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All,
I understand that acyl glucuronides are typically unstable at basic
pH. I am interested in monitoring acyl glucuronides found in human
urine obtained from a clinical study. Is there a recommended
additive ( e.g. citric acid ) that can lower the pH and is easy to
handle (i.e. tablet form) that can be added to the collection vessels?
Where might one find such an additive?
Many thanks,
Damon
--
Damon Papac, Ph.D.
Director, Discovery ADME
Myriad Pharmaceuticals, Inc.
320 Wakara Way
Salt Lake City, UT 84108
dpapac.-at-.myriad.com
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Damon
I remember many years ago having a similar problem and so we developed a
method of reacting the acyl glucuronide with hydroxamic acid soon after
collection. Hydroxamic acid does not react with phenolic glucuronides
and this was what we were trying to achieve, distinction between the two
metabolites. The hydroxamates were then extracted and determined. As
the hydroxamates were very stable the samples could be stored and
analysed at your leisure. Did not publish the method and so can't give
you a reference or any other details because it was a long time ago.
I am sure that there are more modern ways of doing this.
regards
Brian
--
Damon
Correction to my previous E-mail, it was hydroxylamine that was added to
the urine to form the hydroxamic acid.
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I have used glacial acetic acid (about 5 mcl) per 500 mcl sample of
plasma.
Melanie S. Joy, Pharm.D., FCCP
Associate Professor
UNC School of Medicine
Division of Nephrology and Hypertension
UNC Kidney Center
CB #7155, 7005 Burnett Womack Building
Chapel Hill, NC 27599-7155
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This is a suggestion regarding stabilizing a glucuronide in a urine
sample
On one occasion we had an unstable glucuronide in urine (human), and
we provided sample tubes that we had prepared by lyophilizing a
measured amount of fairly concentrated phosphate buffer in them. I
don't remember the pH we chose, but it was a pH region where there
was good buffer capacity. It worked just fine.
-Tom
Thomas L. Tarnowski, Ph.D.
Bioanalytical Development
Elan Pharmaceuticals
700 Gateway Boulevard
South San Francisco, CA 94080
thomas.tarnowski.-at-.elan.com
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In a recent study we used the following conditions:
"A 150 uL portion of the resultant plasma/urine will be transferred
to uniquely labeled, clear polypropylene tubes containing 3.75 uL of
0.48 g/mL citric acid, mixed thoroughly, and frozen immediately. A
ratio of 25 uL of 0.48 g/mL of citric acid to 1 mL plasma/urine must
be maintained to prevent hydrolysis of any potential acyl glucuronide
metabolites of XXXXX."
Hope this will help
Best regards,
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Dear Damon,
The addition of a citrate buffer as solution is probably most
suitable. You
can also add a concentrated phosphate buffer sufficient to adjust the pH
(e.g. 6.5). Think of the problem that your urine pH can vary
significantly
and that your sample is trapped for a while in the bladder. Some
stability
studies at different pH could also be useful. I would not recommend to
strongly acidify your samples (stability of other metabolites).
Kind regards
Thomas
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