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Dear member,
We are developing a Bioanalytical method for Valganciclovir by using
human plasma in UPLC-MS/MS (quatropremier XE) method, but we are not
able to develop a method because of its low recovery in all the
extraction methods.
(a) Internal standard > Aciclovir.
(a) mobile phase > 0.1% formic acid: methanol:: 10:90.
(b) following were the experiments we have undergone for the
extraction methods:
(1) Precipitation method: Precipitate with methanol and
Acetonitrile respectively.
(2) LLE : Using solvents; ethyl acetate, n-hexane, methyl
tertiary butyl ethyl ether (TBME), Diethyl-ether
(3) SPE : catridge: oasis, conditioning: 100% methanol>
water, washing method: 0.5ml water, eluting , 100% methanol.
Results: recovery were below10% in LLE and 20-30% recovery in
precipitation and SPE methods,and inconsistency.
Can any one suggest the alternate methodology for the same which may
give a good recovery.
with lots of hope
regards,
Muthu swamy.C,
BIOANALYTICAL DEPT,
GVK Biosciences Pvt. Ltd., (CPU)
7th Floor, Swarnajayanthi Commercial Complex,
Ameerpet,
Hyderabad - 500 038
Email: muthuswamy.chinnaiah.-at-.gvkbio.com
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The following message was posted to: PharmPK
Muthu-
If it is albumin bound, then do ultrafiltration at ~30KD. Then add
guanidine chloride to release the drug from albumin and filter again (it
will go through the filter). Finally consider adding hydroxypropyl beta
cyclodextrin to bind it in the infranatant prior to doing the sep pack
extraction. See Rogatsky et al 2007 for a similar procedure.
Good luck,
Dan
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The following message was posted to: PharmPK
Dear Muthu swamy.C,
Could the stability of the prodrug be the problem? You may want to
try stabilizing the compound in plasma using sodium fluoride (various
concentrations) to inhibit enzyme activity. Val-aciclovir may be a
better internal standard than just aciclovir. Also, look for
ganciclovir recovery to see if you are clipping your compound. It is
tricky to come up with an assay procedure that will stop the
degradation of labile compounds after blood collection.
Tom
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Dear Sir:
Regarding the method for valganciclovir, here is the reference to the
method we used for valganciclovir determination during its
development, in case you don't have it.
R. Chan, J. LaFargue, R. Reeve, Y. Tam, and T. Tarnowski, J. Pharm.
Biomed. Anal. 1999, 21, 647-656. "An HPLC Method for the
Determination of Diastereomeric Prodrug RS-79070-004 in Human Plasma."
It is for HPLC, but not LC/MS detection, so buffers may need to be
changed to be compatible with the MS. I assume you are trying to
determine the diastereomeric prodrug valganciclovir, and not
ganciclovir itself.
Note that valganciclovir is a mixture (approximately 50:50) of two
different stereoisomers that interconvert (slowly or quickly debending
on conditions) in solution. Note also that valganciclovir is a valine
ester of ganciclovir and is somewhat prone to cleavage during sample
collection and processing. Keeping things cold and working quickly
can help assure an adequate recovery.
There may be more recent methods; I haven't worked on this compound
for a number of years.
Tom
Thomas L. Tarnowski, Ph. D.
Bioanalytical Development
Elan Pharmaceuticals
800 Gateway Blvd.
South San Francisco, CA 94080
thomas.tarnowski.-a-.elan.com
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The following message was posted to: PharmPK
Dear Muthuswamy,
The pka of valganciclovir is 7.6 and the
octanol/water partition coefficient at Ph 7.0 is 0.0095 which is very
low to perform LLE. As the Pka of the molecule is 7.6 you can try LLE by
adding PH 10.0 Base prior to adding the organic solvents such that the
molecule will be in unionized form.
As the protein binding for this molecule is 1-2% you cannot try it out
with Protein Precipitation.
As far SPE is concerned you can use MCX cartridges and first ionize your
molecule by adding some acid at around pH 4.5 and put it into cartridge.
Washing step you can do it with methanol, water, acetonitrile etc..,
Elution step you have to add some basic solution for the molecule to be
unionized and get eluted from the cartridge.
Hope so I answered your query. Any doubts do revert back.
With Regards,
Veeravalli Vijaya Bhaskar,
Research Associate,
Aurigene Discovery Technologies Limited,
Bioanalytical Division,
Bangalore,
Email: vijayb.aaa.aurigene.com
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