Sample Preparation Using

Solid Phase Extraction

Dr. Shulamit Levin
Analytical Department

Medtechnica
Email: [email protected]
[email protected]

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

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)

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

Differences Between HPLC and SPE

Solid Phase Extraction (SPE) Technology

Comparison of Efficiency - HPLC vs. SPE

HPLC

SPE

~5 m
high
low
5-30 cm
~10,000

40-80 m
low
high
~1 cm
< 50

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

10

15

20

SPE:
poor efficiency

1
0.8
0.6
0.4
0.2
0
0

Waters 1998

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

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

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

Prepare: Homogenize, suspend,

centrifuge, etc.

Load onto conditioned cartridge

Prepare: Homogenize, suspend,

centrifuge, etc.

Wash off weakly retained interferences

with weak solvent

Load onto conditioned cartridge

Elute product with strong solvent

Wash off weakly retained interferences

with weak solvent

Elute product with strong solvent

Sample

Analyze: HPLC, GC, etc.

Waters 1998

Analyze: HPLC, GC, etc.

Waters 1998

Dr. Shulamit Levin, Medtechnica

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
[g/mL]

Saline Sample

Serum Sample

Serum
Sample

Naproxen

1.0

96

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

Prepare: Homogenize, suspend,

centrifuge, etc.

Load onto conditioned cartridge

Wash off weakly retained interferences

with weak solvent

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

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."

4
Drying Time
(minutes)

HLB (1cc/30mg)
* No Impact of
Sorbent Drying
* No Silanol
Interaction
* No Breakthrough of
Polar Analytes
0

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
[g/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

Solid Phase Extraction (SPE) Technology

Solid Phase Extraction (SPE) Technology

Step 3 - Wash
Sample

Step 3 - Wash

Prepare: Homogenize, suspend,

centrifuge, etc.

Load onto conditioned cartridge

Wash off weakly retained interferences

with weak solvent

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

minutes

5% Methanol
in Water

Water

Concentration [g/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

Solid Phase Extraction (SPE) Technology

Step 4 - Elute

Solid Phase Extraction (SPE) Technology

Step 4 - Elution

Sample

k=0

Prepare: Homogenize, suspend,

centrifuge, etc.

k=1

k=2

Load onto conditioned cartridge

Wash off weakly retained interferences

with weak solvent

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

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
[g/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

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

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 [g/mL]

0.001
0.000
10.0

20.0

Minutes

Evaporation
to 100 L

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

0.04
AU

Sample in Water

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:

10

15 Min.

Sample in 80%
MeOH, 2% HAc

0.04
AU
1
4 g/ml

Woods, Cheng

Salicylic Acid

5 g/ml

10 g/ml

10

SymmetryShield RP18,
3.5 m, 3.9 x 150 mm
Guard Column: Sentry Guard Column
SymmetryShield RP18,
5m
Temperature: 30C
Mobile Phase: 0.1% TFA:Methanol
(60:40)
Detection:
UV at 210 nm
Flow Rate:
1 mL/min
Inj. Volume:
30 L

Extraction on
Oasis HLB,
96-well, 10 mg/well

S/N=38

S/N=37

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

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

0%

Load
1 mL spiked sample solution

Faster Method Development

More Sensitive Methods
Shorter Processing Times
Reduced Cost Per Analysis

5%
% Organic

Wash
1 mL 5% methanol in water

100%

Load
no organic to retain analytes

Elute
1 mL methanol

Wash

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 g/mL

94.8

1.4

Tetracycline

2.5 g/mL

104

0.55

(CH 3 )2N

Comparison: Tetracyclines

N(CH 3)2
OH

Oasis HLB
Cartridge

0.020

0.016

HO

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

HO

Compound
Conc.
[g/mL]

O
O
H

CONH

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

2.5

OH

30.0
HO

HO

O
O
H

CONH

DemeclocyclineWaters
(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 g)

Naltrexone (1 g)

Salbutamol (2 g)

Oxycodone (1 g)

Propranolol (4 g)

Ranitidine (0.5 g)

Caffeine (0.5 g)

Procainamide (0.5 g)

Theophylline (0.5 g)

Theobromine (0.5 g)

Paraxanthine (0.5 g)

Sulfadiazine (10 g)

Sulfamerazine (10 g)

Acetaminophen (0.5 g)

Naproxen (2 g)

20

Salicylic Acid (5 g)

40

Ibuprofen (2.5 g)

% 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