3M Empore™ Sample Preparation Made Easy Supelco Seminar Series, June 2011

Transcription

3M Purification Inc.
3M Empore™ Sample Preparation Made Easy
An Innovative Look at Using Empore™ for Solid Phase Extraction
Supelco Seminar Series, June 2011
Jennifer Heitkamp, M.S.
1
Introduction
 Empore™ membranes have been around for more than 20 years and
used for thousands of applications.
 A proprietary process that entraps small absorbent particles into a
matrix of PTFE fibrils that creates a mechanically stable membrane.
 Membrane format as disks, cartridges, or 96-well plates
 The membrane has a high exposed surface area of active particles.
 This characteristic property makes the membrane extremely useful
for many applications that may not be typical in many laboratories.
 Examples of ways that the Empore™ membrane format has been
used to prepare samples of large volumes used in environmental
analysis to very small volumes used in drug discovery.
2
Empore™ Products History

1970’s and 1980’s


Early 1990’s







Polypropylene Extraction Disk Plates
Rad Disks
1999



SDB-based sorbents
EPA approvals granted
Extraction Disk Cartridges for biological extractions
Prefilter for disk cartridges
1997


TLC membranes
First products were designed for environmental extractions
1992-1996


Started out as an idea to immobilize molecular sieves to dry organic solvents
Filter Plate
Field Sampling Products
2001

Universal Resin Plate
3
© 3M 2011. All Rights Reserved.
3M Empore™ Solid Phase Extraction Membranes
What is Empore ?
SPE Particle-loaded membrane
Sorbent particles tightly held together within an inert matrix of polytetrafluoroethylene (PTFE)
90% sorbent, 10% PTFE (by weight)
Resin Particles
Silica Particles
Features of Empore ™ membranes
Benefits
Sorbent particles tightly enmeshed in PTFE Eliminates particle fines that plug frits
Dense particle packing with no void space
& uniform particle distribution
High separation efficiency
Thin membrane, small bed volume
Can filter large volumes (typically up to 4 L of
sample with 47 mm discs with pre-filtration)
4
How Does Empore™ Membrane Work?
 Empore membrane is a thin membrane based
chromatography.
 Extraction/separation, purification and concentration of
analytes from an aqueous sample.
 It may look like a filter but filters are only capable of
capturing particles.
 Empore membrane is capable of capturing analytes and
releasing them for analysis.
5
EmporeTM Extraction Kinetics
Independent of sample flow rate
Extraction of dye at 700 mL/minute
6
Traditional SPE Methods for Sample Preparation
 Disk extraction methods

Water (multiple EPA methods for monitoring drinking water)

Maple Syrup (quantify lead)

Animal Urine (Isolate large quantities of metabolites for future
studies)
 Cartridge extraction methods

Small elution volumes reduce the need for solvent
evaporation step
 96-well plate extraction methods

UR plate for clean up of human serum samples for drug and
metabolite quantification
7
EPA Water Extraction Methods

C8 Bonded Silica


C18 Bonded Silica










8
EPA Method 529 Explosives Residues
Anion Exchange


EPA Method 515.2 Chlorinated Acids
EPA Method 526 Semivolatile Organic Compounds
EPA Method 527 Pesticides and Flame Retardants
SDB-RPS Polystyrenedivinylbenzene Reverse Phase Sulfonated


EPA Method 506 Phthalate & Adipate Esters
EPA Method 508.1 Chlorinated Pesticides, Herbicides, & Organohalides
EPA Method 525.2 Organic Compounds
EPA Method 550.1 Polynuclear Aromatic Hydrocarbons
EPA Method 608 ATP 3M0222 Organochlorine Pesticides and PCB’s
EPA Method 1613B Dioxins and Furans
SDB-XC Polystyrenedivinylbenzene


EPA Method 549.1 Diquat and Paraquat
EPA Method 548.1 Endothall
EPA Method 552.1 Haloacetic Acids and Dalapon
Oil & Grease

EPA Method 1664 Rev. A n-Hexane Extractable Materials
Cartridge Elution Volume Comparison
Traditional
100 mg/1 ml cartridge
4 mm /1 ml EmporeTM
Disk Cartridge
More concentrated
Enhanced sensitivity
Time savings
9
LC/MS/MS Quantitation of Parent Drug, Desethyl Metabolite,
3-OH Metabolite, and 4-OH Metabolite in Human Serum
Using Empore™ 96-Well Universal Resin (UR) Plate
10
 Universal resin is a terpolymer based on styrenedivinylbenzene
 Designed to provide good retention of a wide range of analytes during solid
phase extraction of hydrophilic (acidic or basic) and moderately hydrophobic
analytes
 Designed for processing 96 samples simultaneously
 Collar around plate tip helps prevent contamination from sample to
sample during processing
 Method development time is saved by eliminating the need to screen
a variety of sorbents.
 Each well contains a proprietary prefilter to enhance sample flow
 Thin bed mass of the disk allows for minimized solvent and sample volumes
 High sample throughput
 Small elution volumes
 Ability to eliminate the evaporation step
 Sample processing at maximal flow rates without channeling
Method Description
Bioanalytical method to quantitate a drug and 3 metabolites in human
serum.
Sample preparation
dilution with buffer
addition of internal standard (IS)
clean-up using a Universal Resin Empore Extraction Disk Plate
Analysis
Agilent® HPLC system
C18 column
Sciex API 4000 triple-quadrupole mass spectrometer operated under
positive-ion mode using a Turbo V™ electrospray interface.
The linear range for quantitation of drug and metabolites was 0.0500 to
25.0 ng/mL
Sample 0.100-mL human serum
11
Sample Preparation
 Aliquot the samples (0.100 mL)
 Add 300 µL of 0.1 M TRIS (Base)
 Add 50.0 µL methanol
 Add 50.0 µL IS Spiking Solution
 Cover and vortex for 1 minute
12
SPE Extraction
A Speedisk® pressure processor was used with a Universal Resin
Empore™ 96-well extraction disk plate
 Condition with 250 µL of methanol
 Condition with 250 µL of water
 Transfer the entire sample to pre-defined wells on the extraction plate
 Wash with 500 µL of water
 Wash with 200 µL of 20% methanol in water
 Elute the samples with 200 µL of 1% formic acid in methanol
 Remove the solvent with a stream of nitrogen gas at 35 °C
 Add 200 µL of reconstitution solution to each sample well
 Cover the 96-well plate with a cap mat and vortex.
 Analyze by LC\MS\MS.
13
LLOQ Sample Chromatograms of Parent Drug (Top Left),
Desethyl Metabolite (Top Right), and IS (Bottom)
Int. Type:
Retention Time:
Area:
Height:
Start Time:
End Time:
No
1
15.00
75.00
1
30.0
3.90
0.20
0.01
5.00
4.00
3.00
No
2800
2600
cps
cps
2400
sec
min
2200
2000
Base To Base
3.88
min
12569
counts
3300.
cps
3.79
min
4.01
min
Intensity, cps
Modified:
Bunching Factor:
Noise Threshold:
Area Threshold:
Num. Smooths:
RT Window:
Expected RT:
Sep. Width:
Sep. Height:
Exp. Peak Ratio:
Exp. Adj. Ratio:
Exp. Val. Ratio:
Use Relative RT:
Sample Name: "3 025 9196.93 Validation LLOQ 0.050 1 1" Sample ID: "25"
Peak Name: "S28371" Mass(es): "287.0/197.0 amu"
Comment: "none" Annotation: ""
Sample Index:
25
Sample Type:
QC
1370
Concentration:
0.0500
ng/mL
Calculated Conc: 0.0515
ng/mL
1300
Acq. Date:
03/03/04
Acq. Time:
08:48:53 PM
1200
Modified:
No
Bunching Factor:
1
1100
Noise Threshold:
15.00
cps
Area Threshold:
75.00
cps
1000
Num. Smooths:
1
RT Window:
30.0
sec
Expected RT:
2.81
min
900
Sep. Width:
0.20
Sep. Height:
0.01
Exp. Peak Ratio:
5.00
800
Exp. Adj. Ratio:
4.00
Exp. Val. Ratio:
3.00
700
Use Relative RT:
No
1800
1600
Int. Type:
Retention Time:
Area:
Height:
Start Time:
End Time:
1400
1200
1000
Base To Base
2.80
min
3048
counts
945.
cps
2.73
min
2.88
min
Intensity, cps
Sample Name: "3 031 9196.93 Validation LQC 0.150 1 1" Sample ID: "31" File: "9196-93-Validation-Batch-03.wiff"
Peak Name: "R848" Mass(es): "315.0/197.0 amu"
Sample Index:
31
3.88
Sample Type:
QC
3313
Concentration:
0.150
ng/mL
3200
Calculated Conc:
0.157
ng/mL
Acq. Date:
03/03/04
3000
Acq. Time:
09:33:16 PM
File: "9196-93-Validation-Batch-03.wiff"
600
500
400
800
300
600
200
400
0
100
3.68 3.76
3.7
3.8
Sample Name: "3 031 9196.93 Validation LQC 0.150 1 1" Sample ID: "31"
Peak Name: "S-28645" Mass(es): "275.0/203.0 amu"
Sample Index:
31
Sample Type:
QC
3.3e5
Concentration:
1.00
ng/mL
3.2e5
Calculated Conc:
N/A
Modified:
Bunching Factor:
Noise Threshold:
Area Threshold:
Num. Smooths:
RT Window:
Expected RT:
Sep. Width:
Sep. Height:
Exp. Peak Ratio:
Exp. Adj. Ratio:
Exp. Val. Ratio:
Use Relative RT:
03/03/04
09:33:16 PM
No
1
10.00
50.00
1
30.0
3.32
0.20
0.01
5.00
4.00
3.00
No
3.9
4.0
4.1
4.2
4.3
4.4
Time, min
4.52
4.62 4.71 4.84
4.5
4.6
4.7
4.8
4.89 4.96
0
4.9
3.32
2.8e5
2.6e5
cps
cps
2.4e5
sec
min
Int. Type:
Base To Base
Retention Time:
3.32
min
Area:
839580
counts
Height:
333000.
cps
Start Time:
3.27
min
End Time:
3.47
min
2.2e5
2.0e5
1.8e5
1.6e5
Int. Type:
Base To Base
Retention Time:
3.32
min
Area:
858058
counts
Height:
346000.
cps
Start Time:
3.26
min
End Time:
3.50
min
1.4e5
1.2e5
1.0e5
14
2.5
3.0
3.5
3.32
1.4e5
1.2e5
1.0e5
8.0e4
6.0e4
6.0e4
4.0e4
4.0e4
2.0e4
1.0
1.5
2.0
Time, min
1.6e5
8.0e4
0.0
0.5
Sample Index:
25
Sample Type:
QC
3.4e5
Concentration:
1.00
ng/mL
Calculated Conc:
N/A
3.2e5
Acq. Date:
03/03/04
Acq. Time:
08:48:53 PM
3.0e5
Modified:
No
Bunching Factor:
1
2.8e5
Noise Threshold:
10.00
cps
Area Threshold:
50.00
cps
2.6e5
Num. Smooths:
1
RT Window:
30.0
sec
2.4e5
Expected RT:
3.32
min
Sep. Width:
0.20
2.2e5
Sep. Height:
0.01
Exp. Peak Ratio:
5.00
2.0e5
Exp. Adj. Ratio:
4.00
Exp. Val. Ratio:
3.00
1.8e5
Use Relative RT:
No
3.0e5
Intensity, cps
Acq. Date:
Acq. Time:
4.06 4.13 4.22 4.30 4.39
Intensity, cps
200
2.0e4
0.5
1.0
1.5
2.0
Time, min
2.5
3.0
3.5
0.0
0.5
1.0
1.58
1.5
2.0
Time, min
2.65
2.5
3.07
3.0
3.5
LLOQ Sample Chromatograms of 3-OH Metabolite (Top Left),
4-OH Metabolite (Top Right), and IS (Bottom)
Int. Type:
Retention Time:
Area:
Height:
Start Time:
End Time:
Acq. Date:
Acq. Time:
Yes
1
40.00
100.00
1
10.0
3.89
0.20
0.01
5.00
4.00
3.00
No
550
cps
cps
500
sec
min
450
Base To Base
3.87
min
1846
counts
495.
cps
3.81
min
3.94
min
Modified:
Bunching Factor:
Noise Threshold:
Area Threshold:
Num. Smooths:
RT Window:
Expected RT:
Sep. Width:
Sep. Height:
Exp. Peak Ratio:
Exp. Adj. Ratio:
Exp. Val. Ratio:
Use Relative RT:
400
350
300
Int. Type:
Retention Time:
Area:
Height:
Start Time:
End Time:
250
200
500
cps
cps
450
sec
min
Base To Base
4.08
min
2227
counts
574.
cps
3.99
min
4.16
min
400
350
300
250
200
100
100
50
50
3.7
3.8
3.9
4.0
4.1
4.2
4.3
4.4
4.5
Time, min
4.6
4.7
4.8
0
4.9
1.6e5
Int. Type:
Base To Base
Retention Time:
3.32
min
Area:
858058
counts
Height:
346000.
cps
Start Time:
3.26
min
End Time:
3.50
min
1.4e5
1.2e5
1.0e5
3.8
4.6
4.7
4.8
4.9
3.32
1.4e5
1.2e5
1.0e5
8.0e4
6.0e4
6.0e4
4.0e4
4.0e4
2.0e4
3.9
4.0
4.1
4.2
4.3
4.4
4.5
Time, min
1.6e5
8.0e4
0.0
3.7
Sample Index:
25
Sample Type:
QC
3.4e5
Concentration:
1.00
ng/mL
Calculated Conc:
N/A
3.2e5
Acq. Date:
03/03/04
Acq. Time:
08:48:53 PM
3.0e5
Modified:
No
Bunching Factor:
1
2.8e5
Noise Threshold:
10.00
cps
Area Threshold:
50.00
cps
2.6e5
Num. Smooths:
1
RT Window:
30.0
sec
2.4e5
Expected RT:
3.32
min
Sep. Width:
0.20
2.2e5
Sep. Height:
0.01
Exp. Peak Ratio:
5.00
2.0e5
Exp. Adj. Ratio:
4.00
Exp. Val. Ratio:
3.00
1.8e5
Use Relative RT:
No
3.32
550
150
Sample Index:
25
Sample Type:
QC
3.4e5
Concentration:
1.00
ng/mL
Calculated Conc:
N/A
3.2e5
Acq. Date:
03/03/04
Acq. Time:
08:48:53 PM
3.0e5
Modified:
No
Bunching Factor:
1
2.8e5
Noise Threshold:
10.00
cps
Area Threshold:
50.00
cps
2.6e5
Num. Smooths:
1
RT Window:
30.0
sec
2.4e5
Expected RT:
3.32
min
Sep. Width:
0.20
2.2e5
Sep. Height:
0.01
Exp. Peak Ratio:
5.00
2.0e5
Exp. Adj. Ratio:
4.00
Exp. Val. Ratio:
3.00
1.8e5
Use Relative RT:
No
Intensity, cps
No
1
20.00
150.00
2
30.0
4.12
0.20
0.01
5.00
4.00
3.00
No
150
0
Int. Type:
Base To Base
Retention Time:
3.32
min
Area:
858058
counts
Height:
346000.
cps
Start Time:
3.26
min
End Time:
3.50
min
03/03/04
08:48:53 PM
Intensity, cps
Modified:
Bunching Factor:
Noise Threshold:
Area Threshold:
Num. Smooths:
RT Window:
Expected RT:
Sep. Width:
Sep. Height:
Exp. Peak Ratio:
Exp. Adj. Ratio:
Exp. Val. Ratio:
Use Relative RT:
Peak Name: "S32899" Mass(es): "331.0/213.0 amu,331.0/267.0 amu"
Sample Index:
25
Sample Type:
QC
622
Concentration:
0.0500
ng/mL
600
ng/mL
08:48:53 PM
Intensity, cps
Acq. Time:
Intensity, cps
Peak Name: "S32483" Mass(es): "331.0/267.0 amu,331.0/213.0 amu"
Sample Index:
25
Sample Type:
QC
642
Concentration:
0.0500
ng/mL
ng/mL
600
Acq. Date:
03/03/04
2.0e4
0.23 0.31
15
0.60
0.5
1.03 1.19
1.0
1.58 1.72 2.12
1.5
2.0
Time, min
2.50 2.65 2.97 3.07
2.5
3.0
3.5
0.0
0.23 0.31
0.60
0.5
1.03 1.19
1.0
1.58 1.72 2.12
1.5
2.0
Time, min
2.50 2.65 2.97 3.07
2.5
3.0
3.5
E xamp le Cali brati on Cu rves
2.00
Pa re n t D ru g
1.80
1.60
1.40
De s e th y l
1.20
M e ta b o il te
1.00
4 -O H
3 -O H
0.80
0.60
0.40
0.20
0.00
0. 0
5.0
10. 0
15.0
N o m i na l C o nc en t r at i o n ( ng / m L )
16
20.0
25. 0
Method Validation Parameters
 Accuracy and Precision
 Response Linearity
 Specificity and Interference
 Sample Dilution Analysis
 Freeze/Thaw Sample Stability
 Room Temperature Processed Sample Stability
 24-Hour Room Temperature Matrix Stability
 Short and Long Term Matrix Storage Stability (-20 °C)
17
Non-Traditional Uses for Empore Disks
 Sorbent material for passive sampling device
 Capture of volatile compounds from air
 Construction of small-volume protein purification
extraction columns (StageTips)
 Layering of different sorbent phases
18
Passive Sampling Devices - Chemcatcher™
 Importance of representative samples
 Body of holder made of PTFE
 Constructed to hold an Empore™ disk
 Permeable membrane covers the Empore disk to
prevent fouling of the sorbent material
 Measures time weighted average (TWA) of pollutants
in water
 Deployment time from days to months depending on
the goal of the experiment
19
Researchers using the Chemcatcher™ Passive
Sampling Device for Water Contaminants









United Kingdom
Australia
Spain
Germany
Sweden
South Africa
Switzerland
Slovakia
The Netherlands
20
Why Use Passive Sampling?
 Very low level pollutants can be missed
 Discrete water samples give a snapshot of concentrations
 Very low levels require large volumes of water sample


Transportation
Storage
 Can miss episodic pollution events
 Contaminant concentrations in surface water vary over time due to
weather events
 Extractions from organisms to monitor bioaccumulation can be
complex
 Extraction from sediment assumes that the levels in the water and
sediment are in equilibrium
21
Considerations
 Physical Chemical properties of analytes will dictate the
absorptive material






Organophosphate pesticides
Non-polar organic compounds
PAH’s
Organometallic complexes
Metal ions
Pharmaceuticals and personal care products
 Water turbulence
 Water quality


High particulate material
Salt content
 Biofouling potential
 Permeability reference compounds (QC)
 Accumulation time frame
22
Passive Sampling Device Development - Milestones
inorganic
organic
Total number of publictions per year
60
1- first passive sampler for organic micro pollutants in water
2- first publication of semi-permeable membrane device
3- detection of compounds in water at pg/L
4- first publication on Chemcatcher™
50
40
30
20
10
11
23
2
3
4
Vrana, B., Mills, G.A., Allan, I.J., Dominiak, E., Svensson, K., Knutsson, J., Morrison, G., Greenwood, R.
Trends Anal. Chem. 2005(24), pp. 845-868
04
20
03
20
02
20
20
01
00
20
99
19
98
19
97
19
96
19
95
19
94
19
93
19
92
19
91
19
90
19
89
19
19
19
87
88
0
Air Sampling with Empore™
 Why is air sampling important?


Identify and quantify health hazards
Correct problems with unhealthy air
 Compounds




Pesticides
Volatile and semi-volatile organophosphate esters
Explosive residues
Chemical warfare agents
 Why use Empore disks?





24
Higher air flow than cartridges (up to 20 L/min)
Small particles give high sample capacity
Active sorbent particles retain absorbed compounds during sample
collection and store easily
High extraction recoveries
Higher stability of compounds stored on disks
Empore™ Disk as Sorbent for Air Borne
Pyrethroid Insecticides
 Some affect humans' central nervous system or have endocrinedisrupting effects
 18 pyrethroids chosen based on use in Japanese homes as
insecticides
 Samples collected by pumping air through a glass fiber disk and an
Empore™ C18FF disk
 Collection device shielded from light to prevent photodegradation
 Sample collected for 24 hours at a flow rate of 3.0 L/min.
 Total volume of air through the absorbents was 4.32 m³
 Disks removed form collector and placed together in sealed tube for
storage at 4 °C until analysis
25
Yoshida, T. "Simultaneous Determination of 18 Pyrethroids in Indoor Air by Gas Chromatography/Mass
Spectrometry." Journal of Chromatography A. 1216 (2009) 5069-5076
Experimental Method Validation Parameters
 Retention and breakthrough of compounds during sampling
tested


Retention efficiency >93%
Breakthrough <0.2%
 Efficiency of extraction of pyrethroids tested

Desorption efficiencies >91% for spikes at 5 and 0.5 µg/compound
 Detection limits

0.4 - 1.7 ng/m³
 Precision and accuracy


RSD <10%
Accuracy range of 35 ng/m³ to 1.3 µg/m³
 Storage stabilities at 4 °C on disks

26
Up to 1 month storage showed no degradation if kept shielded from
light
Airborne Pyrethroid Concentrations Found
Formulation
Mothproof repellent for
clothes
Mosquito Repellents
used with electrical
devices
Sample #
Compound
Concentration (µg/m³)
1
2
Empenthrin
Profluthrin
2.3
1.0
3
4
5
6
7
8
9
Prallethrin
Furamethrin
Allethrin
Furamethrin
Transflthrin
Parallethrin
Metofluthrin
Daytime
Nighttime
34
39
148
5.0
12
69
0.24
17
24
122
4.1
9.3
23
0.15
ACGIH recommended threshold limit (8-hour workday and 40-hour workweek)
for Pyrethrum at 5 mg/ m³
27
Preparation of Single-Disk Stop and Go Extraction
Tips (StageTips)
 Empore™ 47 mm disk for various functionality









28
C18
C8
Cation exchange
Anion exchange
SDB-XC
Self-made tool for customization
Ease-of-use
Good recovery
Reproducible and robust
Rappsilber, J., Mann, M., and Ishihama, Y. “Protocol for Micro-purification, Enrichment, Pre-fractionation and
Storage of Peptides for Proteomics using StageTips.” Nature Protocols 2, 1896-1906 (2007) Published online:
2 August 2007
StageTips Preparation
 Small disk is stamped out using a blunt-ended
syringe needle (cutter)


Particles held together for easy handling
Size of disk adjusted by using different gauge needle
 Place the cutter inside a pipette tip and release the
disk using a correct size needle plunger
 Press the disk gently into place using the plunger
 Remove cutter
 Additional disks can be layered to provide multiple
functionality
29
Stage Tip Preparation
30
Multiple Layers for Additional Separating Ability
 C18 allows for desalting of
the protein
 Fractionation done on the
cation exchange disk.
 Can be followed up by
another desalting step with
C18
 Very clean sample for mass
spec analysis
Ishihama, Y., Rappsilber, J., and Mann, M., “Modular Stop and Go
Extraction Tips with Stacked disks for Parallel and Multidimensional
Peptide Fractionation in Proteomics.” J. Proteome Res., 2006, 5 (4),
pp 988–994
Publication Date (Web): March 07, 2006 (Technical Note)
31
C18
SCX
In Summary  PTFE fibrils immobilized particle provide a unique medium
for SPE
 Disk shape allows for mounting in a variety of
configurations and sample holders
 Can be layered to combine functionality
 Can be cut to fit desired holder
 Enhanced chemical stability of Empore™-adsorbed
compounds during storage
 Next new idea ?
32

3M Empore™ Sample Preparation Made Easy Supelco Seminar Series, June 2011

Transcription

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