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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 Womens 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

TimeWaiting
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
LiquidLiquid
q
q
Microextraction
(DLLME)
Based upon a three
three-component
component solvent system
system.
Extraction vessel is usually a centrifuge tube
Mixture of immiscible organic
g
extraction solvent ((e.g.10s-L
g
of
tetrachloroethylene) and a dispersive solvent (e.g. 1-mL
acetone) injected rapidly into an aqueous solvent (~ 5 mL) with a
syringe.
Forms cloudy mixturedue to finely dispersed extraction
solvent droplets
Extraction is instantaneous; no shaking is needed
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 2730, 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 2730,
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 2730, 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-L 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 2730, 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, Mercks 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
methodaccuracy, precision, linearity, carryover, matrix effect,
recovery and selectivityin less than a day
LC-MS/MS
MS/MS was used for analysis
LC
Only 325-L/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 NH4OAca 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

extraction
clean-up

quantitation
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

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.

-Lactam Analysis

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)


Adsorbent sealed in pipette tip but loosely

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)

Samples as small as 3.6-uL


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-wont deactivate/dewet if dried out
Wide pH range
rangecan
can use more dramatic washing conditions for interference
removal and elutions
Sphericalhomogeneous 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

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


-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--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 L 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

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

1000

2000

3000

Number of Publications
* University of Waterloo, Waterloo, ON, Canada
Agilent Technologies

4000

Gerstels 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-L


SBSE ~ 25-125-L (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 m 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

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


With tandem LC-MS and GC-MS systems, sample prep steps may be reduced
(e.g. QuEChERS, salting-out LLE)
New formats handle smaller samples for LLE and SPE and are amenable to
automation; chip-based SPE miniaturization is now available
Polymeric SPE sorbents bring many advantages including higher capacity, reduced
bed mass and more rugged performance
Specialty phases like mixed mode SPE, MIPs, immunoaffinity, RAMs can be used
to remove specific interferences or specific analytes
SPME and SBSE are into their next generation formats
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

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