Improved Speed and Confidence in Toxicology …...- Scan = best confirmation of targets and...

Bruce QuimbyApplication ScientistApplication ScientistAgilent TechnologiesOctober 28, 2009

For Forensic Use.

Tools For Improving GC/MS Analysis

• Retention Time Locking (RTL)

• Method Translation• Method Translation

• Synchronous SIM/Scan

• Column effluent split to an NPD

• Column backflushing

• Deconvolution Reporting Software (DRS)

For Forensic Use.

Retention Time LockingGet precisely the same retention times:• After cutting or replacing column• On all instruments in a lab and other labs (method sharing)

Get precisely the same retention times:

( g)• Greatly reduces method maintenance for updating:

– peak recognition (RT) windows– SIM group start times– integration events

• Reduces data review timeReduces data review time• Makes management of large screening databases much easier• Simple procedure restores all RTs to original calibrated values• Process moves RTs back to calibrated values, does not change calibrated

value to current (different) RT• Look for new targets in archived data, since RTs always the same

g , y

For Forensic Use.

How Does RTL Work?

For each method:• A set of five runs of retention time vs inlet pressure for a• A set of five runs of retention time vs inlet pressure for a

single locking compound is collected (only once per method)• The calibration is stored with the method• When locking a new instrument, the locking compound is run and its RT is

measured• RTL software calculates new inlet pressure that makes the RT of the

locking compound and all analytes precisely match that of the originalmethod

• U

•

pdating RTs of individual cal compounds and timed events unnecessaryp g p y

If a table of hundreds of RTs is collected under RTL conditions, anyoneanywhere can lock to the same conditions and get the same RTs. This isbasis of Agilent screening databases for Forensic Tox compounds, pesticides, etc.

For Forensic Use.

RTL Example

RTL keeps the retention times of all analytes typically within 0.030 min absolute. Locking is a simple procedure using 1 compound. After pressure adjustment, all compounds fall within their RT recognition window.

Initial run 4.72 psi4.296 min.

Original locked method

Column trimmed lockingTrim 1 meter 4.72 psi4.064 min. Column trimmed, locking

compound run at original pressure (RT is too short)

Relock 4.42 psi4.297 min.

RTL calculates new inlet pressure to restore RTs

For Forensic Use.

Agilent’s Method Translation SoftwareA technique invented by Agilent that calculates the inletA technique invented by Agilent that calculates the inletpressure and oven temperature program for changing:

– Analysis speed (example: 4 x faster)Analysis speed (example: 4 x faster)– Column dimensions (with same phase ratio)– Carrier gas type (example: Changing from He to H2)– Column outlet pressure (MSD vs NPD vs CFT Splitter)

Determines the required conditions with a calculator instead of trial and error GC runs in the laboratorytrial and error GC runs in the laboratory

Key benefits:Relative elution order is maintained• Relative elution order is maintained.

• Retention times with new method are accurately predicted• Substantial saving in method development time, especially for screening

methods with large numbers of analytes.

For Forensic Use.

Speed Scaling Constant Pressure RTL MethodsAllows method to be adjusted to optimal speed on a

Use MTL software to calculate changes in pressure and

Allows method to be adjusted to optimal speed on aspecific system.

Use MTL software to calculate changes in pressure andtemperature ramps corresponding to desired speed gain

Generate new RTL calibration for the new methodGenerate new RTL calibration for the new method

Peaks will elute in same relative order as before

L k t i t t d tLock system using target compound to:(original target time) (speed gain)

N ll t ti ti i th d ill b l tNow all retention times in new method will be equal to:original RT speed gain

For Forensic Use.

1-5 ng Test Mix: DB-5MS, 1x MSD Direct10C/min and 1.8 mL/min initial flow1 Amphetamine2 Phentermine3 Methamphetamine4 Nicotine Tetrahydrocannabinol

15

18

Lorazepam16 Diazepam17 Hydrocodone

10C/min and 1.8 mL/min initial flow

4 Nicotine5 Methylenedioxyamphetamine(MDA)6 Methylenedioxymethamphetamine(MDMA)7 Methylenedioxyethylamphetamine8 Meperidine9 Phencyclidine

Tetrahydrocannabinol1819 Oxycodone20 Temazepam

22 Diacetylmorphine23 Nitrazepam17

21 Flunitrazepam

y10 Methadone11 Cocaine12 SKF-525a (RTL Compound)13 Oxazepam14 Codeine

p24 Clonazepam25 Alprazolam26 Lysergide (LSD)27 Strychnine28 Trazodone

9

1012

17

1816

23

4

6

7

811

1415

1921

22

25 27

1

5

6

13

20 23 24 2628

TIC: 25mix_340C_2.D\data.ms

4 8 12 16 20 24 28

For Forensic Use.

DB-5MS: Comparison of Different Speeds on MSD4X method RTs are precisely the same as 1X divided by 4

1 x30 m, 10C/min and 1.7 mL/min: 120V oven and Standard Turbo

2 x15 m, 20C/min and 1.2 mL/min: 120V oven and Standard Turbo

4 8 12 16 20 24 28

2 x

15 m, 30C/min and 2.7 mL/min: 240V oven and Perf Turbo2 4 6 8 10 12 14

3 x 15 m, 30C/min and 2.7 mL/min: 240V oven and Perf Turbo

1 2 3 4 5 6 7 8 9

4 x15 m, 40C/min and 5.9 mL/min: 240V oven and Perf Turbo

1 2 3 4 5 6 7

1 2 3 4 5 6 7

For Forensic Use.

Why Use Agilent’s Synchronous SIM/Scan?

Get SIM and Scan Data in a single run which saves time

SIM maximum sensitivity for target compounds- SIM = maximum sensitivity for target compounds

- Scan = best confirmation of targets and identification of unknownsunknowns

- For most methods, changing to SIM/Scan results in little, if any, degadation in signal-to-noise compared to SIMany, degadation in signal to noise compared to SIMonly or Scan only modes

For Forensic Use.

How Does Synchronous SIM/Scan Work?

SIM data pointsChromatographic Peak SIM and Scan data

points are alternatelyScan data pointsSIM data points points are alternately

collected

At end of run, two separate data signals are constructed

Each signal can be d lik SIM

Scan timeSIM

Scan

SIM

Scan

SIM

Scan

SIM

Scan

SIM

Scan

processed like SIM or Scan collected separately

For Forensic Use.

Why Collect NPD and MSD Simultaneously?

NPD gives sensitive nitrogen selective detection:

Identity confirmation (if it is a nitrogen drug it should- Identity confirmation (if it is a nitrogen drug, it shouldrespond on NPD)

- Faster data review alternate to MSD for quantitation- Faster data review, alternate to MSD for quantitation

- Highlights nitrogen drugs that are not in target list

S litt d l ll b kfl hi d h i l- Splitter used also allows backflushing and changing columnswithout venting MSD

For Forensic Use.

Example of Synchronous SIM/Scan/NPDFentan l

Ion 245 Scan S/Npk-pk = 8

Fentanyl Real whole blood tox sample:

Screen for 278 drugs in Scan mode

Ion 146 Scan

Ion 189 Scan

mode.

Screen for low level targets in SIM mode

S/Npk-pk = 77Ion 245 SIMFentanyl found by SIM at trace level because of 10 fold better S/N

Ion 146 SIM

Ion 189 SIM

NPD trace shows nitrogen peak at same RT as fentanyl in MS dataMS data

NPDS/Npk-pk 42

5.52 5.60 5.68 5.76

For Forensic Use.

How Does the Splitter Work?Liquidq

Injector

XEPC NPDVent

New splitter technology

7890A GC

Column 5975CMSD

7890A GC

Column effluent split between MSD and NPD

Reliable, inert, easy to use two way splitter with solvent venting

Auxillary EPC control allows no vent column changing and post run backflushing

backflushing

For Forensic Use.

Why Use Agilent’s Column Backflushing?

Backflushing removes heavy material from column after elution time of last analyte:y

- Prevents heavy matrix compounds from eluting in later runs

- Reduces column trimming and detector maintenance- Reduces column trimming and detector maintenance

- Significantly reduces postrun bakeout times (example: 2 min backflush vs 10 min bakeout)backflush vs 10 min bakeout)

Heavies past end of Whole blood tox sampleacquisition

TIC

p

1 2 3 4 5 6 7 8

For Forensic Use.

How Does Backflushing Work?

Auxillary EPC provides constant pressure makeup

t t l d i

During GC Run

A EPC

SplitVent

gas to post column device.During run, makeup flow is low (~ 1 mL/min)S/S Inlet

Splitter or

4 psi

MSD

Aux EPCTrap

Flow

Splitter orQuickSwapColumn

25 psi

After GC Run A EPC i d

45 psiAux EPC

SplitVent Trap

After GC Run Aux EPC is programmedto high pressure and inlet to low pressure. Reversed flow quickly sweeps

S/S Inlet

Column1 psi

45 psi

MSDFlow

Splitter or QuickSwap

flow quickly sweepsheavies out to split vent trap

Column p

For Forensic Use.

Why Use Deconvolution Reporting Software?

Deconvolution of mass spectra removes/reduces interferences from chromatographically overlapped peaks:g p y pp p• Better identification and confirmation of analytes in high matrix samples • Reduces data review time, especially for large screening methods• Reduces false positives and false negatives in dirty samples• Identification based on matching entire spectrum cleaned of interferences

against library. Much more reliable than target/qualifier ratio method.against library. Much more reliable than target/qualifier ratio method.

For Forensic Use.

How Does Deconvolution Work?Ions with the same abundance vs time profile are groupedIons with the same abundance vs time profile are groupedtogether to create spectra “cleaned” of interferences from overlapped peaks

Three Overlapped Chromatographic Peaks

DeconvolutionInterference 1

TIC

Interference 2

m/zInterference 1Interference 2 Time

Time

Interference 2Target

Time Time

Target

m/z

For Forensic Use.

AMDIS Looks at Apex Retention Time and Peak Shape

28p

50170

280

3118516

075160

Extracted Ion

160 – wrong shape

Chromatograms(EIC)

Ions whose EICs have the:

50170

g p1. same apex RT

2. same shapeThese have same shape and same apex RT280

75 – wrong rt185 – wrong rtare grouped to form

cleaned (extracted) spectra

apex RT

g310 – wrong rt

spect a

For Forensic Use.

Ions With Same RT and Shape Are Grouped and Called Components (Spectra)p ( p )

50170

280

This spectrum isExtractedspectrum This spectrum is

compared to msl for target hits.

spectrumcleaned of interferences

ComponentExtracted Ion

ChromatogramsPeak heights of EICs give

l ti

50170

Chromatograms(EIC)relative

abundancevalues for

t 170280

spectra

For Forensic Use.

Example:Spectral Confirmation of Carisoprodol in Blood Extract With High Level of Fatty AcidsBlood Extract With High Level of Fatty Acids

Apex spectrum 180000

200000

Abundance

Average of 3.564 to 3.601 min.: 120C_1MIN_GB20_VIAL2.D\DATA.MS54.9

#1 NIST Hit is Oleic acid.

Carisoprodol is 80000

100000

120000

140000

160000

96.9 Spectrum at Carisoprodol RT, not deconvoluted

Carisoprodol isnot in top 100 hits

40 60 80 100 120 140 160 180 200 220 240 260 280 300 3200

20000

40000

60000

m/z-->

158.0128.9

185.0

284.1213.0 241.1

75.8 261.9 340.0311.9

58

DeconvolutedSpectrum

0

50

100

44

58

71 86

97104

114132

149

158

184205 223

245257 288 327

Deconvoluted Spectrum at Carisoprodol RT Match = 80

Carisoprodol is #1 Hit in NIST05a

0

50

100

43

55

83

97

114127 144

158

184202 217

245260

Library Reference Spectrum of Carisoprodol

40 70 100 130 160 190 220 250 280 310 340

55

For Forensic Use.

Steps in DRS Process

Software goes through entire GC/MS scan file and extracts all components (spectra).p ( p )

Each component is automatically searched against the target spectral library.

If a component has both:• a spectral match factor > than user defined minimump• a retention time matching the table RT within a user defined limit• It is reported as present.

Data reviewer inspects the results and confirms compounds present. Those compounds are then quanted and report generated.

g

For Forensic Use.

Example: Design 725 Compound Fast Tox Screening SystemScreening SystemRetention Time Locking (RTL)-

use Agilent’s New G1674AA Forensic Toxicology RTL Library– use Agilent s New G1674AA Forensic Toxicology RTL Library

Method Translation– Run 4x faster version (40C/min) of method– Run 4x faster version (40C/min) of method

Synchronous SIM/Scan and Split to NPD– Collect 3 signals Scan SIM and NPD in one short runCollect 3 signals Scan,SIM, and NPD in one short run

Column backflushing– Saves bakeout time, gives cleaner baselines, less maintenanceSaves bakeout time, gives cleaner baselines, less maintenance

Deconvolution Reporting Software (DRS)– Biggest time saver. Data reviewed in ~10 min instead of ~1

gghour/sample

For Forensic Use.

Fast Tox Screening System Post column device provides:

LiquidI j t

pA) 1.4:1 MSD:NPD effluent splitB) Solvent ventingC) BackflushingD) No vent column changingInjector

XEPC NPDSolvent

D) No vent column changing

XEPC3.8 psig

NPD

S/S Inlet

14 9 i

SolventVent

Column

14.9 psig

(nom)

7890A GC

Column

10 m X 0 25 mm id X 0 25 um DB 5ms

5975CMSD

10 m X 0.25 mm id X 0.25 um DB-5ms

For Forensic Use.

Method Parameters for DB-5ms, 4X, NPD/MSD Split on 7890on 7890

Ramp 'C/min 'C Hold minInitial 100 0.25

MSD Agilent 5975CSolvent Delay 0.5 min

Ramp 1 40 325 1.25Runtime 7.13 min

Inlet Split/Splitless

Scan Range 40 to 570Threshold 0Sampling 1Quad Temp 180 'C

Postrun 325 'C/min for 0.5 min

Acquisition Mode SIM/Scan

p pTemp 280 'CMode Splitless, Constant PressurePressure 14.90 (adj to lock)

Quad Temp 180 CSource Temp 300 'C

Purge Flow 50 mL/min

Transfer Line 300 'C

P ti 0 4 i S litt 2 litt / l t t

Tune Gain Normalized 1X

Purge time 0.4 min SplitterPressure 3.8 psig

Column DB-5ms part # (custom) MSD Restictor 0.694 m x 0.15 mm id

Split ratio 1.4:1 MSD:NPDInitial Flow 2 52 mL/min

2-way splitter w/ solvent vent

NPD Restictor 0.361 m x 0.15 mm id10m x 0.25 mm id x 0.25 um film

Injection volume 1 uL

Split ratio 1.4:1 MSD:NPDInitial Flow 2.52 mL/minVent time range 0-0.75 minOutlet SplitterBackflush Time 0.5 minOutlet Pressure 3.8 psig

Backflush Temp 325Backflush Press 76RT locked to Proadifen at 4.285 min

For Forensic Use.

SIM Groups Acquired With SIM/ScanSIM Group Peak Name RT min Tgt Q1 Q2p g

1 Amphetamine 0.600 44 91 652 Methamphetamine 0.700 58 91 653 Methylenedioxyamphetamine(MDA) 1.319 136 135 514 Methylenedioxymethamphetamine(MDMA) 1.431 58 135 774 Ecgonine Methyl Ester 1 481 94 82 964 Ecgonine Methyl Ester 1.481 94 82 964 Ethylecgonine 1.482 94 82 965 Meperidine 1.884 246 218 2476 Ketamine 2.092 180 182 2096 Phencyclidine 2.166 243 242 2006 Tramadol 2.259 58 263 596 Tramadol 2.259 58 263 597 Methadone 2.578 72 57 1657 Dextromethorphan 2.597 271 212 2708 Cocaine 2.695 182 82 948 Cocaethylene 2.783 196 82 949 Diazepam 3.065 258 286 2579 Tetrahydrocannabinol 3.111 299 300 2319 6-Acetyl-Morphine 3.182 268 327 32810 Oxycodone 3.201 315 230 11510 Temazepam 3.281 271 273 27210 Diacetylmorphine 3.328 310 268 32710 Fentan l 3 451 245 146 18910 Fentanyl 3.451 245 146 18911 Zolpidem 3.555 235 236 21911 Clonazepam-M (amino-) 3.622 285 258 28612 Alprazolam 3.753 308 279 28012 Zaleplon 3.797 305 263 24813 Zopiclone 3 937 112 99 139

13 Zopiclone 3.937 112 99 13913 Lysergide (LSD) 4.000 323 324 222

For Forensic Use.

Screening A Real Blood ExtractBlood extracts are very complex. There are more than 400 detectable peaks in y p pthis sample. The problem is to find the compounds of toxicological interest.

Fast method: 9.75 min injection to injection (including backflush)

Scan TIC

SIM TIC

NPD

1 2 3 4 5 6 7

For Forensic Use.

Target Compounds Identified in Sample By DRS1 Nicotine2 Nicotinamide

7 Carisoprodol8 Methadone

7 9 11

2 Nicotinamide3 Carisoprodol artifact4 Cotinine5 Meprobamate6 Caffeine

8 Methadone9 Cyheptamide (ISTD)

10 Oxycodone11 Cholesterol

Scan TIC3 8

7 11

3

215

4

8

610

3 4 7

10 NPD

21

56 8 9

11

1 2 3 4 5 6 7

For Forensic Use.

Deconvolution of Spectra Eliminates Review Bottleneck For Large Screening MethodsBottleneck For Large Screening MethodsConventional quant approach (~ 1hour / sample review time):

Must examine every peak with response at target ion (367 here)– Must examine every peak with response at target ion (367 here)– Takes ~ 1 hour to review, delete false positives, quant real ones

DRS approach (~ 10 min / sample review time)– Gives list of compounds present in 1-2 minutesGives list of compounds present in 1 2 minutes– List has many fewer compounds to review. (12 instead of 367)– Very few false positives and false negatives– In the current example, all compounds identifiable by QEDIT or Screener

approach were found immediately with deconvolution– Can find compounds not identifiable by conventional approach when

p y ppsevere overlap with matrix exists

For Forensic Use.

DRS Report From Scan Data of Sample

For Forensic Use.

Caffeine: Is It There?

All 3 qualifiers have problems. It would be good to have more info lik t l t h >

Interference

Caffeine?

TIC Scan

like a spectral match >60 to confirm ID, since qualifiers are questionable.q

194 Scan

Q1 and Q2 have interference overlap

67 S

109 Scan

82 Scan

Q3 has very low S/N

NPD confirms nitrogen at caffeine RT

67 Scan

NPD

Q3 has very low S/N

3.00 3.05 3.10

For Forensic Use.

Caffeine Spectrum Has Interference Problem With Matrix CompoundMatrix Compound

Match = 51Caffeine Apex Spectrum194 Caffeine Apex Spectrum(no deconvolution)

50

100

4355

67 109

194

Target Library

0

42

71 82 91

94 122

124

136

137 154 165

165

207 221 235 250260 273 284

Target LibraryReference Spectrum of Caffeine

50

100

5567

82

109

194

38 58 78 98 118 138 158 178 198 218 238 258 278

194

For Forensic Use.

Caffeine Component Searched Against Target LibraryLibrary

Deconvolution improves spectral match quality by removing interfering compound spectrum

Match = 70

Component Found at RT of Caffeine

100194

0

50

42

55 6772 82

94

97

109

122 136 165

Target Library Reference Spectrum of Caffeine

50

100

42

5567

82

109

194

38 58 78 98 118 138 158 178 198 218 238 258 278

194

For Forensic Use.

Methadone: Is It There? Scan and NPD Data

SIM i hi h S/N 72 Scan

57 Scan

SIM gives high S/Nconfirmation of ratios, but 57 still doubtful

57 Scan165 Scan

NPD confirms N

NPDcontaining peak at Methadone RT

3.8 3.9 4.0

For Forensic Use.

Methadone Spectrum Has Interference Problem With Octadecanoic Acid in MatrixWith Octadecanoic Acid in Matrix

Match = 42100

72

Methadone Apex Spectrum

(no deconvolution)50

100

43 55

Target Library Reference

0

50

42

43 55

57 77

83

91

97 111

115 128

129 143151

165

165 178

185

191199 223

236

241 253 264 284

294

Target Library ReferenceSpectrum of Methadone

38 58 78 98 118 138 158 178 198 218 238 258 278 298 318

50

100 72

38 58 78 98 118 138 158 178 198 218 238 258 278 298 318

For Forensic Use.

Methadone Deconvoluted Spectrum Searched Against Target LibraryAgainst Target Library

Deconvolution significantly improves spectral match quality by removing interfering octadecanoic acid spectrum

Component found at RT of Methadone

Match = 80

50

100 72

Methadone

042

42

56

57 77

85

91

115

115 128130 143

151165

165

178

178 189195 223

236294

294

Target Library Reference Spectrum of Methadone

50

100 Spectrum of Methadone38 58 78 98 118 138 158 178 198 218 238 258 278 298 318

100 72

For Forensic Use.

Alprazolam: Is It There?Deconvolution and conventional quant are both limited byDeconvolution and conventional quant are both limited bysignal-to-noise ratio of scan data. As S/N drops, ions disappear from deconvoluted spectrum

Deconvoluted Spectrump

Forward Match 57.5

Reverse 57.5

Second Hit in NIST 05a50

100 204

273

279

308

Second Hit in NIST 05a

0

50

3951

63 89102

116137

151 163177

190 219245

253293

38 58 78 98 118 138 158 178 198 218 238 258 278 298 318 338

50

100

77

204

273

279308

For Forensic Use.

Alprazolam at Limit of Detectability in Scan ModeDeconvolution and conventional quant are both limited by

279 Scan

signal-to-noise ratio of scan data

204 Scan

308 Scan

9 ScaBoth deconvolution and standard quant are at the detection li it h

203 Scan

308 Scanlimit here

279 SIM

308 SIMSIM and NPD data aid in confirming very

280 SIM

NPD

in confirming verylow level of Alprazolam

5.55 5.60 5.70 5.80NPD

For Forensic Use.

Time Savings

Conventional Method:Scan run: 35 min– Scan run: 35 min

– SIM run: 35 min– NPD run: 35 min– Data Review: ~1 hour– Total: 165 min/sample

F t T M th dFast Tox Method:– Scan, SIM, and NPD: 9.75 min

Data Review: ~10 min– Data Review: ~10 min– Total: 19.75 min/sample

For Forensic Use.

Summary

Retention Time Locking (RTL)-the same RTs from now onthe same RTs from now on

Method Translationspeed scale to new column sizes carrier gases analysis speedsspeed scale to new column sizes, carrier gases, analysis speeds

Synchronous SIM/Scan and Split to NPDCollect 3 signals Scan SIM and NPD in one runCollect 3 signals Scan,SIM, and NPD in one run

Column backflushingSaves bakeout time, gives cleaner baselines, less maintenanceSaves bakeout time, gives cleaner baselines, less maintenance

Deconvolution Reporting Software (DRS)Data review time reduced while quality is improved

q y p

For Forensic Use.

Get started quicklyGet started quickly

Application kitColumns, consumables & checkout mixApplication Note and Quick start guide

Application kit

Application Note and Quick start guideDVD with method parameters729 compound DRS database for fast screening729 compound DRS database for fast screeningVideo tutorials with step-by-step instructionsPre- configured and pretested for your applicationOn-site application checkout

Page 41

FY09 Solution PlanAgilent Restricted

Feb. 2009

For Forensic Use.

Thanks For Attendingg

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Improved Speed and Confidence in Toxicology …...- Scan = best confirmation of targets and...

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