Technology Transition Workshop DART Workshop – Ames, IA August 2008 Use of the AccuTOF-DART system...

Technology Transition Workshop

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

[email protected]


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


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 < 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


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

GLOSSARY OF TERMS:


DART METHODOLOGY

How do I use the AccuTOF-DART for drug samples?

Types of samples

Best sampling methods

Function Switching

Calibration considerations


HEADSPACE SAMPLING

1. Start acquisition

2. Open container near DART gas stream

3. Remove container

Can’t get simpler than that!!!!!!


SAMPLING OF SOLIDS

Drawbacks:

1. Hard to hold

2. MESSY

3. Non-homogeneous samples

Hold solid material in DART gas stream using tweezers


MORE SOLID SAMPLING DRAWBACKS

Coated tablets!

GAS STREAM IS HOT!!!

4-chloro-2,5-dimethoxyamphetamine


“DRIED ON TUBE” METHOD

Deposit sample onto tubes with syringe

Can measure how much sample is deposited

Turned out not so good!


“DRIED ON TUBE” METHOD

Loading cap tubes with sample

Even 1 uL “runs down” tube

Inconsistent results obtained


Injecting sample into gas stream with syringe

Draw up samples from ALS vials

Can measure amount injected

Turned out not so good!


LIQUID SAMPLINGDissolve 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!!!


DART sampling by hand is an

ART!

Consistency from one person to the next is difficult to obtain!!!

Buy an AutoDART???


In general, ionization of molecules with DART produces hydride addition or subtraction products

of the molecular ion.

Methyl stearate spectrum showing [M+H]+ ion at 299.2931 Da.

No ‘diagnostic’ ions

FUNCTION SWITCHING


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/Collision-Induced_Dissociation


Orifice 1 = 15V

EFFECT OF RAISING ORIFICE 1 VOLTAGE

Orifice 1 = 30V


Orifice 1 = 45V

EFFECT OF RAISING ORIFICE 1 VOLTAGE

Orifice 1 = 60V


Function Switching allows collection of data at SEVERAL orifice 1 voltages AT ONCE!


Function Switching results showing fragmentation at various orifice 1 voltages

Orifice 1 = 20

Orifice 1 = 30

Orifice 1 = 60

Orifice 1 = 90

Noscapine


RUNNING A SAMPLE ON THE AccuTOF-DART

TIC[2]; / ESI+ / cal7908

0 0.5 1.0 1.5 2.0 2.5Time[min]

0

500

Intensity (715231)x103

1.1571.007 2.5540.674 2.4382.2210.258

1.8390.425 1.656 1.972

PEG

Lock and chk stds

Sample

More PEG

Technology Transition WorkshopMass calibrations

Calibration at acquisition – PEG+H

Set at tune (after cleaning)

“Global” mass calibration

Internal mass calibration – temp_PEG

Within your data file

“Fine tunes” your calibration


TIC[2]; / ESI+ / jhp_310a_311a

0 1.0 2.0 3.0

Time[min]

0

500


3.085 3.3172.486

0.7050.971

0.3891.920

1.3871.637

PEG

PEG

“Run PEG before AND/OR after samples within your datafile! Intensity matters!!”

Peggy the Dimetrodon

Technology Transition WorkshopGOOD PEG vs BAD PEG

Technology Transition WorkshopTIC[2]; / ESI+ / cal1308

0.30 0.40 0.50 0.60 0.70

Time[min]

0

500


0.474

0.6740.291 0.5580.524

MS[2];0.442..0.493;-1.0*MS[2];0.361..0.415; / ESI+ / cal1308

100 200 300 400 500 600m/z

0

5000

Intensity (6120)

459.27946371.22813

503.30537327.19959

239.14916 283.17463 547.33420195.12221133.0862989.05961

MS[2];0.442..0.493;-1.0*MS[2];0.361..0.415; / ESI+ / cal1308

100 200 300 400 500 600m/z

0

10

20

Area (23621)x103

415.25460 459.27969

503.30555327.19988

283.17499 547.33415239.14967195.12257

87.04568 133.08689

profile

centroid

Where to average your peak for best PEG intensity?

TIC

30V data

Technology Transition WorkshopTIC[2]; / ESI+ / cal1308

0.30 0.40 0.50 0.60 0.70

Time[min]

0

500


0.474

0.6740.291 0.5580.524

MS[2];0.429..0.571;-1.0*MS[2];0.356..0.412; / ESI+ / cal1308

100 200 300 400 500 600m/z

0

2000

Intensity (3373)

415.25622 503.30599

547.33324327.20019

239.14952 283.17523195.12242

133.0864789.05965

MS[2];0.429..0.571;-1.0*MS[2];0.356..0.412; / ESI+ / cal1308

100 200 300 400 500 600m/z

0

10

Area (14357)x103

415.25632 503.30621

547.33319327.20052

283.17553239.15015195.12281

87.04657 133.08721

profile

centroid

TICLess intensity!


MS[2];3.532..3.699;-1.0*MS[2];3.367..3.512; / ESI+ / jhp_272a_274a

133.00 133.10 133.20m/z

0

500

Intensity (659)

133.09083

Centroiding peaks = difficult with low intensity ions

Interfering peak or noise

MS[2];3.532..3.699;-1.0*MS[2];3.367..3.512; / ESI+ / jhp_272a_274a

415.20 415.40 415.60

m/z

0

2000

Intensity (3161)

415.25405“tailing” due to kinetic energy spread in TOF


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


LOCK MASS AND CHECK STANDARDS

0

20

40

60

80

100

Rel

. Ab

un

dan

ce

120 160 200 240 280 320 360 400 440 480

m/z

150.1296

304.1549

470.2362

Meth 150.1283

Cocaine 304.1549

Nefazodone 470.2323


MS[2];2.225..2.317;-1.0*MS[2];2.042..2.192; / ESI+ / af070701

100 150 200 250 300 350 400m/z

0

10

Area (15434)x103

324.20773

192.08031

325.21241

192.24602192.17013 324.48445 340.22848223.0989287.10397

TIC[2]; / ESI+ / af070701

2.0 2.5 3.0Time[min]

0

200


2.2702.919

2.5372.853

Sample signal is averaged, centroided and calibrations are applied for EACH orifice voltage

Spectra are saved in “JEOL-DX” format

SAMPLE DATA

Technology Transition WorkshopSearchFromList Program – by Dr. Robert “Chip” Cody


Load data file

Load empirical formula library

Adjust these to suit your search

Match spectra search

Empirical formula search

Add or subtract from empirical formula


From empirical formula library must be < 5 mmu

Lot number of std used to create lib spectrum

Spectrum file name

SearchFromList empirical formula search result


SearchFromList empirical formula search result

Spectrum imported from Mass Center

Search result label


Match spectra search

DOUBLE CLICK on library entry to load all spectra!

Choose library to match orifice 1 voltage for the spectrum you are searching

Search!


Match spectra search results

Click on entries to view comparison spectra

Blue = original spectrum

Red = comparison spectrum


Suggested Data for casefiles

• Print out: TIC[2] 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


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


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!


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.


AccuTOF-DART Validation at DFS

1. DART screening method - LLOD determined for 7 drugs; daily calibration data to show stability of instrument

2. DART sampling methods – why we use methanol and MP tube ‘wands’

3. 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!

4. Selectivity study – can you tell the difference in DART spectra of drugs with the SAME empirical formula??

5. “Rugustness” – same result on different days with different people??


Conc. Drug Name Exact Mass measured mass difference

0.5 mg/mL Alprazolam 309.0907 309.0906 0.0001

Butalbital 225.1239 225.1240 0.0001

7/13/2007 Cocaine 304.1549 304.1526 0.0023

Heroin 370.1654 370.1636 0.0018

Methamphetamine 150.1283 150.1268 0.0033

Testosterone Propionate 345.2430 345.2409 0.0021

Trazodone 372.1591 372.1553 0.0038

0.1 mg/mL Alprazolam 309.0907 309.0888 0.0019

Butalbital 225.1239 225.1246 0.0007

Cocaine 304.1549 304.1545 0.0004

7/13/2007 Heroin 370.1654 370.1670 0.0016

Methamphetamine 150.1283 150.1295 0.0012

Testosterone Propionate 345.2430 345.2427 0.0003

Trazodone 372.1591 372.1626 0.0035

LLOD DETERMINATION

and INSTRUMENT STABILITY


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


SELECTIVITY STUDY

119.0872

Easily distinguish methamphetamine from phentermine!

Ori1 30V


SELECTIVITY STUDY

Cocaine and scopolamine ori1 30V


SELECTIVITY STUDY

Cocaine and scopolamine ori1 90V

Technology Transition WorkshopSELECTIVITY STUDY

LSD and LAMPA ori1 90V


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


GHB SCREENING

Ocean Spray Cranberry Juice - blank

Ocean Spray Cranberry Juice – 2 mg/mL spike with GHB

blank

[GHB-H]-


DART Spectral Interpretation


Molecular Weight Information

• M+.

•Unstable – leads to extensive fragmentation

•Usually the peak @ highest mass, excluding isotope peaks

•Look for “logical neutral losses”

• Sometimes hard to find!

Electron Impact

Ionization

DART Ionization

[M+H]+ or [M-H]-

Very stable

Very little fragmentation (without help!)

Proper terms: Protonated or deprotonated molecule

Loss of stable neutrals

Use SearchFromList to find


40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 4400

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

7000

7500

8000

8500

9000

9500

m/z-->

Abundance

#372: VERAPAMIL--DIP303

15158

260

84 107 177130 218 453

EI - DIP

DART

verapamil – MW 454.61 Da

DART Spectral Characteristics


Oxytetracycline

MW 460.44 Da

Stable neutral loss of H2O



All the usual isotope clusters!!



CH2

CH

CH3

N

CH3

H

H

+

- NH2CH3

Methamphetamine



CH2

C

CH3

CH3

NH2

H

+

- NH3

Phentermine



O

CH CH2

CH2

CH2

N

CH3

OO

CC

CH3 CH3

OO

H+

Heroin

OH C

CH3

O

_

OH C

CH3

O

CH2 C O+_

NOT!



DIMERS and

TRIMERS and

ADDUCTS!!!

OH MY!!!!!!!!!

Toto, too!!

Technology Transition WorkshopDimers – hydrocodone/APAP (Lortab)

Ori1 = 20V

Ori1 = 20V

dimer

[M+H]+


NH

C

CH 3

O

O

NH

C

CH3

O

OH

H

+

H

Acetaminophen dimer

19.1%


Ori1 = 30V

Dimers – hydrocodone/APAP (Lortab)

Ori1 = 60V

Dimer disappears!

Technology Transition WorkshopElemental Composition Calculation

1/3/2008 10:08:11 AM

Element Limits:C 0/40 H 0/50 N 0/10 O 0/10 Tolerance: 5.00 mmuEven or odd electron ion or both: BOTHMinimum unsaturation: -0.5Maximum unsaturation: 100.0

Meas. mass Abund. Diff. Unsat. Compositions u % mmu451.227203 0.00 3.91 12.5 C26 H31 N2 O5

WHAT’S THIS?

C18H21NO3 Hyd or Cod

+ C8 H9 N O2 APAP

[C26H30N2O5 + H] adduct!!


Salsalate (disalicylic acid) – analgesic, anti-inflammatory

In MEOH, ori1=20V

OH

C

O

O

C

OHO

121

137

MW = 258.0528 Da


Salsalate – ammonium adduct

NH4OH on swab in DART sampling area

MW of Salsalate = 258.0528 Da

Emp Form: C14H10O5

Charge carrier +NH4 in SFL

[M+NH4–H2O]+ = 258.0766 Da [M+H]+

[salicylic acid+NH4–H2O]+


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]+




Meas. mass Abund. Diff. Unsat. Compositions u % mmu138.053833 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

[C14H10O5+H]+

NOT M+. but

[C14H10O5+NH4–H2O]+

[C7H6O3 + NH4 – H2O]+

Elemental composition



“Salicylate” trimer [3(C7H4O2) + H]+

[3(C7H4O2)+ NH4]+

[3(C7H4O2) + H2O + NH4]+

“Salicylate” dimer [2(C7H4O2) + H]+

“Salicylate” [C7H4O2 + H]+

OH

C

O

O

C

OHO




Meas. mass Abund. Diff. Unsat. Compositions u % mmu361.068268 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)+H2O+NH4]+



What is this???CH

N

N

Cl CH2

CH2

O

CH2

CH2

OH

Hydroxyzine (Vistaril)

[M+H]+: 375.1839 Da

In MEOH, ori1 20V

Chlorinated!


12/12/2007 11:25:13 AM

Element Limits:C 0/40 H 0/50 N 0/10 O 0/10 Cl 1/1 Tolerance: 5.00 mmuEven or odd electron ion or both: BOTHMinimum unsaturation: -0.5Maximum unsaturation: 100.0

Meas. mass Abund. Diff. Unsat. Compositions u % mmu537.233582 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?????



CH

N

N

Cl CH2

CH2

O

CH2

CH2

OH

Hydroxyzine empirical formula

C21H27N2O2Cl

Look at composition list and see if any are “terribly unreasonable”

Technology Transition WorkshopElemental Composition Calculation 12/12/2007 11:25:13 AM


Meas. mass Abund. Diff. Unsat. Compositions u % mmu537.233582 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

Not enough C!

Not enough N or O or too many C

Too many N or O

Apply Chemistry logic!!

Hydroxyzine empirical formula C21H27N2O2Cl


12/12/2007 11:25:13 AM


Meas. mass Abund. Diff. Unsat. Compositions u % mmu537.233582 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

Too many nitrogens!

OK. NOW WHAT??

From 2003 PDR:

Vistaril (hydroxyzine pamoate)

Inert ingredients:

Magnesium stearate

Sodium lauryl sulfate

Starch

Sucrose

LOOK HERE!!

Sucrose is C12H22O11

Hydroxyzine empirical formula C21H27N2O2Cl


Sucrose – ori1=20V

289.0942

Sucrose peaks!


BEST GUESS:

C21H27N2O2Cl + C6H12O6 = C27H39N2O8Cl (not quite!)

But, if subtract H2O:

C27H37N2O7Cl (Still off by one!)

Ionized to:

[C27H37N2O7Cl + H]+ = 537.2367 Da (calculated)

Hydroxyzine empirical formula:C21H27N2O2Cl

Target compound: C27H38N2O7Cl

Hydrolyzed sucrose

Technology Transition WorkshopMIXTURES

Codeine std ori 1 30V

Codeine:APAP 20:1

Magically disappearing ions!


Codeine:APAP 10:1

Codeine:APAP 5:1

MIXTURES


Codeine:APAP 2:1

Codeine:APAP 1:1

MIXTURES


“Today is

done!”

Technology Transition Workshop DART Workshop – Ames, IA August 2008 Use of the AccuTOF-DART system...

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Transcript of Technology Transition Workshop DART Workshop – Ames, IA August 2008 Use of the AccuTOF-DART system...