1. No category

Sample Preparation Using
Solid Phase Extraction
Dr. Shulamit Levin
Analytical Department
Medtechnica
Email: levins@medtechnica.co.il
shulal@zahav.net.il
Tel: 03-9254040
Cell: 052-448632
Fax: 03-9249977
Home page:
http://shulalevin.tripod.com
http://www.forumsci.co.il/HPLC
Based
Based on:
on:
Yung-Fong
Yung-Fong (Henry)
(Henry) Cheng
Cheng
Waters
Corporation
Waters Corporation
34
34 Maple
Maple Street
Street
Milford,
MA
Milford, MA 01757
01757
Present
Present at
at EAS'98
EAS'98 Workshop
Workshop
Outline
Troubleshooting of Sample
Preparation Methods Using
Solid-Phase Extraction
Based
Based on:
on:
Yung-Fong
Yung-Fong (Henry)
(Henry) Cheng
Cheng
Waters
Waters Corporation
Corporation
34
34 Maple
Maple Street
Street
Milford,
Milford, MA
MA 01757
01757
Importance of Sample Preparation
Principle of Solid-Phase Extraction
(SPE)
Typical Problems in SPE
detail steps of SPE
examples
Summary
Present
Present at
at EAS'98
EAS'98 Workshop
Workshop
©Waters 1998
Sample Preparation
Why Sample Preparation?
• Typically the most time-consuming step
Analyte in
matrix
• Typically the most difficult
• Typically the least amount of effort spent
developing a rugged sample preparation
method
Extraction
Magical
Method
Analysis
©Waters 1998
©Waters 1998
Dr. Shulamit Levin, Medtechnica
1
Wouldn't It Be Nice --
Why Perform Sample Preparation?
If We didn't have to Prepare Samples
Remove interferences
e.g. Analysis of drug and metabolite in plasma.
Need to remove protein interferences
Before Injection into the Instrument
Concentrate sample
e.g. Pesticides in drinking water
- Processing Steps needed to get Sample Ready
Before Injecting into the Instrument
^ HPLC
^ GC
^ LC/MS
^ GC/MS
^ AA
^ Others
©Waters 1998
©Waters 1998
Sample Prep Techniques
Method
Liquid-Liquid Extraction (LLE)
Basis for Selectivity
Chemical Technique
• Precipitation
Solubility
• Liquid-Liquid Extraction
Partitioning in one of
two liquid phases
Adsorption/partitioning
onto solid sorbent
Molecular weight/size
Charge
Boiling point/vapor pressure
• Solid-Liquid Extraction (SPE)
• Dialysis / Ultrafiltration
• Electrophoresis
• Distillation/Evaporation
• Supercritical Fluid Extraction
Partitioning into
supercritical fluid
©Waters 1998
Dr. Shulamit Levin, Medtechnica
Where an Immiscible Solvent
is Added to the Sample which
then Separates into 2 Distinct
Liquid Phases. Some Sample
Analytes will go into the
Bottom Phase (Aqueous),
Some will Separate into the
Top Phase, (Organic)
2
Disadvantages of LLE
Advantages of SPE vs.
Other Extraction Techniques
Large solvent consumption
Time/Labor intensive
May require an evaporation step prior to
analysis to remove excess solvent
When one needs to assay for several
analytes, it may be difficult to find proper
solvent/conditions for all analytes,
requiring more than one extraction per
sample
Problematic samples - emulsions
Contamination issues
Cleaner extracts
Easier to automate
Higher recoveries
for polar compounds
©Waters 1998
©Waters 1998
Solid Phase Extraction (SPE)
- Formats and Configurations
Solid Phase Extraction (SPE)
- Chromatographic Particles
- Packed-Bed Column Cartridges
- 1st Commercialized In 1978
- Well Established Technology
- Many Thousands of Literature References
Cartridge
Bed
Bed
Disk
Disk
Coated
Coated
Fiber
96 Well
Plate
Empore™ Disk
SPME
©Waters 1998
©Waters 1998
Dr. Shulamit Levin, Medtechnica
3
Differences Between HPLC and SPE
Solid Phase Extraction (SPE) Technology
Comparison of Efficiency - HPLC vs. SPE
HPLC
SPE
~5 µm
high
low
5-30 cm
~10,000
40-80 µm
low
high
~1 cm
< 50
1
Normalized concentration
Particle size
Packed bed efficiency
Extra-column volume
Column length
Number of plates (N)
Bottom line: HPLC can separate similar compounds. SPE requires
a significant selectivity difference between compounds
for separation. Compounds not well resolved by
HPLC cannot be separated by SPE with a similar
retention mechanism.
HPLC:
higher efficiency
0.8
0.6
0.4
0.2
0
0
5
10
15
20
SPE:
poor efficiency
1
0.8
0.6
0.4
0.2
0
0
©Waters 1998
5
10
Elution volume (mL)
15
20
©Waters 1998
Solid Phase Extraction (SPE) Technology
Solid Phase Extraction (SPE) Technology
Sample Must be in Liquid State
Driving Forces
^ Gravity
^ Pressure
Vacuum Manifolds
^ Vacuum
©Waters 1998
©Waters 1998
Dr. Shulamit Levin, Medtechnica
4
Solid Phase Extraction (SPE) Technology
Manufacturer
Brand Name
Waters
Varian
SEP-Pak
OASIS
BondElute
Baker
BakerBond
International Sorbent
Technology
3M
Isolute
Supelco
Supelclean
Solid Phase Extraction (SPE) Technology
SPE Strategies
• Elute the product of interest, retain interferences
• want k ➠ 0 for analyte
• want k large for interferences
• Elute interferences, retain product
• want k ➠ 0 for interferences
• want k large for analyte
Empore
* Concentrate product of interest
+ Many Others
©Waters 1998
^ want k large for analyte / load large sample volume
^ elute concentrated analyte
^ enhanced sensitivity
©Waters 1998
Solid Phase Extraction (SPE) Technology
Methods Development Approach
Most Common TYPES OF CHROMATOGRAPHY
- Normal
Determine Nature of Analytes, and
Sample Matrix
Similar to Existing Method in Lab?
Phase
^ The "Original" Type - Used By Tswett
^ Non-Polar Mobile Phase
^ Polar Stationary Phase
- Reversed-Phase
Most Common
^ Polar Mobile Phase
^ Non-Polar Stationary Phase
- Ion Exchange
^ Buffer/Ionic Mobile Phase
^ Cationic/Anionic Exchanger Stationary Phase
Try Conditions - Evaluate for
Capacity/ Breakthrough,
Recovery
Reproducibility, Robustness
and Ruggedness
Meets Goals?
Yes
©Waters 1998
Validate
Method
Dr. Shulamit Levin, Medtechnica
No
Yes
No
Yes
Review SPE Bibliography,
and Literature References
for Exact or Similar
Applications
Any?
No
Determine Method
Goals, and Strategy
Call SPE Vendor
Chromatography Mode
Develop Method
Conditions
5
Outline
Solid Phase Extraction (SPE) Technology
Importance of Sample Preparation
Principle of Solid-Phase Extraction
(SPE)
Typical Problems in SPE
detail steps of SPE
examples
Summary
Common Problems in SPE
•
Incomplete Removal of Interferences
•
Low Recovery of Analyte(s)
•
High Variability (RSDs)
©Waters 1998
©Waters 1998
Solid Phase Extraction (SPE) Technology
SPE Procedure
Solid Phase Extraction (SPE) Technology
Step 1 - Sample Preparation
Sample
➀
Prepare: Homogenize, suspend,
centrifuge, etc.
➁
Load onto conditioned cartridge
❶
Prepare: Homogenize, suspend,
centrifuge, etc.
➂
Wash off weakly retained interferences
with weak solvent
➁
Load onto conditioned cartridge
➃
Elute product with strong solvent
➂
Wash off weakly retained interferences
with weak solvent
➃
Elute product with strong solvent
Sample
Analyze: HPLC, GC, etc.
©Waters 1998
Analyze: HPLC, GC, etc.
©Waters 1998
Dr. Shulamit Levin, Medtechnica
6
Sample Pretreatment: Effect of Acid on
Recovery
% Recovery
Solid Phase Extraction (SPE) Technology
Step 1 - Sample Preparation
Typical problems
Analytes
adsorpted to test tube walls
adsorpted to or inclusion in matrix solids
bound to proteins in matrix
Possible solutions
use silanized or plastic test tubes
homogenize more completely
add acid to sample solution
Phosphoric
Acid, 2%
No Acid
No Acid
Compounds
Concentration
[µg/mL]
Saline Sample
Serum Sample
Serum
Sample
Naproxen
1.0
96
4
89
Ibuprofen
10.0
94
19
87
CH 3
OH
H3CO
O
Naproxen
CH3
CH3
H3C
OH
O
Ibuprofen
©Waters 1998
Solid Phase Extraction (SPE) Technology
Step 2 - Sample load
Step 2 - Sample Load
Sample
➀
Prepare: Homogenize, suspend,
centrifuge, etc.
❷
Load onto conditioned cartridge
➂
Wash off weakly retained interferences
with weak solvent
➃
Elute product with strong solvent
Solid Phase Extraction (SPE) Technology
Possible problems
Solutions
•Improper conditioning of
cartridge
•Condition cartridge as appropriate. Do not
let dry, if silica based C18
•Poor analyte retention
•Dilute with weaker solvent, use stronger
sorbent, use larger cartridge
Analyze: HPLC, GC, etc.
©Waters 1998
•Matrix variability
•Buffer sample to constant pH, ionic strength
•Volume overload
•Decrease load volume, use larger cartridge
•Mass overload
•Decrease load volume, use larger cartridge
©Waters 1998
Dr. Shulamit Levin, Medtechnica
7
Incomplete Conditioning of
Cartridges Effect on Recovery:
Incomplete Conditioning of Cartridges
J. D. MacNeil, V. K. Martz, G. O. Korsrud, C. D. C.
Salisbury, H. Oka, R. L. Epstein, C. J. Barnes, J. AOAC
Intl., 79(2) (1996), 405-417
C1818 vs. Oasis® HLB Cartridges
C18 (1cc/100mg)
100
100
80
80
60
60
40
40
20
20
Percent recovery
"Note: Do not dry SPE cartridge
between initial methanol
conditioning wash and completion
of addition of sample and sample
wash. Monitor elutions closely to
ensure that cartridges do not dry."
0
0
4
Drying Time
(minutes)
8
0
HLB (1cc/30mg)
* No Impact of
Sorbent Drying
* No Silanol
Interaction
* No Breakthrough of
Polar Analytes
0
5
Drying Time
(minutes)
Procainamide
Ranitidine
Acetaminophen
Propranolol
10
Doxepin
©Waters 1998
Effect of the Sample pH on Recovery
Load at pH 7
Compounds
Solid Phase Extraction (SPE) Technology
Load at pH <2
Concentration
[µg/mL]
Recovery
(%)
Recovery
(%)
10
62.5
101
Sample Loading
COOH
OH
Salicylic Acid
Normalized concentration
1
Salicylic Acid in Saline
pKa 2.97, 13.4
k=10
0.8
k=20
k=30
0.6
higher k,
less breakthrough
(continuous loading)
0.4
Assume:
N = 40 plates
V0 = 1 mL
0.2
0
0
10
20
30
40
50
Load volume (mL)
©Waters 1998
Dr. Shulamit Levin, Medtechnica
8
Solid Phase Extraction (SPE) Technology
Solid Phase Extraction (SPE) Technology
Step 3 - Wash
Sample
Step 3 - Wash
➀
Prepare: Homogenize, suspend,
centrifuge, etc.
➁
Load onto conditioned cartridge
❸
Wash off weakly retained interferences
with weak solvent
➃
Elute product with strong solvent
Possible Problems
Solutions
•Poor analyte retention
•Use stronger sorbent, use larger cartridge
•Matrix variability
•Buffer sample to constant pH, ionic strength
•Volume overload
•Decrease load volume, use larger cartridge
•Mass overload
•Decrease load volume, use larger cartridge
Analyze: HPLC, GC, etc.
©Waters 1998
©Waters 1998
Effect of Incomplete Wash
Washing Procedure:
Effect of Wash Solvent on Recovery
Interferences
96 inj.
USP Tailing Factor:
1st wash:
40% MeOH,
2% NH4OH
1.67
111 inj.
6.00
5.00
4.00
3.00
Minutes
2.00
0.01 au
1st wash:
40% MeOH,
2% NH4OH
USP Tailing Factor:
1.07
0.00
0.01 au
Cheng
2
minutes
4
6
5% Methanol
in Water
Water
Concentration [µg/mL]
Recovery
(%)
Recovery
(%)
Theobromine
0.5
87
99
Paraxanthine
0.5
67
92
Theophylline
0.5
75
106
Caffeine
0.5
92
105
Compounds
2nd wash:
5% MeOH,
2% HAc.
©Waters 1998
Dr. Shulamit Levin, Medtechnica
9
Solid Phase Extraction (SPE) Technology
Step 4 - Elute
Solid Phase Extraction (SPE) Technology
Step 4 - Elution
Sample
➀
k=0
1
Prepare: Homogenize, suspend,
centrifuge, etc.
k=1
k=2
➁
Load onto conditioned cartridge
➂
Wash off weakly retained interferences
with weak solvent
❹
Elute product with strong solvent
Normalized concentration
0.9
higher retention
larger elution volume
0.8
0.7
0.6
0.5
0.4
Assume:
N = 40
V0 = 1 mL
0.3
0.2
0.1
0
0
1
2
3
4
5
6
Elution volume (mL)
Analyze: HPLC, GC, etc.
©Waters 1998
©Waters 1998
Effect of Elution Solvent on
Recovery and Reproducibility
Methanol
Compound
Testosterone
benzoate
Evaporation and Reconstitution
Methylene Chloride:
Methanol 50:50
Concentration
[µg/mL]
Recovery
(%)
RSD
(%)
Recovery
(%)
RSD
(%)
2.0
92
5.1
102
0.49
6.6
13.3
<0.50
First milliliter of
elution solvent
Second milliliter
of elution solvent
O
Increase Assay Sensitivity
Increase sample concentration
Inject larger sample volume
Improve HPLC Peak Shape
Dissolve in mobile phase or weaker solvent
Disadvantages
O
CH 3
Advantages
Loss of more volatile analytes
Poor solubility
C
CH3
Testosterone Benzoate
O
Dr. Shulamit Levin, Medtechnica
©Waters 1998
10
HPLC Analysis:
Effect of Sample Solvent
0.006
Effect of Evaporation
on Sample Recovery
Sample in MeOH
0.005
Evaporation
to Dryness
0.004
Compounds
Minocycline
AU 0.003
Demeclocycline
Tetracycline
0.002
Concentration [µg/mL]
0.001
0.000
10.0
20.0
Minutes
Evaporation
to 100 µL
Recovery
(%)
RSD
(%)
Recovery
(%)
RSD
(%)
30.0
Benzoic Acid
5.0
62.8
9.1
87.6
3.0
Salicylic Acid
5.0
93.6
5.1
91.3
5.0
Sample in HPLC Mobile Phase
0.006
0.005
(0.1% TFA, 4%ACN and 5%MeOH in Water)
Minocycline
0.004
Tetracycline
AU 0.003
Demeclocycline
0.002
COOH
0.001
COOH
OH
0.000
10.0
Minutes
20.0
30.0
Benzoic Acid
Eliminating the Evaporation and
Reconstitution Step: Effect of Sample Solvent
1
2
0.04
AU
Sample in Water
3
Sample Identification
1.EDDP
2.Diphenhydramine(IS)
3. Methadone
Strategy of Signal-to-Noise (S/N)
Enrichment Comparison of S/N for Dilution (1:3 with
water) of Urine Sample Solution after SPE Extraction
1:3 Dilution
25 uL injection
Column:
5
10
15 Min.
Sample in 80%
MeOH, 2% HAc
0.04
AU
1
4 µg/ml
Woods, Cheng
Salicylic Acid
5
2
3
5 µg/ml
10 µg/ml
10
SymmetryShield™ RP18,
3.5 µm, 3.9 x 150 mm
Guard Column: Sentry™ Guard Column
SymmetryShield RP18,
5µm
Temperature: 30°C
Mobile Phase: 0.1% TFA:Methanol
(60:40)
Detection:
UV at 210 nm
Flow Rate:
1 mL/min
Inj. Volume:
30 µL
Extraction on
Oasis® HLB,
96-well, 10 mg/well
S/N=38
S/N=37
5
10
0.02
AU
15 Min.
1:3 Dilution
S/N=88
S/N=99 50 uL injection
5
S/N=145
2-D SPE Method
10
S/N=164
S/N=89
0.02
AU
15 Min.
S/N=147
1:3 Dilution
100 uL injection
15 Min.
0.02
AU
At this dilution
(1:3 with water);
achieve
- better peak shapes
- higher S/N
Extraction on Oasis® HLB,
96-well, 10 mg/well
2-D SPE Method
©Waters 1998
5
Dr. Shulamit Levin, Medtechnica
S/N=42
10
15 Min.
11
Solid Phase Extraction (SPE) Technology
Generic Reversed-Phase, 1-D,
SPE Method (Oasis® HLB Sorbent)
A One-Dimensional (1-D)
Method- changing only the
percent organic
Prepare Sample Solution
Impact On Today's Analytical Chemist
Condition/Equilibrate
1 mL methanol/1 mL water
1
0%
Load
1 mL spiked sample solution
Faster Method Development
More Sensitive Methods
Shorter Processing Times
Reduced Cost Per Analysis
3
2
5%
% Organic
Wash
1 mL 5% methanol in water
1
100%
Load
no organic to retain analytes
Elute
1 mL methanol
2
Wash
3
Elute
5% MeOH to remove
proteins in matrix
high organic to
elute the analytes
Evaporate and Reconstitute
©Waters 1998
©Waters 1998
Results: Tetracyclines
Compound
Concentration
% Recovery
% RSD
Minocycline
2.5 µg/mL
94.8
1.4
Tetracycline
2.5 µg/mL
104
0.55
(CH 3 )2N
1
H
H
Comparison: Tetracyclines
N(CH 3)2
OH
Oasis® HLB
Cartridge
0.020
2
3
0.016
HO
O
CONH
O
HO
O
H
Minocycline
CH 3 H
HO
0.012
OH
0.008
sample
0.004
blank
0.000
10.0
Minutes
20.0
Tetracycline
Cl HO
H
O
H
HO
Compound
Conc.
[µg/mL]
O
O
H
CONH
2
N(CH 3)2
C18
Cartridge
Recovery
(%)
RSD (%)
n=6
Recovery
(%)
RSD (%)
n=6
Minocycline 2.5
94.8
1.40
40.7
0.82
Tetracycline
104
0.55
67.4
0.44
N(CH 3 )2
AU
HO
2
2.5
OH
30.0
HO
O
HO
O
O
H
CONH
2
Demeclocycline©Waters
(IS)1998
Cheng et. al. Chromatographia 1997,
44 (3/4), p 187
©Waters 1998
Dr. Shulamit Levin, Medtechnica
12
Results of 1-D SPE Method
Acids
Neutrals
Bases
Solid Phase Extraction (SPE) Technology
100
Successful Tips
80
Collect all Fractions (= Mass Balance)
Load
Wash(es)
Elute
2nd Elute
Doxepin (4 µg)
Naltrexone (1 µg)
Salbutamol (2 µg)
Oxycodone (1 µg)
Propranolol (4 µg)
Ranitidine (0.5 µg)
Caffeine (0.5 µg)
Procainamide (0.5 µg)
Theophylline (0.5 µg)
Theobromine (0.5 µg)
Paraxanthine (0.5 µg)
Sulfadiazine (10 µg)
Sulfamerazine (10 µg)
Acetaminophen (0.5 µg)
0
Naproxen (2 µg)
20
Salicylic Acid (5 µg)
40
Ibuprofen (2.5 µg)
% Recovery
60
Spiked Serum on 1 cc 30 mg Oasis™ HLB Cartridges
RSD < 5.0%
Capparella, Cheng,
Phillips
©Waters 1998
©Waters 1998
Solid Phase Extraction (SPE) Technology
Summary
Sample preparation is a necessary step prior to
the analysis
perception was/is time consuming and
tedious
Solid-Phase Extraction (SPE) provides
cleaner extracts
simpler protocol
Successful Tips
perform mass balance
Ideal SPE Method
one method, one good result for a wide range
of compounds
©Waters 1998
Solid-Phase Extraction (SPE) Technology
Acknowledgments:
Dr. Uwe Neue
Dr. Edouard Bouvier
Dr. Dorothy Phillips
Dr. Patrick McDonald
Dr. Tom Walter
Dr. Michael Young
Joe Arsenault
Pamela Iraneta
Mark Capparella
Bonnie Alden
We gratefully acknowledge all
the Trademarks used in this presentation,
which are the property of their respective owners.
©Waters 1998
Dr. Shulamit Levin, Medtechnica
13