INTRODUCTION:

Sample preparation is an essential stage in the analysis process. It takes place between sample

taking and measuring the prepared sample by means of a chromatographic or other analytical

method to quantify the amount of analyte in the sample. Depending on the type of sample,

there are various ways to prepare the sample for measurement. Basically the sample

preparation in pharmacokinetic studies can be done by following techniques.

1. Solid phase extraction

2. Liquid- liquid extraction

3. Protein precipitation

PURPOSE OF SAMPLE PREPARATION:

1. Sample Concentration: Frequently, the component of interest is present in too low levels

for detection. Sample preparation can concentrate the component to adequate levels for

measurement.

2. Contaminants: The presence of interfering matrix elements can mask the analysis of the

component of interest. Sample preparation can remove excess contaminants to yield

clean, informative chromatograms.

3. Bring to solution: For most analyses the sample must be properly prepared in solution for

subsequent analysis.

SOLID PHASE EXTRACTION (SPE)

It is a separation process by which compounds that are dissolved or suspended in a liquid

mixture are separated from other compounds in the mixture according to their physical and

chemical properties. SPE was developed by Zhang and coworkers for the extraction of pure

organic compounds from aqueous samples Analytical laboratories use solid phase extraction to

concentrate and purify samples for analysis. It can be used to isolate analytes of interest from a

wide variety of matrices, including urine, blood, water, beverages, soil, and animal tissue. The

general procedure is to load a solution onto the SPE phase, wash away undesired components,

and then wash off the desired analytes with another solvent into a collection tube.

Solid phase extractions use the same type of stationary phases that are used in liquid

chromatography columns. The stationary phase comes in the form of a packed syringe-shaped

cartridge, a 96 well plate, a 47- or 90-mm flat disk, or a Micro Extraction Packed sorbent

(MEPS) device, each of which can be mounted on its specific type of extraction manifold. The

manifold allows multiple samples to be processed by holding several SPE media in place and

allowing for an equal number of samples to pass through them simultaneously. A typical

cartridge SPE manifold can accommodate up to 24 cartridges, while a typical disk SPE manifold

can accommodate 6 disks. Most SPE manifolds are equipped with a vacuum port. Application of

vacuum speeds up the extraction process by pulling the liquid sample through the stationary

phase. The analytes are collected in sample tubes inside or below the manifold after they pass

through the stationary phase.

Solid phase extraction procedures are used not only to extract traces of organic compounds from

environmental samples but also to remove the interfering component of the complex matrices in

order to obtain a cleaner extract containing the analytes of interest. The SPE technique is widely

applied for isolation of analytes from a liquid matrix and purified extracts.

THE IDEAL SOLID PHASE EXTRACTION METHOD

1. High and reproducible recoveries for acidic, basic and neutral analytes

2. Isolates and allows quantification to g/ml concentrations of drugs and metabolites from

biofluids.

3. Rugged and easy to automate for large volumes of samples.

4. Fast and cost efficient.

SOLID PHASE EXTRACTIONTHEORY

1) Normal Phase(Nonpolar liquid phase, polar modified solid phase)

Hydrophilic interaction polar-polar interactions hydrogen bonding pi-pi interactions dipole-dipole interactions dipole –induce dipole interaction

2) Reversed Phase (Polar liquid phase, nonpolar modified solid phase)

Hydrophobic interactions

Non polar-nonpolar interactions vander Waals or dispersion forces

3) Ion Exchange

Electrostatic attraction of charged group on compound to a charged group on the sorbent’s surface.

4) Adsorption(Interactions of compounds with unmodified materials)Hydrophobic and hydrophilic interactions may apply depending on which solid phase is used.

1) NORMAL PHASE SPE

Normal phase SPE procedure typically involve a polar analyte, a mid- to non polar matrix (e.g. acetone, chlorinated solvents, and hexane) and a polar stationary phase. Polar-functionalized bond silica and polar adsorption media typically are used under normal phase conditions. Retention of an analyte under normal phase condition is primarily due to interaction between polar functional groups of the analyte and polar groups on the sorbent surface. These include hydrogen bonding, pi-pi interactions, dipole-dipole interactions and dipole-induced dipole interactions among other.

NORMAL PHASE: GENERAL ELUTION PROTOCOL

1. Condition the cartridge with six to ten hold-up volumes of nonpolar solvent, usually the sample solvent.

2. Load the sample in to the cartridge.3. Elute unwanted components with a nonpolar solvent4. Elute the first component of interest with a polar solvent.5. Elute remaining components of interest with progressively more polar solvents.6. When recovered all the components, discard the used cartridge in an appropriate manner

2) REVERSED PHASE SPE

Reversed phase SPE separates analytes based on their polarity. The stationary phase of a

reversed phase SPE cartridge is derivative of hydrocarbon chains, which retain compounds of

mid to low polarity due to the hydrophobic effect. The analyte can be eluted by washing the

cartridge with a non-polar solvent, which disrupts the interaction of the analyte and the stationary

phase. It involves a polar or moderately polar sample matrix and a non polar stationary phase.

The analyte of interest is typically mid to non polar. Retention of organic analytes from polar

solutions onto SPE materials is due to attractive forces between the carbon-hydrogen bonds in

the analyte and the functional groups on the silica surface. These nonpolar-nonpolar attractive

forces are commonly called vander Waals forces.

REVERSE PHASE: GENERAL ELUTION PROTOCOL

1. Solvate the bonded phase with six to ten cartridge hold-up volumes of methanol or

acetonitrile. Flush the cartridge with six to ten hold-up volumes of water or buffer. Do

not allow the cartridge to dry out.

2. Load the sample dissolved in strongly polar solvent.

3. Elute unwanted components of interest with a less polar solvent.

4. Elute more tightly bound components with progressively more nonpolar solvents.

5. When recovered all the components, discard the used cartridge in an appropriate manner.

3) ION EXCHANGE SPE

Ion exchange sorbents separate analytes based on electrostatic interactions between the analyte

of interest and the positively charged groups on the stationary phase. For ion exchange to occur,

both the stationary phase and sample must be at a pH where both are charged.

ANION EXCHANGE

Anion exchange sorbents are derivative of positively charged functional groups that interact and

retain negatively charged anions, such as acids. Strong anion exchange sorbents contain

quaternary ammonium groups that have a permanent positive charge in aqueous solutions, and

weak anion exchange sorbents use amine groups which are charged when the pH is below about

9. Strong anion exchange sorbents are useful because any strongly acidic impurities in the

sample will bind to the sorbent and usually will not be eluted with the analyte of interest; to

recover a strong acid a weak anion exchange cartridge should be used. To elute the analyte from

either the strong or weak sorbent, the stationary phase is washed with a solvent that neutralizes

the charge of the analyte, the stationary phase, or both. Once the charge is neutralized, the

electrostatic interaction between the analyte and the stationary phase no longer exists and the

analyte will elute from the cartridge.

CATION EXCHANGE

Cation exchange sorbents are derivative of functional groups that interact and retain positively

charged cations, such as bases. Strong cation exchange sorbents contain aliphatic sulfonic acid

groups that are always negatively charged in aqueous solution, and weak cation exchange

sorbents contain aliphatic carboxylic acids, which are charged when the pH is above about 5.

Strong cation exchange sorbents are useful because any strongly basic impurities in the sample

will bind to the sorbent and usually will not be eluted with the analyte of interest; to recover a

strong base a weak cation exchange cartridge should be used. To elute the analyte from either the

strong or weak sorbent, the stationary phase is washed with a solvent that neutralizes ionic

interaction between the analyte and the stationary phase.

ION–EXCHANGE: GENERAL ELUTION PROTOCOL

1. Condition the cartridge with six to ten hold-up volumes of deionized water or weak

buffer.

2. Load the sample dissolved in a solution of deionized water or buffer.

3. Elute unwanted weakly bound components with a weak buffer.

4. Elute the first component of interest with a strong buffer(change the pH or ionic

strength).

5. Elute other components of interest with progressively stronger buffers.

6. When recovered all the components, discard the used cartridge in an appropriate manner

4) ADSORPTION SPE

It is the interactions of compounds with unmodified materials.Hydrophobic and hydrophilic

interactions may apply depending on which solid phase is used.The polar adsorption material is

modified silica gel commonly used as the base of all of the bonded phases. The functional groups

that are involved in the adsorption of compound from non polar matrices are the free hydroxyl

group on the surface of the silica particles.That may be used to adsorb polar compounds from

non polar matrices with subsequent elution of the compounds in an organic solvent , more polar

than the original sample matrix.

REFERENCE1)Ferenc Z, Biziuk M,Solid phase Extraction technique-Trends,opportunities and application,Polish Journal of Environment Studies,2006,15,677-6902) Guide to Solid Phase Extraction,Supelco,Bulletin 910