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Most of the things around me here are water resistant and dirt repellent.
We also have stain-resistant clothing, non-stick cookware, fire-fighting foams,
coatings on food packagings and many other items,
all of which owe their protection to man-made chemical compounds.
They're known as perfluorinated compounds or PFCs
and we've been using them in all sorts of products for over 50 years.
They're very effective, but there's a potential problem.
They don't just stay in the product.
PFCs have been detected all over the world in the air, water, soil, biota,
blood and breast milk, so we need to monitor them.
The most commonly known and persistent PFC is perfluorooctanesulfonate or PFOS.
It binds to proteins and accumulates in the blood, liver and gall bladder,
so repeated exposure to it can cause health problems for humans.
from toxic and persistent chemicals.
It's the Stockholm Convention
on Persistent Organic Pollutants, or POPs,
and one of the compounds on its list is PFOS.
As a result, there's now a worldwide effort to ban its use.
There is a Global Monitoring Plan in place
to keep track of the effectiveness of the Convention.
Countries are encouraged to collect human and environmental samples
and monitor the concentrations of POPs over time.
But not everyone is set up and ready to perform the necessary analyses.
This training film, produced for the Chemicals Branch
of the United Nations Environment Programme,
will help laboratories to analyse PFOS.
We'll talk you through the various stages of the analyses of PFOS, PFOSA
in air, mother's milk, water and serum
and also four precursor compounds, two foses and two fosas in air.
Before we get started, remember that Teflon contains PFCs,
so don't use it during any of the extraction phases.
We recommend that all Teflon tubings and materials
in the ultra-pure water system be replaced.
"HPLC grade water" is used in all solutions in water.
The blank contamination of solvents and materials used during the analysis
must also be tested to prove that they do not contain
PFOS or any other PFC of interest.
These should be cleaned if necessary and stored covered with aluminium foil.
Remember all steps may be slightly different in your laboratory
so always optimise and validate to suit your own set-up.
Let's begin with analysis in air.
To collect PFOS compounds from air we use polyurethane foams (PUFs)
that have been placed in a passive sampler for three months
at an outdoor sampling location.
These PUF's are suitable for all of the target compounds
including the FOSAs and FOSES but not for the FTOHs.
First, take the PUF out of the sampler.
Perform a Soxhlet extraction with methanol for 12 hours.
You should add an internal standard of labelled PFC before the extraction.
To concentrate the extract we have a choice of using
either a rotary evaporator or, as seen here, a Kuderna-Danish.
When the volume has dropped to 1 millilitre, filter the methanol extract
through a 0.2 micrometer hydrophilic polypropylene filter or GHP.
Concentrate to 200 microlitres under a gentle stream of nitrogen.
Now add 100 microlitres of a mix of labelled PFC compounds
as an injection standard,
and 300 microlitres of a 2 milimolar solution of ammonium acetate.
We're now ready for the final analysis using LC-MS/MS
to identify and quantify them.
But first, let's look at the extraction of PFCs
from water, mothers' milk and serum.
The various stages are the same but the volumes are different.
For milk, water and serum, after we've weighed our samples,
we add a mix with labelled PFCs as an internal standard.
We add 2 millilitres of 50% formic acid
in water to the milk and serum samples
and put them in an ultrasonic bath for 15 minutes.
Then we put the extracts in a centrifuge at 3,000 revolutions per minute
for another 15 minutes.
After which, we do a solid phase extraction (or SPE) clean up.
Water samples don't need to pass through the sonication and centrifugation stages
but may need to be filtered if they contain particles.
They go straight to the SPE stage.
For all samples, we use a special glass container connected to a vacuum pump.
We put the 150mg wax cartridges on the valves on top of it.
To condition the SPE column, we add 4 millilitres
of a solution of 0.1% ammonium hydroxide in methanol
to the cartridges.
When the solutions have sunk in completely,
we add 4 millilitres of methanol.
After the methanol's gone, we add 4 millilitres of ultra-pure water.
For milk and serum we change the amounts
to 2 millilitres of 2% ammonium hydroxide in methanol,
2 millilitres of methanol and 2 millilitres of water.
When this has also completely sunk in, we add the sample extracts.
The flow rate is very important.
The cartridges should be loaded with a flow rate
with a maximum of one drop per second.
After the sample's sunk in the SPE column,
we'll wash the cartridges with a solution
of 4 millilitres of sodium acetate pH4 for the water samples,
2 millilitres of a solution of sodium acetate pH4
followed by 2 millilitres of a solution of 40% methanol in water
for the milk samples,
and 2 millilitres of a solution of 40% methanol in water for the serum samples.
Next, we dry the cartridge by switching on the vacuum pump,
without adding any solvents.
The next phase is to extract the PFC compounds from the cartridges.
For the water sample, we use two fractions.
The first is extracted with 4 millilitres of methanol
and the second with 4 millilitres of a solution
of 0.1% ammonium hydroxide in methanol.
For the milk and serum samples,
we use 1 millilitre of a solution of 2% ammonium hydroxide in methanol.
We place the tube under a gentle stream of nitrogen,
which allows the methanol to evaporate to dryness.
Next, we add a volume of ammonium acetate solution to the milk extract
and a mix of labelled PFC compounds
to all three extracts as an injection standard.
And put them in a centrifuge for centrifugation
at 3,000 revolutions per minute for ten minutes.
We'll then transfer the upper liquid part, the supernatant, to an LC-vial
and we're ready to perform the final analyses for all our samples,
including the air sample, on an LC-MS/MS.
But before we can do the actual analysis,
the instrument needs to be prepared.
For the detection part, we strongly recommend
the use of the triple quadrupole mass spectrometer
together with a negative electrospray ionisation.
A Time-of-Flight mass spectrometer is another option
but unfortunately an ion-trap mass spectrometer doesn't work well for PFOS.
The mass spectrometer conditions need to be optimised
for these compounds.
We need to prevent any PFCs which may have originated in the HPLC system
from interfering with your target compounds,
so we put an extra column between the pump and the injector.
There are several types of suitable analytical columns
but particularly recommended are C8 and fluorinated C8 columns.
The solvents used for the separation and the gradient used
depend on the type and size of analytical column.
These need to be optimised before starting analysis.
One of the mobile phase solvents, the ammonium formate solution,
needs to be filtered before using it
to prevent bacteria growing in the machine.
The other mobile phase is 100% methanol.
The final task is to identify the compounds
and quantify the concentration in the samples.
To do this, we inject standard solutions
containing different known concentrations
to create a calibration curve.
We use the areas of the peaks belonging to the right retention time
and "m over z" transitions to establish the concentration of the PFCs.
To guarantee correct results we need to apply
a number of quality control methods
such as the use of certified analytical standards
and certified reference materials, and an internal reference material,
plotting a QC chart, and including at least one blank in each sample series.
It's important that we get correct statistics
on the incidence of PFCs everywhere in our environment.
By following these procedures in sequence,
we can provide the data which will help to accurately determine these POPs.
It's an essential first step to manage
the risks associated with these contaminants. It's an important job.