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Dear Members
We validated one method for estimation of drug (NSAID molecule) in
human plama by LC-MS/MS using Internal Standard Compound (whis is not
structurally similar nor same pharmacology active group). The
validation range was so high in ug level (1-300 ug/ml ) so we worked
in low recovery (about 30%) but consistence (CV <8%) for drug as well
as Internal Standard.
We selected the internal standard based on chromatographic
suitability, extractability and availbility. Even we tried some
compounds of same catagory but the result was not positive.
I know the fact that the Internal standard should be analogue/same
theraputic classs or similar structure.
The method was validated as per USFDA guideline and during validation
we observed that all the parameters were in acceptance range.
One of our presitigious sponsor pointed out on selection of IS and
low recovery?
I need the opinions on the reliability of method as well as is there
need to change the internal standard. What is the effect on result if
IS belongs to different catagory when the purpose of adding IS to
minimise possible processing errorss. Even for LC-MS method we need
not to think about their separation.
Regards
Manish Jain
Group Leader
Bio-analytical
Wellquest Clinical Reserach
GK Marg, Lower Parel
Mumbai- Maharashtra (India)
Phone: 91 22 5660 8566
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The following message was posted to: PharmPK
Dear Manish,
Although it is ideal to use internal standard (IS) that is structurally
similar to analyte, it is not an obligation. Although it is possible to
have structurally similar analogues (to select as IS) in the discovery
setup, it is not the same when you have to develop methods for marketed
drugs, especially for compounds that are first in their class. Take for
eg. Troglitazone which was first compound in thiazolidinedione class
that was introduced into market under Rezulin brand name for the
treatment of diabetes. It is not possible to get a structurally similar
analogue for some one who wants to develop a bio-analytical method for
that compound.
However, it is necessary to have an IS that behaves like analyte in
terms of:
1. Extraction recovery (similar to analyte)
2. UV/FL properties (if using US/FL detection modes)
3. Chromatographic elution (ideal if IS elutes after the analyte)
4. MS fragmentation (different transition pair)
In fact, with MS methods use of structurally similar (same chemical
class) analogues may some times result in similar transition pairs hence
cross talk.
Considering this, low recovery may be an issue rather than IS type in
your case. It is ideal to have extraction methods with recovery of >80%
since chances of variation are <20% which is the acceptable limit for
bio-analytical methods. But, when you work with methods with 30%
recovery, you have 70% margin for error. In such cases, although you use
IS with similar recovery, it is possible to have large variations unless
you are highly consistent in your sample processing.
Good luck!
Kasiram
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Copyright 1995-2010 David W. A. Bourne (david@boomer.org)