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Dear all,
I was wondering if any of you does routine solubility assays for
new drug candidates. Not owing a nephelometer, I relied on the
classic shake-flask approach. As the drugs I was testing weren't
stable in the chosen media (water or buffer) for 24h, I had to
consider the kinetic solubility assay employing a certain
percentage of DMSO (1, 2%) and shorter times of equilibration.
The trouble came with quantification via HPLC of the dissolved fraction.
I tried either centrifugation or filtration. The first wasn't able to
give
reproducible results. As for filtration, I employed different
materials for filters (Nylon, PTFE, policarbonate) and 0.2 um
porosity, but I was
never able to get to a point of convergence between the pre-
filtration and post-filtration solution, even lowering tremendously
the concentrations. I suspect a non specific interaction of my
substances with the filters, but does it happen also with the inert
PTFE?
Could anyone comment on that?
I have searched for chiarifications in literature
but no one seems to be esplicit on that (especially on the use of the
materials).
Federica
Dipartimento Farmaceutico
Viale Usberti 27a
43100 Parma
ITALY
federica.vacondio.-at-.unipr.it
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Dear Federica,
There may be others with more extensive experience than I, however, I
worked
for several years evaluating new drug candidates for development and we
routinely tested the solubility of the candidates in several different
solvents. Commonly, we would weigh 10-20 mg (when available) and add
solvent 100 micro liters at a time initially, then larger volumes as the
test progressed. The test was just a visual inspection, and the samples
were left overnight to see if any material precipitated. We used very
little shaking, and of course there are kinetic considerations. I
have to
ask about the utility of a drug candidate that is so unstable as to fall
apart in just a few hours (although I have definitely been in board
meetings
where such candidates have been discussed). Some PTFE filters are not
compatible with aqueous solutions - but of course you can look to the
manufacturer's web sites for compatibility with your dissolving
medium. And
nylon are notorious for protein binding. Were you able to run your HPLC
assay of all samples in time to negate stability considerations? If
so, you
might consider a different method of analysis - and of course there
will be
caveats associated with those as well. How pure is the substance to
begin
with? What about an extraction method?
I am sorry, I am beginning to ramble - and I fear that I have not
helped as
much as I would have liked. :-(
James
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Dear federica,
The following article, particularly addressing the adsorption of
rifampcin to filters during analysis may be useful for you.
S. Agrawal, R. Panchagnula. In vitro analysis of rifampicin and its
effect on quality
control tests of rifampicin containing dosage forms Pharmazie 59
(2004) 10, 775-781.
Cheers!!!
ashokraj
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The following message was posted to: PharmPK
Dear Federica,
In my experience with kinetic, turbidimetric, UV and HPLC solubility
methods development, I found that filtering only confuses the issue
further. Centrifugation in discovery-level assays usually works well,
but you in some cases have to be quick and precise with aspirating the
supernatant if the pellet is not very dense and dissipates with time.
This last effect will give you results that would not reproduce. If you
would like to avoid centrifugation, I'd recommend trying turbidimetric
measurement. You don't have to have a nephelometer: absorbance at 620 nm
I believe in a plate reader would work.
-Katya
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The following message was posted to: PharmPK
A small tip:
Centrifugation step may be repeated 2 more times, before processing the
samples for analysis.
Vinayak Nadiger
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The following message was posted to: PharmPK
Dear federica,
You can do a routine aqeous solubility assay as
routine screening within 90 minutes instead of a
shaker flask method for NCE. You can get a quick read
out of the solubility of the compounds and rank order
them. We use a multiscreen solubility plate and we use
a maximum of 5% DMSO and incubate the drug in the
buffer for 90 minutes and then do the vaccum
filtration.Later we take a UV reading with the
spectrophotometer. I think this assay may be used for
NCE for determining aqueous solubility of NCE. We
have done for many compounds and it really works.
with regards
savithiri shivakumar
Dr.Savithiri shivakumar
Head- Biology
Alembic Ltd, vadodara.India
09374986394
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Dr.shivakumar
There is any specific reason to use only 5% DMSO or is it because of
DMSOs' toxicity to cell or there is any guideline for this limit?
Thanking you in anticipation
Regards
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The following message was posted to: PharmPK
Dear Yogesh,
In this assay using a multiscreen solubility plate, we
can use DMSO for the compounds as most of the
compounds have the property to precipiatate and this
method is mainly uses a particular pore size and based
on the filtration, you find out the concentration upon
filtration. Actually since you use DMSO, actually you
may get a enhanced solubility of the compounds. But
for NCE, this is a HTS method and give you a more or
less a similar results as obtained by shaker flask
method.
regards
savithiri shivakumar
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The multi screen solubility plate method is not compatible for all
the highly lipophilic molecules. There are certain loopholes in the
protocol mentioned by Millipore in this regard especially in dilution
protocol. So I feel it is better if you can modify the dilution and
do the similar study by analyzing HPLC where one may not have
limitations of sensitivity. Ofcourse the method has to be optimized
with a short run time that ultimately gives accurate solubility in
screening it self.
Regards,
KANTHI KIRAN
V.S.KANTHI KIRAN VARANASI, M.Pharm, MBA
Head-Pharmacokinetics and Drug Metabolism
--
Glenmark Pharmaceuticals Ltd.,
INDIA
Tel: +91-22 - 56132491/92 Ext. 315
Fax: +91-22 - 27781199
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