Dear all we are working on a NCE which is present as glucuronide in theBack to the Top
blood. We are giving the NCE to healthy humans and analysing the drug
in plasma by Solid Phase Extraction (SPE) procedure. During the
processing conditions, we are using buffer pH 4.5 and washing with
solvents and eluting the drug with 2% ammonia in methanol.
My query is that is it possible that during the processing using SPE,
glucuronide is back converted into parent drug. Has some one
experienced similar thing. Comments on this are welcome from the
Dept. of Pharmaceutical Medicine
Tausif,Back to the Top
Glucuronides can definitely display pH-dependent cleavage.
Whether this happens or not, the rate and the optimum pH (acid or
alkali) all depend on the aglycone and the nature of the linkage.
You indicate that in your case cleavage yields the original NCE
so I assume that your NCE already contains a point of attachment
(e.g. a free hydroxyl) or can undergo direct N-glucuronidation.
When working with clinical samples we try and characterise any
lability in metabolites beforehand, either using equivalent
samples from other species or by generating the metabolites with
microsomal fractions (+UDPGA) or hepatocytes. We then stabilise
the pH during sample extraction.
I hope that this helps.
All the very best,
Bernard Murray, Ph.D.
Senior Research Investigator
Drug Metabolism, PCS, PPD, GPRD, Abbott Laboratories, Chicago, USA
Tausif:Back to the Top
For you inquiry I would say it depends on the stability of your
When you don't have reference standard for your glucuronide metabolite,
until you establish the stability;
1) it is better to analyze your samples with limited clean up steps (use
simple protein pptn)
2) analyze your samples as soon as you collect them as most of the
conjugates are unstable.
3) it is better to use a Mass spec technique since you can selectively
measure the analytes.
And 4) most of the glucuronides are highly polar therefore I suggest you
look at the specificity of your extraction procedure for glucuronide vs
Hope these suggestions would help you.
St. Louis, MO 63146
Dear Tausif,Back to the Top
The glucoronides do have pH dependent stability issues, so it is better
to work with minimum steps involved in sample clean-up. Also if you are
using SPE method followed by drying up of your solvent before
reconstitution and injection to the mass spec, the temperature and time
the sample is exposed to that temperature should also be looked at.
APL Research Center.
Dear Ahmad,Back to the Top
Cleavage of glucuronides needs some rough conditions (something like 1
M hydrochloric acid at 100 degrees centigrade for 1 hour).
Since you have just weakly acidic conditions in your SPE procedure for
a short time, nothing should happen. Basic conditions in elution are
P.S. Since the acidic cleavage needs such a 'hostile environment'
generally it's performed by enzymes.
Helmut Sch\0xB8tz Biokinet GmbH / Dept Biostatistics
Neubaugasse 36/11 Nattergasse 4
A-1070 Vienna/Austria A-1170 Vienna/Austria
tel/fax +43 1 9713935 tel +43 1 4856969 62
no cell phone ;-) fax +43 1 4856969 90
Hello Tausif,Back to the Top
You have already received great inputs from Naveen, Murray and Tata. In
to optimize your analytical procedure, you may also like to check the
stability of conjugate by processing the postdose sample immediately
collection and to process a portion of the same after the usual storage
and storage temperature you are following in the clinial setting. You
also divide this portion of postdose sample and adjust the pH (in
ranges)to find optimum pH of buffer for processing. Hope it helps
Vijay V. Upreti.
Graduate Student, Department of Pharmaceutical Sciences
HSF-II, University of Maryland. 20 Penn St., Baltimore. MD 21201
Mildly acidic conditions are the best to minimize degradation (pH 3-4).Back to the Top
away or minimize time exposed under basic conditions (pH 8 or greater)
the conjugates degrade/isomerize and these processes accelerate the
pH is (degradation is a factor of pH & time). The ring isomerization
may or may not be important to you and occurs in addition to basic
degradation to parent. You'll get multiple chromatographic peaks in
MRM channel (provided you have enough separation power to see the
isomers if they form).
There is a nice acyl-GA review (bio implications of these reactive
and discussion of experimental conditions) by
M. Bailey in 2003 in Chemico-Biological Interactions p. 1-21
M. Shipkova in Therapeutic Drug Monitoring, v. 25 (2003) p. 1-16 also
Daer Ahmad,Back to the Top
in order to reduce enzymatic cleavage of the glucuronides you can as
use the glucuronidase inhibitor saccharolactone at 10mM.
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