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Dear All,
I am currently working on development of analytical method for an
ester prodrug and its active metabolite by LC-MS/MS. The QCs for ester
prodrug are consistently negative because the drug is getting
hydrolyzed to active metabolite in plasma (I observed active
metabolite after addition of only the parent drug into blank plasma).
I was wondering if someone could suggest me the ways to prevent this
process during sample preparation. For example use of esterase
inhibitor that is compatible with MS? I found out some esterase
inhibitors like EDTA, sodium fluoride and paraoxon etc. but i) I am
not sure about the ion suppression or other interferences caused by
these agents in mass spectroscopy and ii) What concentration would be
adequate to use?
Or Is there any other way to deal with this problem?
I would greatly appreciate any help in this regard.
Thanks,
Lokesh
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Dear Lokesh-
I have had experience with many such examples of hydrolytic
instability in ester prodrugs, including mycophenolate mofetil,
valganciclovir, and others that have yet to be registered. We often
had some but still inadequate suppression of hydrolysis by addition of
esterase inhibitors, and many are very toxic and troublesome to work
with. Generally, the most successful approaches involved a
combination of keeping the sample cold, making the extraction fast,
and adequately controlling the pH of the sample and extracts.
Tom
Thomas L. Tarnowski, Ph.D.
Bioanalytical Development
Elan Pharmaceuticals, Inc.
800 Gateway Boulevard
South San Francisco, CA 94080
thomas.tarnowski.at.elan.com
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Hello:
Albeit it's not uncommon to use esterase inhibitors such as dichlorvos
(200 ug/mL plasma), paraxoan etc., to prevent the ex-vivo hydrolysis
of esterases in plasma, you may choose to use alternatively fluoride-
oxalate tubes (available at sizes ranging 0.5-5 mL from Teklab; www.teklab.co.uk
).
Fluoride-oxalate collection tubes are reported to be as effective as
the use of the esterase inhibitors in preventing ex-vivo hydrolysis of
esterases but I am not sure about its compatibility with MS/MS.
I too appreciate expert's comments on this topic.
Regds,
SYED MUSTAFA,
Advinus Therapeutics Pvt. Ltd.,
International Biotech Park,
Genesis Square, Bio Resource Centre,
2nd Floor, Phase II, Hinjewadi,
Pune- 411 057
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The following message was posted to: PharmPK
Dear Lokesh,
In one such case we succesfully stabilised the prodrug by acidifying
the sample. EDTA was used as anticoagulant and blood was kept below
4*C and processed quickly (which was suffucient to prevent hydrolysis
at that stage), then formic acid (1% final) was added to the plasma
before storage. This should not have much effect on analysis - but
matrix effect will still need to be carefully checked - and does not
introduce toxic inhibitors. It does however create some practical
challenges - fast blood processing and precise addition of acid to
plasma - which are easily dealt with in a research lab facility with
few animals, but may be more difficult in the clinic.
You do not give information on the species concerned. An important
point is that esterase activity is high in rodent plasma (was this
your case), but much lower in other species, however prodrugs being
designed to be cleaved they are normally unstable in human plasma as
well. But esterase activity is generally higher in whole blood than
plasma, so for a true evaluation of ester stability, and choice of
strategy to stabilise the prodrug, it should be verified in whole
blood and not only in plasma.
Best of luck
Patrice
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Dear Lokesh,
You can try with the combination of Sodium fluoride and PMSF
(Phenylmethylsulphonylfluoride) at different acidic PH with Acetic
acid to prevent the hydrolysis.
I hope its helpful for you
Dr Venkatesham Akena
Nektar
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You may find the following reference useful. Also you may want to
control the pH (slightly acidic) of plasma.
International journal of Pharmaceutics Vol. 301, (1-2); 2005 Page
262-266.
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The following message was posted to: PharmPK
Lokesh,
TO avoid use of toxic inhibitors we optimised a method involving an
ester prodrug with EDTA / Na F anticoagulant tubes and rapid chilled
collection and handling (we flash freeze plasma within one hour of
collection). In our case EDTA / Na F actually provided further stability
in comparison to Na F / K oxalate and these tubes were available from
Teklab.
Jonathan Scott
Sanofi-aventis
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The following message was posted to: PharmPK
The concentration adequate to completely arrest enzyme activity! Use
serum cholinesterase inhibition as a possible guide. If you are using
negative both cationic and anionic inhibitors are available so you can
tailor to your mode.
--
Ed O'Connor, Ph.D.
Laboratory Director
Matrix BioAnalytical Laboratories
25 Science Park at Yale
New Haven, CT 06511
Web: www.matrixbioanalytical.com
Email: eoconnor.-a-.matrixbioanalytical.com
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