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A drug forms a conjugate with glutathione. We need to
analyze it in blood and/or plasma. I will appreciate
if someone can suggest method(s) to extract the drug
from blood and plasma. Do we need to use
dithiothreitol (DTT) in the process?
Thanks
Ananda
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Hello,
if your drug and metabolites are stable in acid, the easiest way is an
acid
precipitation of the proteins/RBCs, e.g. by 1M HClO4. However, you have
to
be careful with acid precipitation of whole blood samples, if your drug
is
easily oxidable: The oxygen bound to hemoglobin is known to yield
reactive
oxygen species which might produce artefactual metabolites (cf. eg.
Anal.
Biochem. 191,347 (1990); Biol Chem Hoppe Seyler 371, 881 (1990)).
If your drug is highly lipophilic you have to use e.g. alcohols for
plasma
extraction and Tsuchihashi reagent for whole blood samples ( icecold
water
added to blood sample (1:1) and precipitated with mixture of ethanol /
chloroform (63/37, v/v, icecold), thoroughly vortexed and centrifuged at
12000 rpm for 5 min ). However, recovery is mostly a big problem with
lipophilic drugs which can be hardly solved.
Hope that helps.
Regards
Dieter
Dr. Dieter Gallemann
Merck KGaA
Institute of Drug Metabolism and Pharmacokinetics
Am Feld 32
D-85567 Grafing
Germany
Tel.: +49 (0) 8092-7008-11
Fax: +49 (0) 8092-7008-7011
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I remember an ISSX poster on this subject last year. It was presented
by
Andy Baker of Waters. Andy_Baker.aaa.waters.com Perhaps you could contact
him
for a copy.
Mike
Michael D. Cameron Ph.D.
Senior Scientist
CellzDirect, Inc.
8609 Cross Park Dr, Suite 100
Austin, TX 78754
Phone: 512-615-2286
Fax: 512-834-7767
michaelc.-at-.cellzdirect.com
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I worker on the same problems and I used DTT with success.
Best Regards
Fabio Dr. Macchi
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Dear Dr. Machi,
Can you please let me have some protocol or method on
drug glutathione conjugation studies and its reduction
with DTT. Any references will be very helpful.
Thanks
Ananda
Dear Ananda,Back to the Top
DDT is quite easy to use, some problems can occur if the detection
system follows the -SH group (alone or coupled with a derivatizating
agent such as mBBr).
Some information can be found in the following reference
Determination of the new dual ACE-NEP inhibitor Z13752A in human plasma by HPLC with fluorescence detection : F. Macchi, A. Cattaneo, S. Taino, Dept. of Pharmacokinetics and Metabolism, Zambon Group S.p.A., 20091
Bresso, Milan, Italy =95 ABSTRACT
Best regards
Fabio
European Journal of Pharmaceutical Sciences, Volume 6, Supplement 1,
August 1998, Page S43
[I've removed the abstract - db]
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dear sen,
1. If You need to extract the drug alone, then you can
use NEM which will break the thiol reaction and
extraction of the drug can be done as usual procedure
followed for your drug.
2. If you wish to extract the conjugated drug, you can
try to use choloroform:ethanol(75:25)and try to
extract the conjugate. Since the drug-conjugate is
more polar than the drug as such, you can use any of
the procedure of extraction of the polar drug.
regards
sivakumar
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