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Automation of Forensic Toxicology
Sample Prep and Analysis WorkflowsDr Oliver Lerch
GERSTEL GmbH & Co. KG, Muelheim, Germany
Dr Eleanor Miller, Dr Camilla Liscio, Mr Dan Carrier
Anatune Ltd., Cambridge, United Kingdom
LTG/UKIAFT Joint Meeting London, March 9th 2018
GERSTEL History
Founded in 1967 in Muelheim
an der Ruhr, Germany (HQ)
Family owned and operated
Agilent Value Added Reseller
(VAR): partnership since 1986
5 subsidiaries, >70
distributors, ~200 employees
world-wide
Complete solutions for GC-,
LC- and SIFT-MS
Automated sample
preparation and introduction
Anatune - GERSTEL Distributor in UK
and Republic of Ireland
Anatune Ltd.,
Unit 4, Wellbrook Court, Girton Road
Cambridge, CB3 0NA
Develop, sell and support analytical solutions –
chromatography, mass spectrometry and automation
Dan Carrier
Lab Manager
Eleanor Miller
Forensics Market Specialist
Camilla Liscio
Applications Chemist
Oliver Lerch, PhD
• Senior Application Scientist
• GERSTEL employee since 2005
• Analytical Services Department
• Strategic applications
• Presentations at seminars, workshops and
conferences
• Customer demos & Apps training
• Support for R&D
• etc.
Why automate your toxicology
sample preparation workflows?
Chain of Custody and Sample Tracking
Robotic autosampler does not mix up
vials
Optional barcode reader gives
increased confidence in the results for
court presentation
R² = 0.9994
0
1000000
2000000
3000000
0 50 100 150 200 250 300
Pe
ak
Are
a
pg/µL
Benzo[a]pyrene
R² = 0.996
0
1000000
2000000
3000000
4000000
0 50 100 150 200 250 300
Pe
ak
Are
a
pg/µL
Benzo[a]pyrene
PAH Standard + benzo[a]pyren-d12 solution + Benz[a]anthracen-d12 solution + acetonitrile
RSD BaP-d12: 9,0%
RSD BaA-d12: 9,4%
RSD BaP-d12: 5,8%
RSD BaA-d12: 2,0%
Improved Accuracy and Reproducibility
Automation can
improve accuracy of
analyses
Bonus - Quality of Results not entirely
dependent on the “Human Factor”
Not all colleagues may
work on the same level
of accuracy and
confidence
Reduced Analyst Time for Sample
Prep – More Time for Thinking Tasks!
More time for:
Data reviewing
Court reports
Method development
Quality assurance
… and drinking a cup of tea or coffee
Analysis on the robot not always faster
Can run with robot and without human supervision overnight and at the weekend (remote log in possible for the super keen!)
Less Exposure to Harmful/Toxic
Solvents
GESTEL Robotic autosampler:
Handles solvents and reagents in closed containers and vials
Does not need human supervision in the laboratory
Solvent top up of vials can be done in fumehood
Multi Purpose Sampler (MPS)
MPS automation platform: “Lab on a Rail”, John Quick, ALS Environmental
Personalised systems for lab needs –flexible coverage of sample prep method or high sample throughput methods e.g. basics screen
Prep of cals/QCs (dilution), mixing, centrifugation, SPE or ITSP, LLE, evaporation, agitation, reconstitution
Online coupling to Agilent GC or LC (-MS) systems or as standalone
GERSTEL Maestro Software
Integrated into, or
coupled to, Agilent
Technologies software
packages
Examples: Toxicology Applications
Blood alcohol determination
• Automated static headspace analysis coupled to GC-FID
Determination of THC and metabolites in blood serum
• Automated LLE, evaporation, derivatisation, GC-MS
Determination of THC and metabolites in hair
• Automated hair digestion, SPE, evaporation, derivatisation, GC-MS
Determination of opioids and cocaine in serum and blood
• Automated SPE or ITSP, evaporation, derivatisation, GC-MS
Blood Alcohol Example
MPS-HS-GC-FID System
Static headspace analysis of whole
blood sample
Dual column GC-FID system
Agitator Samples
MPS with heated and
gas-tight syringe
GC-FID
Results
Automated analysis meets testing specifications for the California Department of Justice Blood Alcohol Operating Procedures (Title 17), resolution for EtOH, deviation of response factor etc.
When coupled to a MS, minor alcohols can also be analysed
More details: http://www.gerstel.com/pdf/p-gc-an-2005-01.pdf
THC + Metabolites in Blood Serum Example
Δ9-Tetrahydrocannabinol (THC)
11-nor-9-carboxy-
Δ9-tetrahydrocannabinol (THC-COOH)
11-Hydroxy-
Δ9-tetrahydrocannabinol (THC-OH)
Target Analytes
Automated Analysis Method
500 µL blood serum extracted with hexane/ethyl acetate (9/1)
in QuickMix
Centrifugation for phase separation and removal of organic
extract
Evaporation of extract to dryness at 65 °C
Addition of 1 M HCl to the residual sample (pH 4-5) and
second extraction, phase separation and evaporation
Reconstitution in MSTFA solution and inlet derivatisation
GC/MS
Validation Data
Publications
Sonja Remacle (formerly Heinl)
Freidoon Erdmann, PhD
Florian Veit, PhD
University Hospital Gießen and Marburg
Institute of Legal Medicine
Giessen, Germany
Possible Setup – THC and Metabolites
in Hair
Agitator
SPE
module
Evaporation
station
SPE Cartridges
Standard SPE cartridges modified for automated SPE
11-Nor-9-carboxy-Δ9-
tetrahydrocannabinol
(THC-COOH)
11-Hydroxy-Δ9-
tetrahydrocannabinol
(THC-OH)
Cannabidiol (CBD)Cannabinol (CBN) Δ9-Tetrahydrocannabinol (THC)
Target Analytes
THC-COOH cut-offs
0.2 pg/mg Society of Hair Testing (SoHT) and European Workplace Drug Testing Society (EWDTS)
0.05 pg/mg Substance Abuse and Mental Health Services Administration (SAMHSA)
Analysis Method
25 mg washed and powdered hair + ISTD, manually
Automated: Digestion by addition of 1 mL 1 M NaOH at 90 °C for 15 min, pH 14
SPE cleanup on Chromabond HR-XA (Macherey Nagel)
Elution with isohexane/ethyl acetate/acetic acid
Evaporation to dryness 70 °C, 10 min, 80 mbar
Reconstitution in 25 µL MSTFA
Injection 1.2 µL into hot split/splitless inlet at 280 °C
Zebron ZB-5MSi (Phenomenex)
Electron impact ionization (EI) 70 eV, MRM detection mode
2x10
0.4
0.6
0.8
1
Noise (PeakToPeak) = 5.24; SNR (17.096min) = 11.5
* 17.096108
1x10
4
5
6
7
8
9
Noise (PeakToPeak) = 7.88; SNR (17.100min) = 5.5
17.10082
Counts vs. Acquisition Time (min)16.7 16.8 16.9 17 17.1 17.2 17.3 17.4
2x10
0.4
0.6
0.8
1
1.2
1.4
Noise (PeakToPeak) = 5.48; SNR (16.098min) = 17.3
16.098163
4
2x10
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
Noise (PeakToPeak) = 8.96; SNR (16.096min) = 14.5
16.096242
4
Counts vs. Acquisition Time (min)15.9 16 16.1 16.2 16.3 16.4
0.2 pg/mg THC-COOH/THC-OH automatically extracted from spiked blank hair
Analysis by Agilent 7890/7000B triple quadrupole MS
THC-COOH THC-OH
Quantifier
Qualifier
Limits of Detection (LOD)
Validation (GTFCh guidelines)Analyte LOQ
[pg/mg]
LOD
[pg/mg]
RSDr
[%]
RSD(T)
[%]
Bias
[%]
Extr. Eff.
[%]
Cal. range
[pg/mg]
THC-
COOH0.5 0.2
5.5 low
4.7 high
6.3 low
5.7 high
+3.4 low
+10.6 high79 0.5 - 20
THC-OH 0.5 0.23.2 low
4.0 high
4.2 low
4.1 high
+9.5 low
+10.3 high67 0.5 - 20
THC 5 24.3 low
2.9 high
6.6 low
2.9 high
+0.1 low
-2.3 high51 5 - 2000
CBN 5 22.3 low
1.8 high
4.1 low
2.5 high
+5.9 low
-4.3 high70 5 - 2000
CBD 5 23.5 low
2.4 high
4.3 low
2.5 high
+0.7 low
-6.4 high19 5 - 2000
low = 0.8 pg/mg (THC-COOH, THC-OH), 8 pg/mg (THC, CBN, CBD); n = 12
high = 7.5 pg/mg (THC-COOH, THC-OH), 750 pg/mg (THC, CBN, CBD); n = 10
Summary: Cannabinoids and
Metabolites in HairFirst automated on-line solution for determination of THC-COOH, THC-OH, THC, CBN, CBD together in extracted hair
Challenging SoHT testing cut-off met for THC-COOH 0.2 pg/mg as LOD
Last five proficiency tests successfully passed (GTFCh Drugs in Hair by ArveconGmbH, Germany)
Method employed in routine hair analysis (renewal of driving licenses) in the Institute of Legal Medicine in Cologne, Germany
Similar automated SPE method for THC and metabolites in serum, blood, urine and homogenates used there since 2014 with more than 17,000 extracts, cycle time 30 min
Many thanks to Tobias Kieliba, Justus Beike PhD and Hilke Andresen-Streichert from the
Institute of Legal Medicine, University Hospital of Cologne, Germany
Poster at TIAFT 2016, Brisbane, Australia
Samples: Serum, blood,
urine, tissues, stomach
content…
Manual protein precipitation
Validated, manual routine
workflow (SPE) from the
Institute of Legal Medicine
Duesseldorf, Germany
Automated SPE (mixed-
mode cation exchange)
workflow with GERSTEL
MPS
Study: Manual vs. Automated Method
Opioids & Cocaine
Oliver Temme, PhD
Prof. Thomas Daldrup, PhD
University Hospital Düsseldorf
Institute of Legal Medicine
Germany
vs.
Cocaine
0,1
1
10
100
1000
0,1 1 10 100 1000
Reference method institute of legal medicine Düsseldorf, ng/mL
GE
RS
TE
L M
PS
, n
g/m
L
LLOQ - ULOQ
LOD - LLOQ
Serum
Others
Comparison Study – Manual vs. Robot170+ serum sample, 50+ samples of other matrices
1
10
100
1000
1 10 100 1000
Serum
Others
LLOQ - ULOQ
LOD - LLOQ
Reference method institute of legal medicine Düsseldorf, ng/mL
GE
RS
TE
L M
PS
, n
g/m
L
Morphine
Comparison Study – Manual vs. Robot170+ serum sample, 50+ samples of other matrices
Validation Data for manual Method
Open Access article in
Analytical and Bioanalytical Chemistry 406
(2014) 4443
Possible Set-Up – Opioids in Blood
with ITSP Clean-Up
Hazel Torrance
Denise McKeown
Camilla Liscio
CF200
centrifuge
mVAP Wash
station TraysmVorx
Solvent
Reservoirs
ITSP Agitator
Solvent
Filling station
ITSP Cartridges for SPE
Sorbent
Needle Guide
Septum cap
Automated Analysis:
Scale down of manual MethodCleanScreen 10-UDAU-T ITSP 30 mg
CONDITIONING
• 100 µL MeOH
• 100 µL Water
• 100 µL 0.1 M pH 6 Phosphate Buffer
LOADING
Sample: 500 µL
(200 µL whole blood + buffer pH 6)
WASH
• 100 µL Water
• 100 µL 0.1 M pH 4.5 Acetate Buffer
• 50 µL MeOH
ELUTION
250 µL DCM/IPA/NH4OH 78:20:2 (v/v/v)
Evaporate eluate to dryness 10 min 37 °C 500 rpm
Add 50 µL BSTFA 1% TMCS
Incubate 90 °C for 15 min 750 rpm
Inject on GC-MS
ITSP +
Derivatisation
GC-MS
run
Prep Sequence Scheduler
Apart from the initial
pipetting of the blood
sample, all other
sample prep steps
were automated on
an on-line GC-MS
solution!
Summary Opioids ITSP
Automated preparation of calibrators and QCs
Proof of concept study using spiked horse whole blood – needs to be tested in PM
lab
Calibration range 25-500 µg/L for morphine, codeine and dihydrocodeine and 5-
200 µg/L for 6-MAM (R2>0.99)
Extraction efficiencies >90 %
6-MAM LOD determined as 3 µg/L for GC single quadrupole
Some Tox for Thought….
Automation of manual sample preparation workflows is
feasible for a variety of drug and metabolite methods and
biological matrices
GERSTEL automated sample solutions can be flexible or put to
use for high sample throughput assays according to lab needs
Solutions provide high quality lab data consistently when
truth and evidence matter!
Systems are employed routinely in institutes of legal medicine
Thank you for your attention!
Any questions?