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Dear all,
We are doing some in vitro metabolism studies on niacin. We are
facing a peculiar problem in the method development of niacin.
Actually the cofactor used in all metabolism studies (Phase-I
oxidation) NADPH is interferring with the compound Niacin in the
analytical method. We are using pooled human liver microsomes (PHLM)
as our enzyme source. Since NADPH (Nicotinamide adenine dinucleotide
phosphate) is must for oxidative metabolism, we cannot avoid NADPH in
the incubations. As NADPH is containing nictonamide, a metabolic
product of niacin (Nicotinic acid), we are facing difficulty in
separting NADPH interferrence from niacin. We have even tried several
sample clean up procedures like SPE but not able to eliminate NADPH
interferrence. Has anybody came across such a similar practical
problem with niacin, especially involving metabolism studies? I
appreciate any valuable suggestions from the group. Any literature
references will be highly appreciated.
Thanks in advance.
With regards
SK. BASHA
--
SK. Jafar Sadik Basha
Junior Scientist
Drug Metabolism and Pharmacokinetics,
Discovery Research,
Dr.Reddy's Research Foundation,
Bollaram Road, Miyapur, Hyderabad-49
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Dear Basha,
Follow the method as was used previously, but see that you leave the
dry extract on the benchtop at temp above 40 deg C for more than 4
hrs before the reconstitution step. this causes the enzyme to
degrade, eventually resulting in no or little interference.
This method was successful previously.
Regards,
Santosh Tata
Bioanalytical Division, BEC
Apotex Inc, Bangalore
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Dear Basha,
Have you paid attention to chromatographically separate this
interefering peak? Since your interfering peak has same chemical nature
as analyte, it will get extracted even in SPE. Either you device a clean
up procedure specific for your analyte or consider chromatographic
separation. Many time we tend to neglect chromatography part in method
development.
You can also consider derivatising your analyte by phenyl hydrazine
which can derivatise acids, aldehydes and ketones. Look at this
reference : R. Peters et al. / J. Chromatogr. A 1031 (2004) 35-50
Good luck
Vinayak
E Mail: vnadiger.-at-.combinatorx.com
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Basha,
you can go with direct precipitation followed by keeping the
supernatent in a water bath at 60 deg C and then inject the same into
the LC system.
Santosh Tata
Bioanalytical Division, BEC
Apotex Inc, Bangalore
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