PharmPK Discussion - SPE - total or only free drug detected?

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On 14 Jan 2004 at 13:11:31, "Noelle Williams" (Noelle.Williams.-a-.UTSouthwestern.edu) sent the message

Hi,
I am interested in following a novel drug in mouse blood following
injection. I would like to use LC-MS for detection and am looking into
SPE (probably C18) as a means of cleaning up my plasma samples. Will I
be detecting only free drug or total drug (plasma protein bound +
free)? Will this vary with the conditions I employ for washing and
eluting from the column?
Thanks,
Noelle Williams

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On 14 Jan 2004 at 19:23:03, Jimmy Coulton (jimmy_coulton.-at-.yahoo.com) sent the message

Hi Noell,
I think it depends upon the lipophilicity of your compound of interest.
Depending on the lipophilicity you may try out C8 or C18 columns. You
will be getting the free drug in your case if you are not precipitating
the protein by means of an organic solvent.And you may get varying
results,particularly in recovery,as you change the extraction
conditions

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On 15 Jan 2004 at 09:45:53, "Parton, Ted (SLH)" (Ted.Parton.aaa.slh.celltechgroup.com) sent the message

Dear Noelle,
Using SPE or protein precipitation you will recover a proportion (you
hope
for >80%) of the total.  The reason for protein precipitation is that
denaturing the protein destroys the binding sites and at least using
solvent
precipitation, the solvent competes with the analyte for non-specific
binding.
During SPE, bound drug dissociates from the protein. Dissociation rate
constants do vary from compound to compound and are one of the great
underexplored areas of pharmacokinetics, mainly because there is not an
easy
or cheap way of measuring off-rates.  Of the few that are known and
published, some have off-rate half-lives around 1 second or shorter and
a
few are around 10 seconds (likely to be very highly bound compounds at
equilibrium).
Loading a plasma sample onto an SPE cartridge should take 1 minute or
more,
so there is plenty of time for bound drug to come off and partition
into the
stationary phase.  Usually there is a great excess of stationary phase
over
plasma protein (this may limit the sample size to sorbent ratio), so
extraction and later recovery should be good when you get the conditions
right.
Good luck with your method.
Ted

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