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AUTOMATION OF LC/SPME-COUPLING IN
96-WELLPLATE FORMAT
Dietmar Hein PAS Technology Deutschland GmbH
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
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)
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
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
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.
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
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!!!
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)
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)
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
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!!
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
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
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
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 -
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
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!!
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
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
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)
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
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
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
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!!
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)
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.
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• 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”
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
