Sample Preparation Using

Solid Phase Extraction

Dr. Shulamit Levin

Based
Based on:
on:
Yung-Fong
Yung-Fong (Henry)
(Henry) Cheng
Cheng
Waters Corporation
Waters Corporation
34
34 Maple
Maple Street
Street
Milford,
Milford, MA 01757
MA 01757

Present
Present at
at EAS'98
EAS'98 Workshop
Workshop
 Outline
Troubleshooting of Sample
Preparation Methods Using Importance of Sample Preparation
Solid-Phase Extraction Principle of Solid-Phase Extraction
(SPE)
Typical Problems in SPE
detail steps of SPE
Based
Based on:
on:
Yung-Fong
Yung-Fong (Henry)
Waters
(Henry) Cheng
Cheng
Waters Corporation
Corporation
examples
34
34 Maple
Maple Street
Milford,
Milford, MA
Street
MA 01757
01757
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 --
If We didn't have to Prepare Samples
Before Injection into the Instrument
Sample Prep Techniques
Method
• Precipitation
• Liquid-Liquid Extraction
• Solid-Liquid Extraction (SPE)
• Dialysis / Ultrafiltration
• Electrophoresis
• Distillation/Evaporation
• Supercritical Fluid Extraction
Dr. Shulamit Levin, Medtechnica
Why Perform Sample Preparation?
Remove interferences
e.g. Analysis of drug and metabolite in plasma.
Need to remove protein interferences
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
©Waters 1998
^ AA ^ Others
©Waters 1998
Liquid-Liquid Extraction (LLE)
Basis for Selectivity
Solubility
Chemical Technique
Partitioning in one of
two liquid phases
Adsorption/partitioning
onto solid sorbent
Where an Immiscible Solvent
Molecular weight/size
is Added to the Sample which
Charge
then Separates into 2 Distinct
Boiling point/vapor pressure
Liquid Phases. Some Sample
Partitioning into Analytes will go into the
supercritical fluid Bottom Phase (Aqueous),
©Waters 1998 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
Cleaner extracts
May require an evaporation step prior to
analysis to remove excess solvent Easier to automate
When one needs to assay for several Higher recoveries
analytes, it may be difficult to find proper for polar compounds
solvent/conditions for all analytes,
requiring more than one extraction per
sample
©Waters 1998

Problematic samples - emulsions

Contamination issues
©Waters 1998

Solid Phase Extraction (SPE)

- Formats and Configurations

Solid Phase Extraction (SPE)
Cartridge Disk
Disk Coated
Coated
- Chromatographic Particles Bed
Bed Fiber
- Packed-Bed Column Cartridges
- 1st Commercialized In 1978 96 Well
- Well Established Technology Plate

- Many Thousands of Literature References

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 1

Particle size ~5 µm 40-80 µm 0.8 HPLC:

Normalized concentration
Packed bed efficiency high low 0.6
0.4
higher efficiency
Extra-column volume low high
0.2
Column length 5-30 cm ~1 cm 0
Number of plates (N) ~10,000 < 50 0 5 10 15 20

1
SPE:
Bottom line: HPLC can separate similar compounds. SPE requires
0.8
poor efficiency
0.6
a significant selectivity difference between compounds
0.4
for separation. Compounds not well resolved by
HPLC cannot be separated by SPE with a similar 0.2

retention mechanism. 0
0 5 10 15 20
©Waters 1998
Elution volume (mL)
©Waters 1998

Solid Phase Extraction (SPE) Technology

Solid Phase Extraction (SPE) Technology

Sample Must be in Liquid State

Driving Forces

^ Gravity

^ Pressure

^ Vacuum Vacuum Manifolds

©Waters 1998

©Waters 1998

Dr. Shulamit Levin, Medtechnica 4

 Solid Phase Extraction (SPE) Technology
Solid Phase Extraction (SPE) Technology
Manufacturer Brand Name
SPE Strategies
Waters SEP-Pak
OASIS
Varian BondElute • Elute the product of interest, retain interferences
• want k ➠ 0 for analyte
Baker BakerBond • want k large for interferences
• Elute interferences, retain product
International Sorbent Isolute
• want k ➠ 0 for interferences
Technology
• want k large for analyte
3M Empore
* Concentrate product of interest
Supelco Supelclean
^ want k large for analyte / load large sample volume
^ elute concentrated analyte
+ Many Others ©Waters 1998 ^ enhanced sensitivity

©Waters 1998

Solid Phase Extraction (SPE) Technology

Methods Development Approach
Most Common TYPES OF CHROMATOGRAPHY Determine Nature of Analytes, and
Sample Matrix
- Normal Phase Similar to Existing Method in Lab?
^ The "Original" Type - Used By Tswett
^ Non-Polar Mobile Phase Yes No
^ Polar Stationary Phase
Review SPE Bibliography,
Try Conditions - Evaluate for No and Literature References
- Reversed-Phase Most Common Capacity/ Breakthrough,
for Exact or Similar
Recovery
^ Polar Mobile Phase Reproducibility, Robustness
Applications
Any?
^ Non-Polar Stationary Phase and Ruggedness Yes
Meets Goals?
No
- Ion Exchange
^ Buffer/Ionic Mobile Phase Determine Method
Yes Goals, and Strategy
^ Cationic/Anionic Exchanger Stationary Phase
Call SPE Vendor
©Waters 1998
Chromatography Mode

Validate Develop Method

Method Conditions

Dr. Shulamit Levin, Medtechnica 5

 Outline
Solid Phase Extraction (SPE) Technology
Importance of Sample Preparation
Principle of Solid-Phase Extraction
(SPE) Common Problems in SPE
Typical Problems in SPE
• Incomplete Removal of Interferences
detail steps of SPE
examples •
Low Recovery of Analyte(s)
Summary • High Variability (RSDs)

©Waters 1998

©Waters 1998

Solid Phase Extraction (SPE) Technology

SPE Procedure Solid Phase Extraction (SPE) Technology
Sample Step 1 - Sample Preparation
➀ Prepare: Homogenize, suspend,
centrifuge, etc.
Sample
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

Analyze: HPLC, GC, etc.

➃ Elute product with strong solvent

©Waters 1998
Analyze: HPLC, GC, etc.

©Waters 1998

Dr. Shulamit Levin, Medtechnica 6

 Sample Pretreatment: Effect of Acid on
Solid Phase Extraction (SPE) Technology
Recovery % Recovery
Step 1 - Sample Preparation Phosphoric
No Acid No Acid Acid, 2%
Typical problems Compounds Concentration
[µg/mL] Saline Sample Serum Sample Serum
Analytes Sample

adsorpted to test tube walls Naproxen 1.0 96 4 89

adsorpted to or inclusion in matrix solids
Ibuprofen 10.0 94 19 87
bound to proteins in matrix
Possible solutions CH 3 CH3
use silanized or plastic test tubes OH CH3 OH
homogenize more completely H3CO O
H3C
O
add acid to sample solution Naproxen Ibuprofen
©Waters 1998

Solid Phase Extraction (SPE) Technology

Step 2 - Sample load Solid Phase Extraction (SPE) Technology

Sample Step 2 - Sample Load

➀ Prepare: Homogenize, suspend,
centrifuge, etc.

❷ Load onto conditioned cartridge Possible problems Solutions

•Improper conditioning of
➂ Wash off weakly retained interferences
with weak solvent
cartridge
•Poor analyte retention
➃ Elute product with strong solvent
•Matrix variability
Analyze: HPLC, GC, etc. •Volume overload
•Condition cartridge as appropriate. Do not
let dry, if silica based C18
•Dilute with weaker solvent, use stronger
sorbent, use larger cartridge
•Buffer sample to constant pH, ionic strength
•Decrease load volume, use larger cartridge

©Waters 1998
•Mass overload •Decrease load volume, use larger cartridge

©Waters 1998

Dr. Shulamit Levin, Medtechnica 7

 Incomplete Conditioning of Cartridges Incomplete Conditioning of
"Note: Do not dry SPE cartridge
Cartridges Effect on Recovery:
between initial methanol C1818 vs. Oasis® HLB Cartridges
conditioning wash and completion C18 (1cc/100mg) HLB (1cc/30mg)
100
of addition of sample and sample 100 * No Impact of
wash. Monitor elutions closely to 80 80 Sorbent Drying
ensure that cartridges do not dry." 60 * No Silanol

Percent recovery
60
Interaction
40
40 * No Breakthrough of
J. D. MacNeil, V. K. Martz, G. O. Korsrud, C. D. C. 20 20 Polar Analytes
Salisbury, H. Oka, R. L. Epstein, C. J. Barnes, J. AOAC
Intl., 79(2) (1996), 405-417 0 0
0 4 8 0 5 10
Drying Time Drying Time
(minutes) (minutes)
Procainamide Ranitidine Doxepin
Acetaminophen Propranolol

©Waters 1998

Effect of the Sample pH on Recovery

Solid Phase Extraction (SPE) Technology
Load at pH 7 Load at pH <2
Compounds

Concentration Recovery Recovery

Sample Loading
[µg/mL] (%) (%)

1
Salicylic Acid in Saline 10 62.5 101
pKa 2.97, 13.4
0.8 k=10
k=20 higher k,

Normalized concentration
k=30
COOH
0.6 less breakthrough
OH
(continuous loading)
0.4

Salicylic Acid Assume:

0.2 N = 40 plates
V0 = 1 mL

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.
Possible Problems Solutions
➁ Load onto conditioned cartridge
•Poor analyte retention •Use stronger sorbent, use larger cartridge

❸ Wash off weakly retained interferences

with weak solvent
•Matrix variability
•Volume overload
•Buffer sample to constant pH, ionic strength
•Decrease load volume, use larger cartridge
•Mass overload •Decrease load volume, use larger cartridge
➃ Elute product with strong solvent

Analyze: HPLC, GC, etc.

©Waters 1998

©Waters 1998

Effect of Incomplete Wash Washing Procedure:

Interferences
Effect of Wash Solvent on Recovery
96 inj.
USP Tailing Factor:
1st wash: 5% Methanol Water
1.67 in Water
0.01 au 40% MeOH, Compounds
2% NH4OH Concentra- Recovery Recovery
tion [µg/mL] (%) (%)

Theobromine 0.5 87 99
Minutes
2.00

3.00

4.00

5.00

6.00

111 inj. USP Tailing Factor:

1st wash: Paraxanthine 0.5 67 92

40% MeOH,
1.07 2% NH4OH Theophylline 0.5 75 106

0.01 au Caffeine 0.5 92 105

2nd wash:
5% MeOH,
2% HAc.
0.00

Cheng 2 minutes 4 6
©Waters 1998

Dr. Shulamit Levin, Medtechnica 9

 Solid Phase Extraction (SPE) Technology
Solid Phase Extraction (SPE) Technology
Step 4 - Elute Step 4 - Elution
Sample 1
k=0 k=1 k=2

➀ Prepare: Homogenize, suspend,

centrifuge, etc.
0.9
0.8
higher retention

Normalized concentration
0.7 larger elution volume
➁ Load onto conditioned cartridge 0.6
0.5
0.4

➂ Wash off weakly retained interferences

with weak solvent
0.3
0.2
Assume:
N = 40
V0 = 1 mL
0.1

❹ Elute product with strong solvent 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
Evaporation and Reconstitution
Methanol Methylene Chloride:
Compound Methanol 50:50
Advantages
Concentration Recovery RSD Recovery RSD Increase Assay Sensitivity
Testosterone [µg/mL] (%) (%) (%) (%)
benzoate Increase sample concentration
First milliliter of Inject larger sample volume
elution solvent 2.0 92 5.1 102 0.49
Second milliliter
Improve HPLC Peak Shape
of elution solvent 6.6 13.3 <0.50 Dissolve in mobile phase or weaker solvent
O
Disadvantages
CH 3 O C Loss of more volatile analytes
CH3 Poor solubility
Testosterone Benzoate
©Waters 1998
O

Dr. Shulamit Levin, Medtechnica 10

 HPLC Analysis:
Effect of Evaporation
Effect of Sample Solvent
0.006
Sample in MeOH
on Sample Recovery
0.005
0.004 Evaporation Evaporation
AU 0.003 Minocycline Compounds to Dryness
0.002 Tetracycline Demeclocycline to 100 µL
0.001 Concentra- Recovery RSD Recovery RSD
0.000
tion [µg/mL] (%) (%) (%) (%)
10.0 20.0 30.0
Minutes
Benzoic Acid 5.0 62.8 9.1 87.6 3.0

0.006
Sample in HPLC Mobile Phase
0.005 (0.1% TFA, 4%ACN and 5%MeOH in Water) Salicylic Acid 5.0 93.6 5.1 91.3 5.0
0.004
Minocycline

AU 0.003 Tetracycline
Demeclocycline
0.002
0.001
COOH COOH
0.000
OH
10.0 20.0 30.0
Minutes

Benzoic Acid Salicylic Acid

Eliminating the Evaporation and

Reconstitution Step: Effect of Sample Solvent Strategy of Signal-to-Noise (S/N)
1
3 Sample Identification
Enrichment Comparison of S/N for Dilution (1:3 with
2 Sample in Water
0.04
1.EDDP
2.Diphenhydramine(IS)
water) of Urine Sample Solution after SPE Extraction
AU 3. Methadone
1:3 Dilution
25 uL injection 0.02
Column: SymmetryShield™ RP18, S/N=38 S/N=42 S/N=37 AU
3.5 µm, 3.9 x 150 mm
Guard Column: Sentry™ Guard Column
SymmetryShield RP18,
At this dilution
5 10 15 Min.
5 10 15 Min. 5µm
Temperature: 30°C
(1:3 with water);
Mobile Phase: 0.1% TFA:Methanol
(60:40)
achieve
Sample in 80% Detection: UV at 210 nm 1:3 Dilution - better peak shapes
Flow Rate: 1 mL/min
MeOH, 2% HAc S/N=88 S/N=99 50 uL injection S/N=89 0.02
Inj. Volume: 30 µL
- higher S/N
AU
0.04
Extraction on
AU 5 15 Min.
10
Oasis® HLB, Extraction on Oasis® HLB,
2 3 96-well, 10 mg/well S/N=145 S/N=164 S/N=147
96-well, 10 mg/well
1 5 µg/ml
10 µg/ml
4 µg/ml 2-D SPE Method 1:3 Dilution 2-D SPE Method
0.02
100 uL injection AU
5 10 15 Min.
Woods, Cheng ©Waters 1998

5 10 15 Min.

Dr. Shulamit Levin, Medtechnica 11

 Generic Reversed-Phase, 1-D,
Solid Phase Extraction (SPE) Technology
SPE Method (Oasis® HLB Sorbent)
Prepare Sample Solution A One-Dimensional (1-D)
Method- changing only the
Impact On Today's Analytical Chemist percent organic
Condition/Equilibrate
1 mL methanol/1 mL water 1 2 3

Load 0% 5% 100%
Faster Method Development 1 mL spiked sample solution % Organic

More Sensitive Methods Wash 1 Load

no organic to retain analytes
1 mL 5% methanol in water
Shorter Processing Times Elute
2 Wash
5% MeOH to remove

Reduced Cost Per Analysis

proteins in matrix
1 mL methanol
3 Elute
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
Comparison: Tetracyclines
Tetracycline 2.5 µg/mL 104 0.55

(CH 3 )2N N(CH 3)2

1 H H
OH
0.020 Oasis® HLB C18
CONH
2 3 O 2
Compound Cartridge Cartridge
HO O HO O
0.016 H Minocycline
Conc. Recovery RSD (%) Recovery RSD (%)
0.012 HO CH 3 H
N(CH 3 )2 [µg/mL] (%) n=6 (%) n=6
OH
AU
0.008
O
CONH 2
Minocycline 2.5 94.8 1.40 40.7 0.82
HO O HO O
H
0.004 sample Tetracycline
blank Cl HO N(CH 3)2
Tetracycline 2.5 104 0.55 67.4 0.44
0.000 H H
OH
10.0 20.0 30.0
Minutes
CONH 2
O
HO O HO O
H 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)

60
Load
Salicylic Acid (5 µg)

Theobromine (0.5 µg)

Theophylline (0.5 µg)

Sulfamerazine (10 µg)

Acetaminophen (0.5 µg)

Salbutamol (2 µg)
Propranolol (4 µg)

Doxepin (4 µg)
Sulfadiazine (10 µg)

Oxycodone (1 µg)

Naltrexone (1 µg)
Ibuprofen (2.5 µg)

Paraxanthine (0.5 µg)

Naproxen (2 µg)

Caffeine (0.5 µg)

Procainamide (0.5 µg)

Ranitidine (0.5 µg)

Wash(es)
% Recovery

40
Elute
2nd Elute
20

0
Spiked Serum on 1 cc 30 mg Oasis™ HLB Cartridges
Capparella, Cheng,
RSD < 5.0% ©Waters 1998

Phillips ©Waters 1998

Solid Phase Extraction (SPE) Technology

Summary
Sample preparation is a necessary step prior to
the analysis Solid-Phase Extraction (SPE) Technology
perception was/is time consuming and
tedious Acknowledgments:
Solid-Phase Extraction (SPE) provides
cleaner extracts Dr. Uwe Neue Dr. Michael Young
simpler protocol Dr. Edouard Bouvier Joe Arsenault
Successful Tips Dr. Dorothy Phillips Pamela Iraneta
perform mass balance Dr. Patrick McDonald Mark Capparella
Dr. Tom Walter Bonnie Alden
Ideal SPE Method
We gratefully acknowledge all
one method, one good result for a wide range the Trademarks used in this presentation,
of compounds ©Waters 1998
which are the property of their respective owners.

©Waters 1998

Dr. Shulamit Levin, Medtechnica 13