Solid-phase Microextrac2on (SPME), Green Sample prepara2on

Technique
• possibly requiring low sample amount
Sample selec*on • high throughput
• representa*veness, specificity
including biological cycles and factors Instrumental analysis
influencing sample proper*es i.e. drinking, • sensi*vity/linear dynamic range
ea*ng • broad analyte coverage (MW, chemistry…)
• accessibility and invasiveness • high throughput
• stability (during transport & storage)
Data processing
Sample prep “best sample prep is no sample • peak detec*on
prep” but instrumental • peak alignment (RT correc*on)
analysis requires good sample clean-up • noise reduc*on/baseline correc*on
(reproducibility!) • matching peaks across samples
• unselec*ve or very selec*ve
• effec*ve isola*on and quenching of Data analysis
analytes • sta*s*cal tests
• reproducible • iden*fica*on

Sample Prepara*on

The way the sample is treated prior to the analysis, it is important because error will
affect the results of analyses.
Goal: clean up the sample, especially biological and preconcentrate the analytes
especially the endogenous ones.
One type of sample prepara=on is the Extrac=on, is a separa=on process.
Par==on theory, distribu=on of the analyte in 2 phases in the equilibrium.

Modern
Tradi*onal SPME, Solid-Phase Microextrac=on
SPE, Solid Phase extrac=on MEPS, Microextrac=on In Packed
LLE, Liquid-Liquid Extrac=on Syringe
PPt, Protein Precipita=on DLLME, Dispersive Liquid-Liquid
Microextrac=on
Used many years but they use large SDME, Single Droplet Microextrac=on
amount of organic solvents
Miniaturiza=on of the tradi=onal ones

SPE, Solid-Phase Extrac*on

Sorbent based extrac=on technique where compounds are dissolved or suspended in a

liquid mixture and separated from other compounds in the mixture according to their
physical and chemical proper=es.
Objec=ve: concentrate and purify samples for analysis. Isola=ng the analytes of interest
from wide variety of matrices, inclusing urine, blood, water, beverages, soil and animal
=ssues.
Steps, CLWE:
C-Condi=oning (solid phase moisturize with the matrix)
L-Load (sample)
W- Washing (to eliminate the interfering components)
E-Elute (analyte)

1) Column pack with the par=cles and first they have to be ac=vated, to make the
par=cles wet.
2) Load of the analytes and then
3) Wash the solvent for the analytes than can have stayed in the column and lastly
4) Elute

Advantages of the tradi=onal:

Isola=on and preconcentra=ng of analytes in a selec=ve way
Extracted analytes may be kept on the sorbents with no degrada=on
Reduced consump=on of organic solvents (in comparison to LLE)
No forma=on of emulsions
Broad range of available sorbents
Automa=on
One-site analysis

Cons:
- Analyte loss (incomplete desorp=on)
- Low repeatability
- Time consuming

Protein precipita=on Is the most common technique with the SPE the extracts are
much more clean.

SPME, Solid-Phase Microextrac2on

Microextrac=on, miniaturized format of extrac=on that u=lizes small extrac=on devices
and samples sizes. Non-exhaus=ve microextrac=on approaches have valuable
proper=es that create excellent opportuni=es, but also have some key limita=ons. To
op=mize methos performance, these limita=ons must be properly addressed via
appropriate device construc=on and experimental design, which rely on a deep
understanding of extrac=on fundamentals.

A greener (miniaturiza=on of SPE) and convenient sample prepara=on alterna=ve, fast

and easy that allows to enrich the analysis, concentra=ng the compounds of interest.
Quan=ta=ve analysis and customizable geometry (can be adjusted) with a variety of
sorbents. Compa=ble with LC, GC, CE and direct MS, The result can be analyse with this
techniques.

SPME free frac=on (free concentra=on) of analyte, the ones bound to proteins cannot
be extracted. Instead, if we add organic solvents, we can destroy the connec=ons of
the compound and obtain all the molecules in the sample.

You can do in-vivo sampling with the needle to easier the process.
➢ integrates sampling and sample prepara=on in a single step
➢ minimum or no need of sample pretreatment
➢ different formats of SPME devices (fibers, blades (easier to handle), tubes (covering
the inner wall with the solvent or pack with the solvent)
➢ reusable devices
➢ large and small sample volumes can be analyzed
➢ suitable for automated and high-throughput analysis (in clinical lab)
➢ facilitates on-site determina=ons (in the opera=ng room)
➢ biocompa=ble coa=ngs (in vivo sampling)

SPME, provides minimum invasiveness (in vivo) and negligible deple=on (free
concentra=on) vs TFME (Thin-Film Microextrac=on) provides enhanced sensi=vity
without sacrificing the throughput (short extrac=on =me), more surface area and more
volume of extrac=on phase.
They use well-plates that allows to work with mul=ple samples (96) at the same =me.
Steps are similar to SPE:
Precondi=on or organic and water solvents Methanol/water 50/50, ac=vate the
extrac=on phase)

Op=miza=on has 3 main points, extrac=on condi=ons, matrix modifica=on (our sample
can op=mise) and desorp=on condi=ons to obtain the most efficient extrac=on and
improve the process and data obtained.

1- Extrac)on Condi)ons
• Coa=ng (extrac=on phase) select the coa=ng
• Extrac=on Mode, op=mize the extrac=on mode
• Agita=on method
• Extrac=on Time
• Volume of the sample.

SPME Coa*ng
Coa=ng, when we one to select we must keep in mind the proper=es of the compound,
polarity, voli=vity, solubility.

LC applica=ons:
- C18 ideal for hydrophobic compounds, lipids or drugs hydrophobic.
If they are compound with both proper=es beeer:
- DVB
- Mixing mode (C18+C8+benzenesulfonic acid)
- Polar-modified polystyrene/divinylbenzene (PS-DVB)
- Hydrophilic-Lipophilic Balanced (HLB)

GC applica=ons (high temperatures should be considered):

▪ Polydimethylsiloxane (PDMS)
▪ Polyacrylate (PA)
▪ PDMS/DVB
▪ Carboxen (Car)/PDMS
▪ DVB/Car/PDMS
▪ Carbowax (CW)/DVB
▪ PDMS/DVB/PDMS (overcoated), Recently overcoated fibers, that protects the fiber
from contamina=on from the sample.

Precondi=on of the coa=ng before the extrac=on

LC analysis, to wet the pores and ac=vate the coa=ng with 50/50 MeOH/Water
generally, 30 min and before extrac=on a water rising to remove the excess of organic
solvent (pre-extrac=on rising).
GC analysis, expose coa=ng in the GC injector using manufacturer recommended
precondi=oning temperature and =me, and ac=vate split fow (to avoid contamina=on
of the GC).

Extrac*on mechanism
Absorp*on vs Adsorp*on
+ Single-phase coa=ngs (liquid coa=ngs), Absorp=on mechanism.
PDMS, PA
Molecules solvated by the coa=ng and tend to penetrate the whole volume of the
coa=ng.

Mixed-phase Coa=ngs (solid), Adsorp=on mechanism.

PDMS/DVB, Car/PDMS, DVB/Car/PDMS, HLB/PAN, PS-DVB/PAN
Sorp=on occurs only on he porous surface of the coa=ngs, and displacement and carry-
over effects may occur.
Kes = Cee /Cse
Kes = Distribution constant
Cse = Sample concentration at equilibrium
Cee = Equilibrium concentration in extraction phase

The molecules of the analyte can be small hydrophobic molecules that have high Kfs,
low free concentra=ons and high protein binding or small hydrophilic molecules with
low Kfs, high free concentra=ons ad low protein binding.

Displacement effect, compound in high concentra=on goes to the fibber and displaces
other compounds present in the coa=ng. We can shorten the =me of extrac=on to
reduce the displacement effects.

Extrac*on Modes
- HeadSpace mode, for Vola=le compounds, we place the device above the sample
and extract the vola=le compounds present in the atmosphere above the sample.
- Direct Immersion mode, followed by a quick washing step and solvent or thermal
desorp=on. It can be membrane protected too.

Agita*on methods
Agita=on of the sample par=cipate in mass transfer between the sample and the
coa=ng, shortening extrac=on =me, mass transfer rate is faster and non-equilibrium
extrac=ons have higher sensi=vity.
- Magne=c s=rring, no sophis=cated equipment. S=rbar must be inserted in the
sample and s=rring plate can cause hea=ng of vial.
- Orbital Agita=on
- Intrusive s=rring, good performance. Difficult to seal the sample properly and not
useful with vola=les extrac=on.
- Needle vibra=on, useful for trace analysis and applicable only to rela=ve small
sample volumes. Excessive stress of the needle.
- Vortex s=rring, the most used, speed up the extrac=on =me without change any
other parameter, like temperature. Useful for trace analysis and small samples
volumes limita=on do not apply.
- Flow-through s=rring , excellent agita=on. Addi=onal equipment and risk of cross
contamina=on.
- Sonica=on, very efficient to but it can destroy the fibber so reduced fibber life=me.
Temperature of water bath changes.
The only op=on if we place the fibber in the =ssue there is no need of agita=on, Sta*c
extrac*on, in this case the extrac=on =me will be longer.

Extrac*on *me

Depending on the porpoise of the analysis the op=mum extrac=on =me varies.
- High Throughput, pre-equilibrium condi=ons
- High Sensi=vity, extrac=on at equilibrium, because during equilibrium extrac=on
the rela=ve error is less.
- Good Repeatability, extrac=on at equilibrium, in pre-equilibrium to obtain
repeatable =ming and automated system should be selected.
The shorter the extrac=on =me and the steeper the slope of extrac=on curve, the
larger the rela=ve error on the amount of analyte extracted.

Sample volume

Exhaus*ve extrac*on , in this case the amount of analytes extracted increases with the
sample size up to a point, ajer which the sensi=vity does not increase with further
increase in Vs (volume of the sample). Ne +number of moles of analyte extracted at
equilibrium
ne =VsC0

Microextrac*on, microextrac=on techniques u=lizes a smaller amount of extrac=on

phase; the isola=on of analytes is performed based on the equilibrium between the
sample matrix and the extrac=ve coa=ng.
In this case the amount extracted is not related to the sample volume. (Ve, Volume of
the extrac=on phase, fiber coa=ng)

ne = KesVe C0

2- Matrix Modifica)ons
pH
pH modifica=ons can improve the method sensi=vity because of the full conversion of
analytes into neutral forms. SPME is an extrac=on of neutral or non-dissociated
species.
Acid species pH ≤ pK -2
Basic species pH ≥ pK +2
Low and high pH levels can damage the fibber coa=ng.
Ionic Strength
Addi=on of salt special for GC analysis, we can improve the signal strength, but always
when you modified the sample with the salt it affects too other compound present in
the sample so at the same =me you can be extrac=ng other substances, and this is not
ideal.
The sal=ng-in effect consists in the increasing of the solubility of proteins when adding
salt to a solu=on. This is because lower concentra=ons of the salt will decrease the
electrosta=c energy between the proteins increasing the solubility. If the [salt] is too
high, the opposite phenomenon occurs, sal=ng out is the phenomenon observed when
the solubility of a nonelectrolyte compound in water decreases with an increase in the
concentra=on of a salt, as the salt competes with the protein for the interac=on with
water.

❖ Mainly for HS applica=ons

❖ Increases the Kes constant and improves sensi=vity in most applica=ons
Kes(Distribu*on constant) = Cee (equilibrium concentra*on in extrac*on phase)/Cse (sample concentra*on at equilibrium)
❖ Sodium chloride (NaCl) is the most used salt to adjust ionic strength.
However, other salts such as ammonium chloride (NH4Cl), sodium sulfate
(Na2SO4),or sodium hydroxide (NaOH) may have different sal=ng out
capabili=es, par=cularly when dealing with complex matrices such as
food.
❖ It is important to note that while salt may improve the SPME extrac=on of
the desired analytes, it could also cause co-extrac=on of more matrix
interferences or undesired compounds.
❖ The addi=on of salt to the sample matrix may lower the par==on
coefficient for some target analytes, thus decreasing the concentra=on
of analytes in the headspace and amount of analyte extracted

Sample dilu4on
Sample dilu=on, avoid destroying the fibber. Releasing the compounds bound to some
components, increasing the free concentra=on of compounds.
But it depends on the goal, if it is to extract compound that are low level we could lose
them, this is a limita=on.
+ helps minimize the fouling of the coa=ng (in DI mode-longer coa=ng life=me)
+ enhances the concentra=on of analytes in the sample
+ helps avoid satura=on of solid coa=ng, especially for complex matrices
But excessive dilu=on of the sample is not recommended because it could lead to
reduc=on in extrac=on efficiency, with slower diffusion process, slower adsorp=on of
target analytes in heterogeneous samples, and decrease of free concentra=on of
analytes due to binding.

Organic Solvent content

The addi=on of small amount of an organic modifier can help release some analytes
bound to matrix components.

Analyte deriva4za4on
Prior/during-extrac*on, improving the extrac=on, chromatographic behaviour and
detec=on or post-extrac*on, improving the chromatographic behaviour and detec=on.
+ enhances extrac=on efficiency
+ enhances detec=on sensi=vity
+ makes compound more amendable to a par=cular mode of analysis
+ usually in GC analysis
Deriva=za=on reagents could be a source of interferences and error into the system,
and should only be carried out when strictly necessary.
Sample Temperature
Increase of T:
+ Analyte diffusion coefficient
+ HS capacity
+ extrac=on rate and Speed (shorter equilibrium =me)
- Par==on coefficients
- Amount extracted at equilibrium
3- Desorp)on Condi)ons LC
Problems typical during LC based desorp=on are: carry-over (should be tested during
op=miza=on of desorp=on step), band broadening In chromatograms, longer
desorp=on =me.
Desorp=on Solvent composi=on
We want, highest recovery, lowest carry-over, shorter desorp=on =me and LC
compa=bility.
Solvent volume
Affects the sensi=vity and it could be recons=tuted o a smaller volume ajer
evapora=on.
Desorp=on Time
Kine=cs of desorp=on, slower than in GC and depends on the composi=on of
desorp=on solu=on.
Agita=on

In vivo SPME/sampling Living System

✓ no need to prepare blood and =ssue samples (complex and tedious)
✓ no sample collec=on
✓ collec=on of a substance at the site of ac=on
✓ monitoring living systems in their natural state
✓ no disturbance of system equilibrium
✓ no enzyma=c degrada=on of extracted analytes if the method restrict access of
extracted small molecules from enzyme
✓ in vivo metabolism quenching enables capturing of elusive por=on of the
metabolome (unstable, short-lived species, intermediate metabolites)
✓ reduced number of animals used for pre-clinical studies
✓ the animal can also be used as its own control reducing the variance between
different animals, thus improve the quality of the data

Challenges:

• Difficult to mimic system in vitro for valida=on. And quan=fica=on

• Difficult to control all influencing factors
• Variable Blood flow
• Heterogeneous matrix
• Requieres biocompa=ble materials
• Risk of infec=on and inflma=on of the animal

1. Steriliza=on (autoclaving)
 2. Precondi=oning (ACN/Water)
3. Extrac=on
4. Washing (water)
5. Desorp=on (ACN/water)
6. LC-MS/MS

Ex vivo SPME method development for In vivo Lung Tissue sampling

❖ selec=on of fiber coa=ng
❖ selec=on of extrac=on =me (equilibrium)
❖ extrac=on =me profile in sta=c condi=ons
❖ op=miza=on of precondi=oning and desorp=on condi=ons (incl. carry over)
❖ verify the effect of fiber steriliza=on on the extrac=on efficiency of the coa=ng
❖ lack of internal standard, you cannot add the standard to the sample because if the
lancet with the =ssue sample that is treated directly.
❖ extrac=on from intact (non-homogenized) and homogenized =ssue

In vivo SPME
• SPME is flexible tool for sampling/sample prepara=on of different biological matrices
including *ssues of various origin
• SPME is minimum invasive tool suitable for in situ and in vivo =ssue extrac=on
without any =ssue sample collec=on or consump=on
• biocompa*bility, autoclaving and single-use of the SPME devices enables u=liza=on
of in vivo SPME in plant, animal and human studies
• fast sampling and direct SPME-MS couplings could be used as an intra-opera*ve
rapid diagnos*c tool in the near future
• in vivo SPME may provide insight into the metabolism of analytes in the living system
of plants, animals and humans
• The strength of SPME technology is that SPME devices can take many different
shapes and use a variety of extrac=on phases that have been op=mized for specific
analytes. Furthermore, the design of SPME devices can be op=mized for a given
applica=on (e.g., on-site deployment or laboratory automa=on) due to its ability to be
coupled to a range of analy=cal instrumenta=on for the determina=on of the extracted
species.

Pros: Cons:
+ rela=vely short extrac=on - Coated of fibbers
+ minm. consump=on of organic - Limited life=me of the coa=ng
solvents - Degrada=on of analytes at higher T
+ possibility of op=miza=on of several - extrac=on of contaminats
parameters
+ high sensi=vity
+ automa=on
+ on-site and in vivo analysis
+ small size of SPME devices
+ Coa=ng with different proper=es
+ analysis of complex samples