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The following message was posted to: PharmPK
Hi All,
I am currently validating a bioanalytical method by LC/MS, and am
currently
searching for an appropriate internal standard. I would like some
advice as to
how close the internal standard retention time needs to match with that
of the
drug/compound of interest.
Your advice will be greatly appreciated,
Kashyap
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The following message was posted to: PharmPK
Dear Kaskyap,
In an ideal world there would be no difference between the tRs of the
internal standard and analyte. The most suitable internal standard
would be a deuterated analogue of the compound itself. This would
guarantee that it would behave exactly like the analyte in the LC-MS
(chromatographic, ionisation, fragmentation (if MS/MS)
characteristics). Matrix effects should also be the same (although
this still needs to be assessed if a regulatory assay is being
performed).
If this is just not possible, a close analogue would be the next bet.
Choose one that will elute just after your compound of interest e.g.
an ethyl subst. in place of a methyl subst. etc. etc.
cheers,
Iain
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The following message was posted to: PharmPK
Dear Kashyap:
Ideally, the peak of internal standard should be as close as
possible to the compound of interest, of course, always having two
well separated peaks. The behaviour of the standard must be similar
to the problem compound in order to compensate the errors of the system.
C.I.Colino
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Dear Kashyap,
In case of LC-MS/MS assays, retention time is not a very critical
parameter. Even if there is no chromatographic resolution between the
analyte peak and the IS, it should not cause much trouble. The IS
should behave in such a way that whenever there is a variation of
peak area response for the drug, IS response also should vary
accordingly and the Drug/IS ratio should remain within range so that
the standards and control samples meet the acceptance criteria. It is
usually observed that individual drug area response varies a lot over
a period of time and if your batch size is really big then you may
have problem in getting reproducible area response for both the drug
and IS from the beginning of the run to the end of the run and in
such cases a suitable IS is very much important. The separation of IS
from the analyte of interest sometimes become crucial in cases where
you need to quantify the active metabolites, in which case it is
preferable to have a different RT for the metabolite as well.
Hope it helps.
Regards
Neel Kamal Mohan
Pharmacokinetics Division
Glenmark Research Center
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Dear Kaskyap,
In an ideal situation, one should use an analogue of the compound
under test (CUT).
Here, the main consideration of selecting an analouge is its
retention time, which should elute just after CUT. This ensures
similar recoveries for both IS and CUT as the chemistry and
lipophilicity are matching.
However, it is not always posible to go for synthising analouges only
for this purpose. In an industrial setting, this is especially true,
when you are developing bioanalytical methods for evaluating the
forumations of existing drugs. In these situations, better opt for a
compound which is chemically similar to CUT and has a lipophilicity
profile nearly matching, esp in the solvent mixtures used for
extraction. This is to ensure that the IS behaves similar to that of
CUT in extraction process and show similar recoveries.
Regards
Varma Manthena
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