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Dear all,
The metabolism of my compound seems to yield to an N-Oxyde metabolite (RR1R2-N to RR1R2N-O).
What is the retention time expected for this type of metabolites (before or after the parent, near or far from the parent) ?
Is the N-O binding is stable in LC-MS/MS ?
Acidic mobile phase (HCOOH) and C18 column, ESI mode.
Many thanks.
Fabrice Guillet
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For many metabolites, metabolism will improve their polarity so that they are easy to get out and then result in shorter retention time on RP-HPLC.
Feng
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From my personal experience, I would be careful to make conclusions of
N-oxides based purely on retention time. The inclination is to rely on
it being a more nonpolar metabolite, but that is not always the case.
You should try a TiCl3 incubation; there is an excellent recent paper on
selective reduction of N-oxides using this reagent in biological samples
and I use the procedure extensively in my lab.
Jim Schmidt
Sr Scientist, DMPK
Lexicon Pharmaceuticals, Inc
The Woodlands, TX USA
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unhindered by too much experience, one could imagine that the oxidation of an amine moiety drastically changes ionization (reducing the likelihood of protonation), and the ability to engage in hydrogen binding. I would not be surprised if it results in a slightly longer retention on reversed phase columns.
Kind regards, Jan
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I have come across with a metabolite (N-oxide) which had higher retention time than the parent compound on RP-HPLC. I found it interesting at the time.
Try changing the pH of your mobile phase and see how their retention (parent and metabolite) time is changing. At different pH it will ionize differently and the retention time will be affected accordingly.
It is still a metabolite and will excrete accordingly regardless of later retention time compared to parent on RP-HPLC. Increased retention time of a metabolite is a chromatography theory and not physiology based.
I hope this helps.
Regards,
Anila
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Dear Fabrice,
In our experience aliphatic N-oxides run shortly after the parent. A
good example is AQ4, AQ4M (mono-N-oxide), and AQ4N (di-N-oxide) with
retention times in that order. Yes, they tend to be fairly stable with
ESI.
For example, see:
A preclinical pharmacokinetic study of the bioreductive drug AQ4N.
Loadman PM. Swaine DJ. Bibby MC. Welham KJ. Patterson LH.
Drug Metabolism & Disposition. 29(4 Pt 1):422-6, 2001 Apr.
Hypoxia-activated prodrugs: substituent effects on the properties of
nitro seco-1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one (nitroCBI)
prodrugs of DNA minor groove alkylating agents.
Tercel M. Atwell GJ. Yang S. Stevenson RJ. Botting KJ. Boyd M. Smith E.
Anderson RF. Denny WA. Wilson WR. Pruijn FB.
Journal of Medicinal Chemistry. 52(22):7258-72, 2009 Nov 26.
With aromatic N-oxides it is quite a different story. For example,
tirapazamine is a triazine-di-N-oxide and it runs before the
nor-N-oxide, which runs before the 1-N-oxide (mono-N-oxide, if you
like).
For example, see:
Pharmacokinetics and bioreductive metabolism of the novel benzotriazine
di-N-oxide hypoxic cell cytotoxin tirapazamine (WIN 59075; SR 4233; NSC
130181) in mice.
Walton MI. Workman P.
Journal of Pharmacology & Experimental Therapeutics. 265(2):938-47, 1993
May.
Obviously, and as always, chromatography conditions play an important
role.
The bottom line is that the relative retention time might give you a
clue but no more than that and you still have to confirm the structure
ID.
HTH
Frederik Pruijn
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