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Dear all,
I am working on a compound that have a metabolite involving O-
glucuonidation at -N. This metabolite has retention time of two
minutes greater than the parent. This information is from human
urine sample. I have two questions:
1. What is the reason for an increase in retention time on C18 column
with formic acid/ACN/water mobile phases, other than more non-polar
metabolite and retaining longer on the column compared to parent?
Any other theory behind?
2. Based on retention time could it be same in the in-vivo, meaning
take longer to excrete from the body compare to parent.
Typically glucuronidated metabolites tend to be more polar and have
seen eluting before parent on C18 RP column.
On another occasion I have seen N-OH metabolite had increased in
retention time compared to parent as well.
Please share your thoughts with N-OH and N-O-Gluc metabolite
retention. There has to be scientific rationale behind this.
Thanking you, Sue
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Sue,
Thank you for your interesting message.
As for your apparent elution order condundrum, I have observed the exact
same behavior (i.e., glucuronide elutes after parent) in my experiments
and analyses with trifluoperazine (a well-studied UGT1A4 substrate)
using a mobile phase and columnh that are similar to the ones you
described. There are other examples in the literature of glucuronides
eluting after the parent compound. For example, see:
Remmel RP, Crews BC, Kozak KR, Kalgutkar AS, Marnett LJ, "Studies on the
metabolism of the novel, selective cyclooxygenase-2 inhibitor
indomethacin phenethylamide in rat, mouse, and human liver microsomes:
identification of active metabolites," Drug Metab Dispos. 2004
Jan;32(1):113-22.
I would use caution in making additional conclusions about the excretion
or PK of the GLU-metabolite, or concluding that it is a "nonpolar
metabolite," based simply on elution order on an HPLC system. I wonder
if specific analyte interactions based on column chemistry and
selectivity, mobile phase composition, pH, etc. could be responsible for
the "non-intuitive" elution order.
I look forward to other people's thoughts on Sue's questions.
Best Regards,
Jim Schmidt
Research Scientist, DMPK
Lexicon Genetics
The Woodlands, TX USA 77381
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The following message was posted to: PharmPK
Dear Sue,
I had similar experience where I have seen metabolites eluting before or
after the parent depending on the mobile phase composition. In my case,
the glucuronide metabolite eluted after the parent when acetonitrile was
used and before the parent when methanol was used with other factors
kept constant.
This could be due to involvement of multiple mechanisms in
chromatographic retention of solute. In RP column, they include polar
interactions with mobile phase and non-polar interactions with
stationary phase and the actual retention depends on the net effect. It
could be possible that the glucuronide metabolite may have more sites
interacting with stationary phase resulting in higher retention.
However, it is interesting to note similar observation with N-OH
metabolite which seems hard to explain in the similar lines.
With regard to the excretion of glucuronide metabolites in vivo-
regardless of their polarity excretion of these metabolites is mediated
by efflux transporters (mainly MRP2). As a result these metabolites are
readily excreted into bile and urine.
Hope this helps.
Kasiram.
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