Method for Sample Pretreatment and Preparation in Bioanalysis

Biomedical Analysis 2012

EXTRACTION :

1.Liquid-liquid extraction
2.Solid phase extraction

Principles
Why do sample preparation?

Remove interferences from sample More accurate results Concentrating analytes to improve detection Protecting equipment to reduced costs

Sample preparation
Sample preparation for the analysis of drugs in biological fluids consists of:
(i) release of the drug from a conjugate or biological matrix (ii) removal of endogenous compounds that could interfere with the assay (iii) techniques for liquid handling

Introduction to Extraction Processes

When separation by distillation is ineffective or very difficult e.g. closeboiling mixture, liquid extraction is one of the main alternative to consider.

What is Liquid-liquid extraction (or solvent extraction)?

Liquid-Liquid extraction is a mass transfer operation in which a liquid solution (feed) is contacted with an immiscible or nearly immiscible liquid (solvent) that exhibits preferential affinity or selectivity towards one or more of the components in the feed. Two streams result from this contact:

a) Extract is the solvent rich solution containing the

desired extracted solute.

b) Raffinate is the residual feed solution containing little solute.

Introduction to Extraction Processes

Liquid-liquid extraction principle

When Liquid-liquid extraction is carried out in a test tube or flask the two immiscible phases are shaken together to allow molecules to partition (dissolve) into the preferred solvent phase.
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An example of extraction:

Extract Acetic acid in H2O +

Organic layer contains most of acetic acid in ethyl acetate with a small amount of water.
Raffinate

Ethyl acetate

Aqueous layer contains a weak acetic acid solution with a small amount of ethyl acetate.

The amount of water in the extract and ethyl acetate in the raffinate depends upon their solubilites in one another.

Comparison of LLE vs SPE Disk

LLE
Uses 200 - 500 ml solvent Shaking / continuous process Forms emulsions Little selectivity Takes 1 - 2 hours / sample

SPE DISK
2 - 20 ml solvent Filtration process No emulsions formed Wide selectivity (adsorbent) Takes 10 - 20 min. / sample
Uses

Problems with the LLE Procedure

Tedious and time-consuming

Shaking and separation time Evaporation time

Expensive-labor and materials

Time factor Solvent cost and exposure Solvent disposal

Poor results
Forming of emulsions Irreproducible extractions Low recoveries

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Principles of SPE
SPE is an extraction process
whereby an aqueous sample is filtered through a thin bed of
sorbent particles, the analytes of interest are removed from the liquid matrix,

and concentrated onto the sorbent.

Once concentrated, the analytes are removed by an eluting solvent.

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What are the Benefits of SPE?

SPE uses less solvent than LLE

SPE is faster (at least 5 times)

High capacity Total SPE costs are considerably less than LLE

High selectivity: broad choice of bonded phases and solvents

Automation much easier

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SPE Column

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Sorbent in SPE has to :

Sorption process are reversible Sorbent are porous with larger surface area Free from leachable impurities Exhibit stability toward the sample matrix and the elution solvents Have good surface contact with sample solution

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Types of Base Materials for SPE Packings

Silica NA and K Silicates Fluorosil Mg Silicates Alumina Carbon Polystyrene Polystyrene Divinyl benzene Polystyrene N-Vinylpyrrolidone Cellulose Hydroxyapatite Fullerenes Cyclodextrin Agarose
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4 Steps in SPE

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Types of Sorbent-Analyte Interactions

Polar Non-polar Ion-exchange Copolymeric

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Polar Extractions
Also called hydrophilic or normal phase Unequal distribution of electrons Involves hydrogen bonding, and dipole/ dipole interactions Sorbents : silica, diol, diethylamino, cyanopropyl Applications - lipids, oil additives, carbohydrates, phenols, oil soluble vitamins Analytes - amines, hydroxyls, carbonyls, heteroatoms (O, S, N, P) Matrix : non-polar, organic Elution solvents : medium to high polarity
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Interaction between analyte and polar sorbent

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Representation of unbonded silica

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Non-Polar Extractions
Also called hydrophobic or reverse phase Interactions between sorbent C-H bonds and analyte C-H bonds Involves van der Waals / dispersion forces Sorbents - C2, C3,C4, iC4, tC4, C5, C6, C7, C8, C10, C12, C18, C20, C30 phenyl and cyclohexyl, polymeric Applications - drugs of abuse, TDM, pesticides Analytes : protonated / neutral state, aromatics & alkyl chains Matrix : biologicals, water, aqueous buffers Elution solvents : typically non-polar to moderately polar
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Sample pH vs. Recovery

CH3 (CH3) 2CHCH2 C C O H OH

Ibuprofen
Copolymeric DAU Column A/N Extraction Hydrophobic Retention
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Sample pH vs. Recovery of Ibuprofen

3 4 5 2 1 3 4 5

Sample added at pH 6

Ibuprofen Sample added at pH 5 2 Meprobamate 3 Glutethimide 4 Phenobarbital 5 Phenytoin

1

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Chain Length Effect

(Recovery & Extract Cleanliness)
4 4

3.0e4
2

3 1

3.0e4
2

3 1

2.0e4 1.0e4 0 0 2

2.0e4 1.0e4 0

C18
endogenous peaks: area = 71,628
96% 94% 94% 96% 98%
1 2 3 4 5

C2
Butabarbital Amobarbital Pentobarbital Secobarbital Glutethimide 64% 87% 88% 89% 78%

endogenous peaks: area = 11,257

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Chain Length Effect

(Recovery & Extract Cleanliness)
4 4

3.0e4
2

3 1

3.0e4
2

3 1

2.0e4 1.0e4 0 0 2

2.0e4 1.0e4 0

C18
endogenous peaks: area = 71,628
96% 94% 94% 96% 98%
1 2 3 4 5

C2
Butabarbital Amobarbital Pentobarbital Secobarbital Glutethimide 64% 87% 88% 89% 78%

endogenous peaks: area = 11,257

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Ion Exchange Mechanisms

Ionic interactions occur between charged sorbent & analyte of opposite charge pH is manipulated to ionize analytes functional group Ionic bonds are strong & retain analyte Hydrophobic interferences washed away with organic solvents Polar interferences removed with aqueous or weak aqueous / organic washes Elute solvents containing stronger counterions or by changing pH For ionic/hydrophobic analytes, elute by simultaneously disrupting both interactions
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Specialty Anion Exchange Columns

Ion exchange columns possess charged functional groups which allow analytes to bind upon sample application. Prior to column use, these groups require counter ions at these charged sites. The standard counter ion for cation exchangers is the hydronium ion and for anion exchangers is the chloride ion . From time to time during sample application, a charged analyte is not strong enough to displace the counter ion & therefore does not bind to the column. In cases such as these, a weaker counter ion is required. Two such columns with weaker counter ions (Quaternary amine with acetate counter ion) & (Quaternary amine with hydroxide counter ion) are commercially available. In terms of strength, the acetate ion is stronger than the hydroxide ion.

CAQAX

Silica Backbone Quaternary Amine anion exchanger Acetate counter ion (Standard anion exchanger carries Cl- )

CHQAX

Silica Backbone Quaternary Amine anion exchanger Acetate counter ion (Standard anion exchanger carries Cl- )

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Relative Counter ion Selectivity

Cations
Larger numbers reflect greater ability of the ion to displace other ionic materials from the bonded surfaces.
Strong Cation Exchanger
Ba2+

Anions
Benzene Sulfonate Citrate IPhenateHSO4CIO3NO3BrCN HSO BrO NO2 CI HCO3IO33Formate Acetate Propionate FOH 500 220 175 110 85 74 65 50 28 27 27 24 22 6.0 5.5 4.6 3.2 2.6 1.6 1.0

SO-3 Si
Benzenesulfonic Acid (BCX)

Strong Anion Exchanger

- Si - (CH2)3 N (CH3)3
Quaternary Amine (QAX)

Ag Pb2+ Hg2+ Cu+ Sr2+ Ca2+ Ni2+ Cd2+ Cu2+ CO2+ Zn2+ Cs2+ Rb+ K+ Fe2+ Mg2+ Mn2+ NH4+ Na+ H+ Li+

2+

8.7 7.6 7.5 7.2 5.3 4.9 3.9 3.0 2.9 2.9 2.8 2.7 2.7 2.6 2.5 2.5 2.5 2.3 1.9 1.5 1.0 0.8

Standard cation exchange counter ion

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Cation Exchange Extractions

Cation exchange sorbents negatively charged
Basic analytes manipulated to carry positive charge Opposites attract forming strong bonds Sorbents
Benzenesulfonic acid (strong) Propylsulfonic acid (strong) Carboxylic acid (weak)

Applications include basic drugs, catecholamines, pharmaceuticals, herbicides Analytes

Amines Pyrimidines (cations)

Matrix - aqueous Basic elution solvents to neutralize analyte

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Interaction between cationic exchange sorbent and analyte

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Anion Exchange Extractions

Anion exchange sorbents positively charged Acidic analytes manipulated to carry negative charge Opposites attract forming strong bonds Sorbents
1, 2 amine Aminopropyl (weak) Quaternary amine (strong) Diethylamino (weak)

Applications include phosphates, acidic drugs, organic acids, fatty acids, vitamins Analytes
Phosphates Carboxylic acids Sulfonic acids (cations)

Matrix - aqueous Acidic elution solvents to neutralize analyte

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Interaction between anion exchange sorbent with analyte

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I.

ROBINUL (GLYCOPYRROLATE)
FROM EQUINE URINE BY LCMSMS
(CLEAN UP CUCCX-2) 500 mg / 14 mL

CONDITION SPE COLUMN 1. Wash with 2 x 2.5 ml MeOH 2. Wash with 2 x 2.5 ml phosphate buffer (0.1m, pH 7.0)

II. SAMPLE PREPARATION 1. Buffer 5 ml of urine to pH 7.0 by adding 3 mL of 0.1M phosphate buffer (pH 7.0) 2. Add (12.5 ng) of mepenzolate (internal standard) 3. Add 5 ml of water to the sample 4. Vortex or shake thoroughly 5. Centrifuge for 5 min at 800g 6. Apply supernatant to SPE column III. WASH COLUMN 1. Wash column with 5 ml of MeOH 2. Wash column with 5 ml of H2O IV. DRY COLUMN 1. 5 min. with vacuum at 25 mm Hg

V.

ELUTE OF GLYCOPYRROLATE 1. Elute with 4 ml of methanol 0.5M ammonium acetate buffer pH 3.00

VI.

BLOWN DOWN 1. Blown down eluent at 60C under nitrogen and reconstitute with 0.1 mL of MeOH
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Solubility
Best wash solvents are those in which the compound of interest is insoluble.

Example:
Soluble in H2O

Vancomycin

Insoluble in Methanol
Wash: 100% methanol Elution: 80:20 methanol/H2O
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SPE Sorbent-Analyte Interaction

Primary Interaction Mechanism Sorbent Energy of Interaction (Kcal/mol)

Van Der Waals Polar/dipole-dipole Hydrogen Bonding Electrostatic

Octadecyl, Octyl, Ethyl Cyano, silica, alumina Amino, diol Cation exchange, anion exchange

1-10 1-10 5-10 50-200

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METHOD SELECTION GUIDELINES

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PRETREATMENT SOLID SAMPLES

Ultrasound Microwave assisted Extraction Solid phase micro extraction (SPME) Matrix Solid Phase Dispersion (MSPD)

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