fundamentals of spe - waters corporationthe new spe “textbook”. part #: 715003405. 212 pages,...

Fundamentals of Solid-Phase Extraction (SPE):

Principles and Practical Tips for Developing

Sample Preparation Methods

WELCOME!

Agenda

The Importance of Solid Phase Extraction

How SPE Works

Device Design and Tips for Processing Samples

Chromatographic Sorbent Choices

Typical SPE Strategies and Starting Protocols

Was My SPE Method Successful?

Appendix

The New SPE “Textbook”

Part #: 715003405

212 pages, paperback

Size: 8.25 x 11”

>150 full color figures and diagrams

Chapter Titles

– Benefits of SPE in Sample Preparation

– SPE is LC

– Key Terms and Calculations

– In the Lab

– Method Development

– Troubleshooting

– Appendix: Glossary of SPE and LC Terms

– Appendix: Oasis Sorbent Technology for SPE

– Appendix: Applications

– Appendix: Additional Reference Materials

Why do Sample Preparation?

Issue: Plugging

Cause: solid particulates

Impact: may limit instrument/system up-time

Sample Prep Tools

For solid particulates:

– Filters

– Centrifuge

For sample matrix components:

– Precipitation

– Liquid-Liquid Extraction (LLE)

– Solid-Phase Extraction (SPE)

Why do Sample Prep?

Issue: Complicated analytical results / too much variability and

potential resolution issues

Cause: analytes contained in a complex sample matrix

Impact:

Quantitation errors: slight changes might resolute in loss of

resolution of critical pairs

May reduce instrument/system up-time

Minutes

0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00

Improve Analytical Results: Example #1

Minutes

0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00

Minutes

0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00

Only need to analyze these peaks

Improvement: Remove unnecessary peaks

Sample Prep

0 10 5

Sample

Minutes

Improvement: Remove Baseline interferences

Spiked Sample

Blank Sample Matrix

3 0.004 AU

10 8 6 4 2 0 min.

Spiked Sample

Blank Sample Matrix

2 0.004 AU

Peak 1 has baseline contamination from sample matrix

Peaks 2 and 3 are clean

Peak 1 is clean

With SPE Clean-up

Improvement: Remove Baseline interferences

Why do Sample Prep?

Issue: not enough sensitivity

Cause:

Analyte concentration in original sample matrix

TOO LOW to measure by instrument

Impact:

Quantitation errors

Concentration of Analyte in Original Sample

TOO LOW (difficult to quantitate)

Time 0 1 2 3 4 5

Concentration of Analyte in

Original Sample TOO LOW

(difficult to quantitate )

Time 0 1 2 3 4 5

Utilize SPE Chromatographic Bed to Trace Concentrate the Original Sample

for that Analyte - Obtain Good Response

Why do Sample Prep?

Issue:

– Not enough sensitivity

Cause:

– For MS detection: minimize ion suppression/enhancement

Impact:

– Quantitation Errors

260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0

Scan ES+

354.4 518.5

472.6 485.5 609.6

Scan ES+ 354.4

485.6472.6

411.5591.6

50/50 Water/ACN + human plasma supernatant

50/50 Water/ACN

260.2 - 97 %

291.2 - 96 %

354.4 - 86 %

411.4 - 93 %

472.6 - 93 %

485.6 - 95 %

591.6 - 89 %

609.5 - 93 %

260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0

Scan ES+

354.4 518.5

472.6 485.5 609.6

Scan ES+ 354.4

485.6472.6

411.5591.6

50/50 Water/ACN

260 280 300 320 340 360 380 400 420 440 460 480260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0

500 520 540 560 580 600 620m/z0

Scan ES+

354.4 518.5

472.6 485.5 609.6

Scan ES+ 354.4

485.6472.6

411.5591.6

50/50 Water/ACN

260.2 - 97 %

291.2 - 96 %

354.4 - 86 %

411.4 - 93 %

472.6 - 93 %

485.6 - 95 %

591.6 - 89 %

609.5 - 93 %

Analytes in human plasma with only Protein Precipitation Standards in aqueous solution give acceptable response

What is Ion Suppression?

260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0

Scan ES+

354.4 518.5

472.6 485.5 609.6

Scan ES+ 354.4

485.6472.6

411.5591.6

50/50 Water/ACN

260.2 - 97 %

291.2 - 96 %

354.4 - 86 %

411.4 - 93 %

472.6 - 93 %

485.6 - 95 %

591.6 - 89 %

609.5 - 93 %

260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0

Scan ES+

354.4 518.5

472.6 485.5 609.6

Scan ES+ 354.4

485.6472.6

411.5591.6

50/50 Water/ACN

260 280 300 320 340 360 380 400 420 440 460 480260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0

500 520 540 560 580 600 620m/z0

Scan ES+

354.4 518.5

472.6 485.5 609.6

Scan ES+ 354.4

485.6472.6

411.5591.6

50/50 Water/ACN

260.2 - 97 %

291.2 - 96 %

354.4 - 86 %

411.4 - 93 %

472.6 - 93 %

485.6 - 95 %

591.6 - 89 %

609.5 - 93 %

Analytes in human plasma with only Protein Precipitation

Analyte standards in aqueous solution

% Loss

Complex Sample Matrix: Ion Suppression

Do all Sample Preparation Techniques Give the Same Result?

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

100 MRM

472.2 > 436.4 1.27e6

MRM 472.2 > 436.4

1.27e6 1.91

80% ion suppression

Minimal ion suppression B (SPE) (Oasis® MCX)

A (PPT)

Note: These samples are dried and reconstituted. Gradient time = 1.5 min

No loss in signal observed for analytes when the interferences, which cause the suppression,

are removed by SPE.

MRM for Terfenadine

The Ultimate Goal is to make your analytical lab more productive

- Lower Limits of Detection

- Run more samples with less time

- Minimize costs in manpower and equipment maintenance

Goals of Sample Preparation: Summary

To remove interferences for

– Better chromatography

– More confident analytical results

– Longer column lifetime

– Less instrument downtime

To enrich sample for

– Higher detection sensitivity

To make sample more compatible for separation and detection

– Matching solvent strength

– Eliminating ion-suppression in LC/MS analysis

Agenda

How SPE Works

Appendix

Yellow is the earliest eluting analyte “band”, it “likes” the mobile phase,

has very low ‘k” (little retention)

Blue is well retained, it “likes” the particles, has high “k” (high

retention - “captured”)

Mix Yellow, Red and Blue Dyes together to create what appears,

to our eyes as a “Black” Sample

Food Dyes

How a Chromatographic Column Works -- “BANDS”

Yellow is the earliest eluting analyte “band” - it “likes” the mobile phase,

has very low ‘k” (little retention)

Blue is well retained - it “likes” the particles,

has high “k” (high retention - “captured”)

How a Chromatographic Column Works -- “BANDS”

Band Migration on an SPE Cartridge

SPE follows the same principles as LC

Blue Red

Yellow

Sample

Agenda

How SPE Works

Appendix

SPE Configurations from Waters

Syringe Barrel Cartridge

Standard 96-Well Plate

On-line SPE Devices

melution 96-Well Plate

Sample Processing: Gravity

Stop Cock flow control valves- to prevent drying – out effect

Male-male Adapter

Syringe Adapter

Vacuum Manifold

Sample Processing: Glass Vacuum Manifold

Vacuum Manifold

Sample Processing: Large Liquid Volumes

Sample Processing: 96-well Plate

Set-up for typical 96-well plates Under vacuum, a properly seated well-plate should move down and the tips will extend into the wells of the collection plate.

Collection Plate

Sample Processing: Positive Pressure

Tip: Cartridge Stacking

Multiple cartridges can be stacked for a mixed-mode approach

2 of the SAME for More Capacity

2 Different Sorbents

for 2 Dimensional

Selectivity

Sample Processing: Positive Pressure Manifold

Agenda

How SPE Works

Typical SPE Strategies

Appendix

SPE Terminology

Sorbent -- Chromatographic packing material (Stationary Phase)

Retention Mechanisms: 1. Reversed Phase (RP) 2. Normal Phase (NP) 3. Ion-Exchange (IEX)

Syringe Barrel Style

Sorbent

Filter/Frit

Sorbents for SEP-PAK® Products Silica and Alumina Based

Sorbents for SEP-PAK® Products Reversed-Phase

Sorbents for SEP-PAK® Products Normal Phase

Sorbents for SEP-PAK® Products Ion-Exchange (Silica Based)

Sorbents for SEP-PAK® Products Specialty Phases

Hydrophilic

monomer

Lipophilic

monomer

Reversed-phase Retention

Hydrophilic-Lipophilic Balanced Copolymer

Polymeric SPE Sorbent Chemistry: Oasis® HLB

• Water wettable

• Polar retention

• Stable across pH 1-14

• No silanol interactions

• High recoveries for acids, bases and neutrals

Retention of Polars

Polymeric SPE Sorbent Chemistries: Oasis® Mixed-mode Ion Exchangers

Agenda

How SPE Works

Appendix

SPE Strategies

Typical SPE Strategies and Protocols –Pass Through

–Clean-up (retention of analytes of interest)

–Fractionation

–Trace Concentration

SPE Strategy #1: Pass Through Purification

Pass Through Approach:

Load: want k = high for interferences (max retention)

want k = 0 for analytes -- no retention

Advantages: clean-up is easy, fast, with little or no method

development

Disadvantages: no concentration of analytes; no solvent switch

Analyte is a

Purple Compound

Pass Through SPE: Load and Collect

Same Sample Solvent

k=0 for analyte

Ostro™ Plate Technology Pass Through Strategy

Specifically Designed for

Phospholipid Removal which, when present,

results in Significant

Ion-Suppression

Methodology

Precipitate

Proteins

Held up by

Filters

Phospholipids

Retained

by SPE

Sorbent Bed,

Analytes

Pass Through

Ostro™ Plate Technology Pass Through Strategy

It is possible to work with lower sample volumes (such as

25µL). When doing so you will need a higher

organic solvent to sample ratio, such as

10:1 or 20:1.

The well volume is 1.9 mL, however in order to mix by aspiration, the

maximum volume is 1.4 mL. This translates to a maximum sample size

of 350µL.

Place Ostro plate onto collection plate

Pipette 50-200µL of plasma into wells

Forcefully add 2% formic acid in acetonitrile,

3:1 solvent:plasma

(methanol not recommended)

Mix thoroughly by aspirating 3x with pipette

Filter samples using vacuum manifold or

positive pressure manifold

Analyze samples

Competitive Techniques: Phospholipids Remaining

MRM of m/z 184-184

Time 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

100 184.4 > 184.4 (Lipid 184) 2.00e8

2.88 2.29 2.21

2.60 2.78 2.72

184.4 > 184.4 (Lipid 184) 2.00e8

2.80 2.27

2.62 2.56 2.68

184.4 > 184.4 (Lipid 184) 2.00e8

1.96 1.90 1.77

184.4 > 184.4 (Lipid 184) 2.00e8

2.84 2.21 1.96 1.75 1.42 1.38

1.63 1.51

Ostro™

LLE with MTBE

LLE with 5% NH4OH in MTBE

SPE Strategy #2: Capture Retain Analytes of Interest

Capture Approach:

Load: want k = high for analyte (max retention – capture)

Wash: want k = 0 for interferences -- no retention

want k= high for analyte so we don’t lose to waste

Elution: want k = 0 for analyte (no retention - strong solvent) to

elute and collect

Advantages: clean-up, desalting, concentration, solvent switching

= clean extracts

Disadvantages: requires method development

Capture SPE Method (RP): Conditioning

Capture SPE Method (RP): Equilibration

Proper Wetting Conditioning

and Equilibration

Good Capture

Capture SPE Method (RP): Load

Capture SPE Method (RP): Wash

Wash Solvent

Capture SPE Method (RP): Elution

Capture SPE Method (RP): Dry down and reconstitute

These steps are performed for some applications where the mobile phase conditions, for the actual analytical

separation, will require a change in the sample solvent. Typically, the strong

elution solvent from the SPE Protocol is carefully evaporated away and the analytes

are reconstituted with the mobile phase used in the instrument.

Optimizing Peak Shape Poor Peak Shape Sample Solvent TOO Strong

Tip: Solvent Strength

Use the correct solvent strength to optimize peak shape

Sample: 80% Organic Mobile Phase: 40% Result: Poor Peak Shapes

Same Column, Same Mobile Phase Weaker Sample Solvent

Tip: Solvent Strength

Sample: 0% Organic Mobile Phase: 40%

Use slower flow rate to increase

contact time

Tip: Contact Time

Tip: Solvent Volume

Sorbent per

Maximum Mass

Capacity

Typical Sample

Volume (Pre-

Dilution)

Typical Elution

Volume

2 mg 0.03 to 0.05 mg 5 to 200 µL ≤ 50 µL

5 mg 0.15 to 1 mg 10 to 200 µL ≤ 150 µL

10 mg 0.35 to 2 mg 50 to 400 µL ≤ 250 µL

30 mg 1 to 5 mg 100 µL to 1 mL > 400 µL

60 mg 2 to 10 mg 200 µL to 2 mL > 800 µL

Solvent volume is directly proportional to bed mass

SPE Protocol: Reversed-Phase

Condition cartridge – strongest solvent first (DCM, MTBE, ethyl

acetate)

– intermediate solvent next (methanol)

– weak solvent last (water)

Load Sample – dissolve in or exchange to weak solvent (water

or water/methanol)

Wash Cartridge – use strongest possible solvent without eluting

analyte (methanol/water)

Elute Cartridge with strong solvent* – methanol, IPA,MTBE, DCM

*Oasis HLB – elute solvent should have at least 5 % methanol or IPA as polar modifier

Prepare Sample

Condition/Equilibrate 1 mL methanol, 1 mL water

Load Sample

Wash 1 mL 5% methanol/water

Elute 2 mL methanol

Evaporate, Reconstitute

Oasis® HLB

SPE Protocol

Conditions for 3 cc 60 mg cartridges

SPE Protocol: Reversed-Phase vs. Mixed-Mode IEX

Prepare Sample

Condition/Equilibrate methanol, water

Load Sample

Wash 5% methanol/water

Elute methanol

Evaporate, Reconstitute

Oasis® HLB

SPE Protocol

Prepare Sample

Condition/Equilibrate

Load Sample

2% Formic acid

Wash 2:

100% MeOH

Elute:

5% NH4OH in MeOH

Oasis® MCX

SPE Protocol

Polymeric SPE Sorbent Chemistries: Oasis® Mixed-mode Ion Exchangers

Strong Cation

Strong Anion

Weak Cation

Weak Anion

Reversed Phase

Oasis® 2x4 Method: Acids and Bases

Neutrals

For Bases:

pKa 2-10

Use Oasis® MCX

For Strong Acids

pKa <1.0

Use Oasis® WAX

For Strong Bases

pKa >10

Use Oasis® WCX

For Acids

pKa 2-8

Use Oasis® MAX

Prepare Sample

Load Sample

5% NH4OH

Elute 1:

100% MeOH

Elute 2:

2% Formic Acid in MeOH

Protocol 2 Prepare Sample

Load Sample

2% Formic acid

Elute 1:

100% MeOH

Elute 2:

5% NH4OH in MeOH

Protocol 1

Bases Strong

Strong

Bases Acids

Oasis® 2x4 Method: Neutrals

Neutrals

For Bases:

pKa 2-10

Use Oasis® MCX

For Strong Acids

pKa <1.0

Use Oasis® WAX

For Strong Bases

pKa >10

Use Oasis® WCX

For Acids

pKa 2-8

Use Oasis® MAX

Prepare Sample

Load Sample

5% NH4OH

Elute 1:

100% MeOH

Elute 2:

2% Formic Acid in MeOH

Protocol 2 Prepare Sample

Load Sample

2% Formic acid

Elute 1:

100% MeOH

Elute 2:

5% NH4OH in MeOH

Protocol 1

Bases Strong

Strong

Bases Acids

Reversed-phase Backbone

SPE Strategy #3: Fractionation of Analytes

Capture and Fractionation:

Load: k = high for analytes (max retention)

Elution 1: k = 0 for one analyte class only [slightly stronger solvent]

(remaining k’s = high)

Elution 2: k = 0 for second analyte class only [slightly stronger solvent]

(remaining k’s = high for remaining)

Elution 3: etc…

Advantages: separates classes of compounds, if they have

different polarities and hydrophobicities

Grape Kool-Aid®

Sample Matrix

Polar Compounds Polar Water

Red Dye # 40 8%IPA

Blue Dye # 1 35%IPA

Non-Polar Oils Non-Polar 70%IPA

{Reversed-Phase Conditions}

Polarity Elution

Solvent

Solution LOOKS Purple

Dry Powder Fruit Flavored

Mix – add Water

Capture and Fractionation

Capture and Fractionation: Load

Capture and Fractionation: Elute #1

Capture and Fractionate: Summary

Polars Red Dye Blue Dye Non- Polars

SPE Strategy #4: Trace Enrichment

Trace Concentration/Enrichment:

Load: k = high for analytes (max retention)

and load large sample volume

Elution: k = 0 for analytes

Advantages: concentration of very low level analytes

Trace Enrichment: Load

Trace Enrichment: Continue Loading

Trace Enrichment: Elution

Elute the analtyes, which now have been concentrated, by using a stronger solvent to

release them from the device (k = 0)

Determine the concentration of

THIS NEW sample -- and then calculate the

ORIGINAL Sample Concentration by dividing by the volume of

ORIGINAL sample processed by the

SPE Device

Reverse Flow

Direction

Adapter Tube

Elute Band in Less Solvent

More Concentrated

Trace Enrichment: Elution (Reverse Cartridge)

Reverse Flow

Direction

Adapter Tube

Elute Band in

Less Solvent

Concentrated

Greatly Enhanced Sensitivity

Trace Enrichment: Elution (Reverse Cartridge)

Agenda

How SPE Works

Appendix

Determining % Recovery: Calculation

Analytical Result

= 9.5 ng/mL

Determining % Matrix Effect: Calculation

Analytical Result =10 ng/mL

Importance of % Recovery and % Matrix Effect

% Analyte Recovery in plasma

% Matrix Effects in plasma

PPT PLR Plate

LLE RP SPE

Oxycodone

Oxycodone d6

PPT PLR Plate LLE RP SPE

Oxycodone

Oxycodone d6

Conclusions

SPE is a powerful sample preparation technique that

utilizes extraction selectivity with the tangible

benefits of:

– Clean-up: the simplification of a complex sample matrix

– Reduce ion suppression (or enhancements) for MS

applications

– Fractionate to analyze distinct classes of compounds

– Trace concentration (enrichment) of samples with very low

concentration levels

– Solvent switch

Additional SPE Seminar Topics

Food Compound Specific Analysis of Food

Food Safety Screening

Environmental Environmental Sample Preparation

BioAnalytical Small Molecule Fit for Purpose Bioanalytical Method Development

2x4 Mixed Mode SPE: Troubleshooting and Optimization

BioAnalytical Large Molecule Intro to peptide Bioanalysis

Thank you for your attention

Questions?

Contact Info: James_Teuscher@waters.com

Agenda

How SPE Works

Typical SPE Strategies and Protocols

Appendix

Technology Capability Literature: Primers

www.waters.com

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