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Dear all,
I am working on statins regarding the bioanalytical method development.
As the internal standard is necessary for bioanalytical. I tried the
all statins which is available. But the statins which i used that will
elute befor the analyte or there is so much difference in the retention
time of the analyte and IS.
IF anyone suggest me which internal standard we can use that will be
very useful.
THANKING YOU
PRASHANT
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Hello Prashant,
As far as the bioanlaytical method development goes, you need to
select an IS which preferably elutes after the main analyte. This is
because, metabolites being more polar than the main analyte, elutes in
HPLC before the main analyte. There is another reason for having the RT
of IS after parent drug. In Reverse Phase chromatography, most of the
compounds of the biological origin/matrices elute in the begining of
the chormatographic run time. In many instances, based on the study
design, we also need to quantify the active metabolites. And in such
instances, if there is not enough chromatographic resolution between
the IS and metabolite then proper quantification of the metabolite gets
complicated. So, it is always preferable to select an IS which elutes
after the parent drug within a set of LC conditions. When we select IS
for bioanalytical applications, we prefer the deuterated IS and if not
possible to have the deuterated one, then a compound of similar
structure is also preferred. You can, if available, also go for a drug
intermediate if it is of good purity. However, it is not always
mandatory for the IS to be eluted prior to the parent drug. If your
method does not require quantification of the metabolite(s) or if there
is no acive metabolite, then you can also go for an IS which elutes
before parent drug provided your chromatography is not affected.
And to answer your second part, any resolution of 3-5 minutes between
the main analyte and the IS is considered to be optimum, what you
basically need to see that there is proper baseline separation between
the adjacent peaks. You can also improve resolution by changing the
column dimensions.
Hope this helps.
Regards
Neel Kamal Mohan
Department of Pharmacokinetics,
Glenmark Research Centre,
Mumbai, India.
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The following message was posted to: PharmPK
Hi Prashanth,
As neel told it necessary to have an IS for
bioanalytical quantification.
And as far as statins are considered you cannot
quantify on HPLC because of very low concentrationsin
ng/ml) for biostudies, and if you go to LC-MS-MS
method development you will good results by uring
Deutoriated compuonds, intermediate product or
analogues of that compound.
i think this will help you
Srinivas.K
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Regarding internal standards: an internal standard can only correct for
errors that affect both the internal standard peak and the analyte peak
in the same way. When this type of error dominates the precision
problem, internal standardization helps. When an error affects the
internal standard and the analyte peaks independently (integration
problems due to baseline noise, for example), then internal
standardization will hurt.
Regards,
Stan Alekman
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Hi Prashant
I agree partly to what Neelkamal said. It all boils down to the kind
of analysis you really want to do. To be more specific, if the method
of detection is by MS/MS then i would prefer the IS to elute at the
same retention time of as that of the parent (that is where you want
o use a C13 labeled IS). The main reason for the retention times to
be identical is because MS detectors have been shown to display a
higher degree of fluctuation in their baseline sensitivity. You could
also use a Deuterium labelled IS. I gave this example as most of the
analysis performed these days is by LC-MS/MS. Hope that make sense.
Manish Issar
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Hi prashant,
I agree with what manish said. I would only like to add one more
thing. Try using IS of similar Pka if you don't get Deuterium
labelled IS. It will cartainly help you for methods in LC/MS/MS
analysis.
Arpana Prasad
B. A. Research
India
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