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The following message was posted to: PharmPK
Hi Group
I am working on the development of a LC/MS/MS method for a very
polar analyte .I did observed very low recovery due to high matrix
effect( about 85%).I am doing SPE as the analyte is basic in nature
and has few phenolic OHs which make is very unstable in basic
conditions so it need to be worked under acidic conditions. Also due
to its high polarity it starts eluting even with 5% organic during
the wash step.
On proceeding with this method as such it shows a lot of matrix
effect. Matrix effect is very prominent with tissues. So could
anybody suggest anything to reduce the matrix suppresion from tissue
phospholipids?
Any help in this regard will be greatly appreciated.
Thanks
Lakshmikant Bajpai
Scientist
CV Therapeutics
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The following message was posted to: PharmPK
Dear Lakshmikant,
My suggestion for LCMS analysis of your very polar compounds would be
trying mixed mode chromatography - reverse phase/ion exchange. I used
Primesep columns (SIELC Inc.).
If your compound has amino-groups you can try Primesep C, which forms
weak complexes with polar amines helping to retain those better.
Acidic conditions are optimal for this column. It also retains
inorganic ions (Na+, K+) responsible for most of ion suppression by
matrix helping to reduce it for less retained species.
There are other types of Primesep columns. For example Primesep AB
has both weak cation- and anion-exchange properties in addition to
reverse phase. These columns should work well with common LCMS
buffers like NH4OAc and others.
Good luck!
Yelena Vasina
excimer.-a-.bellsouth.net
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Dear Lakshmikant,
I wondered how feasile something other than reverse phase might be
for your compound, such as ion exchange or normal phase. You also
might contact Dionex to see if they have a possible ion exchange
method that might address your problem. Perhaps you have already
considered these things.
-Tom
Thomas L. Tarnowski, Ph. D.
Associate Director, Bioanalytical Development
Elan Pharmaceuticals
800 Gateway Blvd.
South San Francisco, CA 94080
650 794-4235
thomas.tarnowski.-a-.elan.com
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Hi Lakshmikant,
In SPE method, which type of sorbent did you use? You can try
cationic or anionic exchange cartridges to have high selectivity for
basic or acidic compounds respectively.
regards,
Jignesh Kotecha
Torrent Research Centre.
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The following message was posted to: PharmPK
Hi all
First of all I would like to thank you all for your kind and genuine
suggestions.
After going through all the replies it seems everybody is agreeing to
the use of ion exchange methodology.
Now the problem is that my compound is basic in nature so will
defenitely need the cation exchange cartridge/column and for elution
from that will need the basic elution solvent.In the analyte there
are few phenolic groups as I said already they are highly unstable
and lead to maximum decgradation as soon as I add base to the
medium.I did observed this during the HPLC opt. by adding ammonium
hydroxide to the MP and confirmed later by adding base wash during
the SPE. Also I need very low sensitivity so cant afford to loose the
compound during the processing itself.
Thanks again for everyone's inputs.
Bajpai
(Lakshmikant Bajpai)
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The following message was posted to: PharmPK
Dear Bajpai:
Not knowing the structure of your compound and what you had tried; do
you have to use SPE for sample preparation? In your case, SPE may not
be the best sample preparation approach.
Can you use protein crush at a pH environment that your compound is
stable for better recovery, concentrate and reconstitute for
sensitivity and optimum separation, then use diverting valve to divert
endogenous and negative mode MRM for detection (if necessary) to
minimize ion interference from endogenous?
Good luck.
Ta Kung
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The following message was posted to: PharmPK
How is molecular weight?
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The following message was posted to: PharmPK
Hi,
You may try weak cation exchage cartridge for the
clean up step, then you can use acidic condition
rather than basic condition to elute the analyte, if I
am not wrong.
For HPLC part, you can definitely try Waters Atlantis
HILIC silica column, using 85% ACN, 15% 5mM Ammonium
aceate, pH 4 or 5, or using 90% ACN with 10mM Amoonium
acetate, pH 4 or 5,something like that. This column
can very well retain the polar basic compounds and it
gives no tailing.
Good luck.
Xiaodong Shen
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The following message was posted to: PharmPK
Dear Lakshmikant Bajpai,
You could try the polar-modified LC column for your LC separation, which
it can be commercial available from several LC column suppliers. And
also, cyano-column with high organic phase contents is deserved to try.
Hope it helpful!
Fred Li
RRDCC
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