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Hi!
I would like some information about in vivo recovery in
microdialysis. I've performed in vitro recovery at 3 different
concentrations. There were no differences among the levels analyzed,
but there were between loss and gain. Now, I would like to determine
the in vivo recovery. Which method is more suitable to evaluate probe
recovery? Zero no net flux and retro-dialysis method can not be
applied, because loss and gain in vitro were not the same. So, could
zero flow rate method suitable to determine in vivo recovery?
Best regards,
Prof. Doutorando Leandro Tasso (UFRGS - BRAZIL).
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Dear Prof. Tasso,
I was forwarded this mail by a colleague from the pharmacokinetics
department at Schering AG, in Berlin, since I am just finishing a PhD on
the subject of microdialysis and pharmacokinetics. My thesis
concentrates
on the in vitro testing of probes which are intended to be used in
vivo for
quantitative microdialysis, but I have also done some work in vivo,
and I
am most happy to help you with your question.
Firstly, the problem of differing recovery and delivery has been rarely
described in the literature, but below is one excellent article I came
across. A wide range of compounds were tested, and the recovery did not
differ significantly from the delivery for any of them. Therefore, I
have
some questions about your in vitro tests first. You said you tested 3
different concentrations and found constant recoveries. I presume
that you
have taken three consecutive samples at each concentration to take
the mean
of. If you calculated the standard deviation as well, is it less than 5%
per concentration? If not, the samples may not have been taken at steady
state, in which case plotting the collected individual data against time
(recovery versus sample number) will show an increasing slope. If
that is
the case, then your observed recovery is lower than the true
recovery, and
your delivery experiment is going to give different 'recovery'-
values. The
same is of course also true if the samples from the delivery
experiment are
not taken at steady-state. If you find this is the problem, have a
look at
the poster of mine for some other materials. Only materials that reach
steady-state straight away are suitable for pharmacokinetic studies!
If you really do have a discrepancy between your recovery and
delivery, the
delivery method may still be applicable if you use the recovery/delivery
ratio found in vitro as a correction factor. Also, the no net flux
method
may still be applicable, since you are determining the concentration at
which the amount recovered equals the amount delivered. I have no
experience with this method at all, maybe you get an in vivo recovery
value
somewhere between the true delivery and recovery this way, but you
can test
this in vitro; it would certainly make an interesting paper! The
perfusion
rate method you asked about also uses delivery to determine the
recovery.
Below an article comparing these last two methods.
Alternatively, you could carry out the entire study at zero flow (< 0.5
uL/min), ergo at 100% recovery (test this perhaps in vitro first!). This
however poses great problems with sample volumes and collecting enough
material for analysis. One suggestion has been to use a pulse like
microdialysis, where you stop the flow for a few minutes (whatever time
resolution you want), then flush out that fraction and collect it. The
problem here is that the exact volume at the membrane must be known,
which
however becomes greater as your time interval increases (you get
diffusion
into the inlet and outlet as well). Then the sample is diluted by
flushing
it out, having already little compound in the original fraction (as flow
rate decreases, the relative recovery increases, but the absolute amount
recovered decreases), which may give you problems with detection limits.
Another approach is to add a mixing chamber to the probe, using a 0.1
uL/min flow rate, and constantly adding another 0.9 uL/min to the
dialysate, giving you a resulting recovery of 10%. If this is enough for
your analytical method, perhaps Mr. Cremers from the Netherlands can
help
you (T.I.F.H.Cremers.-at-.rug.nl); he is the only one who has made these
kind of
probes so far!
I hope this will give you a start, I would be very interested in your
progress, if you could please keep in touch.
Good luck, and with kind regards,
Beatrix von Meier-Ince
[Attached files removed - db]
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Dear Prof Tasso,
It appears that attachments aren't passed on, therefore the missing
references here:
Zao, Liang and Lunte: Comparison of recovery and delivery in
vitro for
calibration of microdialysis probes. Analytica Chimical Acta 316
(1995),
403-410.
Stahle, Seversvardt and Ungerstedt: A comparison between three
methods
for estimation of extracellular concentrations of exogenous and
exogenous compounds by microdialysis. Journal of pharmacological
methods
25 (1991), 41-52
For poster ask me.
With kind regards,
Beatrix von Meier-Ince
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Copyright 1995-2010 David W. A. Bourne (david@boomer.org)