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Dear ALL
I am working on plasma protein binding studies for discovery
compounds.
My question is
After performing the protein binding studies with
equilibrium dialysis method, the sample from buffer compartment is
behaving differently from the buffer of same composition. It means
when we analysed sample in LCMSMS, we are getting consistent IS
concentration(peak area) in CC and plasma compartment samples but when
comes to buffer compartment samples, IS peak area is exactly half from
CC & QC IS area, but sometimes area ratio is also varying from buffer
sample to buffer sample in one study, but in some it is consistent. If
IS is coming only 50% of the initial IS value, which means where is
the problem? why IS peak area is half?
We changed chromatography and even system also showing same
effect, whether any one can give kind suggestion and mechanism for
this behaviour.
with regards
Hari
[Dilution? How long are you equilibrating? Stability in the buffer? -
db]
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IS could be sticky in the dialysis device, if it is not equilibrated
or saturated first.
Steven
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Dear Hari,
Of course, if this is only occurring for certain compounds, it could
mean that you have ion suppression. Make sure your compound peak is not
eluting at the same time as IS. It sounds like you've already tried
working on the chromatography though.
What is your sample preparation method? It's best if you dilute the
buffer side with plasma and/or plasma side with buffer to ensure a
uniform matrix before further sample preparation (protein crash) and
analysis. If the buffer side is behaving inconsistently, it could also
indicate solubility issues. Do you use the same IS for all compounds or
does it differ by compound?
Thanks,
Beverly Knight
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Dear Hari:
Did you add the IS prior to the dialysis as part of the protein
binding dialysis or after the dialysis as part of analytical process?
TK
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Dear sir
yes sir, there is suspection of ion supression, but the buffer
which is used for preparing CC & QC has no effect, only the sample
buffer behaves differently.
Even when compound peak is eluted after the retention time of
analyte also reproducing this effect, but for some compounds it is
rectified by simply changing the column brand from C18 BDS Hypersil to
Kromasil.
In some cases it is consistent regardless of change in
chromatography, we are working with retention time above void volume.
Can you clearly explain little more regarding how to mix the plasma and
buffer sample in this experiment.
We are using different IS but in some cases same IS used for the study.
we are also suspecting solubility issues, but i like to ask
According to our experiment, we are adding 100mM buffer on one side
and plasma on other side of compartment, but as both different phases
are seperated by permeable membrane.
1. Whether this effect has any reasons like (problem from permeable
membrane or its make or composition) any cases available
2. Since 100mM added on one side, but on other side it is plasma which
obviously dilute the buffer (approx 50mM) which has any effect.
3. As the buffer ions mix with plasma ions seperated by membrane has
causing any change in the composition and concentration of buffer
samples in another compartment.
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It is or is it not the same buffer?
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Probably surfactants in the plasma cross the membrane into the buffer
compartment and cause interference with analyte and/or IS in the
analysis. You could check what is in the plasma - blood tubes with
heparin often contain wetting agents which have PEG oligomers (eg Tween)
in them and these can interfere. Two things you can do:
1 Perform a full scan and look for a series of peaks with 44amu
difference in parent ion
2 Try EDTA as anti-coagulant - it does not require wetting agents.
The natural surfactants in plasma (lecithin-like) tend to be very late
eluting. Do you always run a gradient to high organic content?
Ted
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