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Hi, All,
I met serious non-specific adsorption when I used Amicon YM-10 to
test the protein binding. Could you please give me any suggestion to
overcome it if ultrafiltration is used? Or I have to use other
methods to test the protein binding?
Many thanks.
Regards!
Jian Li
Jian Li
Centre for Pharmaceutical Research
School of Pharmaceutical, Molecular & Medical Sciences
University of South Australia
City East Campus, Adelaide
Australia SA5000
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The following message was posted to: PharmPK
You may try equilibrium dialysis.
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The following message was posted to: PharmPK
Hello Li
This is a very good paper which deals with the same problem of nonspecific
adsorption. This will definetly help you.
This paper uses charcoal adsorption method.
Khurana M, Paliwal JK, Kamboj VP, Gupta RC.
Binding of centchroman with human serum as determined by charcoal adsorption
method.
Int J Pharm. 1999 Dec 10;192(2):109-14.
Hope this helps
Atul
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Hi,
Even while using equilibrium dialysis technique make sure you are
siliconising dialysis cells and other relevant glasswares to minimise
nonspecific binding.
Best wishes
hamim
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Anyone else have any ideas, desides dialysis?
I have had problems before with non-specific binding to the MPS device
(Amicon), but the YMT (30k cut off) membranes were fine. Amicon had some
literature available for preventing the non-specific binding to the device
itself (soaking in BSA or milk powder solutions), but not the membrane as I
recall. You may want to try the YMT membranes if you are just looking a
plasma protein binding.
I myself am about to investiagte the binding to microsomal proteins to tidy
up some in vitro-in vivo scaling work using the YM10 membranes (YMT 30k cut
off is far to large)...I hope I dont have the same problem (but I fully
expect to!).
David Foster
David Foster, PhD
Department of Clinical and Experimental Pharmacology
Faculty of Health Sciences
Adelaide University
Adelaide, South Australia 5005
Tel: +61 08 8303 5985
Fax: +61 08 8224 0685
Email: david.foster.at.adelaide.edu.au
http://www.adelaide.edu.au/Pharm/index.htm
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The following message was posted to: PharmPK
Classical equilibrium dialysis is a good alternate and takes care of
moderate degree of non-specific binding with dialysis membrane. But if the
drug in question has severe non-specific binding to dialysis membrane too,
the equilibrium times are very long. Hence other methods like
ultracentrifugation or charcoal adsorption might be more useful. Hope it is
useful.
Dr. Jyoti Paliwal
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Hi,
in case of relevant non-specific binding at therapeutic plasma concentrations,
I would recommend using another method, e.g. distribution method:
J. Schuhmacher et al. Determination of the free fraction and relative free
fraction of drugs strongly bound to plasma proteins.
J Pharm Sci 89 (8), 1008-1021, 2000.
good luck
Elke
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The following message was posted to: PharmPK
Jian:
If your compound is binding to the membrane, you may also encounter this
problem using equilibrium dialysis. If this is the case, you could try
ultracentrifugation which doesn't involve the use of a membrane. You'd
have to check the extent of binding of your compound to the centrifuge
tube.
Regards
Dave
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If you haven't got a problem with stability of your compound, try
ultracentrifugation. Of course with that, there's always the possibility of NSB
to the ultracentrifuge tubes!
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We encounter similar problem frequently. When the drug has very high
non-specific binding using Amicon's Centrifree (YMT membrane), we use an
alternative method of Biological Equilibrium Dialysis (BED)
see: Hinderling, P. H., Therapeutic Drug Monitoring 9:331-336 (1987)
It works very well for us when we observe the limitations closely, as
discussed in the paper.
Regards,
Elena
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We had the same problem with amiodarone, and tried a couple of
indirect methods which you might like to have a look at.
Cheers,
Stuart McLean
Tasmanian School of Pharmacy
Veronese ME, S McLean and R Hendriks. (1988). Plasma protein binding
of amiodarone in a patient population: measurement by
erythrocyte partitioning and a novel glass-binding method. British
Journal of Clinical Pharmacology. 26 721-731.
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Go to eqilibrium dialysis. No way you will get any meaningful results with
ultracentrifugation. Another method is the use of charcoal.
Stanley Cotler
Non-Clinical Drug Safety
Nutley, NJ 07110
Phone 973-235-2857
Fax 973-235-7010
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Dear Dr. Cotler,
You wrote:
> Go to eqilibrium dialysis. No way you will get any meaningful results
> with ultracentrifugation. Another method is the use of charcoal.
Your statement on ultracentrifugation is quite clear, but lacks
convincing arguments. Could you provide some arguments or
references? And what about ultrafiltration?
Best regards,
Hans Proost
Johannes H. Proost
Dept. of Pharmacokinetics and Drug Delivery
University Centre for Pharmacy
Antonius Deusinglaan 1
9713 AV Groningen, The Netherlands
tel. 31-50 363 3292
fax 31-50 363 3247
Email: j.h.proost.at.farm.rug.nl
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If you have non-specific binding with the amicon device, the results you get
are not valid.
Stanley Cotler
Non-Clinical Drug Safety
Nutley, NJ 07110
Phone 973-235-2857
Fax 973-235-7010
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Stanley -could you give details as to why the results are "not valid". As
Hans asked before -why??
David
David Foster, PhD
Department of Clinical and Experimental Pharmacology
Faculty of Health Sciences
Adelaide University
Adelaide, South Australia 5005
Tel: +61 08 8303 5985
Fax: +61 08 8224 0685
Email: david.foster.at.adelaide.edu.au
http://www.adelaide.edu.au/Pharm/index.htm
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Copyright 1995-2010 David W. A. Bourne (david@boomer.org)