By : Dr. Sarhan Ali Salman

ANALYZING DOA WITH GCMS AND GCMSMS

TECHNOLOGIES

Toxicology milestones

The Thermo legacy

1939: DDT Excellent insecticide= excellent lifesaver

1956: Minimata Mercury poisoning

1961: society of toxicology founded

1970:EPA founded and ban of cocaine in the US

1972: DDT is banned 1937: ban on marihuana

1928: ban on doping

1984: Bhopal 1995: Sarin attack in Tokyo

1999 Dioxine crisis in Belgium

1948: Foundation ATLAS MAT

1958: start of Carlo Erba SID and MAT CH4 first commercial succesful GCMS

1969: first splitless injection by Grob on Carlo Erba GC

1969: Finnigan Model3000GCMS 1975: first capillary GC by Carlo Erba

1987: MAT90 first PC controlled GCMS

1990s: MD800 and GC8000

Joined company forces

1920 1950 1968 1980 1990

YOUR NEEDS TODAY ARE STILL ADDRESSED Sample prep: as less as possible; as quick as possible

Software: Complete with data validation

Instrumentation: Rugged and robust; fast and reliable

Total workflow: Optimized production with high quality results

DRUGS OF ABUSE TESTING LABORATORY WORKFLOW

•Sample preparation reagents and solutions for various matrices

•Blood, Urine, Oral Fluid hair and other bodily fluids

•Accurate separation of components analogues

•Avoid misinterpretation of target compounds

•Avoid metabolic product exclusion or interference

•Opioids - Keto-moeity derivitization

•Removal of noise associated with sample matrix components

•Higher recovery, Better reproducibility

•Less noise = Better chromatography

•Hyper Sep Verify - CX

•Enhance target compound stability

•Better detection

SAMPLE PREP: INJECTING BLOOD, SWEAT AND TEARS ON A

GC Sample Matrix

Derivatization

Solid Phase Extraction

Multiple Derivitization

THE TRIPLUS RSH AUTOSAMPLER

KEYPOINTS AND SOLUTIONS FOR THE PRODUCTIVE LAB a large number of samples in the trays

Up to 648 2-mL vials and multiple 100 mL solvent bottles stations

Unattended, injection modes changes in a sequence: liquid – liquid Large Volume - HS - SPME

ATC (Automatic Tool change) Station for automatic recognition and syringe exchanges during

operation

Reliable, reproducible injections with microvolume samples using the bottom sensing sensor

<2%RSD and up to 3 injections possible with 5 ml sample volume in vial.

Advanced sample handling and sample preparation

Dilution, derivatization, standard addition, internal standard additions, etc.

Barcode reading for avoiding writing mistakes

Set up possible for two GC’s

Completely adaptable to your wishes:

cooled/heated trays;

larger washing stations;

HS ovens;

vortex for mixing;

and more……….

just ask us for implementation of your idea!

THE TRACE1300 SERIES GC

KEYPOINTS FOR THE LAB: Modular approach:

change injectors and detectors in 3 minutes only

disassemble and sonicate the injector parts

Injection modes include:

SSL

SSL with backflush

PTV

PTV with backflush

all are compatible with Large Volume without any hardware change

Fast cooling of the oven and PTV for fast run to run times;

from 350C to 40C in approximately 4 minutes

extra fast cooling with cryo option for oven and injector

Methodology is completely compatible with third party vendors

little RT difference

same injection parameters

Quick start up; Fast methodology; Easy maintenance

Generate results shortly after installation;

Ensure production over a long period of time

SINGLE QUAD: THE ISQ

THE ISQ: SINGLE QUAD MS

Heated source: ion volume, repellor, lenses, RF prefilter

Solid, highly inert material ensures reliable performance in all

ionization modes, including EI and CI

Dual filament cartridge

Two filaments in same magnetic orientation

Filament lens assures that both filaments will remain protected, for

longer life even with redundancy

The S-Shaped Ion Guide

Reduces neutral noise created by excited helium neutrals striking

the detector, resulting in lower detection limits and better selectivity

Autotune:

Complete and standardized autotune algorithm for excellent day to

day variability

THE ISQ: TRUE FAST SCANNING d:\training\fs2802 28/02/2010 11:33:35

new 1pgOFN, at scan rate 2500 amu/s, factory spec

RT: 3.502 - 3.753 SM: 5B

3.55 3.60 3.65 3.70 3.75

Time (min)

0

10000

20000

0

10000

20000

0

5000

10000

15000

20000

Re

lative

Ab

un

da

nce 0

5000

10000

15000

624 625623

621 627

620 628619

1201 12031200

12061198

12091196

1212

2262 226322602267

22572270225522732251

45314530 4533

454145224546451645544512

NL:1.79E4

m/z= 271.500-272.500 MS fs2802

NL:2.04E4

m/z= 271.500-272.500 MS fs2803

NL:2.28E4

m/z= 271.500-272.500 MS fs2806a

NL:2.40E4

m/z= 271.500-272.500 MS fsre2801

Theoretical

(scan/sec)

Actual

(scans/sec)

9.62 9.67

18.52 18.58

35.00 35.00

70.13 70.00

THE WIRELESS SOURCE: EASY MAINTENANCE

Step 1. Insert removal tool

Step 2. Remove source

Step 3. Hot source is held in tool Step 4. Push source out of tool

COCAINE AND MAJOR METABOLITES IN URINE Total ion chromatogram of cocaine and it metabolites from an extracted urine sample at 150 ng/mL

Confirmation

analysis

THC IN URINE

Validated based on guidelines published by EWDTS

- Linearity, Carryover, Inter/Intraday Precision and Robustness

Confirmation

analysis

THC in urine

Precision

- Intra-and inter-batch precision of less

than 4% CV at 6 and 18.75ng/mL

Robustness

-100% of all QC samples at passed

across a study spanning 7 weeks in over

100 batches.

Minimal Instrument Maintenance

-Daily septa replacement

-Daily tune

-liner replacement - as needed

-1 Ion source for the duration of the

experiment

Confirmation

analysis

GHB AND GLYCOLS

THC IN SERUM THC SIM

THC MS-MS

THC in serum at 1 ng/ml

COTININE IN SERUM

Cotinine FS

Cotinine MS-MS

THE TSQ8000

TRIPLE QUADRUPOLE MS – PRINCIPLE OF

OPERATION Quantitation of target compounds in matrix samples

Q1 selects the precursor ion TCDD ion: m/z 319.90

Q3 selects the product ion fragments to m/z 256.90

Argon

Collision Gas

Select Fragment Detect

Ion S

ourc

e

Dete

cto

r

SRM: selected

reaction monitoring

RT: 25.70 - 26.46 SM: 5G

25.7 25.8 25.9 26.0 26.1 26.2 26.3 26.4

Time (min)

0

20

40

60

80

100

0

20

40

60

80

100

Re

lative

Ab

un

da

nce

0

20

40

60

80

10026.36

26.3226.18 26.4025.83 25.89

25.9825.72 25.79 26.0926.04 26.25

RT: 26.03SN: 18RMS

RT: 26.11SN: 9

26.35

26.17 26.4525.79 26.0625.70 25.97 26.3026.2025.83 25.92

RT: 26.02SN: 136880RMS

RT: 26.11SN: 108979RMS

NL: 6.62E8

TIC F: MS 2407-14

NL: 2.54E6

m/z= 291.5-292.5 F: MS 2407-14

NL: 1.37E5

m/z= 256.5-257.5 F: + c SRM ms2 292.00@3.25 [ 190.00-300.00] MS ICIS 2407-06

MS/MS vs SIM for detecting pcb’s in soil samples.

RT: 19.80 - 43.03 SM: 5G

20 22 24 26 28 30 32 34 36 38 40 42

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

26.94

26.36

32.12

27.75 31.8633.24

30.6633.94

34.31

28.07

28.6325.6334.63 36.1330.0925.57

37.1624.3740.42

38.4823.54

23.30

40.9221.45

41.47

21.39

NL:7.79E8

TIC F: MS 2407-14

PCB’s in MS/MS

s/n 14000

Complete FS

chromatogram

Selective ion mode

s/n 18

PCB’s in FS

Increased S/N:

-Lower detection limits

- automatic integration by

software much more repeatable

and reliable

SHORT OVERVIEW

Dual Filaments

for Prolonged

Uptime

Repellor for

longer source

uptime

Heated ion guide

protects main

quadrupole sets so

they never have to be

cleaned or replaced.

Curved ion

guide enables

off-axis optics

to effectively

eliminates

neutral noise

Choose between

nitrogen or argon for

collision gas.

Full source

removal without

venting, including

ion guide surface.

No wire

connections are

necessary, and

never vent to clean

your instrument.

Super fast analyzers switch

between Full Scan, SIM and

MRM in milliseconds.

KEY POINTS FOR THE PRODUCTIVE LAB

•Repeller

•Allows effective tuning even when parts get contaminated

•Increased ion abundance stability and tune-ability

•Dual filament cartridge

•Two filaments for reducing instrument downtime

•Both filament operate in the same magnetic orientation for an increased life cycle

•Alignment insure to get same ionization performance while switching filaments

•Increased temperature all along the ion path

•Reduces residence of the matrix that form the basis of ion burns

•Dual heated zone for more ruggedness

•Increased diameter prefilter and lenses

•Dilutes effect of contamination

The TSQ 8000 features the ExtractaBrite ion source, the first

ever source on a GC-triple quadrupole in which all parts

that require periodic maintenance can be removed and

inserted while maintaining vacuum, greatly reducing

downtime for source maintenance.

UNSTOPPABLE PRODUCTIVITY – VENT-FREE

MAINTENANCE

DOA IN ORAL FLUID

Results of analysis: DOA in oral fluid

Confirmation

analysis

• Methadone D9

• Methadone

• Cocaine D3

• Cocaine

• BZE D3

• BZE

• Nordiazepam D5

• Nordiazepam

• THC D3

• THC

• Oxazepam D5

• Oxazepam

• Dihydrocodeine

• Codeine D3

• Codiene

• Lorazepam D4

• Lorazepam

• Diazepam

• Morphine D3

• Morphine

• 6am D3

• 6am

• Temazepam D5

• Temazepam

• THC Acid D3

• THC Acid

Run time 9.5 mins

METHADONE AT 5 NG/ML

6 AM AT 0.5NG/ML

THC IN ORAL FLUID

Agilent application note 2D GCMS: selectivity in chromatography Sample prep: - 400 ul oral fluid - SPE clean up - derivatization with BSTFA and TCMS - evaporate to dryness - add 25 ul acetonitrile - 4ul injection

- injected on column 11ng

Thermo solution GC-HSRM: selectivity in detection Sample prep: - 200 ul oral fluid - SPE clean up - derivatization with BSTFA - evaporate to dryness - add 50ul toluene - 2ul injection

- injected on column 16ng

Results of analysis: THC in oral fluid at 0.2ng/ml

Confirmation

analysis

THC IN ORAL FLUID

TN10306 for more information

Confirmation

analysis

DETERMINATION THCA IN HAIR •Delta 9 THC (THC) is the most commonly abused illicit drug.

•The metabolite THCA indicates the ingestion of THC as opposed to passive contamination of the

hair through the air.

•Hair is a very complex and variable matrix even after sample cleanup

Small Samples

typically 20mg

Low Concentrations

40 fg on column

Low Level Detection

Cutoff of 0.05 pg/mg

Overcome Matrix Interference

Accurate Quantitation

Detection limit of 20% of cutoff

Confirmation

analysis

•Sensitivity of GC Quantum XL to THCA

•On-Column Injection of 8fg

•Use of a 0.05 pg/mg cutoff

•Demonstrates the sensitivity sufficient for published cutoffs

DETERMINATION THCA IN HAIR

MSTFA Derivatized, EI, SRM Transition: 371 > 289

• NCI - Specific transitions can be defined in NCI (Methane used as reagent gas),

enhancing selectivity for both THC-COOH and the corresponding ISTD D3

Confirmation

analysis

•Demonstrates very low noise level relative to the signal

•Zoom in the baseline by 100 times before we start seeing significant noise

•Possible to drop the concentration by a factor of 10 and still achieve the selectivity to accurately quantitate THCA

THCA IN HAIR - NCI TRANSITIONS

PFAA Derivatized, Negative Ion Chemical Ionization - 50 fg THCA and ISTD-D3

Confirmation

analysis

ETG IN HAIR:

PTV BACKFLUSH-GC-NCIMSMS

THOMAS FREY

METHODOLOGY: SHORT DESCRIPTION •Hair strand is washed with deionized water and acetone, dried and cut into small pieces;

•20 – 30 mg hair snippets are powdered in a ball mill

•1ng EtG-D5 as internal standard is added

•15 min ultrasonic extraction with 1.5ml deionized water

•SPE clean up

•Derivatization with PFPA

•PTV injection: 250C (5.5min)-14.4C/sec-280C- backflush on 5min

•GC: 80C (3min)-25C/min-270C (4min)

•He: 2ml/min

•Sample volume 1ul

•Transferline: 200C

•Source Temperature: 140C

•CI gas: methane 2ml/min

DETERMINATION OF ETG IN HAIR

Maintenance Classic injection with SSL and

single quad

‘Smart’ injection with PTV backflush and

Quantum

Improvement

Liner Every 20 inj 100 inj 5

Precolumn 20 inj 100 inj 5

Analytical column 400-500 inj 1000-1500 inj 3

Ion volume 400-500 inj 1000-1500 inj 3

Added bonus: • GC Run time: 2 times shorter • LOD: 10 times better

GC/MS

Confirmation and Quantitation of an Expanded Panel of Sympathomimetric Amines in Urine

Using Single Quadrupole GC/MS

High-Throughput Confirmation and Quantitation of a THC Metabolite in Urine Using Single

Quadrupole GC/MS

Confirmation and Quantification of Phenclidine in Urine Using the ISQ Single Quadrupole

GC-MS

Confirmation and Quantification of Benzoylecgonine in Urine Using the ISQ Single

Quadrupole GC-MS

Confirmation and Quantitation of Cocaine and Major Metabolites in Urine Using the ISQ

Single Quadrupole GC-MS

Quantum (TSQ) XLS

Confirmation and Quantitation of THC in Oral Fluid Using MRM Method

Quantitation of EtG in Hair using PTV backflush-GC-NCIMSMS

APPLICATION NOTES

HOT LIST: MEPHEDRONE - NEW DESIGNER

DRUG • Mephedrone is illegal since 2010 across Europe.

• The drug is very popular as a party drug and so far little analytical experiments have been performed.

• Recently published by Westphal and Junge

• Massenspektrometrische, infrarotspectroskopische und NMR spektroskopische Daten von Mephedron, butylon und

Methylon sowie einigen ihrer Derivate

• System: TSQ7000. Predecessor of QuantumXLS

SOFTWARE SOLUTIONS

TRACEFINDER: METHOD DEVELOPMENT • Import from Xcalibur processing method, or from

a datasheet; or through automated library search

• Automatic input for quan and qual ions

• Add quality controls for the method:

• Linearity demands

• Internal standard recovery

• Blancs and negatives

• Quality control samples

• Ion ratio demands

• LOD,LOQ and LOR

• Add automated library search for metabolites

Data evaluation

TRACEFINDER: PRODUCTION • Batches and batch templates for quick sample

list set up

• Sample types include specimens, negatives,

calibrators

• Colour coded flags for QC controls as set uo in

method development

• Active link between reporting and data

• Update RT; integration; ion ratio; QC settings in

• Local method: this set of data

• Master method: all following set of data

• Choose between over 30 report types

• Export to LIMS

• All results, data, methodology in one directory

Data evaluation

CONCLUSION: A TOTAL SOLUTION FROM PREP

TO REP

• Fast and easy sample preparation procedures are available

• A robust and sensitive instrumentation is provided

• Coupled with complete method information for fast start up

• Data handling is fast and the software provides fully embedded qualtity controls