Back to the Top
Hi,
I am currently working on in vitro permeability study, using Millipore
multiscreen 0.4 micron PCTE, 5% hexadecane in hexane as the artificial
membrane, on a series of hydrophilic drugs (clogP ranging from a -1.5
to -3.5). My analytical method involves use of LC-MS/MS in positive
ESI mode. My concern is the ion suppression seen in my assay due to
presence of phosphate buffer (from PBS pH 7.4) which I use for the
permeability assay. This leads to my question of using just water
instead of PBS for the permeability studies. Will this still be
relevant physiologically to estimate the passive intestinal
permeability? Any work around to reduce the interference or any
relevant references will be highly appreciated.
Thanks,
Pavan.
Back to the Top
The following message was posted to: PharmPK
So how will you insure Physiological pH?
Back to the Top
The following message was posted to: PharmPK
Dear Pavan,
Your PAMPA assay might be the wrong in vitro method to study
permeability of low logD drugs if your goal is to determine relevance
to the in vivo situation. Most low logD drugs have such low passive
transcellular permeability that the paracellular path (which is also
slow) can become very significant. Naturally, the PAMPA assay does not
measure paracellular permeability so your experiment does not mimic
the in vivo condition.
You can calculate the paracellular contribution and add that
to the PAMPA transcellular permeability to get an estimate of human SI
permeability as described in:
Sugano K, Int. J. Pharmaceut. 241:241 (2002)
Sugano K, Int. J. Pharmaceut. 257:245 (2003)
Also, a recent paper describes a similar approach to your's for
measuring the passive transcellular permeability of hydrophobic ion-
pairs. If that is your goal then you might be on the right track.
Quasi-equilibrium analysis of the ion-pair mediated membrane transport
of low-permeability drugs.
Miller JM, Dahan A, Gupta D, Varghese S, Amidon GL.
J Control Release. 2009 Mar 2. [Epub ahead of print]
Sorry, but I don't have the expertise to comment on your assay
conditions.
Mike
Back to the Top
Hi, Pavan:
Can you remove the phosphate with the LC?
David PPL, Inc.
Back to the Top
Pawan,
Have you incorporated a diverter configuration between column and mass
spec? That will help to send initial eluate containing salts to waste,
so that your source will not deteriorate due to salt deposition. You
may wish to push the analyte RT a bit far away as permitted by your
method. (Hope you are using a gradient method.) You may consider using
bicarbonate buffer in same pH as phosphate buffer. This may need some
validation.
Others may wish to comment whether we can replace phosphate buffer by
bicarbonate buffer.
Regards,
Vinayak
Vinayak Nadiger
Manager, Bioanalytical Chemistry
11 Biopolis Way, Helios #08-05
Singapore 138667
E Mail: vnadiger.-at-.combinatorx.com
Website:www.combinatorx.com
Back to the Top
Dear Pavan,
Play around column chemistry to retain compound with high organic
content in mobile phase. This would help you to achieve better
sensitivity and divert valve would help to take away initial LC eluent
to waste. You can use volatile buffers (e.g. ammonium acetate)
adjusted to physiological pH, but true representation of permeability
model is by using inorganic buffers.
regards,
Jignesh
Back to the Top
The following message was posted to: PharmPK
Hi Pavan,
I feel you can not use simple water as it is not replicative of
physiological pH and also composition. I don't have reference for
using water for permeability studies but instead of that you can
easily take care of matrix effect. Many people around the globe works
with analysis of phosphate buffer (PBS pH 7.4) samples using LC-MS
with few techniques.
1. As pointed out by Vinayak, try using post column switching
technique wherein initial waste (containing high amount of salt) is
drained off and analyte is not subjected to suppression.
2. Increase the amount of aqueous content in your mobile phase to push
the retention time of analyte bit far away from solvent
front(providing good chromatographic peak parameters)
4. You may try to work at less flow rates.
5. You may also look into tryout some liquid-liquid extraction (LLE)
technique as it removes the salt and provides cleaner extract. LLE
tedious but as the number of samples in permeability studies is less,
this can be worked out.
6. Try to reduce injection volume or dilute the samples if your assay
sensitivity is good as it may reduce matrix effect.
7. Use SILIS (stable isotope labeled internal standard, its difficult
to produce but if you have) which elutes at the same retention time of
analyte accounting for matrix effect.
8. Finally (not preferred but can be looked in) use alternative
ionization source like APCI/EI which is less sensitive to matrix effect.
Thanks & Regards
Sivacharan Kollipara
Research Associate
Metabolism & Pharmacokinetics
Ranbaxy Research Laboratories
Gurgaon-122015, India
Back to the Top
Pawan
How are you processing your samples before injecting onto the massspec?
Manish Issar, Ph.D
Applied Biopharmaceutics
Back to the Top
Pavan,
Most probably not! Permeability of an ionizable compound is not a
function of logP but logD. You are mentioning hydrophilic compounds
so I am assuming that they are ionizable. If you dissolve in water
and the resulting pH is different from physiological pH, most probably
you will find differences in permeability. Please consider ionization
into experimental set up.
Ayyappa Chaturvedula
Want to post a follow-up message on this topic?
If this link does not work with your browser send a follow-up message to PharmPK@boomer.org with "Permeability and phosphate buffer ion suppression" as the subject | Support PharmPK by using the |
Copyright 1995-2011 David W. A. Bourne (david@boomer.org)