automation of lc/spme-coupling in 96-wellplate format · asms 2011 poster, „biocompatible and...
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AUTOMATION OF LC/SPME-COUPLING IN
96-WELLPLATE FORMAT
Dietmar Hein PAS Technology Deutschland GmbH
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Plunger
Barrel
Z-slot
Plunger Retaining Screw
Hub-Viewing Window
Commercial Design of the SPME Device by Supelco
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Adjustable NeedleGuide/Depth Gauge
Hub-Viewing Window
Tensioning Spring
Sealing Septum
Septum Piercing Needle
Fiber Attachment Tubing
Coated Fused Silica Fiber
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Schematic of Manual Interface
Commercial SPME fibre assembly
Valco tee (SS) (0.75 mm ID)
fingertight nut
Inner needle
Outerneedle
Inner needle
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ferrule (VG) 0.4 mm ID
sorbent
From pump
To column
Six-port valve
1/16” tubing (0.03” ID)
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LC-SPME coupling
SPME-LC INTERFACES AND AUTOMATION IN THE PAST
SPME-LC INTERFACE AUTOMATIONSAMPLE
THROUGHPUT
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THROUGHPUT
MANUAL INTERFACE LOW LOW
IN-TUBE SPME HIGH LOW
OFF-LINE DESORPTION LOW HIGH
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LC-SPME coupling
ACHIEVE HIGH DEGREE OF AUTOMATION AND HIGH SAMPLE-THROUGHPUT
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PERFORM EXTRACTION AND DESORPTION OF MANY SIMILAR
SAMPLES IN PARALLEL
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Fibre Multiwell System
a)
Multi-fibre top plate
a) For simplification one row and one column of SPME fibres are shown to be inserted into the top plate.
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PDMS-coated flexible wire
96-well plate
b)
b) The multi-fibre top plate can then be placed into a commercial multi-well plates for extraction and desorption.
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Well filled with sample
Insert SPME fibre for extraction
Agitate well until equilibrium is reached
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Remove fibre from well
Desorb in well filled with solvent
N2
Evaporate solventReconstitute and inject into LC
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Advantages of SPME
0CVKn ffs=
no further sample preparation!!!
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increasing sensitivity by increasing Vf!!!
Vf = d f x A f
increased area improves sensitivity and extraction rate!!!
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The “Brush”
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• Pre-washing/conditioning of fiber/membrane in well-plate
• Extraction (variable time setting) of analytes using a PAN/C18 coated blade device
• Washing of fiber/membrane prior to desorption
CONCEPT 96
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Washing of fiber/membrane prior to desorption
• Desorption (variable time setting) of analytes from coated blade device into different solvent system
• Evaporation of solvent – pre-concentration/re-constitution step (depends on type of solvent been used for desorption)
• (Injection)
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CONCEPT 96
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• 4 stepper-motor driven axis (X, Y, Z, Z1)Axis max. range step widthMain axis Y 560 mm 0.250 mmArm axis X 170 mm 0.030 mmInjector axis Z 160 mm 0.150 mmSyringe piston Z1 70 mm 0.047 mm
• 2 pneumatically driven axis
CONCEPT 96
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• 2 pneumatically driven axisEXTRACTION-Tool (96 fiber „Brush“)DRYING-Tool („Shower-Head“)
• Up to 4 agitators (Conditioning, Extraction, Washin g,Desorption/Evaporation), optionally heatable, (0 – 1 .700 rpm)
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Available coatings:
• C18-polyacrylonitrile (C18-PAN)
• Polystyryne-divynyl benzene- polyacrylonitrile (DVB-PAN )
• Phenylboronic acid -polyacrylonitrile (PBA-PAN)
CONCEPT 96
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• Phenylboronic acid -polyacrylonitrile (PBA-PAN)
• Methacryloyloxy ethyl phosphorylcholine (MPC)
• Chromabond Easy/PAN
• LC Diol/PAN
• C18SCX/PAN
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CONCEPT 96
Method for determination of reusability and reprodu cibility
PRECONDITIONINGPAN-C18/ -PS-DVB30 min MeOH/water (1/1)
EXTRACTION60 min, 1000 rpm
Wash15 seconds
Purified water
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MeOH/water (1/1)Purified water
DESORPTION40 min120 rpm
Acetonitrile/water (1/1)
Sample Prep. Time: 130 minutes!!
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Reusability and reproducibility
of C18-PAN coating for
extraction from PBS
and plasma
40
80
120
%E
xtra
ctio
n re
cove
ry
Reusability and reproducibility of C18-PAN: extract ion from PBS
Diazepam
Lorazepam
0
1 5 15 25 35 45 55 63 73 85 95 100%E
xtra
ctio
n re
cove
ry
Extraction #
Lorazepam
0
2.5
5
7.5
10
% A
bsol
ute
reco
very
Extraction
Reusability of C18-PAN coating: extraction from pla sma
Diazepam
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Reusability and reproducibility of DVB -PAN coating in PBS
Reusability of DVB-PAN for diazepam in PBS
20
30
40
50
60
70
80
90
100
110
120
1 5 10 15 20 25 30 40
% A
bso
lute
reco
very
DIAZEPAM
20
40
60
80
100
120
7 12 20 25 30 40
% A
bsol
ute
reco
very
Extraction
Reusability of DVB-PAN for caffeine and riboflavin in PBS
Caffeine
Riboflavin
1 5 10 15 20 25 30 40
Extraction
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Efficiency of DVB-PAN for extraction of diazepam fro m plasma
Reusability and reproducibility of DVB -PAN coating in Plasma
0
10
20
30
40
50
1 10 25
ng e
xtra
cted
Extraction #
Diazepam
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Comparison of absolute recovery of the PAN -PS-DVB coating with C18-PAN
Comparison of absolute recovery of the PAN-PS-DVB coating with C18-PAN (extraction from PBS and n=12) Analyte Log P Pka %recovery
for PAN-PS-
%recovery for PAN-C18 coating PAN-PS-
DVB coating
C18 coating
Diazepam 2.82 3.4 96 ±4 97±3 Oxazepam 2.24 12.4 97±3 80±4 Caffeine -0.07 10.4 99±5 40±6 Riboflavin -1.46 10.2 75±4 60±5 Sucrose -3.7 12.6 3±0.2 -
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Limits of detection and quantitation
PAN-PS-DVB SPME-LC-MS/MS detection and quantitation
LOD and LOQ for extraction of benzodiazepines from plasma LOD for extraction from plasma (3×S/N)
LOQ for extraction from plasma (10×S/N)
0.1- 0.3 ng/mL 0.5-1 ng/mL
PAN-PS-DVB SPME-LC-MS/MS detection and quantitationAnalyte LOD (extraction from
plasma) ng/mL LOQ (extraction from plasma) ng/mL
Oxazepam 0.1 0.5 Diazepam 0.3 1 Caffeine 0.3 1 Riboflavin 0.5 1.5 Sucrose 10 25
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CONCEPT 96 Application
ANALYSIS OF BENZODIAZEPINES IN WHOLE BLOOD
PRECONDITIONINGRPA coating
30 minMethanol/water (1/1)
EXTRACTION30 min, 850 rpm
Whole blood or plasma (0.8 mL) + IS
RINSE30 seconds
Purified water
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Methanol/water (1/1) (0.8 mL) + ISPurified water
DESORPTION30 min, 850 rpm
Acetonitrile/water (1/1)
Sample Prep. Time: 90 minutes!!
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CONCEPT 96 Application
ANALYSIS OF BENZODIAZEPINES IN WHOLE BLOOD
5 ng/mL std in whole blood
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5 ng/mL std in whole blood
blank whole blood
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CONCEPT 96 Application
ANALYSIS OF BENZODIAZEPINES IN WHOLE BLOOD
Validation Parameter Diazepam Oxazepam Nordiazepam Lorazepam
LLOQ (ng/mL) 4 4 4 4
LLOQ Accuracy and 102 111 105 102
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LLOQ Accuracy and Precision (%) (11) (17) (9) (14)
LLOQ S/N RATIO 5 19 16 16
Linear Range (ng/mL) 4-1000 4-500 4-1000 4-500
Accuracy (%) 94-103 91-98 98-106 97-106
Intra-batch Precision (%) 2-8 8-20 5-6 7-11
Inter-batch Precision (%) 3-6 7-12 2-4 7-12
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AUTOMATED DRUG-PROTEIN BINDING STUDY
CONCEPT 96 Application
• Determine time required to reach equilibrium between ligand and receptor
• Obtain fibre constant (fC) by calibration using standard solutions containing no receptor
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• Prepare standard solutions containing different amounts of ligand and constant amount of receptor
• Perform SPME-LC to determine the amount of analyte extracted by the fibre (m)
• Calculate free concentration of ligand (Cf)
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AUTOMATED DRUG-PROTEIN BINDING STUDY
CONCEPT 96 Application
BINDING OF DIAZEPAM AND
HUMAN SERUM ALBUMIN
Diazepam - Human Serum Albumin Binding Curve
0.10
0.12
0.14
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ALBUMIN
1-SITE BINDING MODEL
regression coefficient = 0.991
Free Concentration of Diazepam (M)
0.0 2.0e-7 4.0e-7 6.0e-7 8.0e-7 1.0e-6 1.2e-6 1.4e-6 1.6e-6
B
0.00
0.02
0.04
0.06
0.08
Experimental1-site binding model
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AUTOMATED DRUG-PROTEIN BINDING STUDY
CONCEPT 96 Application
ADVANTAGES OF SPME TO STUDY LIGAND-RECEPTOR BINDING
•Ability to study binding under any conditions and for any
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•Ability to study binding under any conditions and for any concentrations of receptors and ligands
•Fast
•Works well for highly bound drugs
•Automation and increased sample throughput
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Ochratoxin A in Human Urine
CONCEPT 96 Application
Ochratoxins:
•structurally related secondarymetabolites, produced by Penicillium verrucosum
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Penicillium verrucosum
•a common storage fungus
•OTA has nephrotoxic, carcinogenic and immunosuppres sive properties
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Ochratoxin A in Human Urine
CONCEPT 96 Application
•Sample volume: 1 mL urine
•Extraction Time: 1 h @ 850 rpm
•Extraction Fiber: 12 mm of Carbon Tape on 0.061 SS -wire
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•Extraction Fiber: 12 mm of Carbon Tape on 0.061 SS -wire
•Desorption Solvent: 0.9 ml MeOH
•Desorption Time @ 15 min
•Injection Volume LC-MS/MS: 20 µl
Sample Prep. Time: 75 minutes!!
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Ochratoxin A in Human Urine
CONCEPT 96 Application
Validation Parameter
Ochratoxin A – Summary of Validation results
LOD (ng/mL) 0.3
LLOQ (ng/mL) 0.7
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LLOQ (ng/mL) 0.7
Linear Range (ng/mL) 0.7-50
1 ng/mL 10 ng/mL 50 ng/mL
Accuracy and Intra-batch
Precision (%)
106(12)
114(2)
93(4)
Accuracy and Inter-batch
Precision (%)
91 (14)
100(5)
109(4)
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References
• Erasmus Cudjoe, Janusz Pawliszyn, PBA-6864, 2008, „ A new approach to the application of solid phase extraction disks with LC– MS/MS for the analysis of drugs on a 96-well plate format“
• R. Vatinnoa, D. Vuckovic, C.G. Zambonin, J. Pawliszy n, Journal ofChromatography, 2008, „Automated high-throughput me thod using solid-phase microextraction–liquid chromatography–tandem mass sp ectrometry for the determination of ochratoxin A in human urine“
• Dajana Vuckovic, Erasmus Cudjoe, Dietmar Hein, Janusz Pawliszyn, Anal. Chem. 2008, „Automation of Solid-Phase Microextraction in H igh-Throughput Format and Applications to Drug Analysis“
• Dajana Vuckovic , Erasmus Cudjoe, Florin Marcel Musteata , Janusz Pawliszyn , Vol.
11/15/2011 29
• Dajana Vuckovic , Erasmus Cudjoe, Florin Marcel Musteata , Janusz Pawliszyn , Vol. 5, No. 1, 2010 Nature Protocols, „Automated solid-p hase microextraction and thin-film microextraction for high-throughput analysis of biological fluids and ligand-receptor binding studies“
• Fatemeh Mirnaghi, Janusz Pawliszyn, Yong Chen, Leon ard Sidisky, Dietmar Hein, ASMS 2011 Poster, „Biocompatible and reusable octad ecyl-polyacrylonitrilecoating for high throughput automated 96-thin-film solid phase microextractionsystem coupled with LC-MS/MS”
• Fatemeh Mirnaghi, Janusz Pawliszyn, ASMS 2011 Poste r „Modified PAN-PS-DVB 96-Thin-FilmSPME System, Capable of Extracting wide polarity range of Analytes from Biological Fluids”
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Acknowledgements• Prof. Pawliszyn and his group members, specifically
Fatemeh Mirnaghi, Erasmus Cudjoe and Dajana Vuckovic , who supplied most of the data
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Vuckovic , who supplied most of the data http://www.spme.uwaterloo.ca/
• Supelco http://www.sigmaaldrich.com/Brands/Supelco_Home/Spotlights/SPME_central.html