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Hi,
I am trying to develop an LC/MS/MS method for a boronated compound
and unable to set up quantitative method for determination in
positive ion or negative ion mode.
Another question is how to identify metabolites? I ran FS and the m/z
values of metabolites absolutely does not make sense at all
considering possible biotransformations.
I would highly appreciate your suggestions.
Thanks very much
Sue
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Sue,
If its a boronic acid many of these de-boronate chemically (ROS
driven?) or in the instrument so this can lead to the impression that
the masses are peculiar.
Depending on the structure they can also dehydrate.
Look at the recent metabolic work on Velcade for an illustration of
some of the possible complexities.
Howard Haspel
Laboratory Sciences
Charles River Laboratories Preclinical Sciences
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Sue,
Free boronic acids are known to undergo dehydration / trimerization
to boroxines at elevated temperatures. The high MS source
temperature is a leading cause for instability of molecular ion /
fragments in LC-MS. A common practice to prevent this thermal
reaction product is to perform a derivatization to form a cyclic
boronate ester, which is stable under elevated temperatures. A
derivatization with an aminopropanediol provides the functionality
for APcI and ESI positive ionization analysis.
Metabolite identification of these compounds can be quite
challenging. I found it to be useful in metabolite identificfation
was usefulness of isotopic pattern of boron. 10B has natural
abundance (atom %) of 20% compared to 11B has 80% natural abundance.
The major metabolic pathway in my case was de-boranation + oxidation
and de-boronation + dioxidation.
Quantitation of PK samples can be done by derivatizing or can also be
done by using 14C labeled parent and use B-RAM for detection.
Listed below please find a reference for detivatization:
1. The analysis of boronic acids by ESI and APCI, positive and
negative ionization.
John A Castoro, DuPont Pharmaceuticals Company, Deepwater, NJ, 08023.
Best regards,
Anila
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Hi Anila,
Thank you for an excellent literature. It will solve my problems.
While I am waiting for reagents to arrive I wanted to ask you if you
could please let me know the ratio of parent compound to derivatizing
agent you used. The literature does not go in that much detail.
Many thanks
Sue
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Hi Sue,
I did a small study to optimize molar equivalent ratio of parent to
derivatizing agent and time required to complete the reaction. You
can try 1:1, 1:1.5, 1:2...and time perhaps 2, 5, 10 15 min. Compare
your peak areas from LC-MS/MS for each time points and ratio. When
you see the consistent peak areas at certain time and molar ratio of
parent to derivatizing agent you can say that the ratio and time are
optimized. Your compound reacted with serinol (91Da) will give mono-
hydration as intermediate 1 and bisdehydration as a final product.
Most likely you will not see the intermediate 1 because it goes to
final product quickly. Your compound should produce an intense (M-H)-
negative ion signal by API.
Cheers
Anila
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Copyright 1995-2010 David W. A. Bourne (david@boomer.org)