Demystifying Solid Phase Extraction for Toxicological ... · PDF fileDemystifying Solid Phase Extraction for Toxicological Analysis of Biological Samples ... SCX / SAX Non-Polar Polar

“ONE Sorbent,

ONE Method,

For ANY SPE application”

Demystifying Solid Phase Extraction for Toxicological Analysis of Biological Samples

Presented byMarc Boggeri

Technical Manager, Sample PreparationPhenomenex USA

Before We Start….a word on Non-commercial Intent.

My goal is to present you with tools to remove some of the mystery surrounding SPE.

• Fact. It’s 2004. New SPE tools do exist. • Fact. Not every manufacturer sells them due to patents and

commercial concerns.• Fact. Phenomenex pays my mortgage, so…• Fact. I will use Phenomenex products in my examples, more

as a practical matter than a commercial matter.• Fact. It’s 2004….I can get sued if I speak too much “on the

record” about other competitors… thank the lawyers for that!

So please bear with me as I try to navigate the minefield !

SDB Polymers

C18E

C18U

C8

Phenyl

CN

SI

NH2

FLPR

SCX / SAX

Non-Polar

Polar

Ionic

Strata X

Strata X-C

THIS….

+

PLUS

96-well Plate(96 little SPE tubes in a microtitre plate

footprint)

THIS

= Confusion!Which one to

choose?

Let me simplify.

Introduction to Solid Phase Extraction

What is “SPE” ?A “Sample Preparation” technique.

For our purposes Sample Preparation is“Manipulations performed to the sample, prior to analysis, that

are designed to facilitate chromatographic analysis.”

Common Sample Preparation Techniques:

HomogenizationDilutionWeighingSettling and DecantingCentrifugationFiltrationEnzymatic HydrolysisEvaporationLiquid / Liquid ExtractionProtein Crash/ PrecipitationSolid Phase Extraction

and many others.


Goals of SPE

“Cleanup”

“Concentrate”

“Solvent switch”


1 Liter Dirty Water

SPE Extraction

1 ml concentrated analtye

1. Clean-up

0 Minutes 100 Minutes 10

Why do we perform SPE?

Before (Liq /Liq)After SPE

2. Concentrate and 3. Solvent Switch


The REAL GOAL !


HOW SPE WORKS

The sample is passed through a SPE device. Analyte(s) is retained onto the sorbent.

The contaminants are washed out of the tube.

Analyte is eluted from the tube.

Note: Step 1: Conditioning / Solvation omitted for visual simplicity.


What Type of Interactions?

SPE relies upon the same basic chromatographic retention mechanisms as

HPLC, GC and TLC.


SPE and HPLC differ in the strength of the interactions between the analyte and the stationary phase.

– SPE: Strong interactions, 100% retention of analyte, or 100% elution.

– HPLC: Weaker partial interactions that allow each of the analytes todifferentially migrate along the length of the column.


4 Main Steps

1. Mechanism2. Chemistry3. Sorbent Mass in tube4. Tube Size / Format

Choosing a SPE Product

1.a. Non-Polar / Reverse Phase: HydrophobicTypical phases: C18, C8, C4, CH

Step 1. Mechanism

1.b. Non-Polar / Reverse Phase: AromaticTypical phases: Strata X, SDBL, styrene divinylbenzene polymers, PH.

Step 1. Mechanism

Reverse Phase: Aromatic Selectivity

2. Hydrophilic / Polar / Normal PhaseTypical phases: Silica, NH2, CN, FL., X

Step 1. Mechanism

3. Ionic / Ion Exchange / (hydrophilic)Typical phases: SCX, SAX, NH2,

Plus: Mixed-mode sorbents: X-C, Screen C & Screen A.

Step 1. Mechanism

SDB Polymers

C18E

C18U

C8

Phenyl

CN

SI

NH2

FLPR

SCX / SAX

Non-Polar

Polar

Ionic

Strata X

Strata X-C

Step 1. Mechanism

SPE offers a wide-range of extraction “tools”!

Beware of The “Hidden” Strong Ion Exchangers

• Strata X-C: SCX on a aromatic resin.“For just about anything!”

• Strata Screen C: C8 + SCX“for screening of cationic compounds”

• Strata Screen A: C8 + SAX“for screening of anionic compounds”

Method Development

Before we get too far: A Practical View

The most common Method Development Mistakes are1. Choosing the wrong SPE product ( sorbent or mass)2. Using the wrong solvents or volumes3. Not setting realistic goals for the sample prep.

Ie, Which is most important to your overall analysis?

– Absolute highest recovery ?– Greatest clean-up?

Typically some compromise is required.

Method Development

Historical Information

• How do you extract now or in the past? Ie, Liquid/Liquid Data. • “Bases” = cationic amines • “Acids” = anionic organic acids• “Neutrals” = general non-polar structure

• Any SPE methods that did not work well? What did not work is almost as useful as knowing what did!

Method Development

Summarizing: Analyte Polarity vs Retention Mechanism

Analyte Polarity RetentionMechanism

Hydrophobic Non-Polar

Hydrophilic Polar

Ionized (+ or -) Ion Exchange

“Like dissolves Like”

For Ion Exchange: “ Opposites Attract”

Method Development

Classify the Matrix / Sample

• Liquid, Solid or Suspension?• Composition: Aqueous, Organic, Solids, Fatty.• Liquid: pH, Ionic strength• Solubility: Aqueous, Organic• Impurities and known interferences • Expected Concentration of Analyte:

= volume needed to extract to reach Method Detection Limit?

But that’s only ½ the Story!

Summarizing Matrix Effects and SPE Mechanism

Sample Matrix Composition Likely SPE Retention Mechanism

Aqueous: Water, biological fluids, aqueous homogenates. Non-Polar

Organic: Non-polar organic solvents Polar

Aqueous or Organic Ion Exchange

Method Development

3. Identify Extraction Mechanism

Putting it all together.

Method Development

3. Identify Extraction MechanismAnalyte + Matrix = Mechanism !

Analyte Polarity Sample Matrix Polarity SPE Mechanism Sorbents

Non-Polar Aqueous Non-PolarC18E, C18U, C8, PH,

SDBL, X "Universal" Polymeric Sorbent

Polar Non-polar organic Polar SI-1, Si-2, NH2, CN, FLPR

Ionic Aqueous or Organic: Ion Exchange (Strong and Weak)

SCX, WCX, SAX, NH2, Mixed-mode w/

IEX

Once you identify the mechanism: then you select a specfic sorbent

Method Development

In reality this happens:

AqueousWaterUrinePlasmaBloodAq. Tissue Preps

Organic H2O Misc.

MeOH

Acetonitrile

IPA

Organic NOT H2O Misc.

Hexane

DCM

Reverse Phase

Strata X, C18E, C8

Mixed Mode-Ion Exchange

Strata X-C, SAX, WCX, etc.

Ion Exchange

Strata SCX, SAX, WCX

Normal Phase

Strata NH2, CN, SI, FLPR

Ion Exchange

Strata SCX, SAX, WCX

+ H2O

+ Hexane

Method Development

Method Development

SDB Polymers

C18E

C18U

C8

Phenyl

CN

SI

NH2

FLPR

SCX / SAX

Non-Polar

Polar

Ionic

Strata X

Strata X-C

4. a) Chemistry

Step 2. Choose Sorbent Chemistry

Mixed Mode sorbents should be classified by their

ion exchange group!

Step 3. Choose Sorbent Mass in Tube

Selection of the proper sorbent mass is critical:

• Not Enough Sorbent = Overloading = Sample BreakthroughOverloading results in Poor Irreproducible Recovery.

• Too Much Sorbent = Sufficient Capacity = Safe but Economically Undesirable.

Larger tubes more expensive, need more solvent and sometimes dirtier extracts.


FACT #1: The sorbent packed inside the tube performs the extraction .

FACT #2: Sorbent mass selection is NOT an exact science. Lots of matrix effects!

Si-based Sorbent Capacity: A Theoretical Approach:– Non-polar and polar sorbents: Equivalent to 5% of the sorbent mass.– Ion Exchange Sorbents: Typically from 0.5 to 1.0 milliequivalents per gram of

sorbent (meq / g ). Values are commonly reported by the manufacturer.

Biological:Blood, serum and plasma:

Resin (Strata X or X-C) 30 mg per 250 ul sampleSilica-based: 50 mg per 250 ul sample.

Urine: Resin (Strata X or X-C) 30 mg per 1 ml sampleSilica-based: 50 mg per 1 ml sample.

Filtered Tissue homogenates: 60 mg Resin or 100 mg Si-based per 100 mg tissue.

Summary: Be safe. Round Up.

Step 3. Choose Sorbent Mass in TubeSorbent Capacity: A Practical Approach

For Example:

“How much sorbent do I need to extract a 500 ul plasma sample?”

For strata X: “ 30 mg per 250 ul sample”

2 x 30 mg = 60 mg strata X

For C18E: “50 mg packing per 250 ul sample”

2 x 50 mg = 100 mg C18E

Do the Math…. scale up or down as necessary.


4. b) Sorbent Mass

Step 4. Choose Tube Format / Size.

• Typically the required sorbent mass is available in a limited number of tube sizes, or volumes. Choose the closest available mass.

• Remember: tube “size” in ml ( or cc) is of secondary importanceunless you have some hardware/robotic requirements. The sorbent does the work.

96-well Plate(96 little SPE tubes in a microtitre plate

footprint)



Sample volume > than Tube ?

Sorbent Mass

Theoretical Minimum 2 bed volumes Not Recommended ! )

Practical Minimum Wash and Elution Volume 8 bed volumes

Recommended Wash and Elution Volume 16 bed volumes ( Safe Starting Point)

10 mg * 50 ul 200 ul 400 ul 25 mg 75 ul 250 ul 500 ul 30 mg * 125 ul 500 ul 1 ml 50 mg 125 ul 500 ul 1 ml 60 mg * 250 ul 1 ml 2 ml 100 mg 250 ul 1 ml 2 ml 150 mg 375 ul 1.5 ml 3 ml 200 mg 500 ul 2 ml 4 ml 500 mg 1.25 ml 5 ml 10 ml 1 g 2.5 ml 10 ml 20 ml 2 g 5.0 ml 20 ml 40 ml 5 g 12.5 ml 50 ml 100 ml 10 g 25.0 ml 100 ml 200 ml

Method Development

* = resin sorbents. Strata X and X-C

Practical Tool: Solvent Volumes for SPE Processing

Introducing Secondary Interactions:

Primary Interaction = Strongest Interaction between analyte and sorbent.

Secondary Interaction = Weaker Interaction…

For ion exchangers: – Ionic = primary & very strong

– Hydrophobic = secondary & weaker… but still there.

Lets’ have a closer look…

Method Development

Secondary Interactions on Ion Exchangers

Primary

Secondary

For Elution: Must disrupt both !

Hydrophobic

Method Development

Practical Suggestion: Ion Exchange Elution Solvents:

Because of Secondary Non-polar interactions.

• The MEOH + acid or base (98:2) may be the best choice.• Efficient Low volume elution.• Volatile.• Simple to make.

For Strata X-C: MeOH : Acetonitrile : NH4OH (sat.) (49:49:2)

Note: For X-C and Screen C mixed-mode ion exchangers… organic is a MUST!

Method Development

Method Development Update: 6 / 04

New Resin Technology Greatly Simplifies Method Development from Bio samples and Aqueous Matrices.

•Strata X. Simple reverse phase SPE method is very simple and rugged.

•Strata X-C mixed-mode cation exchange for super cleanup from dirty biomatrices.

Not Marketing hype!

Feeling BOMBARDED with the following claims?

“Deconditioning Resistant”

“Superior recovery for Acidic,Basic and Neutral Compounds”

“Automation Friendly”

“Water-wettable”

“Hydrophilic-Lipophilic Balance”

“Fast and Simple Method Development”

And the Mother of All Marketing Claims….

“ONE Sorbent,

ONE Method,

For ANY SPE application”

Does it justify such wild claims?

• Short Answer:

Yes !• Long Answer

Let’s have a closer look….

First: Recall the Deficiencies of traditional SPE sorbents

• Residual silanol / uncontrolled secondary interactions (end capping

effect)

• pH limitations of base silica

• Base silica fragile: crushing = fines

•Lot to lot reproducibility

•Must keep “wet”after conditioning

• Narrow selectivity range: limited retention of polar metabolites

With that in mind…

Most Popular are: Styrene-divinyl benzene polymers modified by the addition of a “ polar”

functional group. N-vinylpyrrolidone.

– Phenomenex Strata X : surface coated resin– Competitor OW: copolymerized resin

Phenomenex Strata X is a unique patented sorbent chemistry only available from Phenomenex.

What are these new “ wonder” resins?

Strata X and X-C Offer Three Primary Benefits Over Silica Based Sorbents.

1. Aromatic selectivity2. Worry-free processing. 3. Non-halogenated Elution Solvent

Plus:– Exceptional cleanup– Superior Lot to lot reproducibility

How does Strata X compare to traditional Si-based ?

Structure of Strata X


Polar / HydrophillicSelectivity

The Strata X resin family.

SDBL X

X-C+ SCX group

+ SAX group X-A ( ? )

X-CW

+ WCX group

(coming soon !)

X-WA

+ NH2 group

( ? )

Benefit #1. Unique SelectivityMulti-mode Retention Mechanism: π - π, hydrogen bonding and

hydrophobic

Styrene divinylbenzene: Aromatic reverse phase selectivityPlus

N-Vinylpyrrolidone: Hydrophilic – polar.

Think about it. How many of your analytes have aromatic regions?How many have some polar/charged functional groups?

The majority.

Some common analyte structures.

O

O H

H

H

O

O H

THC-COOH

NH2

Amphetamine

O

O N

Acidic Probes

X

X=OH (Salicylic acid)X=OCOCH3 (Aspirin)

COOH

CH3O

Naproxen

COOH

Ketoprofen

Basic ProbeC2H5

C2H5H2NProcaine

COOH

OHOH

OOH

O

H

H

H

Prednisolone

Example of basic compounds

O

N

OH

O

H

NH

OH

NH2

NH

O

OH

NH

O

NH2

OH

C18 “De-Conditioning” / Chain collapse reduces (destroys!) sorbent efficiency.

Unconditioned Conditioned

> 2 min @ 5 in Hg Vacuum

Benefit #2. Worry-free Processing.“ Deconditioning”: What it is and how it can effect your work.

Note dye band on C18 vs Strata X after drying for 10 minutes.

Silica C18 sorbent dried after conditioning with

methanol

Strata X

Benefits of strata-X: resistant to deconditioning

BAD ! Good !

Structure of Strata X


Polar / HydrophillicSelectivity

No “Chains” to collapse !

Polar Modifier “holds” conditioning solvents!

Sorbent Chain Collapse from an Extraction Recovery Perspective.

ProcainamideTheophyllineCaffeine

RanitidineDoxepin

0

20

40

60

80

100

0 2 4 6 8 10Drying time (min)

% R

ecov

e ry

Both dried at 10” Hg after conditioning with MeOH.

0

20

40

60

80

100

120

0 2 4 10

% R

ecov

ery

Drying time (min)

Silica C18 Strata-X

In the Lab: Benefits of Strata X

• No need to use stopcocks with tubes

• Higher throughput. • Turn the vacuum on and leave it on!

• Worry-free automation. • Program vacuum and flow rates for the “slow flow” samples

• Super rugged / Super reproducible

• Best sorbents for 96-well plate SPE!

Fewer Headaches !

Conditionmethanol

Equilibratewater or buffer

Load Sample

Wash1. buffer

2. acidified water3. methanol

Elutemethanol/AcN + base

Halogen-free elution!

Benefit #3. Halogen-free Elution with Strata X-C for Bases

Strata X: So does it really perform?

Bases

Neutrals

Acids

0

20

40

60

80

100

Nor

doxe

pin

Am

itrip

tylin

e

Nor

tript

ylin

e

Dox

epin

Pro

cain

amid

e

Ace

tam

onop

hen

Theo

brom

ine

Theo

phyl

line

Caf

fein

e

Cim

itidi

ne

Ibup

rofe

n

Nap

roxn

Feno

prof

en

Indo

met

haci

ne

% R

ecov

ery

RSD: 2-5%

Clean up of analytes from serum

Recovery of a wide spectrum of analytes

strata-X in pharmaceutical applications: extraction of steroids from plasma

F

OHOH

O

OOH

H

H

OHOH

OOH

O

H

H

H

Betamethasone

Prednisolone

0

20

40

60

80

100

% R

ecov

ery

Pred

nisolo

ne

Betha

met

haso

ne

Greater than 90% Recovery


strata-X in biological applications: selective elution of peptides from a salt matrix

Selective Elution Based on their Acidity by Different Elution Solvents

Eluent ACTH Bradykinin Angiotensin I HGRH

40:60 ACN/water 0.0% 5.1% n/a 101.5%

70:30 ACN/water 0.0% 44.1% 98.4% n/a

70:30 ACN/1.0% formic acid 78.8% 92.5% 98.5% n/a

Absolute Recovery from strata-X, 30mg. Vol of eluents = 250uL.

Basic Neutral Acidic


strata-X-C in veterinary applications: extraction of sulfa drugs from plasma

NH2

S

O O

NH

ONCH3

Sulfamethoxazole

N

N NH

SNH2

O O

Sulfaquinoxaline

0

20

40

60

80

100

% R

ecov

ery

sulfa

thiaz

olesu

lfam

etho

xazo

le

sulfa

quino

xalin

e


strata-X-C in toxicological applications:extraction of diuretics from plasma

S

NH

NH

O O

S

Cl

NH2

O O

NH

OHO

S

ClNH2

O

O

O

0

20

40

60

80

100

% R

ecov

ery

Hydr

ochlo

rothi

azid

e

Furo

sem

ide

hydrochlorothiazide

furosemide


strata-X-C in toxicological applications:extraction of drugs of abuse from urine

020406080

100

% R

ecov

ery

THC-

COOH

Amph

etam

ine

Met

ham

phet

amine

NH2

O

O H

H

H

O

O H

THC-COOH

Amphetamine


strata-X-C

strata-X-C: Your best choice for toxicological analysis :

Bases: strong cation exchanger + powerful secondary interactions including π- π and hydrogen bonding

Acids and Neutrals too ! Utilize the π- π and hydrogen bonding for retention.

No pH limitations

Deconditioning resistant.

No Methylene Chloride needed!

2004…Reverse Phase

New Simplified :

Surface-modified, resin based:

• strata X

Old / Historical:

Predominantly silica based, linear hydrocarbon.

• C18E, C18U, C8, C1, PH.

2004… Ion Exchange and Mixed-mode

Old:

Silica based: SCX, WCX, SAX, NH2

Mixed Mode: Screen C and Screen A.

New Simplified:Resin based:

•Strata X-C (mixed-mode, strong cation exchange)

•Strata X-A ( mixed-mode, strong anion exchange)

Silica based•WCX (weak cation exhange)•NH2 (weak anion exchange)

Thank You !Questions?

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Demystifying Solid Phase Extraction for Toxicological ... · PDF fileDemystifying Solid Phase Extraction for Toxicological Analysis of Biological Samples ... SCX / SAX Non-Polar Polar