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Members,
We have run into a problem (described below) and would
appreciate any insights/info. Thanks!
During clinical trials, we have been adding 0.1M Citrate (pH
4.7) buffer as a "stabilizer" to plasma samples, which are stored at -20
degrees for 4-6 months before analysis. The reason
for this 1:1 dilution is to prevent the hydrolysis of the
glucuronides to the parent drug, thereby, confounding the true drug
concentration at the time of assay.
We will be embarking upon a long term (> 2 years) multicenter
study(>20 sites) and are concerned about the accurate addition of buffer
to accurately measured volumes of plasma
by a variety of personnel. Can anyone share their experience of
such a problem and methods
of resolution to it? Does anyone have any experience working
with powdered buffer? We think this would be a way of avoiding the
"dilution" step.
The obvious suggestion of sending plasma collection tubes
already containing buffer to hospitals presents other problems - will
the buffer stay microbe-free if it has to be stored for more than a
month. We had considered adding a preservative but wish to avoid this as
it will require revalidation of the assay.
Any suggestions would be helpful. Thanks.
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Kanak Singh
Associate Scientist
PDM, Parke Davis
Ann Arbor, MI 48105
Ph. (734) 998-2809
Fax (734) 996-3133
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I have two proposals:
Try to use an internal standard, if possible. We always worked with an
internal standard, mostly in form of addition of a radioactive tracer in
small amounts. Thus you are able to calculate your losses and mistakes.
It is possible to store your stuff in liquid nitrogen? Easy and cheap
for small amounts.
--
Dr. Harald Mertes
Anaesthesiologist
Rastatt/Germany
mailto:harald.aaa.mertes.inka.de
http://www.inka.de/sites/mertes/eharald.html
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This seems to be somewhat difficult problem. I have not heard about powdered
buffered tubes. Have you thought about using a central laboratory for
supplying these special tubes as part of a Kit. With proper instructions and
field training the staff at the hospital laboratory can be trained. If you
supply prepared tubes in bulk, the tubes may be lost. However supplying tubes
in a kit may increase compliance as for as use is concerned. It still does not
solve the problem of variable volumes that might be collected by different
personnel.
Dyal Garg
Clinical Research Services, Inc.
4949 S. Congress Avenue, Suite C
Lake Worth, FL 33461
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Dear Kanak,
I had a very similar problem which was solved slightly differently to those
presented. I am assuming that if the bloods are collected and spun down
using refrigerated centrifuge and that the conjugate is stable under these
conditions. I would then transfer a fixed volume of plasma into
polypropylene tubes containing a fixed volume of buffer. Clearly this
involves a certain degree of dilution and very low levels of the drug may
not be detectable if you are working at the limits of the assay. Otherwise
this is a well tried and tested method and the final result only requires
correction by which your fixed volume of plasma was diluted by the presence
of a fixed volume of buffer.
Faruq H Noormohamed
Imperial College, School of Medicine
Department of Clinical Pharmacology & Therapeutics
Chelsea and Westminster Hospital
369 Fulham Road
LONDON
SW10 9NH
Tel +44 (0)181 746 8141
Fax +44 (0)181 746 8887
email f.noormohamed.aaa.ic.ac.uk
email f.noormohamed.at.cxwms.ac.uk
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Stability of metabolites is a question that needs to be
addressed case by case basis. When you have no idea of this it is a safe
measure to store the samples at -70 or -80. Scheduling the analysis in
batches at 3 -4 months. Personally I doubt the stability of any
conjugates over six months. In your situation I will follow the
following steps to get an accurate measure of glucuronide conjugate.
1. During the sample collection, I add sufficient quantity
of ascorbic acid powder to give desired pH. Ascorbic acid gives two
benefits a) prevents the degradation due to oxidation and b) stability
of the metabolite due to pH. --- This will avoid the problems associated
with dilution.
2. Schedule the sample analysis in 1-2 months duration from
the time point of collection until I have confident data to establish
the stability of the metabolite, both as a neat solution and as in
Matrix.
3. If the above steps are not possible I will document the
concentrations of the phase -I metabolite with and without enzymatic
hydrolysis. This documents the extent of glucuronide formation.
Hope this Helps,
Prasad Tata, Ph.D.
Division of Bionalytical & Drug Metabolism
Otsuka America Pharmaceutical, Inc.
2440 Research Blvd.
Rockville,MD 20850
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Prasad,
One word of caution. Ascorbic acid will break down from UV-Visible light
so take the neccesary precautions. We routinely use ascorbic acid as a
buffer.
Another possible buffer would be citrate. Better pH, I don't know about
it preventing oxidative metabolism.
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Copyright 1995-2010 David W. A. Bourne (david@boomer.org)