensation lock at
  304.1549 Da
• Methamphetamine (150.1283 Da) and Nefazodone
  (470.2323 Da) cover the low and high ends of mass
  range
• Adds another level of calibration applied to
  sample data

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LOCK MASS AND CHECK STANDARDS
Cocaine 304.1549
Meth 150.1283
Nefazodone 470.2323
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SAMPLE DATA
Sample signal is averaged, centroided and
calibrations are applied for EACH orifice voltage
Spectra are saved in JEOL-DX format
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SearchFromList Program by Dr. Robert Chip Cody
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Match spectra search
Load data file
Load empirical formula library
Adjust these to suit your search
Empirical formula search
Add or subtract from empirical formula
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SearchFromList empirical formula search result
Lot number of std used to create lib spectrum
Spectrum file name
From empirical formula library
must be lt 5 mmu
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SearchFromList empirical formula search result
Search result label
Spectrum imported from Mass Center
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Choose library to match orifice 1 voltage for the
spectrum you are searching
Match spectra search
DOUBLE CLICK on library entry to load all spectra!
Search!
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Match spectra search results
Blue original spectrum
Click on entries to view comparison spectra
Red comparison spectrum
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Suggested Data for casefiles

• Print out TIC2 from Chromatogram view
• Print out spectrum of cocaine lock mass and
  check stds
• Save spectra as averaged, background subtracted,
  centroided, calibrated JEOL-DX files
• Print out SearchFromList search result for 20V
  spectrum
• Print out Other spectra or search results at
  other voltages enough to characterize the
  spectrum

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FUNCTION SWITCHING - ADVANTAGES

• FOUR spectra collected every second
• Higher orifice 1 voltages result in more
  fragmentation
• Can lead to greater confidence for
  identification
• Except for mixtures, spectra are reproducible

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FUNCTION SWITCHING - DISADVANTAGES

• Mixture spectra no prior chromatography (can
  be good OR bad!!)
• Less sensitivity splitting ionization between
  four functions
• Finite database for searching library
  databases need to be developed!

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Application of the DART to DX

• Currently being used as a screening tool for DX
  casework.
• Validation took over one year to complete.
• Approved by DFS Scientific Advisory Board and
  full VA Forensic Science Board, May 6-8, 2008.

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AccuTOF-DART Validation at DFS

• DART screening method - LLOD determined for 7
  drugs daily calibration data to show stability
  of instrument
• DART sampling methods why we use methanol and
  MP tube wands
• Comparison of DART to GCMS new technology vs.
  established technology 553 samples run on each
  ALL match in highest Scheduled drug found DART
  typically showed more cmpds!
• Selectivity study can you tell the difference
  in DART spectra of drugs with the SAME empirical
  formula??
• Rugustness same result on different days with
  different people??

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LLOD DETERMINATION and INSTRUMENT STABILITY
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COMPARISON OF DART RESULTS WITH ANALYTICAL SCHEME
553 SAMPLES!
DART result
GCMS confirmation
1161 heroin, papaverine,
6-MAM, quetiapine
1161 heroin
1665 cocaine, diltiazem, naproxen
1665 cocaine
1910 MDMA, caffeine, procaine
1910 MDMA, caffeine, procaine, dimethylsulfone
1976 cocaine, caffeine, benzocaine
1976 cocaine, caffeine
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SELECTIVITY STUDY
Ori1 30V
119.0872
Easily distinguish methamphetamine from
phentermine!
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SELECTIVITY STUDY
Cocaine and scopolamine ori1 30V
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SELECTIVITY STUDY
Cocaine and scopolamine ori1 90V
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SELECTIVITY STUDY
LSD and LAMPA ori1 90V
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GHB SCREENING
Detection of GHB in various drink matrices via
AccuTOF-DART Bennett MJ, Steiner RR. J.
Forensic Sci., in press
Spiked 50 beverages with GHB at impairment
levels to determine if DART could detect GHB
easily seen in all drink matrices Gave better
results than using GHB color test! Done in
NEGATIVE ion mode
slated for publication Jan 2009
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GHB SCREENING
Ocean Spray Cranberry Juice - blank
blank
Ocean Spray Cranberry Juice 2 mg/mL spike with
GHB
GHB-H-
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DART Spectral Interpretation
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Molecular Weight Information
Electron Impact Ionization
DART Ionization

• MH or M-H-
• Very stable
• Very little fragmentation (without help!)
• Proper terms Protonated or deprotonated
  molecule
• Loss of stable neutrals
• Use SearchFromList to find

• M.
• Unstable leads to extensive fragmentation
• Usually the peak _at_ highest mass, excluding
  isotope peaks
• Look for logical neutral losses
• Sometimes hard to find!

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DART Spectral Characteristics
EI - DIP
verapamil MW 454.61 Da
DART
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DART Spectral Characteristics
Oxytetracycline MW 460.44 Da
Stable neutral loss of H2O
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DART Spectral Characteristics
All the usual isotope clusters!!
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DART Spectral Characteristics
Methamphetamine
H

H
- NH2CH3
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DART Spectral Characteristics
Phentermine

H
- NH3
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DART Spectral Characteristics
Heroin
H

_

_
NOT!
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DIMERS and TRIMERS and ADDUCTS!!! OH MY!!!!!!!!!
Toto, too!!
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Dimers hydrocodone/APAP (Lortab)
Ori1 20V
MH
Ori1 20V
dimer
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Acetaminophen dimer

H
H
19.1
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Dimers hydrocodone/APAP (Lortab)
Ori1 30V
Ori1 60V
Dimer disappears!
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Elemental Composition Calculation
1/3/2008 100811 AM Element Limits C 0/40 H
0/50 N 0/10 O 0/10 Tolerance 5.00
mmu Even or odd electron ion or both
BOTH Minimum unsaturation -0.5 Maximum
unsaturation 100.0 Meas. mass Abund. Diff. Unsat
. Compositions u
mmu 451.227203 0.00 3.91 12.5 C26 H31 N2 O5
C18H21NO3 Hyd or Cod C8 H9 N O2
APAP C26H30N2O5 H adduct!!
WHATS THIS?
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Salsalate (disalicylic acid) analgesic,
anti-inflammatory
MW 258.0528 Da
In MEOH, ori120V
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Salsalate ammonium adduct NH4OH on swab in DART
sampling area MW of Salsalate 258.0528 Da Emp
Form C14H10O5
MNH4H2O 258.0766 Da
MH
salicylic acidNH4H2O
Charge carrier NH4 in SFL
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Salsalate ammonium adduct
276.086517 16.63 -2.52 1.0 C1 H12 N10 O7
2.01 9.5 C10 H10 N7 O3
2.00 4.0 C11 H16 N0 O8
0.66 9.0 C12 H12 N4 O4
-0.67 14.0 C13 H8 N8 O0
-0.67 8.5 C14 H14 N1 O5
C14H10O5 NH4
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Salsalate ammonium adduct
Elemental composition
Element Limits C 0/40 H 0/50 N 0/10 O 0/10
Tolerance 5.00 mmu Even or odd electron ion
or both BOTH Minimum unsaturation -0.5 Maximum
unsaturation 100.0 Meas. mass Abund. Diff. Unsat
. Compositions u
mmu 138.053833 15.72 1.01 0.0 C4 H10 N0 O5
-0.33 5.0 C5 H6 N4 O1
-1.67 4.5 C7 H8 N1 O2 258.072021 15.47
-1.93 5.0 C11 H14 N0 O7
-3.27 10.0 C12 H10 N4 O3
-4.61 9.5 C14 H12 N1 O4
3.94 14.0 C18 H10 N0 O2 259.063446 12.03
2.81 15.0 C13 H5 N7 O0 2.80
9.5 C14 H11 N0 O5 1.45
14.5 C15 H7 N4 O1 0.13
14.0 C17 H9 N1 O2
C7H6O3 NH4 H2O
NOT M. but C14H10O5NH4H2O
C14H10O5H
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Salsalate ammonium adduct
3(C7H4O2) NH4
3(C7H4O2) H2O NH4
Salicylate trimer 3(C7H4O2) H
Salicylate dimer 2(C7H4O2) H
Salicylate C7H4O2 H
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Salsalate ammonium adduct
Elemental composition
Element Limits C 0/40 H 0/50 N 0/10 O 0/10
Tolerance 5.00 mmu Even or odd electron ion
or both BOTH Minimum unsaturation -0.5 Maximum
unsaturation 100.0 Meas. mass Abund. Diff. Unsat
. Compositions u
mmu 361.068268 7.36 -2.94 21.0 C20 H7 N7 O1
-2.94 15.5 C21 H13 N0 O6
2.92 24.5 C28 H9 N0 O1
378.095795 15.06 -0.61 15.0 C19 H14 N4 O5
-1.96 20.0 C20 H10 N8 O1
-1.99 14.5 C21 H16 N1 O6
-4.64 19.0 C24 H14 N2 O3
3.89 23.5 C28 H12 N1 O1 396.108185 7.08 -0.14
19.0 C20 H12 N8 O2 -0.14
13.5 C21 H18 N1 O7 -1.50
18.5 C22 H14 N5 O3 -2.82
18.0 C24 H16 N2 O4 -4.15
23.0 C25 H12 N6 O0
Salicylate trimer 3(C7H4O2) H
3(C7H4O2 NH4
3(C7H4O2)H2ONH4
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What is this???
Hydroxyzine (Vistaril) MH 375.1839 Da In
MEOH, ori1 20V
Chlorinated!
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Elemental composition
Elemental Composition Calculation
12/12/2007 112513 AM Element Limits C 0/40
H 0/50 N 0/10 O 0/10 Cl 1/1 Tolerance
5.00 mmu Even or odd electron ion or both
BOTH Minimum unsaturation -0.5 Maximum
unsaturation 100.0 Meas. mass Abund. Diff. Unsat
. Compositions u
mmu 537.233582 5.76 3.53 6.5 C18 H34 N10 O7
Cl1 2.19 6.0 C20 H36 N7 O8
Cl1 0.84 5.5 C22 H38 N4 O9
Cl1 -0.50 10.5 C23 H34 N8 O5
Cl1 -0.50 5.0 C24 H40 N1 O10
Cl1 -1.84 10.0 C25 H36 N5 O6
Cl1 -3.15 15.0 C26 H32 N9 O2
Cl1 -3.19 9.5 C27 H38 N2 O7
Cl1 -4.50 14.5 C28 H34 N6 O3
Cl1 4.05 19.0 C32 H32 N5 O1
Cl1 2.70 18.5 C34 H34 N2 O2
Cl1 -1.32 22.5 C39 H34 N0 O0
Cl1
NOW WHAT?????
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Hydroxyzine empirical formula
C21H27N2O2Cl
Look at composition list and see if any are
terribly unreasonable
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Elemental Composition Calculation
12/12/2007 112513 AM Element Limits C 0/40
H 0/50 N 0/10 O 0/10 Cl 1/1 Tolerance
5.00 mmu Even or odd electron ion or both
BOTH Minimum unsaturation -0.5 Maximum
unsaturation 100.0 Meas. mass Abund. Diff. Unsat
. Compositions u
mmu 537.233582 5.76 3.53 6.5 C18 H34 N10 O7
Cl1 2.19 6.0 C20 H36 N7 O8
Cl1 0.84 5.5 C22 H38 N4 O9
Cl1 -0.50 10.5 C23 H34 N8 O5
Cl1 -0.50 5.0 C24 H40 N1 O10
Cl1 -1.84 10.0 C25 H36 N5 O6
Cl1 -3.15 15.0 C26 H32 N9 O2
Cl1 -3.19 9.5 C27 H38 N2 O7
Cl1 -4.50 14.5 C28 H34 N6 O3
Cl1 4.05 19.0 C32 H32 N5 O1
Cl1 2.70 18.5 C34 H34 N2 O2
Cl1 -1.32 22.5 C39 H34 N0 O0
Cl1
Hydroxyzine empirical formula
C21H27N2O2Cl
Apply Chemistry logic!!
Not enough C!
Too many N or O
Not enough N or O or too many C
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Elemental Composition Calculation
12/12/2007 112513 AM Element Limits C 0/40
H 0/50 N 0/10 O 0/10 Cl 1/1 Tolerance
5.00 mmu Even or odd electron ion or both
BOTH Minimum unsaturation -0.5 Maximum
unsaturation 100.0 Meas. mass Abund. Diff. Unsat
. Compositions u
mmu 537.233582 5.76 -3.19
9.5 C27 H38 N2 O7 Cl1 -4.50
14.5 C28 H34 N6 O3 Cl1 4.05
19.0 C32 H32 N5 O1 Cl1
Hydroxyzine empirical formula
C21H27N2O2Cl
OK. NOW WHAT??
Too many nitrogens!
From 2003 PDR Vistaril (hydroxyzine
pamoate) Inert ingredients Magnesium
stearate Sodium lauryl sulfate Starch Sucrose
Sucrose is C12H22O11
LOOK HERE!!
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Sucrose peaks!
Sucrose ori120V
289.0942
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Hydroxyzine empirical formula C21H27N2O2Cl Targe
t compound
C27H38N2O7Cl
BEST GUESS C21H27N2O2Cl C6H12O6
C27H39N2O8Cl (not quite!) But, if subtract
H2O C27H37N2O7Cl (Still off by one!) Ionized
to C27H37N2O7Cl H 537.2367 Da
(calculated)
Hydrolyzed sucrose
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MIXTURES
Magically disappearing ions!
Codeine std ori 1 30V
CodeineAPAP 201
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MIXTURES
CodeineAPAP 101
CodeineAPAP 51
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MIXTURES
CodeineAPAP 21
CodeineAPAP 11
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Today is done!