SPE Fundamentals and Recent Developmentsll1.workcast.net/10311/0279275158671341/Documents/SPE...

© 2012 Sigma-Aldrich Co. All rights reserved.

sigma-aldrich.com/analytical

SOLID PHASE EXTRACTION:

Fundamentals and Recent Developments Olga I. Shimelis, Ph.D

Principal R&D Scientist

© 2012 Sigma-Aldrich Co. All rights reserved. 2

Time Spent on the Analytical Process

R.E. Majors, LC/GC Magazine, 1992, 1997, 2002


Fit of Solid Phase Extraction in the Analytical or

Chromatography Workflow

SPE is a Sample Prep Technique

It is used to extract analytes from a wide variety of matrices prior to HPLC

or GC analysis.

SPE is form of liquid chromatography used for sample prep of liquid

samples, the particle size for SPE is larger then for HPLC

SPE is selective and versatile: Many different sorbents and elution

conditions are available for different analytes and matrices

3

Sample Prep Separation Detection Analysis

Sample

Collection/

Storage

Fraction

Collection


Important Goals of an SPE Extraction

The characteristics of a good SPE method are:

High analyte recovery

• Recovery measured relative to an internal standard

No interference from sample matrix components

High analyte concentration

Simple method, minimal steps

Reproducible

No waste

• Shortest extraction time that still gives good results

• Minimum wash and elution solvents

4


How Does SPE Work?

There are 2 different elution strategies in SPE. Which one to choose depends on the goal of the extraction.

• Bind-Elute Strategy (shown on next slide)

• Most common

• Bind: Analytes bind to sorbent in the tube, unwanted matrix components are washed off

• Elute: Eluant changed to remove analytes from tube

• Different eluants can be used to fractionate the analytes

• Analytes are concentrated via evaporation prior to HPLC or GC analysis

• Interference Removal

• Bind all unwanted matrix components and allow analytes to pass through during the sample loading stage

• Like chemical filtration

Bind-elute SPE strategy is the most common.

5


Bind-elute Strategy Diagram

6

Shown is a step-by-step bind-elute SPE extraction, beginning with a filtered

sample containing analytes and internal standard (IS) in a matrix and ending

with purified and concentrated analytes and internal standard ready for HPLC

or GC analysis.

Matrix fraction

= waste Analyte

fraction

2) Apply

wash solvent

3) Apply

elution solvent

Original sample

(analytes & IS

in a matrix)

1) Apply

sample to

SPE tube

HPLC or

GC analysis

Purified &

concentrated

analytes & IS

A dilute

solution of

analytes & IS

in the

elution solvent

4) Evaporate

elution solvent,

reconstitute

Must first condition

& equilibrate SPE

tube


Interference Removal Strategy „Chemical Filtration“

Sample with Internal Standard in Matrix Matrix adsorbed Analytes & IS pass

2) Apply

elution solvent

3) Evaporate

elution solvent,

reconstitute

Must first condition

& equilibrate SPE

tube


Understanding Retention Mechanisms


Reversed-Phase SPE

Common Sorbents: C18-functionalized silica, polymeric (DVB), carbon


Normal-Phase SPE

Common Sorbents: Bare Silica, NH2 or Diol-functionalized silica,Florisil, Alumina,


Ion-Exchange SPE

Advantage: Wash with 100% Organic possible very clean Extracts


Useful Ion Exchange SPE Tips

Ion exchange kinetics slower than RP & NP => reduce flow rate

Strong vs. weak ion-exchangers

• Strong = sorbent functional group always ionized regardless of pH

• Weak = sorbent functional group has controllable pKa;

commonly used for extracting strong analytes

Counter-Ion Selectivity


Ionization of Acidic & Basic Molecules- Acids (e.g., carboxylic acids): (e.g., R-COOH R-COO-) HA H+ + A-

(Un-ionized) (ionized) 50% @pKa 50% 100% low pH 0%

0% high pH 100%

Bases (e.g., amines): (e.g., R-NH3+ R-NH2)

BH+ + OH- B (Ionized) (Un-ionized) 50% @pKa 50% 100% low pH 0%

0% high pH 100%

The Critical Role of pH in SPE

Neutral State (Blue) = promotes hydrophobic (RP) interaction

Ionized State (Green) = promotes electrostatic (IOX) interaction

pKa of most acids (e.g. –COOH) is 3-5

• Presence of halogen atom near a carboxy

group strengthens acid effect (electron sink)

• e.g., acetic acid (pKa 4.75), monochloro

acetic acid (pKa 2.85), dichloroacetic acid

(pKa 1.48)

pKa of most amines is 8-11

• Aromatic (electron sink) amines have a

lower pKa than aliphatic amines

• e.g., Aromatic amines- aniline (pKa 4.6),

pyridine (pKa 5.2); Aliphatic amines- (pKa

9.7), dimethylamine (pKa 10.7)


Keys to Successful SPE methods


Consider the analyte(s) of interest

What functional groups may influence the analytes’ solubility, polarity,

ionization state (pKa), etc.?

Hydrophilic Groups:

•Hydroxyl -OH

•Amino -NH2

•Carboxyl -COOH

•Amido -CONH2

•Guanidino -NH(C=NH)NH3+

•4° Amine -NR3+

•Sulfate -SO3-

Hydrophobic Groups:

•Carbon-Carbon -C-C

•Carbon-Hydrogen -C-H

•Carbon-Halogen -C-Cl

•Olefin -C=C

•Aromatic

Neutral Groups:

•Carbonyl -C=O

•Ether -O-R

•Nitrile -C=N


SPE Phase selection

Aqueous (Biological Fluids, water,

aqueous extracts)

Reversed-Phase

Moderately polar to non-polar

compounds

C18, C8, Ph, CN, DPA-6S, Carbon, SupelSelect HLB

Ion-Exchange

Weak cations/anions

SAX, SCX, MCAX

Strong cations/anions

WCX, NH2, PSA

Organic (organic extracts in

hexane, dichloromethane)

Normal-Phase

Polar to moderately

polar compounds

Si, -CN, Diol, -NH2, Florisil,

Alumina

16

YOUR

Sample

matrix is:

Recommended

Retention

Mechanisms:

Analyte

Characteristic:

Recommended

SPE phases:


Key to Successful SPE Methods

Choose the appropriate SPE

phase by understanding the sample

matrix and identifying analyte(s)

functional groups that influence its

solubility, polarity, etc..

Understand how the analyte(s)

behaves on the sorbent in

response to changing extraction

conditions.

Manipulate these conditions to

meet the defined sample prep

objectives

Supel™ Sphere dual-layer

Multiple sorbents beds can be combined in one cartridge to improve

the selectivity of the cleanup method


On the Outside: SPE Formats

Different SPE formats are available. They all have the following in

common: sorbent particles held securely in place to withstand the force of

the liquid flow.

Tubes

96-well plates

Disks

Sorbent particles are

held in an inert,

permeable filter disk.

Allows faster flow

rates and larger

samples

Sorbent particles are

contained inside the

wells, held in place by

frits

Tubes (plastic or glass) are the most

common SPE format. Also called cartridges.

Loose sorbent

A modern format,

called dispersive SPE

where sorbent is loose

in a tube


Using the Appropriate Bed Weight for Sample Size

96-well plates often comes with different bed weights

Sorbent Bed weight Loading volume Retained by

sorbent

15 mg 20-160 µL up to 30 µL

25 mg 75-400 µL 75 µL

50 mg 200-800 µL 120 µL

100 mg 300-1000 µL 250 µL


SPE Method Development: Tricyclic

Antidepressants from Serum on C18 SPE

Wash/Elute Profile Evaluation-

At low pH, complete

elution occurs at 60%

MeOH.

At neutral pH, complete


MeOH.

Under basic pH, complete


MeOH.

At low pH, retention

limit is 10% MeOH.

At neutral pH, retention

limit is 20% MeOH.

Under high pH, retention

limit is 40% MeOH.

Low pH Wash/Elute Profile

0 20 40 60 80 100

% Methanol

Peak A

rea (

mA

U*s

)

Neutral pH Wash/Elute Profile

0 20 40 60 80 100

% Methanol

Peak A

rea (

mA

U*s

)

High pH Wash/Elute Profile

0 20 40 60 80 100

% Methanol

Peak A

rea (

mA

U*s

)

Amitriptyline

pKa 9.76


SPE Products from Supelco

21

Supel™-Select (basic method provided)

• Hydrophilic modified styrene based polymers

• Great generic SPE

HybridSPE®-Phospholipid (method & guidelines provided)

• Simultaneous removal of phospholipids and proteins

• Enhances MS detection by reducing ion suppression

SupelMIP® (method provided)

• Molecularly imprinted polymers

• Highly selective for analytes in difficult matrices

Supel™ Sphere Carbon/NH2 (for Japanese positive system)

• Dual layer tubes for pesticide residue analysis

Supel™ QuE (standardized methods)

• QuEChERS tubes and supplies

• Z-Sep family of sorbents for more challenging matrices cleanup


Triclosan in creek water

22

Supel-Select HLB –

A generic hydrophilic-lipophilic polymer SPE sorbent

Functions under reversed-phase conditions

Compatible with aqueous loading samples

Triclosan

It is commonly known as antibacterial agent in personal care products

Triclosan was first registered as a pesticide with the US EPA in 1969.

Currently, triclosan also has 20 antimicrobial registrations. A more

comprehensive review of triclosan by EPA started in 2013.

Triclosan was previously found to be persistently present in the

environment, it should be “labelled as toxic to fish and other aquatic

animals”

Triclosan was analyzed in a small stream and in a waste-treatment plant

eluent


Supel-Select HLB: triclosan extraction/concentration

• Per EPA method 1692

• Amber glass container should be used

• Sample analyzed within 7 days Sample collection

• Acidify 50 mL or 100 mL sample to pH 2

• Add 25 mg or 50 mg of EDTA-Na4, mix for 60 min Sample Preparation

• 3 mL methanol Supel-Select HLB, 60 mg/3 mL (54182-U)

• 3 mL distilled water

• 3 mL 0.01 M HCl Condition SPE tube

• Load prepared sample (50-100 mL) Load

• 10 mL DI water

• Dry for 10 minutes under vacuum Wash

• 3 mL acetonitrile: methanol (1:1)

• Evaporate to dryness under vacuum

• Reconstitute into mobile phase solvent Elution

23

500 mL per

EPA

method

7 mL wash

was tested

6 mL elution

was tested

88% recovery from spiked water samples at 3500 ng/L


Supel-Select HLB SPE and LC-MS/MS:

Triclosan in creek water

24

0

100

200

300

400

500

600

700

800

0 2 4 6 8 10

MR

M 2

86.8

/35.0

Time (min)

In waste-water plant effluent

-10

0

10

20

30

40

50

60

0 2 4 6 8 10

MR

M 2

86.8

/35.0

time (min)

Triclosan found

at 63 ng/L

In creek water, 1 mile

downstream from discharge

Instrument: Agilent 1100/1200 HPLC and AB Sciex Qtrap 3200

HPLC Column: Ascentis Express C18 2.7 µm 15 x 2.1 mm

Mobile phase: 40% Methanol, 40% Acetonitrile, 20% aqueous 0.1% formic acid

Flow rate: 0.150 mL/min

Temperature: 30˚

Injector volume: 10.0 µL

Detection: Q1/Q3 286.8/35.0 ESI negative

Triclosan found

at 1100 ng/L


Supel-Select HLB SPE: Triclosan in creek water

• SPE method was developed using Supel-Select HLB to clean and pre-

concentrate triclosan from water samples using reversed phase interactions

• The method was successfully applied to creek water and to waste-water using

LC-MS/MS detection

• The creek level of triclosan was found to be 1/20 that in waste-water due to the

dilution of the waste-water plant effluent in the creek.

• For more information, please visit sigma-aldrich.com/supel-select

25

http://sigma-aldrich.com/supel-select







Supel-Select SCX: Isolation and LC-MS

Characterization of Illicit “Bath Salts” in Urine

Phenethylamine and cathinone compounds were being marketed

as “bath salts”, affects similar to heroin and methamphetamine

− there are three sets of isobaric compounds that require

chromatographic resolution for positive confirmation: LC-MS

method required for testing

Supel-Select SCX: is a polymerically based cation exchange

absorbent, containing a strong anion exchange sulfonic acid

functionality.

– Analytes selectively retained via ion exchange

– High organic wash solvents displace endogenous matrix

– Bath salts eluted with basic organic solvent

– Result: Highly clean sample!


Supel-Select SCX: SPE method for bath salts

•Supel-Select SCX, 30mg/1mL (54240-U) •1 mL water/urine spiked with 100 ng/mL bath salt mixture

Loading sample

• 1 mL 1% formic acid in acetonitrile

• 1 mL water Conditioning

• Load 1 mL spiked urine

• 1 mL water

• 1 mL 1% formic acid in acetonitrile

• 1 mL water

Loading and wash

• 2 mL 10% ammonium hydroxide in acetonitrile

• Evaporate to dryness, reconstitute into 100 μL water:methanol

• Inject into LC-MS, HILIC HPLC column

Elution

27

76-90% recoveries from spiked water samples


Bath salts: HILIC chromatography

28

Column: Ascentis Express HILIC (Si), 10 cm x 2.1 mm, 2.7 μm (53939-U)

Mobile phase: (A) 5 mM ammonium formate in acetonitrile, (B) 5mM ammonium formate water (98:2)

Flow rate: 0.6 mL/min; inj. 1 μL; standard at 200 ng/mL; Detection: TOF/ESI+ 100-1000 m/z


Sample (100 ng/mL) Spiked water Spiked Urine

MDVP 79.3 43.7

Buphedrone 90.6 77.4

3-Fluoromethcathinone 76.2 67.

Butylone 89.5 80.8

Ethylone 88.5 76.6

4-Fluoromethcathinone 83.1 69.1

Mephedrone 86.1 78.2

Methylone 89.1 77.9

Methedrone 89.9 80.3

Recoveries of bath salts using Supel-Select SCX

SPE cleanup and HILIC-MS detection


Supel-Select SCX: Isolation and LC-MS

Characterization of Illicit “Bath Salts” in Urine

•The Supel-Select SCX sample prep method allows for efficient urine

matrix removal while maintaining high analyte recovery.

•The combination of the ion-exchange SPE with the HILIC separation

provides a novel approach for the testing of problematic bath salt

compounds.

•By utilizing ion-exchange mechanisms for sample cleanup, and taking

advantage of the unique selectivity of chromatographic modes such as

HILIC, analytical chemists can greatly improve the selectivity and

sensitivity of their difficult bioanalytical applications.

•For more information, please visit sigma-aldrich.com/supel-select

30








HybridSPE-Phospholipid (HybridSPE-PL)

96-well SPE plates and cartridges

Zirconia-coated silica particles

Features:

• Selective removal of phospholipid interferences and precipitated proteins

• Simple 2-3 step procedure

Benefits

• Improved LC-MS sensitivity (reduced matrix effect)

• Enhanced column lifetime

• Gradients not needed to clean column

31


Monitoring Phospholipid Contamination

• PLs major component of cell membranes

• Polar head group, non-polar tail

• Largest subclass (phosphatidylcholine)

monitored using m/z 184 or m/z 104 fragment

ions

• Used as a marker for ion-suppression risk

assessment during LC-MS/MS

• Determine selectivity effectiveness of sample

prep technique

J.L. Little et al. / J. Chromatogr. B 833 (2006) 219–230

polar head group non-polar “tail”

32


Problem: Protein and Phospholipid Accumulation

on HPLC Column

Standard protein ppt technique

Reduces performance

Increases backpressure

Unpredictable carry-over & elution in future injections

Gradients needed to clean column

33

0 10 20Time (min)

02

00

0

Inj. #1, 2020 psi

Inj. #5

Inj. #10

Inj. #20, 2150 psi

Increasing back-

pressure from

protein

precipitation, and

baseline from

phospholipid build

up

HPLC column: Sub-2um C18, 5 cm x 2.1 mm I.D.

Monitoring PLs at

184 m/z


Solution: Phospholipids Selectively Removed

using HybridSPE-PL Technology

34

Phospholipids Proprietary HybridSPE

zirconia-coated silica

• The Zr atom on the particle acts as a

Lewis acid

• The phosphate groups on the

phospholipids are strong Lewis bases

and complex with the zirconium atoms

• Analytes are eluted free of

phospholipids


HybridSPE-PL Method (96-Well Format)

Precipitate

proteins in well

Apply vacuum

Resulting filtrate/eluate is free

of proteins and phospholipids,

ready for LC-MS

Mix

• 100 µL plasma/serum

• 300 µL 1% formic acid in

acetonitrile

• Add I.S. as necessary


Improved Situation: No Protein or Phospholipid

Accumulation Using HybridSPE-PL

Consistent column performance

No increase in backpressure

Eliminates carry-over & elution in future injections

Extends column lifetime

Gradients are not needed to clean column

0 10 20Time (min)

02

00

0

36

0 10 20Time (min)

Inj. #1, 1920 psi

Inj. #20, 1925 psi

No change in

back-pressure

and baseline

Monitoring PLs at

184 m/z 184 m/z


Improved Through-put with

HybridSPE-PL

Elimination of need for post-gradient

HPLC column clean-up improves

sample throughput

Gradient, 20 min.

~ 70 inj./day

Isocratic, 2 min.

~ 700 inj./day

0

100

200

300

400

500

600

700

800

Injs

./d

ay/in

str

um

en

t

HybridSPE-PL

Std. PPT

0.0 1.0 2.0Time (min)

01

00

00

20

00

0

2 min.

20 min.


Overlay of HybridSPE-Small Volume and Protein

Precipitation Samples

Methadone and metabolites from plasma

Sample was extracted using HybridSPE-PL small volume (20 uL of plasma

was used) or standard PPT (100 uL of plasma was used)

High concentration (1200 ng/mL), still shows suppression with standard

ppt method

38

HybridSPE-Small Volume

Protein Precipitation Method

EDDP

Methadone EMDP

Column: Ascentis Express RP-Amide 10 cm X 2.1, mm I.D., 2.7um; ESI+ detection


HybridSPE-PL Technology

For more information, please visit sigma-aldrich.com/hybridspe-pl.

39

• Fast and convenient SPE method uses Interference Removal

strategy

• Complete removal of precipitated proteins and phospholipids for

analysis of pharmaceutical compounds

• Reduces matrix effects, improves HPLC column lifetime and method

throughput

• Can be used to extract and concentrate phospholipids in lipidomics

application

http://sigma-aldrich.com/hybridspe-pl







QuEChERS Method: Pesticides in Food

Pesticides in

• fruit

• vegetable

• further food & feed

Quick

Easy

Cheap

Effective

Rugged

Safe


HOW DOES QUECHERS METHOD WORK?

Weigh 10 g homogenized sample

Add 10 mL acetonitrile, Add I.S.

Add 4 g MgSO4, 1 g NaCl, adjust to pH 5.5 with citrate buffer

Add MgSO4 and PSA (C18) to aliquot

Acidify extract to pH 5

GS-MS/ LC-MS/MS

41

SHAKE

SHAKE AND CENTRIFUGE

SHAKE AND CENTRIFUGE

OPTION: Acidic

pesticides

LC-MSMS

OPTION:

Acid-labile

pesticides

LC-MSMS


QuEChERS Method: the choice of sorbents

New choice of cleanup sorbents for Fat-containing and pigmented

samples:

• Supel Que Z-Sep for hydrophobic analytes

• Supel QuE Z-Sep/C18 (Discovery® DSC-18 + Z-Sep) for samples containing

<15% fat

• Supel QuE Z-Sep+ (C18 and zirconia dual bonded to silica) for samples

containing >15% fat

interference PSA C18 C18/PSA ENVI-

Carb

ENVI-

Carb/PSA

PSA/C18/

ENVI-Carb

Fats X X X

Pigments X X X X

Sugars X X X X

Acids X X X X

Z-Sep Z-Sep+ Z-Sep/C18

X X X

X X X


QuEChERS: Pesticides in avocado by GC-MS

using Z-Sep+ as a cleanup sorbent

Pesticide mix included hydrophobic

compounds (e.g.organochlorines,

hexachlorbenzene) and some other more

polar classes all GC-MS amenable

QuEChERS extraction was performed • Ratio 3 g sample to 25 mL acetonitrile

extraction solvent improved recoveries

• Tested cleanup used Z-Sep+ with and without

PSA and C18

The analysis performed using GC

equipped with single quadrupole MS

detector


12 14 16 18 20 22 24

Time (min)

Z-Sep+ cleanup

12 14 16 18 20 22 24

Time (min)

C18/PSA cleanup

Analysis of avocado extracts Scan mode


QuEChERS: Pesticides in avocado by GC-MS

Results: Pesticide Recovery • Z-Sep+ showed

better recovery

overall.

• PSA/C18: matrix

interference

prevented

analysis of

cyfluthrin,

cypermethrin and

deltametrin.

• Z-Sep+ showed

better

reproducibility

than PSA/C18


Z-Sep and Z-Sep+ as cleanup sorbents for QuEChERS

For more information, please visit sigma-aldrich.com/quechers or

sigma-aldrich.com/zsep

• QuEChERS method is a multiresidue method for analysis of pesticides

• New family of Z-Sep sorbents can provide cleanup advantages

− Provide better removal of fatty and colored matrix components

− Provide better recovery of hydrophobic analytes in comparison to

C18 cleanup

− No change in the QuEChERS procedure is required to try Z-Sep

sorbents

http://sigma-aldrich.com/quechers




http://sigma-aldrich.com/zsep





SUMMARY

47

1. Choose the sorbent and SPE hardware according to

you analysis needs

2. Pay attention to the pH of the loading, washing and

elution solvents as it affects the recovery of the

ionizable analytes

3. Stay informed of the new

SPE developments that make

sample prep easier, faster

and more convenient


Resources

48

• To view the complete SPE product line from Supelco, instructional

product videos/webinars, and technical literature,

Visit sigma-aldrich.com/spe

• To request SPE samples for method development,

Visit sigma-aldrich.com/spe-samples

• To learn more about HybridSPE-PL

Visit sigma-aldrich.com/hybridspe-pl

• To learn more about ZSep

Visit sigma-aldrich.com/zsep

• If you have additional questions related to this presentation,

Contact [email protected]

http://www.sigmaaldrich.com/analytical-chromatography/sample-preparation/spe.html




http://www.sigmaaldrich.com/analytical-chromatography/sample-preparation/spe/samples.html






http://www.sigmaaldrich.com/analytical-chromatography/analytical-products.html?TablePage=20682404






http://www.sial.com/zsep

http://www.sigmaaldrich.com/analytical-chromatography/sample-preparation/spe/dispersive-spe-tubes/supel-que-z-sep.html




SPE Fundamentals and Recent Developmentsll1.workcast.net/10311/0279275158671341/Documents/SPE...

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Transcript of SPE Fundamentals and Recent Developmentsll1.workcast.net/10311/0279275158671341/Documents/SPE...