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Dear all,
Please clarify the following query.
Experimental details:
We have done analytical method development, recovery and linearity of
NCE in biological fluid (rat plasma) using LC/MS/MS (MRM). Here we used
1:1 ratio of plasma:5%TCA for recovery studies.
Followed by, we have carried out PK of a compound in rats at 5mg/kg
(both IV and PO).
Observations:
We observed two peaks in PK samples not in linearity samples (spiked
plasma), both in PO and IV in all time points.
We suspect no geometric isomers or keto enol tautomerism.
Question:
How one can quantify the same.
Thanks in advance,
Samiulla
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Dear Samiulla,
If your drug has a functional group like -OH or -COOH, or if its phase I
metabolism yields to this type of function (that is very common),it's
possible that your drug or its metabolite is O-conjugated with a sugar
(glucuronic acid).
You could obtain a glucuronide with a (M+H)+ at M+176 (glucuronic acid=
194
+ M -H2O (18)= M+176).
In the ionisation source, this ester could gives your native product
(that
is common).
Try a MRM with Parent M+176 and the same daugther as your NCE. You could
probably see a peak at the two transitions (it's the same in negative
ion
mode)
Hope this helps.
regards
Fabrice Guillet
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The following message was posted to: PharmPK
In addition to a phase 2 conjugate fragmenting in the source to give a
second peak in the chromatogram's parent MRM channel, it is possible
that it
is a chromatographic artifact (only one compound in sample giving two
chromatographic peaks). Two possibilities are:
1) I can't tell from your note if you're treating your study samples
different than your standards and QCs. If so and if your injection
solvent
pH or organic percentage is drastically different than your initial
starting
HPLC conditions, then peak splitting can occur. Don't assume that
drying
sample completely and reconstituting in the mobile phase means that the
pH
of that reconstituted sample is the same as the native mobile phase.
Residual acid/base can still be around. I've found it safest to
measure the
pH and compare. Buffering of the mobile phase helps reduce the problem.
2) You might try changing pH of mobile phases. When your mobile phase
pH is
near the pKa of ionizable group, peak splitting can occur sometimes.
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The following message was posted to: PharmPK
Peak Splitting is actually quite common in my experience. The
principal reasons are
1. Incompatible mobile phase and injection solvent (poor miscibility)
2. Ionization effects (as described by Dr. Emary)
3. Insufficient ion-pairing agent (if used)
3. Solid phase saturation.
The last one was quite interesting when I was working with pterin
derivatives (which have the solubility of a rock from the get go!).
But having got them into solution, very well defined pre-peaks to the
main absorption peak would appear with increasing mass. This occurred
at mass levels that I never would have thought would overload an
analytical sized column. The artifactualy peak looked very "real" and
well-defined. It certainly threw me off for a
while.
Steve Dueker
www.vitaleascience.com
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Copyright 1995-2010 David W. A. Bourne (david@boomer.org)