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Trends in Sample Preparation for
Chromatography
P
Presented
t d by
b
Ronald E
E. Majors
Agilent Technologies
Wilmington,
g
DE
USA
© Agilent Technologies
© Agilent Technologies
Sources of Error Generated During
Chromatographic
Analysis
Sources of Error Generated During
Chromatographic Analysis
Introduction
6.0% (6%)
Sample Introduction
Contamination
(4%) Sample
Contamination 4.0%
Integration 6.0%
Chromatography (7%)
Columns 11.0%
Columns (11%)
C
Chromatography
7.0%
Integration (6%)
Instrument
Instrument
(8%)
8.0%
Operator
19.0%
Operator (19%)
Calibration
(9%)
9 0%
Calibration 9.0%
30.0%
Sample
Processing(30%)
Sample
Processing
(R E Majors,
(R.E.
Majors LC/GC Magazine,
Magazine 2002)
© Agilent Technologies
Time Spent on Typical Chromatographic
Analysis
Time Spent on Typical
Chromatographic Analysis
Data Management 27.0%
Data Management (27%)
Collection
6.0% (6%)
Collection
Analysis 6.0%
Analysis (6%)
Sample Processing
61.0%
Sample Processing
(61%)
(R E Majors,
(R.E.
Majors LC/GC Magazine.
Magazine 2002)
© Agilent Technologies
Tribute to Academics Working Primarily
in the Area of Sample Preparation
• K.-S. Boos, University of Munich, Germany
• L.G. Blomberg,, Karlstad Univ., Sweden
• U. Brinkmann (emeritus), H. Irth and H. Lingeman, Free Univ. of
Amsterdam, The Netherlands
• M.F. Burke (emeritus), Univ. of Arizona, USA
• J. Haginaka, Mukogawa Women’s Univ., Japan
• M.-C. Hennion, V. Pichon, ESPCI, France
• J.A. Jonsson and L. Mathiasson (emeritus), Univ. Lund, Sweden
H K Lee
Lee, Univ.
Univ of Singapore,
Singapore Singapore
• H.K.
• J. Pawliszyn, Univ. of Waterloo, Canada
• S. Petersen-Bjergaard and K.E. Rasmussen, U. Oslo, Norway
• B. Sellergren, Tech. Univ. Dortmund, Germany
• Countless others who have devoted research to sample prep
© Agilent Technologies
Outline of Sample
p Prep
p Presentation
• Overview of Trends
• Liquid-liquid extraction
• Solid-phase extraction
• Formats
F
• Chemistries
• Automation
© Agilent Technologies
Trends in Sample Preparation
TREND
Smaller samples
EXAMPLES
•Life sciences, proteomics studies
encountered
IMPLICATION
Lower bed mass, less solvent, faster results,
less evaporation time in SPE
Simpler methods--“Just
•Protein crashing instead of SPE
Cuts down on # of sample prep steps (e.g.
enough” sample prep
•QuEChERS instead of lengthy multi-
less error (better data), faster results, higher
step processes
recovery)
•Simple
Simple LLE
Continued growth of MS-MS for LC, CE and
GC applications
“Greener” approaches
•Reduced use of organic solvent (e.g.
Better for environment, less exposure of
SPME, hot water extractions)
workers to toxic solvents, lower purchase and
disposal costs
High throughput
More selectivity
•SPE pipette tips, 96-well plates
Seamless integration to analysis
• On-line extraction
Favors different formats more attuned to
•Multi-functional autosamplers
automation (e.g. pipette tips, 96-well plates)
•Immunoaffinity sorbents
For use with less specific and less sensitive
MS-MS
MS (e.g. UV)
•Molecularly imprinted polymers (MIPs) detectors than MS
•Molecularly-imprinted
•RAMs
Improved chemistries
© Agilent Technologies
•Mixed mode sorbents
Higher capacity
•Polymeric
P l
i SPE packings
ki
M
More
rugged
d phases
h
Time Consuming and Laborious LiquidLiquid Extraction
Typical Separatory Funnels
Manual labor
labor–
vigorous shaking
Time—Waiting
for layers to
separate
© Agilent Technologies
New Formats for LLE
• Microextractions
— Liquid
q
p
phase microextraction ((LPME))
— Single drop microextraction (SDME)
— Dispersive liquid-liquid microextraction (DLLME )
• Microextractions with addition of hollow fiber membranes
— Supported
pp
liquid
q
membranes ((SLM))
— Electromembrane extraction (EME)
• Supported Liquid Extraction (SLE)
© Agilent Technologies
Schematic of a Single Drop LPME Apparatus
Stir bar
© Agilent Technologies
LPME Can Be Automated
© Agilent Technologies
Dispersive
p
Liquid–Liquid
q
q
Microextraction
(DLLME)
ƒ Based upon a three
three-component
component solvent system
system.
ƒ Extraction vessel is usually a centrifuge tube
ƒ Mixture of immiscible organic
g
extraction solvent ((e.g.10’s-µL
g
µ of
tetrachloroethylene) and a dispersive solvent (e.g. 1-mL
acetone) injected rapidly into an aqueous solvent (~ 5 mL) with a
syringe.
ƒ Forms cloudy mixture—due to finely dispersed extraction
solvent droplets
ƒ Extraction is instantaneous; no shaking is needed
ƒ Mixture is centrifuged (e.g. 1.5 min at 6000 rpm) and extraction
solvent sedimentates to bottom of tube and is removed with
syringe
© Agilent Technologies
Extraction of Pesticides from Water
using DLLME: Extraction Solvent
Selection*
*S
S.S.
S Caldas,
Caldas F.P.
F P Costa,
Costa and E
E.G.
G Primel
Primel, XII Congresso Latino-Americano
Latino Americano de Chromatografia E
Technicas Relacionadas (Colacro XII), Florianopolis, Brazil, October 27–30, 2008, Poster Tu-145
© Agilent Technologies
Optimization of Volume of Extraction
Solvent in DMLLE of Pesticides in Water
Water*
*S.S. Caldas, F.P. Costa, and E.G. Primel, XII Congresso Latino-Americano de Chromatografia E
Technicas Relacionadas (Colacro XII), Florianopolis, Brazil, October 27–30,
27 30, 2008, Poster Tu-145
Tu 145
© Agilent Technologies
Investigation on the Type of Dispersion Solvent*
*S.S.
S.S. Caldas, F.P. Costa, and E.G. Primel, XII Congresso Latino
Latino-Americano
Americano de Chromatografia
E Technicas Relacionadas (Colacro XII), Florianopolis, Brazil, October 27–30, 2008, Poster Tu145
© Agilent Technologies
Overall Results of DLLME Study of
Pesticides from Water*
Water
• The variables in method development included: choice of
dispersion solvent and its volume, choice of extraction solvent
and its volume, pH if necessary, and centrifuge speed
• Volume of water: 5 mL containing phosphoric acid
acid, pH 2
• Extraction Solvent: 60-µL of carbon tetrachloride
• Dispersive solvent: 2 mL of acetonitrile
• For all three pesticides, the linear range was found to be 0.0011.0 mg/L and limit of quantitation (LOQ) was 0.02 mg/L.
• Overall, the DLLME technique is simple, fast, provides good
recovery, is low cost, and provides good enrichment factors.
*S.S. Caldas, F.P. Costa, and E.G. Primel, XII Congresso Latino-Americano de Chromatografia E
Technicas Relacionadas (Colacro XII), Florianopolis, Brazil, October 27–30, 2008, Poster Tu-145
© Agilent Technologies
Microscale Automation of Sample Prep
using Modern Autosampler (Agilent
7693A)
Example, LLE using 2-mL vial
© Agilent Technologies
Steps in Supported Liquid-Liquid Extraction
1) Apply aqueous sample so that it permeates no more than 75% of the bed height of the column
2) Wait 5-15
5 15 minutes
3) Apply a suitable water immiscible organic solvent and collect the effluent
(Courtesy of Biotage)
Product Examples: Varian Hydromax, Biotage Isolute HM-N, Merck’s Extrelut
© Agilent Technologies
Salting Out LLE
• Addition of an inorganic salt into a mixture of water and a watermiscible organic solvent causes a separation of the solvent from
th mixture
the
i t
and
d fformation
ti off two-phase
t
h
system
t
• Sometimes referred to as “salt-induced phase separation”
g acetone,, MeOH,,
• Occurs with manyy common solvents ((e.g.
EtOH, acetonitrile, etc.)
• Salt and salt concentration causes different degree of phase
separation
• Also occurs with saccharide addition (“sugaring out”!) and with
water-soluble polymers with salt addition.
• Useful for polar (as well as non
non-polar)
polar) analytes and often used
for partitioning of metal chelates and other metal complexes
© Agilent Technologies
Recoveries of Nitroaromatics, Nitramines, and Nitrate
Esters from Water by Salting-Out
Salting Out LLE (%)
(%)**
**G.M. Nikolic, J.M. Perovic, R.S.
Nikolic, and M.M. Cakic, Physics,
Chemistry
y and Technology
gy 2 ((5),
) 293299 (2003).
© Agilent Technologies
Salting-out LLE of Pharmaceutical in
Biofluids with Acetonitrile*
Acetonitrile
• Abbott Laboratories authors investigated automated salting-out
LLE for Aids drug Kaletra in human plasma; consists of two
compounds
d Rit
Ritonavir
i and
dL
Lopinavir
i
i
• MgSO4 was used for salting out; ACN was organic solvent
g 96-well p
plate was used to develop
p GLP analytical
y
• A single
method—accuracy, precision, linearity, carryover, matrix effect,
recovery and selectivity—in less than a day
LC-MS/MS
MS/MS was used for analysis
• LC
• Only 325-µL/well (sample + salt solution + internal standard +
organic solvent) was used for entire assay
• Recoveries were in range of 62
62-84%
84% across lots,
lots species
species, and
concentration levels;
• % CV was 2.7-6.5% for Lopinavir and 3.3-6.5% for Ritonair
• In recent publication, authors used NH4OAc—a MS friendly salt
as salting-out reagentfor hydrophobic drug candidate &
metabolite in human plasma
* J. Zhang et al, Biomed. Chromatogr. 23, 419-425 (2009)
© Agilent Technologies
QuEChERS* (Pronouced “Catchers”)
A Low Cost, Highly Effective Sample Preparation Technique for
Multiclass, Multiresidue Analytical Approach for Pesticides in Foods
¾ extraction
¾ clean-up
¾ quantitation
¾ confirmation
QuEChERS
method
GC-MS/MS
LC-MS/MS
Quick
Easy
Cheap
Ch
Effective
Rugged
Safe
*M. Anastassiades,, S. J. Lehotay,
y, D. Stajnbaher,
j
, F.J. Schenck,, Journal of AOAC
International (JAOAC) 86, p. 412-431, 2003
© Agilent Technologies
Flow Diagram of QuEChERS Process
Step 1
Weigh 10 g of sample into a 50-mL Centrifuge-Tube (1)
Step 2
Add 10-mL of Acetonitrile (2)
Extraction
Step 1
Shake vigorously 1 min (3)
Add 4
4-g off MgSO
M SO4 & 1
1-g off NaCl
N Cl (4)
Step 3
Shake vigorously 1 min (3)
Add ITSD Solution
S l ti (5)
Step 4
Shake 30 s and Centrifuge (3,6,7)
St 5
Step
T k aliquot
Take
li
t & add
dd M
MgSO
SO4 (and
( d sorbent)—dSPE
b t) dSPE (8)
Shake 30 s and Centrifuge(9,10)
Step 6
[Add 0.1%
0 1% acetic acid and “analyte
analyte protectants”
protectants (11)]
Step 7
Analyze
y by
y GC-MSD or LC-MS/MS
© Agilent Technologies
Dispersive
SPE Step 2
(co rtes of Ste
(courtesy
Steve
e Lehota
Lehotay, USDA)
Pictorial Representation of the QuEChERS
Sample Prep Process
© Agilent Technologies
Pictorial Representation of the QuEChERS
Sample Prep Process
© Agilent Technologies
Step One: Salting-out LLE
Three Methods Currently Practiced:
1) Original unbuffered method
2) AOAC 2007.01 buffered method
(US)
3) EN15662 method (Europe)
4) All three methods in rest of world
© Agilent Technologies
Step 2:
Dispersive SPE Kits
for QuEChERS
© Agilent Technologies
QuEChERS Advantages
9 A batch of 6-12 extracts can be prepared in 3040 min by a single analyst with ≈$1-3 of
disposable materials per sample and generate
<12
12 mL
L solvent
l
t waste.
t
9 Consistently high recoveries (mostly 90-110%
with
ith RSD
RSDs < 5%) off a wide
id range off GC
GC- and
d LCLC
amenable pesticides are achieved from many
matrices.
matrices
(courtesy
(cou
tesy of
o Steve
Ste e Lehotay,
e otay, USDA)
US )
© Agilent Technologies
Recoveries of 15 Pesticides in Different Matrices
120%
n=40
n=31
Rec
covery
100%
n=43
n=45
n=43
n=45
n=43
n=43
80%
60%
40%
20%
on
d
Al
m
M
ilk
e
Fo
lia
g
n
be
a
So
y
Pe
tF
oo
d
ry
D
C
or
n
O
at
s
lfa
s
Al
fa
M
ol
as
se
Si
la
ge
0%
No differences were found vs. matrix for individual pesticides
or concentration
(courtesy of Steve Lehotay, USDA)
© Agilent Technologies
Analysis
y
of 17 Representative
p
Pesticides in
Apple by QuEChERS (AOAC Buffered
method)) with the Detection by
y LC/MS/MS
17 Representative Pesticides Information
Pesticide
Category
Pesticide
Category
Pesticide
Category
Acephate
Organophosphate
Dichlorvos
Organophosphate
Thiabendazole
Benzimidazole
Carbaryl
Carbamate
Imidacloprid
Neonicotinoid
Carbendazim
Benzimidazole
Cyprodinil
Anilinopyrimidine
Penconazole
Diazinon
Organophosphate
Dichlofluanid
Sulphamid
© Agilent Technologies
Methamidophos Organophosphate
Thiophanatemethyl
Benzimidazole
Tolyfluanid
Sulphamide
Triazole
Ethoprophos
Organophosphate
Propoxur
Carbamate
Kresoxim methyl
Kresoxim-methyl
Strobilurin
Pymetrozine
Pyridine
Apple Matrix Blank
© Agilent Technologies
Chromatogram of Apple Sample Spiked with 5ng/g (5ppb)
Of 17 Pesticides
© Agilent Technologies
Apple AOAC Recovery and Repeatibility
Results ((n = 6))
Pesticides
Low QC (5ppb)
Mid QC (50ppb)
High QC (200ppb)
recovery
RSD % (n=6)
recovery
RSD % (n=6)
recovery
RSD % (n=6)
Methamidophos
77 57
77.57
5 03
5.03
91 79
91.79
2 75
2.75
91 22
91.22
5 54
5.54
Acephate
78.36
4.11
89.06
4.58
97.24
4.94
Pymetrozine
70.67
5.75
77.20
11.54
88.06
12.53
Carbendazim
Ca
be da
83.13
83
3
6.29
6
9
94.05
9
05
4.34
3
91.39
9
39
2.78
8
Imidacloprid
96.16
4.79
90.03
2.76
97.41
1.85
Thiabendazole
70.07
4.84
80.20
2.86
91.28
3.73
Dichlorvos
96.73
6.13
90.97
2.85
85.03
0.95 *
Propoxur
95.23
1.68
94.86
2.33
95.08
2.98
Thiophanate methyl
102.17
13.53
79.47
13.43
89.33
3.74
Carbaryl
94.56
3.56
99.94
2.44
104.48
2.05
Ethoprophos
100.88
2.56
97.93
2.15
92.71
2.09
Penconazole
110.97
2.80
96.93
1.79
102.62
2.63
Cyprodinil
98.90
4.01
93.48
1.54
95.08
2.02
Dichlorfluanid
77.38
9.84
76.29
11.98
89.72
8.71
Diazinon
133.53
0.74
116.50
1.83
108.14
2.26
Kresoxim methyl
97.13
2.28
90.31
2.03
97.25
2.00
Tolyfluanid
114.08
4.77
92.63
7.86
101.37
5.48
© Agilent Technologies
* n = 3.
β-Lactam Analysis
β
y
in Beef Kidney
y
1 g sample in a 50 mL centrifuge
FEP tube tube
add internal standards (PENV,
(PENV CEFD)
add 2 mL water + 8 mL acetonitrile
vortex briefly, shake for 5 min
centrifuge for 5 min at 3450 rcf
supernatant + 500 mg C18 sorbent
mix for 30 s
centrifuge for 1 min at 3450 rcf
evaporate 5 mL supernatant to 0.5 mL
filter 0.5 mL extract with the Mini-UniPrep
Mini
-UniPrepTMTM
LC-MS/MS analysis
Slide adapted from Kate Mastovska, USDA-ARS
© Agilent Technologies
QuEChERS for Acrylamide in Foods
1 g sample in a 50 mL FEP tube
add d3-acrylamide
acrylamide at 500 ng/g
add 5 mL hexane, vortex
add 10 mL water + 10 mL MeCN
+ 4 g MgSO4 + 0.5 g NaCl
shake vigorously for 1 min
centrifuge for 5 min at 3450 rcf
discard the hexane layer
1 mL of the upper layer
+ 50 mg PSA + 150 mg MgSO
4
mix for 30 s
centrifuge
t if
for
f 1 min
i att 3450 rcff
© Agilent Technologies
Slide adapted from
Kate Mastovska,
USDA ARS
USDA-ARS
Extraction/Cleanup for
Acrylamide in Foods
Centrifuge Tube
discard ...... removal of fat
hexane layer
MeCN layer
matrix
take 1 mL aliquot for
dispersive SPE cleanup
water layer
excessive salts
Slide adapted from Kate Mastovska,
USDA-ARS
© Agilent Technologies
Status of QuEChERS
• After AOAC International interlaboratory trials were
s ccessf l and the method is no
successful,
now AOAC
International Official Method 2007.01. In Europe,
Official EN1566.2
EN1566 2 has been published
published.
• Many laboratories have implemented the method
successfully
f ll for
f 200-350
200 350 pesticides
ti id iin ffood
d and
d
lowered costs (4-fold faster, less labor, lower cost).
•Commercial products for QuEChERS have been
introduced
• The streamlining features of QuEChERS are being
used in more applications
pp
(acrylamide;
(
y
; vet. drugs).
g )
© Agilent Technologies
Most Popular SPE Formats: SPE
Cartridges and Disks
Typical SPE Disk Configuration
polypropylene
packing
© Agilent Technologies
frits
PTFE
Disk
or fiberglass
Pre-filter
(Optional)
Solid Phase Extraction Pipette Tip*
* Designed to work with x-y-z liquid handling
systems without needed for modification
*C
Can also
l b
be used
d manually
ll
* No vacuum manifold required
* Flow can be bi-directional
* Disposable, no carryover or cross-contamination
* Recommended for small samples (less than 100-uL)
* Examples:
Agilent Cleanup C18 Pipette Tips
Varian SPEC Plus PT
Millipore ZipTip
DPX Disposable Pipette Extraction
© Agilent Technologies
* Ansys SPEC Plus PT
Disposable Pipette Extraction (DPX)
¾ Adsorbent sealed in pipette tip but loosely
¾
¾
¾
¾
¾
¾
packed
High efficiency extractions
Extractions are rapid (<3 minutes)
Extraction efficiency is flow rate
independent
Use less sorbent, so less solvent
is required
Minimal solvent waste generated
Readily automated
•William E. Brewer, US patent 6,566,145 B2
(courtesy of DPX Labs)
© Agilent Technologies
•Application number of PCT/US08/54584
41
Basics of DPX Extraction Technology
© Agilent Technologies
(courtesy of DPX Labs)
GC/MS Analysis of NIDA-5 Drugs of Abuse using DPX
P
Pre-extraction
t ti
(courtesy of W. E. Brewer, Clemson Veterinary Diagnostic Center and DPX Labs)
© Agilent Technologies
Industry Example - Gerstel Automated
SPE & DPX
• CTC PAL base hardware
• Gerstel
G
l custom SPE module
d l
• LC (LC/MS), GC (GC/MS)
• Maestro Software/interfaces
to ChemStation
• Industry
I d t Standard
St d d SPE Format
F
t
© Agilent Technologies
44
MICRO EXTRACTION BY PACKED
SORBENT (MEPS)
ƒ Samples as small as 3.6-uL
ƒ Can be automated; SPE
steps and injection in
same device
(
(courtesy
t
off SGE)
© Agilent Technologies
Schematic Diagram
g
of 96-Well Extraction
Plate System
Extraction
plate
Polyethylene manifold lid
Polypropylene
manifold
To vacuum
base
pump
O i
O-ring
On / off valve
(in off position)
Collection plate
Vacuum Gauge
Needle valve
((courtesy
y of Agilent)
g
)
© Agilent Technologies
Comparison of Protein Precipitation and SPE
Cleanup of Plasma Using 96-Well Plate Format—
Oasis uElution Plate
(courtesy of Waters)
© Agilent Technologies
LC/MS runs using water
water-ammonia
ammonia
Gradient on Xterra MS column
Chemistries of SPE
© Agilent Technologies
Advantages of Polymers in SPE
((relative to Silica-based))
• Water-wettable-won’t deactivate/dewet if dried out
• Wide pH range
range—can
can use more dramatic washing conditions for interference
removal and elutions
• Spherical—homogeneous packed bed and reproducible flows
• High surface areasareas 600-800
600 800 m2/g; higher loading capacity; can reduce sorbent
bed volumes, less solvent, less sample
• No acidic silanol sites
• Higher retention of polar compounds due to frequent mixed mechanisms
(lipophilic-hydrophilic balanced); allows development of “generic” methods
Disadvantages of Polymers in SPE
• Not as many phase chemistries
• More expensive
© Agilent Technologies
Polymeric SPE Recovery Performance-Dry vs.
Wet Sorbent
140
120
100
80
60
DRY WET
DRY WET
DRY WET
DRY WET
DRY WET
DRY WET
DRY WET
40
20
0
acetaminophen
p
brompheniramine
p
propranolol
1
doxepin
p
mianserin
SampliQ OPT (Agilent Technologies)
© Agilent Technologies
fluoxetine
Dihydroxy
y
y
naphthalene
Selective Chemistries in SPE
• Mixed
Mi d Mode
M d Phases
Ph
(both
(b th silicaili
& polymer-based)
l
b
d)
Phases that contain two (or more) types of functional groups to
allow multiple chemical interactions for selectivity and wettability
purposes
- SAX and PS-DVB
- SAX and SCX (removal of ions when interested in
neutral compounds)
- C18 and SCX
- C18 and SAX
-C
C18
8a
and
d WAX
- C8 and SCX (mainly for drugs of abuse)
- DVB and hydrophilic (drugs in biol. Fluids)
- PS-DVB
PS DVB and
dh
hydrophilic
d
hili (d
(drugs in
i biol.
bi l Fluids)
Fl id )
- RPC and Cyano (matrix removal, alternative to
protein ppt)
- DVB-amide and SCX
© Agilent Technologies
Immunoaffinity Phases for Sample
Preparation
© Agilent Technologies
High Abundance Proteins in Human Plasma
α-1-antitrypsin
p 3.8%
Immunoglobulin G
16.6%
Immunoglobulin A 3.4%
Transferrin 3.3%
1DGE/2DGE
Haptoglobin 2.9%
Other 15%
Albumin
54.3%
Analysis and Identification
Protein Expression
Drug Targets
Disease Markers
© Agilent Technologies
Depletion of High Abundance Proteins in Human
Serum/Plasma
Anti-albumin resin
• Affinity purified polyclonal
antibodies bound to resin.
• Mixed resin bed for simultaneous
removal all six proteins
from serum
• Currently up to 20 proteins can be
depleted
• Robust chemistry
Anti-transferrin resin
Anti-haptoglobin resin
Anti-α-1-antitrypsin-resin
Anti-IgA resin
Anti-IgG-resin
Individual Ab materials are mixed in
selected percentages and packed into a
column format.
Agilent MARS Column
© Agilent Technologies
Multiple Affinity Removal System
H High-Abundant Proteins
(Albumin, IgG, IgA, Transferrin,
Haptoglobin, Antitrypsin)
L Low
Low-Abundant
Abundant Proteins
(Biomarkers for disease and
drug targets)
• Column and optimized buffers are
used to remove
e o e the
t e top six
s most
ost
abundant proteins in human serum
and plasma samples.
• Attach to HPLC instrument and pump
samples through - proteins of
y
interest are collected and analyzed.
© Agilent Technologies
Multiple Affinity Removal System
H H
L HL L
H L H
LL H
H H LH
Crude
C
d Human
H
Serum
H High-Abundant Proteins
(Albumin, IgG, IgA, Transferrin,
Haptoglobin, Antitrypsin)
L Low
Low-Abundant
Abundant Proteins
(Biomarkers for disease and
drug targets)
• Column and Optimized buffers are
used to remove
e o e the
t e top six
s most
ost
abundant proteins in human serum
and plasma samples.
• Attach to HPLC instrument and pump
samples through - proteins of
y
interest are collected and analyzed.
© Agilent Technologies
Multiple Affinity Removal System
H H
L HL L
H L H
LL H
H H LH
Crude
C
d Human
H
Serum
LL
L LL LL
Low-Abundant
Proteins
Free from
Interferences
© Agilent Technologies
H High-Abundant Proteins
(Albumin, IgG, IgA, Transferrin,
Haptoglobin, Antitrypsin)
L Low
Low-Abundant
Abundant Proteins
(Biomarkers for disease and
drug targets)
• Column and Optimized buffers are
used to remove
e o e the
t e top six
s most
ost
abundant proteins in human serum
and plasma samples.
• Attach to HPLC instrument and pump
samples through - proteins of
y
interest are collected and analyzed.
Immunoaffinity Column Elution Profile 50 mm column
• Total column run cycle = 20.00 min, for
injection, elution, and regeneration (4.6 x
50 mm column).
l
)
• Capacity =
15-20 µL serum per injection
1.2 - 1.6 mg total serum proteins
• Multiple Affinity Removal column is
reusable, protein binding capacity is
unchanged after 200 injections of serum
Comparison of Run #20 and Run #200
2500
Flow-through,
Low Abundant
Proteins
2000
1500
Bound,
B
d Hi
Highh
Abundant
Proteins
Injection
0.25 mL/min
1000
Elution
1 0 mL/min
1.0
500
1
2
3
4
5
6
Time
(min)
0.00
9.00
9.01
12.50
12.60
20 00
20.00
Flow
%B
rate
0.00
0.250
0.00
0.250
100.00 1.000
100.00 1.000
0.00
1.000
0 00
0.00
1 000
1.000
Re-equilibration
1 0 mL/min
1.0
End run
(20.0 min).
0
0
2.5
5
7.5
10
12.5
Retention Time (min)
© Agilent Technologies
Max.
Max
pressure
120 bar
120
120
120
120
120
15
17.5
Venture AF Immunoaffinity Column for On-Line
Aflatoxin Analysis
(courtesy of Grace Davison Discovery Sciences)
© Agilent Technologies
R t i t d Access
Restricted
A
Media
M di (RAM)
* Developed for analysis of low MW compounds
in complex matrices, especially drugs in biological
fluids.
* Macromolecular sample compounds have limited
y to sorption
p
sites of column p
packing
g and
accessibility
elute at column void volume; small molecules will
diffuse into matrix and interact with stationary phase.
* Can be used off-line, on-line via column switching,or
as an HPLC column alone.
© Agilent Technologies
Typical Structure of Restricted Access Medium
© Agilent Technologies
Use of Coupled Column RAMRPC System for Analysis of Drugs in Plasma
A.
B.
100-uL plasma
Column A: RAM Column
Mobile phase: water, 0.5 mL/min
C l
B:
B LiChrospher
LiCh
h 60 RP S
Select
l tB
BioTrap C8 Column
Mobile phase: Trichloroacetic acid,
Acetonitrile, 0.1%
Triethanolamine,
pH2, 1.0 mL/min
SPS C8
ISRP C8
Peaks:
1. Epirubicinol
2 Epirubicinal aglycone
2.
3. Epirubicin
4. Epriubicin aglycone
5. 7-deoxyepirubicinal aglycone
(compounds in 5.6-8.2 ng/mL range)
LiChrospher RP-4 ADS
A. Rudolphi and K.-S. Boos, LC/GC 15, 814-823 (1997).
© Agilent Technologies
Molecularly
y Imprinted
p
Polymers
y
((MIPs))
© Agilent Technologies
LC Chromatogram Showing Selective Cleanup of
Clenbuterol from Beef Liver using Molecularly
Imprinted SPE Phase*
H
O
Cl
H2N
H
N
C(CH3)3
Cl
Clenbuterol
a) Blank b) Spiked (1-ng/g) beef liver extract
*Ensing, Berggren, Majors, LCGC
© Agilent Technologies
Peaks
1) Clenbuterol
2) Bromoclenbuterol (template
bleeding)
Most Successful Story for Acceptance of New Sample
Prep Method: Janusz Pawliszyn
Pawliszyn* and coworkers
coworkers’ SPME
SPME GC
SPME-GC
SPME-LC
0
1000
2000
3000
Number of Publications
* University of Waterloo, Waterloo, ON, Canada
© Agilent Technologies
4000
Gerstel’s Twister for Stir-Bar Sorptive
Extraction (SBSE)
© Agilent Technologies
Recovery of Solutes as a Function of OctanolWater Partition Coefficients for SBSE and SPME
Volume of coated polydimethylsiloxane: SPME ~ 0.5-µL
SBSE ~ 25-125-µL (10-mm X 0.5-mm)
(Sandra and David, LCGC, 2003)
© Agilent Technologies
Thin Film Microextraction
1. Rotate thin-film as it
is inserted into the
li
liner
Stainless
Steel Wire
Thin-film
2cm
1cm
2. Place cap
onto liner
Gerstel
Twister
Desorption
Liner
3. Place
liner into
MPS2 Tray
Gerstel
GC Liner
with
Coiled
Thin-film
4. Exchange of liner between the tray and
the Twister Desorption Unit (TDU) via the
MPS2 autosampler arm
(courtesy of J. Pawliszyn, June, 2009)
© Agilent Technologies
How does SPME membrane
d i work?
device
k?
100 μm PDMS fiber
Af: 10 mm2
Vf: 0.61mm3
1 cm x 1 cm PDMS membrane
Am:200 mm2
Vm : 2.55 mm3
Ratio of extraction p
phase volume ((Vm/Vf))=4.5
Ratio of extraction phase surface area (Am/Af)=20
(courtesy of J. Pawliszyn, June, 2009)
© Agilent Technologies
Extraction Time Profiles of Fluoranthene by
Twister and Thin-film Coupled to a Drill
Extraction time profile of fluoranthene
E
Extracted
d amount/ ng
140 00
140.00
120.00
100 00
100.00
thin-film
extraction
t i t
twister
extraction
80.00
60.00
40.00
20.00
0.00
0
100
200
300
400
Extraction time/ min
500
(co rtes of JJ. Pa
(courtesy
Pawliszyn,
lis n JJune,
ne 2009)
© Agilent Technologies
Extraction Efficiency Comparison Between PDMS Fiber and PDMS
Membrane (Extraction of Ice Wine Volatile Constituents)
4.0E+07
3 5E+07
3.5E+07
area
a counts
3.0E+07
2.5E+07
PDMS membrane
2 0E+07
2.0E+07
1.5E+07
1.0E+07
5 0E 06
5.0E+06
PDMS-100 fiber
0.0E+00
0
5
10
15
20
(courtesy of J. Pawliszyn, June, 2009)
© Agilent Technologies
25
30
RT (min)
35
40
45
50
55
60
SPME Multiwell Method
Well filled with
sample
Insert SPME fibre
for extraction
Agitate well until
equilibrium is reached
N2
Reconstitute and
Evaporate solvent
inject into GC or LC
© Agilent Technologies
Desorb in well filled
with solvent
Remove fibre
from well
Alternative Approaches to Stir Bar
Sorptive Extraction
a)
Magnet
PDMS
Glass
Glass
stopper
Conventional PDMS Twister
c)
b)
Magnet
Inner phase
PDMS
Dual-Phase Twister
Solvent
PDMS
tubing
Silicone-membrane
S
Sorptive
ti Extractor
E t t
(courtesy of Prof. Carlo Bicchi, Univ. of Torino, Italy, see May 2009 LCGC Magazine)
Summary and Future Directions
ƒ With tandem LC-MS and GC-MS systems, sample prep steps may be reduced
(e.g. QuEChERS, salting-out LLE)
ƒ New formats handle smaller samples for LLE and SPE and are amenable to
automation; chip-based SPE miniaturization is now available
ƒ Polymeric SPE sorbents bring many advantages including higher capacity, reduced
bed mass and more rugged performance
ƒ Specialty phases like mixed mode SPE, MIPs, immunoaffinity, RAMs can be used
to remove specific interferences or specific analytes
ƒ SPME and SBSE are into their next generation formats
ƒ Automation of many sample prep protocools is readily available; sample prep can
be integrated into analytical system
© Agilent Technologies
Acknowledgements
•
•
•
•
•
•
•
•
Gerstel, Dr. Ed. Pfannkoch
J. Pawliszyn,
y Univ. Waterloo,Canada
Agilent, Limian Zhou and Carol Ball Haney
USDA, Steve Lehotay and colleagues
O h
Orachem
Biotage
DPX Labs
SGE
© Agilent Technologies