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Dear All,
We are using RED inserts For protein binding assay.
I would like to Know reasons For the variation in data in Human Plasma through this procedure.
I have done so many times in our lab and we have got a consistent results with all the QC.
But wen I did last time with Human plasma I have seen a huge variation in the data.
Did anybody got the same problem any time. Iam Unable to figure out the reason. Iam Taking atmost care not to introduce any air bubble if that may be one of the reason. I have done the experiment twice and I have got the same variation.
Waiting for all your Suggestions.
Thank you in advance
Regards,
Indira
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Hi Indira,
If you are getting consistent results for the QC's with human plasma it means there is no problem with the experiment setup. It might be a compound inherent property. Check the solubility profile of the compound in the buffer.
Regards --Naveed Shaik Abdul
Graduate student
MCPHS
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Dear Indira,
Good morning to you,
If your QC's are fine then your plasma is not having any problem.
Please check the "pH" of the plasma and also solubility of the compound in the buffer.
Equilibration time and shaking speed may also affect the results.
Hope you can check all these points and take proper action.
Regards Amit Patil
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Dear Patil
I wonder why shaking speed may also affect the results
Best Regards
KJ Bae
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The following message was posted to: PharmPK
Dear Indira,
We experienced high variability in PPB determinations for lower concentrations of some compound. In our case it was down to analytical problems / aspecific binding on plastics from RED device, collection plates ... The issue essentially is that the compound exhibits high aspecific binding whilst in acqueous medium, even though it did not in plasma. We could point it out by investigating the aspecific binding and bioanalytical part of the experiment.
As pointed out by others insolubility, re-precipitation ... could give similar confounding effects.
Patrice patrice.larger.aaa.rottapharm.com
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I suggest you look into Harvard 96 well plates. They generally gave an overall recovery of greater than 90 %.
Stanley Cotler
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1) There could be chances of freeze thaw effect of human plasma
2) if you are using Human plasma from different sources and or persons, one could expect variations in the results.
Was your variation was significantly different from the previous resuls?
Jitendar Reddy
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The following message was posted to: PharmPK
hi indira,
we had done some compounds using the same equipment RED inserts in our
lab, but surprisingly the results are consistent with
literature.however as others said there could be essentially higher
degree of non specific binding to the equipment.
some times solubiluity, RPM and the Ph of the medium during long hours
of incubation also makes the values to shift on to a lower side or
result in abnormal values,if possible can you breif your experimental
conditions?
vijaybhargav
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Dear Jitender ,
Yes it showing 95% bound In verpamil, Previously it was 90% percent bound which was similar to literature values
I will follow your suggestion, in regard to freeze thaw .
Regards,
indira
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