BIOANALYTICAL APPLICATIONS OF SOLID PHASE MICROEXTRACTION

COUPLED TO LC/MS

Heather Lord, Marcel Musteata, Dajana Vuckovic, Simon Zhou and Janusz Pawliszyn

Department of Chemistry University of WaterlooWaterloo, Ontario, Canada, N2L 3G1

Waterloo

• Financial supports from:– The Natural Sciences and Engineering Research Council of

Canada– Supelco– Varian– Leap Technologies– Firmenich– Eli-Lilly– Merck– NoAb Biodiscoveries– Sciex– Smiths Detection– Convergent Bioscience – Restek

Acknowledgment

Outline

• Introduction• Fundamentals of SPME• Calibration of SPME• Manual SPME-HPLC Interface• In-tube SPME• Multiple Well SPME• In-vivo SPME

Outline

Solid Phase Microextraction

Solid Phase – solid or “rubbery”* sorbent

Microextraction – volume of the extraction phase is small compared to volume of the sample matrix

*“rubber” polymer like PDMS is physicochemical liquid

Sample flow

Fibre

Tube

Vessel

Suspended Particles Stirrer

Extraction PhaseSample

particle

SPME Configurations

Disk

Fiber Attachment Tubing

Plunger

Barrel

Adjustable NeedleGuide/Depth Gauge

Z-slot

Plunger Retaining Screw

Hub-Viewing Window

Tensioning Spring

Sealing Septum

Septum Piercing Needle

Coated Fused Silica

Supelco SPME Device

0

50

100

150

200

250

300

350

400

450P

ubli

cati

on

s

2000 2001 2002 2003 2004 2005

Year

SPME TREND

Total publications 2000-2006: 3000+

SPME Databasehttp://www.spme.uwaterloo.ca/

Features of Microextaction• Selectivity based on differences in K• Sensitivity without evaporation of solvent• All extracted components are transferred to instr.• Flexibility in configuration (SPME)• Convenience of automation (SPME fibre device)• Small size• Convenience in handling on site (SPME)• Integration of sampling and sample preparation• Free concentration determinations• Complexity of optimization

Mass of an Analyte Extracted from a Heterogeneous System

∑=

=

++=

+++++=

ni

isiisffs

sffs

snnsssffs

sffs

VVKVK

VCVKn

VVKVKVKVKVCVK

n

1

0

2211

0

L

This represents challenge in microextraction method optimization,but also substential opportunity to study the interations since the method is sensitivity to multiphase partitioning.

Distribution of Chemicals in Heterogeneous Samples

=

Study of Binding with SPME

SPME coating: silica-based RAM with liquid C18 extraction phase; MWCO~15kDaTopochemically bifunctional surface:• the outer particle surface is modified with hydrophilic diol groups• the inner pore surface is covered by hydrophobic alkyl chains

Comparison between Methods

Method Advantage Disadvantage

Multiple Standard Solutions

• good accuracy• simple calculations for total and free concentrations

• several samples containing receptor are needed

Successive Extractions

• a single sample containing the receptor is necessary• fast, since no supplementary steps for sample preparation or loading with ligand are needed

• the maximum permissible number of successive extractions is somewhat limited by the accumulation of experimental errors

Successive Additions

• allows the study of binding parameters with a single solution of receptor• permits the investigation of a much wider concentration range

• experimental errors may accumulate both in the loading and extraction steps

Chlorhexidine in Saliva -Pharmacokinetic Study

0.E+00

5.E-04

1.E-03

2.E-03

2.E-03

3.E-03

0 1 2 3 4 5 6 7 8 9

time (h)

Cm (m

olar

)

0

50

100

150

200

250

300

350

0 1 2 3 4 5 6 7 8 9

time (h)

Ct (

µg/m

L)

0.00.10.20.30.40.50.60.70.80.9

0 1 2 3 4 5 6 7 8 9

time (h)

Cf (

µg/m

L)

Variation of chlorhexidine concentration in time during the pharmacokinetic study (mouthrinsing with 1.0 mg/mL chlorhexidine base):

(a) binding matrix concentration, molar

(b) total concentration of chlorhexidine, µg/mL

(c) free concentration of chlorhexidine, µg/mL

a

b

c

Study of the Kinetics and Equilibria of Chemical Processes

in Investigated Systems

A B

• Partitioning in Multiphase Systems• Chemical Reaction Kinetics• Speciation

Boundary Layer Model

Distance

Con

cent

ratio

n

SampleBoundary LayerFiber

Coating

0

Fiber Core

δ

When Boundary Layer Controls the Extraction Rate

0 1 2 3 4 50

20

40

60

80

100

Ds t / δKfs(b-a)

n / n

∞[%

] ( )s

fs

DabK

t−

=δ

3%95

sffs

sffs

VVKCVVK

n+

= 0

ss CADdtdn )/( δ=

0

:For

CVKn

VKV

ffs

ffss

=

>>

Calibration in SPME

Equilibrium Distribution constant K

Diffusion Diffusion coefficient Ds

Reaction rate Reaction rate constant k

Exhaustive Volume Vs

Estimation of the Calibration Parameters (K)

•Empirical Formula•Chromatographic Behavior•Experimental

-External Calibration Curve-Internal Calibration

-Standard Addition-Internal Standard-Standards in Extraction Phase

Standards in Extraction Phase

sffs

sffs

VVKCVVK

n+

= 0

=

Standards on the Fibre

'fC

fC

'sC

sC

xfδ sδ

silica fiber liquid polymer coating

sample matrix

C

'C

δ'fC

fC

'sC

sC

xfδ sδ

silica fiber liquid polymer coating

sample matrix

C

'C

δ

Isotropy:

0nn

0qQ

+ = 1

Results – Validate the Isotropy of Absorption and

Desorption for C18-PEG Fibers

10

=−−

+e

e

e qqqQ

nn Compare the time constant, a (-slope),

for absorption & desorption

The experiment demonstrated that the kinetic calibration is valid for in vivo sampling by C18 fibers

Results – Study of the time constant leads to

simplified kinetic calibration for in vivo sampling

Advancement:1. The time constant, a, is independent of the sample concentration.2. One point calibration method3. Non-deuterated standard for calibration

01020304050607080

0.08 0.5 1 1.5 2 3 4 6 8

Time point for SPME sampling (hr)

Q (p

g)

Diazepam (d5) Nordiazepam (d5) Oxazepam (d5)

)exp( 0

atqQ

−=Desorption:

SPME fibers

stem

syringe pump

Microdialysis

Setup for comparison MD with SPME for in vivo sampling of living plants

Concentrations of carbofuran in the leaves of a jade plant. The sampling time was 20 min after 20 days of the pesticide

application to soil

0

10

20

30

40

50

60

70

80

1 2 3 4Sample position

Con

cent

ratio

n (n

g/m

L)

microdialysis

solid-phase microextraction

Design of the Custom-Made SPME/HPLC Interface

Frame Enlarged

HPLC pump

UV-VIS detector

Narrow boreHPLC column

InterfaceSPME Device

6-Port injection valve

(d)Desorptionchamber

(a)

(b)

(c)

(e)

To columnFrom pump

To column

Fibre SPME Desorption chamber

design

From pump

Flush Line

Chromatographic Tee0.75mm ID

0.03” ID tubing to guide needleSPME assembly needle

Sealing hub

Attachment hub

Polymer coating on fibre hosting wire

fibre hosting wire

fibre hosting wire

0.4mm ID GC ferrule

Standard HPLC nuts/ferrules

Waste

Manual Injection Tee

Valco 3-way stainless steel tee

s.s. tubing 0.03” ID

PEEK tubing 0.005” ID

V/G ferrule, 0.3mm ID

0.005” ss wire with PPY coating on last 15 mm

Analysis of PAH Mix 525 by SPME/HPLC

1

2

3

4

5

6+7

8

9

10

11

1213

12

3

4

5

6+7

8

9

10

11

1213

0 20105 15 min

(a)

(b)

(a) 1 µl loop injection, (b) fiber injection, 7 µm PDMS extraction for 30 min from 100 ppb of each compound spiked into water.Chromatographic Conditions: Column: 25cm x 2.1mm ID, 5µm ODS; flow rate 0.2 mL/min; detection: UV 254 nm; solvent program: CH

3CN/H

2O (80/20, v/v) linear gradient to 100% CH

3CN in 15 min.

Peak identification: 1. acenaphthylene, 2. fluorene, 3. phenanthrene, 4. anthracene, 5. pyrene, 6. benz[a]anthracene, 7. chrysene, 8. benzo[b]fluorathene, 9. benzo[k]fluorathene, 10. benzo[a]pyrene, 11. dibezo[ah]anthracene, 12. indeno[1,2,3-cd]pyrene, 3. benzo[ghi]perylene.

Commercial SPME/HPLC Interface Design (Supelco)

Compression union

Needle guideSeptum piercing needle

SPME fib re holder

Double taperedferrule

SPME fib re

Solventdesorption

chamber

Valve

Waste

Frominterface

Mobile phasefrom pump

HPLCcolumn

Tointerface

Static desorption(no flow)

Valve

Waste

Tointerface

Mobile phasefrom pump

HPLCcolumn

Frominterface

Sampleinjection

Solventfrom

syringe

Sample vial

Buffer tubing

Loop

2

6

1

3

4 5

HPLC Pump

Column

Six-port valve

a) Transfer Line In-tube SPME

b) Standard Loop In-tube SPME

Sample vial

Loop

2

6

1

3

4 5

HPLC Pump

Column

Six-port valve

Buffer tubing

From pumpTo column

In-tube SPME capillary

Six-port valve

Metering pump

Injection loop

AutosamplerInjection needle

Column connector

c) Flush Loop In-tube SPME

In-Tube SPME Approaches

Alternative Capillary Coatings

Affinity coatings:

• Antibodies

• MIP

Commercially Available Tailor Made PhasesGC capillaries:(from J & W)

• DB5

• DB-1701

• DB- 210

• DB WAX

Polarity

Polymer Coatings:

• PPY

(Molecularly Imprinted Polymers)

Applications of In-tube SPME

Analyte/Compound Class Matrix Extraction Capillary

Amphetamines Urine Omegawax 250Antidepressants Urine DB-WAX

Aromatics Water PPYβ-blockers Serum/urine MIP, PPY, Omegawax 250

Benzodiazepines Serum/urine Supelco-Q Plotn-butylphthalate Waste water Packed Zylon® fibers

Carbamate pesticides Water Omegawax 250Catechins/caffeine Tea PPY

Chlorinated herbicides River water DB-WAXtrimethyl/ethyl lead Water Supelco-Q Plot

Ranitidine Pharma. tablet Omegawax 250

16000

12000

8000

4000

00 5 10 15 20 min

Urine (non-spike)

Urine (spike, 2 μg/mL)

1

2

3

4

5

67

8 9

(A)

10000

8000

6000

4000

2000

2(A)

Serum (non-spike)

Serum (spike, 0.2 μg/mL)

13

4

5

6 78

9

0 5 10 15 20 min

Urine: diluted 10x with water, filtered through a 0.45 µm syringe filter

Serum: diluted 10x with water, ultrafiltered with Nanosep centrifugal microconcentrator (3K), 1000g, 20 min

1: nadolol

2: pindolol

3: acebutolol

4: timolol

5: metoprolol

6: oxyprenolol

7: labetalol

8: propranolol

9: alprenolol

Analysis of B-Blockers in Spiked Urine and Serum Samples

40000

20000

0

20000

10000

0

300020001000

0

40000

20000

0

12 3

4

5

1

2 3

4

5

TIC

m/z=276

m/z=218

m/z=260

4 8 12 16 min

4 8 12 16 min

4 8 12 16 min

4 8 12 16 min

1: 5-hydroxypropranolol (20 ng/mL)

2: 4-hydroxypropranolol (50 ng/mL)

3: 7-hydroxypropranolol (50 ng/mL)

4: N-desisopropylpropranolol(20 ng/mL)

5: propranolol(200 ng/mL)

In-Tube Extraction of Standard Propranolol and Metabolites

4 8 12 16 min

4 8 12 16 min

4 8 12 16 min

4 8 12 16 min

TIC

m/z=276

m/z=218

m/z=260

2000

1000

300002000010000

0

1600

1200

800

300002000010000

0

5

5

2

3

4

Serum: diluted 10x with water, ultrafiltered with Nanosep centrifugal microconcentrator (3K), 1000g, 20 min

1: 5-hydroxypropranolol (n/d)

2: 4-hydroxypropranolol (7.0 ng/mL)

3: 7-hydroxypropranolol (1.5 ng/mL)

4: N-desisopropylpropranolol(2.3 ng/mL)

5: propranolol(134 ng/mL)

Analysis of Clinical Serum Sample

In-tube SPME Method Performance

Analyte Detection Limit(ng/mL)

% R.S.D. Linearity

β-Blockers 0.1 – 1.2 0.4 – 8.9 % R2 = 0.99892-100 ng/mL

Amphetamines 0.38 – 0.82 0.6 – 6.8 % R2 = 0.99902-100 ng/mL

42

DEVELOPMENT OF MULTIFIBRE SPME SYSTEM

In-tube SPME offers high degree of automation using HPLC autosampler BUT

SAMPLE PRE-TREATMENT such as filtration, dilution and/or centrifugation is required to prevent blockage of capillary column

LOW-THROUGHPUT – samples are processed serially and kinetics of mass transfer in liquid-phase are slow (long extraction and

desorption times)

PERFORM EXTRACTION AND DESORPTION OF MANY SIMILAR

SAMPLES IN PARALLEL

43

Device that will hold 96 fibres and fit in the centres of wells

of 96-well plate

Low cost coating with good inter-fibre reproducibility

Robotic system to manipulate the device

Agitation method

MULTI-FIBRE SPME USING 96-WELL PLATES

O’Reilly, J.; Wang, Q; Setkova, L.; Hutchinson, J.P.; Chen, Y.; Lord, H.L.; Linton, C.N.; Pawliszyn J.; J.Sep. Sci., 2005, 28, 2010-2022

Well filled with sample

Insert SPME fibre for extraction

Agitate well until equilibrium is reached

Remove fibre from well

Desorb in well filled with solvent

N2

Evaporate solventReconstitute and inject into GC or LC

SPME Multiwell Method

Coating Procedure

Slurry of binding agent and chloroform and particles

Pasteur Pipette Tip

Steel wire

• SEM of bare steel wire (diameter 0.005”)

• SEM of steel wire coated with ADS particles (25 µm)

• Comparison of blank and coated steel wire (digital magnification 60:1)

Immobilization of ADS-Particles on Steel with Binding Agent

47

PAS AUTOSAMPLER

Agitation using two orbital-shakers

Manipulation of SPME device

Solvent-evaporation using N2

Solvent reconstitution and injection into

HPLC port

User-programmable using Concept ™

software

AUTOMATED MULTI-FIBRE SPME

48

MULTI-FIBRE SPME DEVICES

Commercially available pin-tool replicator

Small-dimension SPME fibre (0.014″diameter)

Hollow-tubing PDMS (165 µm) coating

Custom-built multi-fibre SPME device

Large-dimension SPME fibre (0.061″ diameter)

Coated-silica particle coatings (C18 and C16-amide)

Automated robot sampler for high throughput SPME sample preparation

Thin film

50

AUTOMATED MULTI-FIBRE SPME REPRODUCIBILITY

51

SPME METHOD

VALIDATION OF HIGH-THROUGHPUT METHOD FOR BENZODIAZEPINES IN WHOLE BLOOD

PRECONDITIONING30 min

Methanol/water (1/1)

RINSE30 seconds

Purified water

EXTRACTION30 min, 850 rpm

Whole blood or plasma (0.8 mL) + IS

DESORPTION30 min, 850 rpm

Acetonitrile/water (1/1)

LC-MS/MS

TOTAL SAMPLE PREPARATION TIME

~ 100 minutes for 96 samples

52

VALIDATION OF HIGH-THROUGHPUT METHOD FOR BENZODIAZEPINES IN WHOLE BLOOD

50 ng/mL std in whole blood blank whole blood

N

O

L

D D

N

O

L

53

Validation Parameter Diazepam Oxazepam Nordiazepam Lorazepam

LLOQ (ng/mL) 4 4 4 4

Accuracy and precision at LLOQ (%)

102(11)

111(17)

105(9)

102(14)

LLOQ S/N RATIO 5 19 16 16Linear 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

VALIDATION OF HIGH-THROUGHPUT METHOD FOR BENZODIAZEPINES IN WHOLE BLOOD

54

BINDING STUDIES USING SPME• Determine time required to reach equilibrium (30 min in this study)

• Prepare standard solutions containing different amounts of ligand and constant amount of receptor

• Perform SPME and analysis to determine the amount of analyte extracted by the fibre (m)

• Obtain fibre constant (fC) by calibration using standard solutions containing no receptor

• Calculate free concentration of ligand (Cf) cfm

fC =

st V

mmC −= 0Calculate total concentration of ligand (Ct)

Fit the results to the appropriate binding model and obtain binding parameters

Musteata F. M.; Pawliszyn J., J. Proteome Res. 2005, 4, 789-800.

55

COMPARISON OF LITERATURE VALUES

Technique Diazepam-HSA binding parameters

Automated SPME-LC K1=9.1x105±3x105 L/mol n1=1

SPME-GC K1=1.02x106 L/mol K1=1.23x106 L/mol

n1=1

SPME-LC K1=1.76x106±6.32x104 L/mol n1=1

Equilibrium dialysis K1= 17.49x105 ±6.26x105 L/mol n1=1

Capillary electrophoresis – frontal analysis

K1= 3.2x104 L/mol n1=1.6

K1= 2.1x105 L/mol n1=1.5

56

SPME METHOD (PRE-EQUILIBRIUM)

VALIDATION OF HIGH-THROUGHPUT SCREENING METHOD FOR ANALYSIS OF OCHRATOXIN A IN URINE

RINSE30 seconds

Purified water

EXTRACTION 60 min, 850 rpm

0.5 mL Urine + IS + 0.5 mL buffer pH 3.0

DESORPTION15 min, 850 rpm100% Methanol

LC-MS/MS

TOTAL SAMPLE PREPARATION TIME

~ 80 minutes for 96 samples

SPME extraction phase: carbon tape

57

Validation Parameter

Ochratoxin A – Summary of Validation results

LOD (ng/mL) 0.3LLOQ (ng/mL) 0.7Linear 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)

HIGH-THROUGHPUT SCREENING METHOD FOR ANALYSIS OF OTA IN URINE

GOOD SENSITIVITYSIMPLERAPID

EXCELLENT QUANTITATIVE RESULTS WITH

SINGLE-USE CARBON-TAPE FIBERS

SPME/MALDI Plate

Analytical Approaches

InvestigatedSystem

Field Sampling

InvestigatedSystem

On-Site SamplingSample Preparation

InvestigatedSystem

On-Site Analysis

Lab

Lab

FieldAnalyticalInstrument

Lab Analysis

Lab SeparationQuantification

Sample representative?Sample change?Time?

Representative?Sample loss?Time?

Sensitivity?

In Vivo Analysis with Generic Sorbent Probes

analysis of antibiotics in anaesthetized pigs

analysis of benzodiazepines in awake dogs

Samplers for In-Vivo SPME

For rats

For dogs

High-throughput Desorption

• Modified 96-well plate for on site sampling, 1: 96-well plate, 2: silicon compression mat, 3: PTFE block with stainless steel clip, 4: sampling device.

SPME device placed into sample

Remove fibre, rinse

Desorption: 0.5 – 1 mL MeOH + IS 5 min

N2

dry wells

Reconstitute in 50 μL MeOH/H2O, inj. 20 μL(LC-MS/MS)

Optimized Method

SPME in vivo

SPME in vitro

Multiwell SPME Sampler

Beagle during SPME probe extraction

Dog is free to move around with probes in place

Probes in vein via cephalic catheter

The last 1.5 cm of probe (coated section) is exposed to flowing blood in vein. During sampling the probes are taped to the white pad, and area is wrapped in ‘Vetrap’(tm)

to prevent movement.

In vivo SPME study on rodents: placement of SPME devices and interface connection to the

carotid artery

Validation – A simple Chemical Method to Analyze Drugs in Whole Blood Sample (in vitro)

Averaged benzodiazepines’ profiles from pharmacokinetic studies (n = 3). Blood draws via the same cephalic catheter. RSDs are within 7%. The results were used for cross-validation with those from the SPME sampling and good agreement was observed.

Method: 500 uL of methanol was applied to 100 uL of whole blood sample to denature the proteins and release all drugs into methanol, vortex it and centrifuge @ 10000g for 10 min. Collect the supernatant and evaporate it, then redissolve in 25 uL of solvent for detection by LC/MS/MS.

Conventional analysis of blood sample

-200

0

200

400

600

800

1000

1200

0 2 4 6 8 10

Time (hr)

Conc

entra

tion

(ng/

L)

DiazepamNordiazepamOxazepam

Blood concentration by ISF (In Vivo sampling)

(200)

-

200

400

600

800

1,000

1,200

0 2 4 6 8 10

Time (hour)

bloo

d co

ncen

tratio

n (n

g/m

L)

DiazepamNordiazepamOxazepam

Blood = SPME extraction from whole bloodPlasma = Standard analysis of plasmaSOF-PPY = Standard in the fiber method with PPY coatingsSOF-PEG = Standard in the fiber method with PEG coatings

Diazepam Pharmacokinetic Profile

Location of SPME fiber used for in vivo sampling in the dorsal-

epaxial muscle of a rainbow trout

Two sewage treatment plants outfalls versus sampling sites in Grand River watershed

2km kmkm kmkm

N

Reference Site:Upstream Control

Sampling Location:Downstream of Outfall for Plant A

Outfall for Plant A

Outfall for Plant B

Sewage Treatment Plant Outfall

Municipal Drinking Water Intake

Direction of water flow (approx. north→south)

Sampling Location:Downstream of Outfall for Plant B

Diltiazem concentrations in the muscle of wild fish collected below Sewage Treatment Plant using SPME for field in vivo sampling

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

whitesucker-1

whitesucker-2

whitesucker-3

johnnydarter-1

johnnydarter-2

johnnydarter-3

Fish

Dilt

iaze

m c

once

ntra

tion

in m

uscl

e (p

g/g)

SPME Databasehttp://www.spme.uwaterloo.ca/