drugs of abuse: simple, swift and selective sample preparation...

Drugs of Abuse: Simple, Swift and Selective Sample

Preparation Strategies

December 2012 Lee Williams Ph.D.

Biotage, UK

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Contents

» Introduction – What is Supported Liquid Extraction? – How does it work? – Benefits of SLE+ compared to conventional sample preparation

» Approaches to method selection – How do we start method development?

» Hydrolysed Urine Approach » Drugs of Abuse Applications for LC-MS/MS

– Benzodiazepines – Amphetamines – Opiates – Cocaine – 11-nor-9-carboxy-Δ9-THC and 11-nor-9-carboxy-Δ9-THC

glucuronide – DOA screening approach

» Summary

Supported Liquid Extraction

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Supported Liquid Extraction

» The technique is analogous to traditional LLE – Same partitioning parameters:

– pKa values – LogP, analyte polarity – Solubility in water immiscible organic solvents

» Uses an inert support material (modified diatomaceous earth)

» High surface area for extraction interface

» Flow through technique but not in the traditional SPE sense

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Supported Liquid Extraction: How does it work?

Collect organic extract

containing drug

Whole sample is absorbed onto

bed

(unlike SPE – no ‘flow though’)

Step 1:

Apply aqueous sample

Step 2:

Wait for 5 min.

Step 3:

Add organic

solvent

Individual well of

96-well plate

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Formats

1 mL – 10 mL 200 µL – 400 µL

Total Volume = sample + buffer

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Benefits of SLE+ Versus SPE

» Simplicity – 3 step process: Load, Wait, Elute – No column conditioning or equilibration required – No wash steps required – Lower solvent usage – Higher productivity

» Low ion suppression – Clean Extracts – Robust – Accurate Analysis

» Cross Functional Analysis (neutral/basic analytes) – Mixed-mode not optimal for neutral analytes

(e.g. benzos) – Cleanliness impact due to non-optimal wash solvents

Simple Method Development Tree

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Method Optimization

Hydrolysed Urine Approach

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Enzymatic Hydrolysis Procedure

» 1 mL Urine – Spike with ISTD; typically 10 µL

» β-glucuronidase – Helix pomatia – 50 µL per mL of Urine – Equivalent to at least 4500 U of Enzyme – Activity pH range from 4.5-5.5

» 100 mM NH4OAc buffer at pH5 – 950 µL – 1 mL

» Hydrolyse at 60˚C for 2 hours

Drugs of Abuse Applications from Urine

Benzodiazepine Extraction from Non-hydrolysed and

Hydrolysed Urine

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Parameters

» Supported Liquid Extraction 200 µL plate – 100 µL of urine (non-hydrolysed)

– Pre-treated 1:1 with various aq buffers across pH range – 1 mL Extraction Solvents

– DCM, 95/5 DCM/IPA, MTBE, EtOAc – Samples concentrated and reconstituted in 30% MeCN

» LC – Waters 2795 Liquid Handling System – Column: Phenomenex Kinetex C18, analytical column

(50 mm x 2.1 mm i.d, 2.6 µ) – Mobile Phase: 0.1% formic acid aq: MeCN – Isocratic 70/30 0.1% formic acid aq and MeCN at 0.3 mL/min – Column Temperature: Ambient – Injection volume: 25 µL

» Mass Spectrometry – Waters Ultima Pt triple quadrupole MS – Electrospray Ionization: Positive ion – Desolvation Temperature: 350°C – Ion Source Temperature: 100°C – MRM using intact protonated molecular ions

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Non-hydrolysed Urine Recoveries

Various Extraction Solvents with a 1% Formic acid : urine load. Approximate loading pH 2.7

Various Extraction Solvents with a 0.1% Formic acid : urine load. Approximate loading pH 3.5

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Non-hydrolysed Urine: Extraction Solvent Screen using

1% Formic acid Pre-treatment

MTBE DCM 95-5 DCM-IPA Ethyl Acetate

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0.1% Formic acid Pre-treatment


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Various Extraction Solvents with a H2O : urine load. Approximate loading pH 6.0

Various Extraction Solvents with a 0.5M NH4OH: urine load. Approximate loading pH 10

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H2O Pre-treatment


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0.5M NH4OH Pre-treatment


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Hydrolysis Procedure: 1 mL of urine, 50 µL of β-glucuronidase (approx. 4500U) and 950 µL 100mM NH4OAc buffer at pH5 was hydrolysed at 60 ˚C for 2 hours

Hydrolysed Urine Recoveries

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Hydrolysed Urine: Extraction Solvent Screen using SLE+

200 µL

MTBE DCM Butyl Acetate Ethyl Acetate

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Hydrolysed Urine Extraction using SLE+ 1mL

Method Robustness

Urine 1 Urine 3 Urine 2 Urine 4

LoQ < 1ng/mL for benzos extracting 100µL of matrix

Optimized Procedure: Hydrolysed urine extracted with DCM

Amphetamine Extraction from Non-hydrolysed Urine

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Parameters


– Pre-treated 1:1 with various aq buffers at the high pH end – 1 mL Extraction Solvents

– DCM, 95/5 DCM/IPA, MTBE, EtOAc – Blow down with 100 µL of 50 mM HCl in MeOH – Samples concentrated and reconstituted in 20% MeCN

» UPLC – Waters ACQUITY UPLC – Column: Waters UPLC BEH C18, analytical column

(100 mm x 2.1 mm i.d, 1.7 µ) – Mobile Phase: 0.1% formic acid aq:0.1% formic acid /MeOH – Isocratic 80/20 at 0.43 mL/min – Column Temperature: 40 ˚C – Injection Volume: 10 µL

» Mass Spectrometry – Waters Quattro Premier XE triple quadrupole MS – Electrospray Ionization: Positive ion – Desolvation Temperature: 450 °C – Ion Source Temperature: 150 °C – MRM using the [M+H]+ precursor

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Initial Urine Recoveries

Blowdown stability: Comparison of blowdown from free base amphetamines with stable HCl salt

pH Investigation: Comparison of extractions using H2O, 0.1M NH4OAc or 0.5M NH4OH pre-treatment

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Blowdown Stability:

Free Base Versus HCl Salt

DCM, no post acidification DCM, post acidification

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Non-hydrolysed Urine: pH and Extraction Solvent Screen using

SLE+ 200 µL

H2O-DCM H2O-EtOAc NH4OAc-DCM NH4OAc-EtOAc NH4OH-DCM NH4OH-EtOAc

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Amphetamine Recoveries using Optimized Protocol

Scale up Reproducibility: Comparison of extractions using 0.5M NH4OH pre-treatment and extraction with DCM on 200 µL 96-well plates and 1 mL columns

Method Robustness: Comparison of extractions using 4 different human samples

LoQ < 0.5 ng/mL extracting 100µL of matrix

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Non-hydrolysed Urine: Extraction using SLE+ 1 mL

Method Robustness

Volunteer 1 Volunteer 2 Volunteer 3 Volunteer 4

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Non-hydrolysed Urine: Method Scale Up from 200 µL to 1 mL

200 Ul FWP 1 mL Columns

Opiate Extraction from Non-hydrolysed and

Hydrolysed Urine

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Parameters


– Pre-treated 1:1 with various aq buffers at the high pH end

– 1 mL Extraction Solvents – DCM, 95/5 DCM/IPA, MTBE, EtOAc

– Samples concentrated and reconstituted in 0.1% formic acid aq


(100 mm x 2.1 mm i.d, 1.7 µ) – Mobile Phase: 0.1% formic acid aq:0.1% formic acid /MeOH – Gradient: 80/20–30/70, 1.2 min at flow rate of 0.43 mL/min – Column Temperature: 40 ˚C – Injection Volume: 15 µL


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pH Investigation: Comparison of extractions solvents using 0.5M NH4OH pre-treatment

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NH4OAc-DCM NH4OAc-95/05 NH4OAc-MTBE NH4OAc-EtOAc

pH Investigation: Comparison of extractions solvents using 0.1M NH4OAc pre-treatment

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NH4OH-DCM NH4OH-95-5 NH4OH-MTBE NH4OH-EtOAc

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Hydrolysed urine Recoveries using Optimized Protocol

Scale up Reproducibility: Comparison of extractions using 200 µL 96-well plates and 1 mL columns

Method Robustness: Comparison of extractions using 4 different human samples

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200 µL FWP 1 mL Column

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Volunteer 1 Volunteer 2 Volunteer 3 Volunteer 4

Hydrolysis Procedure: 1 mL of urine, 50 µL of β-glucuronidase (approx. 4500U) and 950 µL 100mM NH4OAc buffer at pH5 was added and hydrolysed at 60 ˚C for 2 hours Post Hydrolysis: Addition of 10 µL of 25% NH4OH per mL urine to pH > 7

LoQ < 1ng/mL extracting 100µL of matrix

Cocaine Extraction from Non-hydrolysed and

Hydrolysed Urine

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Parameters


– Pre-treated 1:1 with various aq buffers at the high pH end – 1 mL Extraction Solvents

– DCM, 95/5 DCM/IPA, MTBE, EtOAc – Blow down with 100 µL of 50 mM HCl in MeOH – Samples concentrated 40 ˚C and reconstituted in 90/10 H2O/MeOH


(100 mm x 2.1 mm i.d, 1.7 µ) – Mobile Phase: 0.1% NH4OH aq:0.1% NH4OH /MeOH – Gradient: 60/40–10/90, 2 min at flow rate of 0.3 mL/min – Column Temperature: 40 ˚C – Injection Volume: 10 µL


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pH Investigation: Comparison of extractions solvents using 0.5M NH4OH pre-treatment

pH Investigation: Comparison of extractions solvents using 100 mM NH4OAc pre-treatment

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Non-hydrolysed Urine: Extraction Solvent Screen using 100 mM

NH4OAc Pre-treatment

MTBE DCM 95:5 DCM/IPA Ethyl Acetate

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Non-hydrolysed Urine: Extraction Solvent Screen using 0.5M

NH4OH Pre-treatment


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Non-hydrolysed Urine Optimized Protocol

Optimized Extraction: Pre-treatment with 0.1% NH4OH and Extraction with 95/5 DCM/IPA

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Non-hydrolysed Urine: Method Scale up from 200 µL to 1 mL

200 uL FWP 1 mL ColumnsCompound name: BZE Quant

Correlation coefficient: r = 0.999819, r^2 = 0.999638

Calibration curve: 1.02106 * x + 0.0320985

Response type: Internal Std ( Ref 5 ), Area * ( IS Conc. / IS Area )

Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

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Calibration Curves 1-100 ng/mL:

Estimated LOQs 25-200 pg/mL

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Hydrolysed urine Recoveries

Post Hydrolysis: Addition of 10 µL of NEAT NH4OH per mL urine

Hydrolysis Procedure: 1 mL of urine, 50 µL of β-glucuronidase (approx. 4500U) and 1 mL 100mM NH4OAc buffer at pH5 was hydrolysed at 60 ˚C for 2 hours

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Hydrolysed Urine: Extraction Solvent Screen using pH5

Conditions


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Non-hydrolysed Urine Optimized Protocol

Optimized Extraction: Post Hydrolysis: Addition of 10 µL of Neat NH4OH per mL urine

Calibration Curves 1-100 ng/mL:

Estimated LOQs 50-400 pg/mL

Compound name: BZE Quant

Correlation coefficient: r = 0.999740, r^2 = 0.999479

Calibration curve: 1.07691 * x + 0.0364176

Response type: Internal Std ( Ref 5 ), Area * ( IS Conc. / IS Area )

Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

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11-nor-9-carboxy-Δ9-THC and 11-nor-9-carboxy-Δ9-THC

glucuronide Extraction from Non-hydrolysed Urine

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Parameters

» Supported Liquid Extraction 200 µL plate – 100 µL of URINE (non-hydrolysed)

– Pre-treated 1:1 with 25mM dibutyl ammonium acetate (ion pair reagent) – Extract with EtOAc – Samples concentrated & reconstituted in 200 µL 30/70 mobile phase


(100 mm x 2.1 mm i.d, 1.7 µ) – Mobile Phase: 0.1% formic acid aq:0.1% formic acid /MeOH – Isocratic 20/80 at 0.4 mL/min – Column Temperature: 40 ˚C – Injection Volume: 15 µL

» Mass Spectrometry – Waters Quattro Premier XE triple quadrupole MS – Electrospray Ionization: +/- – Desolvation Temperature: 450 °C – Ion Source Temperature: 150 °C – MRM using the [M+H]+ or [M-H]- precursor

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Initial Urine Recovery using pH pre-treatment (200 µL plate)

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Ion Pair Reagent Investigation (200 µL plate)

DoA Multisuite from Non-hydrolysed and Hydrolysed

Urine

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Parameters


– Pre-treated 1:1 with 1% NH4OH – 1 mL Extraction Solvents

– 95/5 DCM/IPA – Blow down with 100 µL of 50 mM HCl in MeOH – Samples concentrated 40 ˚C and reconstituted in 80/20 mobile phase


(100 mm x 2.1 mm i.d, 1.7 µ) – Mobile Phase: 2mM NH4OAc aq: 2mM NH4OAc /MeOH – Gradient: 90/10–10/90, 10 min at flow rate of 0.3 mL/min – Column Temperature: 40 ˚C – Injection Volume: 10 µl


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Optimized Extraction: Pre-treatment with 1% NH4OH and Extraction with 95/5 DCM/IPA

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Non-hydrolysed Urine: Method Scale Up for Amphetamine, Bath

Salt and Opiates

200 uL FWP 1 mLColumns

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Non-hydrolysed Urine: Method Scale Up for Benzodiazepines

200 uL FWP 1 mL Columns

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Non-hydrolysed Urine: Method Scale Up for other Classes


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Hydrolysed Urine Recoveries

Optimized Extraction: Post Hydrolysis: Addition of 10 µL of Neat NH4OH per mL urine Extraction with 95/5 DCM/IPA

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Hydrolysed Urine: Method Scale Up for Amphetamine, Bath Salt

and Opiates

200 uL FWP 1 mLColumns

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Hydrolysed Urine: Method Scale Up for Benzodiazepines


0102030405060708090

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Hydrolysed Urine: Method Scale Up for other Classes


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ISOLUTE SLE+ Summary

» Advantages compared to other sample preparation techniques – Simplicity: 3 step process of load, wait, elute – Fewer number of processing steps (conditioning, equilibration and wash

steps removed) – Lower solvent consumption

» Simple methodology to extract a variety of drugs of abuse with a single extraction product

– Optimized individual protocols – DoA multisuite screen (without giving up cleanliness) – Excellent recoveries and RSDs across the required range

» Clean extracts with low ion suppression

– Robust, reliable – Accuracy and precision

» Technique is fully automation compatible – No manual steps involved

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Acknowledgements

» Biotage Cardiff R&D team:

– Rhys Jones – Adam Senior – Kerry Stephens – Helen Lodder – Geoff Davies – Steve Jordan

Thank you for your attention

Lee Williams Ph. D.

Biotage, UK

Email: [email protected]

www.biotage.com

drugs of abuse: simple, swift and selective sample preparation...

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