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?