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Sample preparation

63 important questions on Sample preparation

Why sample preparation?

- adjustment of the concentration (enrichment or dilution)
- removal of interfering compounds
- phase transfer (make a sample liquid)
- stabilization (stabile analyte)
- Improvement of analyte properties ( react it with another molecule to make it better reactable)

How can you counteract degradation of a compound in urine?

- for example acidification: add an acid to the urine to prevent a basic pH.
- Add BSA or a detergent if it sticks to containers.
- In case of sediment formation ( a bit proteins and a lot of salts): vortex or heat the sample since no sediment will be formed at high temperatures (37 degrees)

What is the principle of stabilization?

minimize degradation of analytes by optimization of experimental conditions
1) electrochemical degradation: oxidation e.g.
2) enzymatic degradation
3) chemical degradation
4) photochemical degradation (reaction with light)

How can electrochemical degradation be counteracted?

- Add an antioxidant like ascorbic acid or bisulfite
- add a component which complexates metal ions. (metal ions are catalyzing oxidation reactions)
- keep sample cold.

How can enzymatic degradation be counteracted?

e.g. esterase activity. (makes from esters acids)
- add an esterase inhibitor like fluoride (NaF) 5 mg/ml in plasma
- pefabloc

- remove enzymes by protein precipitation

- keep sample cold.

The activity of esterases in rat in mouse plasma is often higher than in human plasma.

How to counteract chemical degradation?

e.g. lactones into acids. via catalysis
e.g. acyl glucuronides into free acids

Add acid or base to minimize the reaction.
for lactones the optimal pH is 4-5
for acyl glucuronides the optimal pH is 3-4
- keep sample cold.

How to counteract photochemical degradation?

e.g. nifedipine.
- protect from (UV-light)
- Use amber tubes and glassware
- analyse in dark room (under red light or under yellow light since this light is less energetic)

What is the purpose of homogenization?

liquidify the sample to allow pipetting.

What is the principle of homogenization?

- add liquid and grind.

- This is usually followed by supplementary sample preparation.

What is the practical execution of homogenization?

- A rotor mill (kitchen blender) could be used.
- a mixer mill could be used. This one uses balls to disrupt the solid sample.
- ultrasonic device: no liquid addition. It pulverizes the solid sample.

What are the problems with homogenization?

- precipitation of sample during storage. so before taking a subsample always mix thoroughly
- contamination between samples. problem with a kitchen mixer.
- the preparation of spiked samples that are representative. This means that the solid sample cannot be spiked directly. but the homogenate needs to be spiked. this is indirectly.

Be sure that proteins do not denature during homogenization.

What is the purpose of centrifugation?

Removal of interfering components (particles).
- protein threads/ clots from plasma or sediment from urine.

What is the principle of centrifugation?

It is separation based on difference in precipitation speed at a certain centrifugal force. It is usually followed by supplementary sample preparation

What is the practical execution of centrifugation?

- The larger the centrifugal force, the smaller the particles that are being precipitated.
RPM = SQ(RCF/(1,118x10-6)*r))
r = distance from axis to bottom tube
rcf: relative centrifugal force (g).
10000 g means 10000 earth gravitational force.

it is better to use RCF (or g) because this does not depend on the force of the macine. This means that it is easier to replicate experiments.

What are problems of centrifugation?

- loss of analyte caused by binding to sediment. (so no centrifugation of urine for quantitative studies)
- loss of analyte caused by binding to lipid layer. (so sometimes also no centrifugation of plasma)

What is the driving force of filtration?

1) vacuum/ pressure (filtration)
2) centrifugal force (ultrafiltration)
centrifugal force is around 1000 -2000 g.

The thing here is that only the free (unbound) fraction passes the membrane.

Which 2 applications are there for ultrafiltration?

- sample preparation technique for determination of the total fraction of non-protein bound analytes.
- sample preparation technique for determination of the free fraction of protein-bound analytes.

What is a possible protocol for ultrafiltration?

Depends on the protein binding. If you want to have only the free concentration it might be necessary to ultrafiltrate by LLE after filtration

What are problems of filtration?

- build-up of a protein layer on the membrane (therefore more than 1 ml plasma or more than 2000g is useless.
- binding of analyte to the membrane or the protein layer (be careful with non-polar analytes)
- limited selectivity
- since you might loss something, the callibration curve needs to filtrated as well.

What is the purpose of dilution?

- concentration adjustment
- reduce the effect of interfering compounds.

What is the principle of dilution?

Increase the volume by addition of liquid.

What is the practical execution of dilution?

You have to dilute with a liquid that is compatible with the subsequent analytic method.
for chromatography: add less methanol/acetonitrile than that is present in the mobile phase, proper pH.

for chromatography, here dilution is only applicable for protein-free matrices (so urine e.g.).

What can be a problem with dilution?

- The measurable concentration does not decrease proportionally.
For binding assays, the measurable (non-protein bound) concentration may increase at higher concentrations because of saturation of the binding sites at the proteins.
- limited selectivity

What is the purpose of protein precipitation?

- Removal of interfering components (particles and/or proteins)
- stabilization: de-activation of enzymes

It is mainly used for removal of proteins from plasma/serum to protect the LC system.

What is the principle of protein precipitation?

An addition of denaturing solvent which precipitates proteins followed by centrifugation to remove the precipitate.

It is simple and very quick, but not that selective.

How is protein precipitation carried out?

Normally plasma proteins are soluble at physiological conditions:
- aqueous environment
- neutral pH
- physiological salt concentration
- body temperature

protein precipitation takes place
- in an organic environment
- at strongly acidic pH
- at high salt concentration
- at high temperature

What is the difference in protein precipitation between an acidic environement and an organic environment?

In en acidic environment the charges will change and therefore the protein will denature.
In an organic environment the apolar sides will go to the outside of the protein and therefore the protein denatures.

What is the practical execution of protein precipitation?

Mostly 2 volume equivalents of acetonitrile or methanol are added or 0,4 volume equivalents of 10% trichloroacetic acid is added.

Then, it is diluted with a solvent that is compatible with the subsequent analytical method. This means that for chromatography less methanol/acetonitrile needs to be present than in the mobile phase, proper pH.
For LC-MS volatile salts.

It is often performed in eppendorf cups (manual) or in 96 well plates. It is followed by centrifugation.
It can also be performed in filter plates which is followed by filtration.

What can be protocol for protein precipitation?

- Mix an amount of plasma with a similar amount of the internal standard.
- add methanol (4 times as high as plasma)
- centrifuge at 3000g for 15 min to precipitate the protein.
- mix a small amount of the supernatant with an amount of formic acid.

What are the problems with protein precipitation?

- analyte adsorption to or inclusion in the precipitate. (use methanol to form a fine precipitate and add solvent in several steps.
- limited selectivity (due to phospholipids)

What is the principle of LLE?

Selective transfer of analytes to a (water immiscible) organic solvent.
This is usually followed by evaporation of the organic solvent and reconstitution of the residue.

The extraction yield in LLE depends on?

- pH: only uncharged compounds will be extracted.
- polarity: a higher yield is the case when compound is better soluble in the extraction solvent.
- volume: a higher yield in case of a larger volume of the extraction solvent.

How do you determine the percentage of molecules that is charged?

1- (1/ (1+ 10^(ph-pka)))
This means that a separation of 2 compounds is possible by the selection of a proper pH.

Why LLE is less suitable for amphoteric compounds?

There is very often not one pH value at which the whole compound is neutral. It can be that the overall charge is neutral, but since there are still charges, it will not be extracted.

What is the partition coefficient?

This is the ratio of the concentration of an unionized compound in octanol divided by the concentration of an unionized compound in water. Normally this is expressed by Log P. So the higher the Log P the better the compound will disolve in an organic solvent.

Where does the partition coefficient depend on?

- on analyte
- on solvent
- on temperature
- on ionic strength of sample.

What is an advantage of LLE?

The optimal P can be otained by mixing several organic solvents.

What are some practical considerations of LLE?

- Density of the solvent (the best is if it is lighter than water). (if it is equal, it might be difficult to separate it. If it is heavier, it is more laborious because the aqueous has to be removed.
- boiling point of the solvent: the best is not too high, then a long evaporation step is the result.
- safety of the solvent: the best is not to use a toxic or inflammable solvent.
- purity of the solvent: avoid presence of interfering impurities.

What is the advantage of a washing step?

In this case a new aqeous layer is applied on the organic layer. The result is a more clean extract.

What is the practical execution of LLE?

It is performed in individual tubes (manual) or in 96 wells plates (automated)

What is an example of a protocol for LLE?

1) mix plasma with internal standard solution
2) add buffer and mix
3) extract with an organic solution (e.g. diethyl ether)
4) evaporate extraction solvent and re-dissolve compound.

What are problems of LLE?

- emulsification: phases are not properly separated then.
The reason is a low surface tension of the solvent, because of high viscosity and small difference in density between sample and solvent.

This can be decreased by
- gently mixing
- low temperature
- addition of surface-active compounds
- different extraction solvent

What is the purpose of SPE?

- Removal of interfering components (proteins and small molecules)
- concentration adjustment
- stabilization: de-activation of enzymes

What is the principle of SPE?

A selective isolation of analytes by a solid phase in a cartridge.
It is often followed by an evaporation of the elution solvent and re-dissolution of the residue. It was introduced in 1980s.

When is the selectivity optimal for SPE?

in caes of
- maximum extraction yield of analytes and minimum extraction yield of other compounds.

Where does the extraction yield depends on?

- affinity: higher yield in case of better binding to the solid phase.
- area of the phase: higher yield in case of larger area.

What is ion exchange retention SPE?

This depends on coulomb forces. You have a cationic exchanger (sulphonic and carboxylic acids) or an anionic exchanger (amines)

cation exchangers: exchange positively charged ions.
anion exchangers: exchange negatively charged ions.

What is the difference between a strong ion-exchanger and a week ion-exchanger?

In a strong ion-exchanger: SPE phase is charged over entire pH range. This means that elution is only possible by neutralizing the analyte.

weak ion-exchanger: SPE phase is uncharged at a certain pH. This means that elution is also possible by neutralizing the SPE phase.

What is the practical execution for SPE normal phase?

1) conditioning: the purpose is to activate the phase (solvate) and prepare the phase. This can be done with a non-polar solvent.
2) loading phase: the purpose is to bind the analytes to the phase: this can be done add the sample in a non-polar solvent and add it.
3) washing: to remove the interferences. This can be done with a 1-5% polar solvent.
4) elution: to collect the analytes. This can be done with a 5-50% polar solvent. The elution speed is not critical.

What is the practical execution for ionic exchange SPE?

1) conditioning: with methanol/water/buffer
2) loading: with diluted sample
3) washing: with buffer/ mofifier
4) Elution: with acidic/ alkaline modifier

How does the solvent pass? (SPE)

with positive pressure
with negative pressure (vacuum) or with centrifugation

What kind of configuration columns can be used for SPE?

- syringe
- cartridge (on-line systems)
- 96-well plate
- disk
- miniaturization (elution in a small volume (25 ul - 100 ul). Dilution of eluate through the plate (no evaporation required) (gedaan in leuven ;)

What is the support material for SPE?

- silica or polymers with an area of 250-600 m2/g, a pore size of 60-70 angstrom and a particle size of 50 um.

What are 2 combination phases?

1) certify: contains reversed-phase and cation-exchange groups: for bases
2) certify II: contains reversed-phase and anion-exchange groups: for acids.

What could be a possible protocol for a reverse phase SPE?

conditioning: methanol or water
loading: in diluted plasma
washing:
elute with acetonitrile/ methanol (70:30)

What could be a possible protocol for a normal phase SPE?

- conditioning: hexane
- loading: hexane extract of plasma
- washing: hexane
- elution: ethylacetate/hexane (80:20)

What could be a possible protocol for ion exchange?

1) conditioning: methanol and formic acid
2) loading acidified sample
3) washing: with methanol and formic acid
4) elution: with 1% ammonia in methanol followed by 1% formic acid

What could be a problem of SPE?

- formation of channesl in the column: therefore avoid aspiring or blowing air too long. This does not frequently occur for the newest generations
- secundary interactions with silanol groups: use endcapped material

What is the purpose of derivatization?

it is to improve analyte properties. so for a
- better detection
- better chromatography
- better stability
- better adsorption behavior

What is the principle of derivatization?

It is a chemical reaction.
Usually in combination with supplementary sample preparation.

What are the problems with derivatization?

- laborious (long method development and long sample preparation) so perform only if necessary
- dangerous, often with hazardous chemicals. So perform in fume hood.
- risk of (additional) variation in results. So use a proper internal standard.

Wich methods to choose if you want to have a cheap method?

protein precipitation or LLE.

Which method to choose if you want to have a sensitive method?

LLE or SPE.

The question on the page originate from the summary of the following study material:

introduction

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