Sample Prep Options - Agilent Prep... · sample, add water if needed, spike 2) Add 10ml ACN 3) Vortex 4) Add salt packet 5) Shake 1 minute 6) Centrifuge at 4,000 rpm for 5 minutes

Sample Prep Options forGC users

Tina ChambersTechnical Specialist Sample Preparation

tina.chambers@agilent.com949-842-0493

Why Bother?

• Removal of interferences which would otherwise affect detection of analyte

•Removal of interferences which would otherwise affect instrumentation and/or columns

• Concentration of an analyte to a detectable levels

• Solvent switching into an analytically more compatible solvent

Matching SPP to Analytical Method

All GC based systems require highest degree of cleanliness, regardless of detector!!

Sample Preparation Options

•Dilute and shoot

•Protein precipitation

•Liquid-liquid extraction/solid supported liquid extraction•Liquid-liquid extraction/solid supported liquid extraction

•Solid phase extraction

•QuEChERS

Considerations for LLE/SLE

- Polarity of the analyte – soluble in non-miscible organic?

- Nature of interfering compounds – presence of lipids?

- LODs/LOQs – is concentration necessary?

TOXI-TUBES A & B ®

• Contain solvents and salts ideally proportioned for Clinical/Forensic drug analysis • Ultimate in ease-of-use: just add sample, mix and centrifuge• Can be used with challenging matrices: post-mortem blood, gastric content, liver tissue etc.• Absolutely no method development required!

Sample Preparation – Supported LLE (SLE)

Hydromatrix™ - diatomaceous earth sorbent

• Composed of fossilized diatoms• Purified at high temperatures• Purified at high temperatures• High surface area for water adsorption• Very polar surface

Chem Elut™ - pre-assembled cartridges with HydromatrixCombilut ™- 96-well plate filled with Hydromatrix

The SLE Process

Aqueous layer

Organic layer

Dry sorbent

Before ExtractionApply

SampleExtract with

Organic Solvent

layer layer

The Chem Elut Method

Aqueous samplebeing applied

Solid support adsorbs water ontohigh surface area particles

Organic extraction solvent

Multi-Residue Confirmation of Pesticides in Honey using SLE

App note SI-01002

Recovery comparison of pesticides between solid supported liquid-liquid extraction (SLE) on Chem Elut extraction (SLE) on Chem Elut and classical liquid-liquid extraction (LLE).

LLE/SLE summary

- Polarity of compound and nature of interferences dictate whether this is suitable

- pH adjustments and/or addition of salts can be helpful

- LLE/SLE solvents are often a good choice for GC analysis

Considerations for Solid Phase Extraction (SPE)

- High degree of versatility (over 40 different chemistries available)

- Concentrates dilute samples

- Method development often necessary

Solid Phase Extraction Triangle

Often, both the the analyte and the matrix are known, it is sorbent choice which is the critical component

Four Steps of SPE

• Conditioning: Preparation of the sorbent prior to sample addition

• Retention: Analytes of interest and other interferences adsorb onto the surface of the sorbent during sample additionaddition

• Washing: Elimination of undesired interferences

• Elution: Selective desorption and collection of desired analytes from the sorbent

The Four Steps of SPE – Selective Elution

Sample loading Retention Rinsing Elution

Green = Blue and Yellow

Blue is more non polar than yellow

Blue is retained

Silica VS. Polymer

• “True” polar/ion exchangepossible

• Wide range of chemistries• Inherent hydrophobicity

(conditioning)• Wide range of chemistries• Wide range of established

methods• Can be more selective• Conditioning is crucial

(conditioning)• Higher capacity

(sorbent mass/flow)• Polarity gradient in Plexa

Polymers and Conditioning

450 ng/mL, LCUV

60%

80%

100%

120%

% R

ecov

ery

MethanolAqueous

Non-Conditioned

0%

20%

40%

Atenolol Ranitidine Pseudoephedrine Quinidine Brompheniramine Mianserin Fluoxetine

% R

ecov

ery

Non-Conditioned

Silica and Conditioning

Benefits by Design: Sorbent Quality

Bond Elut Plexa Oasis HLB

Bond Elut Plexa PCX Oasis MCX

See Plexa Advantage Note, SI-1935Confidentiality Label

Size Distribution

May 30, 2013Confidentiality Label

Non-Polar Method Development

Method Development Considerations

• What is the partition coefficient (log P) of the un-ionized compound?

log Poctanol/water = log ([solute]octanol/[solute]water)

• If log P > 1.5, good candidate for hydrophobic method

• If log P unknown, “rule of thumb”: four covalently bound • If log P unknown, “rule of thumb”: four covalently bound carbon atoms or benzene ring minimum requirement

• If compound is ionizable, determine pKa• Load acids at pH<pKa by 2 units minimum• Load bases at pH>pKa by 2 units maximum

Method Development Considerations

Solubility characteristics of target compound?

Method Development Considerations

• Select suitable solvents

• Prepare 0%-100% concentrations

• Plot recoveries

80000100000120000

Elution Profile

020000400006000080000

0% M

eOH

10%

MeO

H20

% M

eOH

30%

MeO

H40

% M

eOH

50%

MeO

H60

% M

eOH

70%

MeO

H80

% M

eOH

90%

MeO

H10

0% M

eOH

Elution Profile

Method Development Consideration

100000120000

Elution Profile

• Highest % organic with low recoveries for wash

• Lowest % organic with high recoveries for elution

• Try acid/base modifiers

020000400006000080000

100000

0% M

eOH

10%

MeO

H20

% M

eOH

30%

MeO

H40

% M

eOH

50%

MeO

H60

% M

eOH

70%

MeO

H80

% M

eOH

90%

MeO

H10

0% … Elution Profile

NH3+

Silica baseOH

SO3-

Interactions on Ion Exchange Sorbents

Electrostatic attraction

CO2H

SO3

CO2-

OCON(CH3)2

+N

STRONG: Ionic group is always charged (+ or -)

WEAK: Ionic group is variably charged (+ or -)

CATIONS: (+) Found in basic compounds

ANIONS: (-) Found in acidic compounds

Ion Exchange Nomenclature

ANIONS: (-) Found in acidic compounds

Extract weak ions with strong exchangers and strong ions with weak exchangers!

Ion Exchange Sorbent Selection

• All bases except quarternary amines can be processed on Plexa PCX or Bond Elut SCX

• Only carboxylic acids can be processed on Plexa PAX or Bond Elut SAXBond Elut SAX

• Phosphates, sulfates and other STRONG acids should be processed with weak anion exchangers (Bond Elut NH2, PSA)

NH3+

Silica base

OH

SO3-

Interactions on Ion Exchange Sorbents

50%

CO2H

SO3

CO2H

NH2If the pKa=9

and the pH=9

50%

Method Development Considerations

What is the pKa of your compound?

pKa= -log Ka

and

Ka = [A-][H+]/[HA]

• If pH=pKa, 50% of the compound is ionized and 50% is neutral

• To ensure full charge or full neutralization, employ the rule of 2

NH3+

Silica base

OH

SO3-

Interactions on Ion Exchange Sorbents

100%

CO2H

SO3

CO2H

NH2If the pKa=9

and the pH=7

0%

NH3+

Silica base

OH

SO3-

0%

Interactions on Ion Exchange Sorbents

CO2H

CO2H

NH2If the pKa=9

and the pH=11

100%

Important Consideration for Ion Exchange

• Reduce ionic strength of “salty” matrices by dilution

• Consider competitive binding when choosing bed mass

• Remember that ALL polymeric exchangers are mixed-mode, elute in organic solventmode, elute in organic solvent

• Some organic should be present even with silica based ion exchangers because of carbon linkers

QuEChERS(Pronounced “catchers”)• Quick, Easy, Cheap, Effective, Rugged, and Safe

• Portmanteau: blend of 2 or more words

• www.quechers.com

• Introduced in 2003: M. Anastassiades, S.J. Lehotay, D. Stajnbaher, and F.J. Schenck, J. AOAC Int 86 (2003) 412F.J. Schenck, J. AOAC Int 86 (2003) 412

• Validated in 2005, with subsequent modification in 2007

• AOAC 2007.01 and European Method EN 15662

• Streamlined approach that makes it easier and less expensive to examine pesticide residues in food

5/30/2013

QuEChERS First step extraction

1) Weigh sample,

add water if needed, spike

2) Add 10ml ACN

3) Vortex3) Vortex

4) Add salt packet

5) Shake 1 minute

6) Centrifuge at 4,000 rpm for 5 minutes

Second Step – Dispersive SPE

7) Choose d-SPE kit based on matrix characteristics

8) Transfer 1-8ml alliquot, vortex 1 minute

Page 37

9) Centrifuge

11) Analyze by GC/MS or LC/MS

QuEChERS Applications• Pesticides• PAHs• Hormones• Antibiotics• Acrylamides • Vet Drug Residues

Page 38

• Animal tissue• Fish and Shellfish• Fruits and Vegetables• Food oils• Soil• Baby Food

Questions?