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Dear all,
I am trying to develope a method for the extraction of a drug from
plasma for subsequent HPLC analysis. This drug have a high binding to
plasma proteins (> 95%).
Two questions:
1. If I treat the plasma to precipitate proteins, do the molecules of
drug lose in the precipitate? Or if I treat the plasma with solid
phase extraction, does the complex proteins-drug remain in the SPE
column or it is eluted ?
2. Can the use of K-EDTA (or K2-EDTA, K3-EDTA, K4-EDTA, heparin or
citrate) as anticoagulant modify the binding proteins-drug ?
Thanks.
Many kinds.
Raffaella Bombelli
Section of Experimental and Clinical Pharmacology
Departement of Clinical Medicine
University of Insubria
Varese, Italy
raffaella.bombelli.-a-.uninsubria.it
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Depends. All of the above none of the above. That is what method
development is all about. Share the structure we may be able to
throw some
light on it. Look at how analogous compounds are handled using
vendors SPE
application notes, etc.
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Raffaella,
Q1. Usually protein precipitation will release the drug from the
protein, but this is not always the case. It will depend on the drug
involved. I would suggest the following experiment:
Prepare standards in plasma/serum. Also prepare standards in isotonic
buffer (ph 7.4) (I think it is 0.01 M phosphate with 0.3 % sodium
chloride - but I suggest you confirm these details). Prepare both sets
of standards as per your proposed extraction method. Inject both sets of
standards. The ratio of the slopes of plasma/buffer (assuming intercept
is zero, and using external standardisation (i.e. no internal
standard)), will let you know how much is going down with the proteins.
Even if you have a high percentage going down with the proteins,
provided your assay is sensitive enough and has good accuracy and
reproducibility there is no reason why you shouldn't still use it (I
have had assays where up to 75 % of the drug went down with the
proteins, but by using an analog as internal standard we still had
excellent accuracy and reproducibility (as deternmined against an
international external control program).
Q2. Again this may depend on the drug. I suggest you test for this if
you are planning on measuring protein binding. Also, if you are
measuring protein binding be aware that plasticisers can influence
protein binding as well as temperature also.
Regards,
Ross Norris,
PhD, MAppSc, BAppSc.
Research Consultant, Australian Centre for Paediatric Pharmacokinetics &
Therapeutic Advisory Service, Mater Pharmacy Services, Raymond Terrace,
South Brisbane, Q 4101, Australia.
Associate Professor, School of Pharmacy, Griffith University, Gold Coast
Qld Australia.
Senior Lecturer, School of Pharmacy, University of Queensland, St Lucia,
Qld Australia.
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Dear Raffaella
If drug is extracted using SPE with polar solvent it will possibly
extract the drug but stability of the drug should be checked in the
extraction solvenr.
Dr Zafar
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Dear Rafaella,
In answer to your question 1, you just need to test by spiking the
compound to plasma (less than 2% spike solvent) to prepare QC samples,
and then process them and determine the recovery of the extraction -
compare with a reference which you spike after extraction. In my
experience, most 'small molecule' drugs, even when high protein bound,
are not lost when you add 2 or more parts of acetonitrile to the plasma
to precipitate the proteins - but you need to test for each compound to
know.
Also, when you choose solid phase extraction, there is a large change
that the protein-drug complex is disrupted, but again you need to run
the recovery experiment to be sure. In case of solid phase extraction,
it helps if the plasma is diluted with buffer or acid prior to
application to the SPE column. This helps break the proteins-drug
complex.
In answer to your question 2, as far as I know EDTA or heparin or
citrate do not significantly modify the plasma protein binding.
I hope this helps.
Best Regards, Ronald
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Dear All:
There was a classical paper published in Journal of Chromatography on
various agents for protein precipitation, Chemists routinely use 1:2
plasma: Methanol or Acetonitrile as precipitating agent. Once protein
are precipitated drug that's bound for these proteins will dissociate
and come into the supernatant (organic aqueous component), majority of
the analysts to improve specificity of extraction and improved
recovery use protein precipitation prior to the solid phase extraction.
If your comment is about determine protein binding choice is K2EDTA as
anticoagulant, through unpublished works many folks prefer EDTA over
heparin except that your drug has any inorganic elements in your
analyte (like cisplatin etc) where heparin is a choice.
Hope this helps.
Prasad
Prasad NV Tata, MS., Ph.D., FCP
Manager-Clinical Pharmacology
Mallinckrodt, Inc.
675 McDonnell Blvd.
Saint Louis, MO 63134
e-mail: prasad.tata.-at-.covidien.com
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Dear Prasad,
Is this the paper you are referring to "Optimization of protein
precipitation based upon effectiveness of protein removal and
ionization effect in liquid chromatography-tandem mass spectrometry".
But, I still doubt of the universatility of the EDTA for the ranges of
the analytes as sometime there will be the specific complexation of
the analytes with EDTA. So, I think the efficiency is case to case
basis.
Thanks for the information.
Amrit
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A 1:2 mixture will often times not be sufficient to crash out all
proteins
and may not be sufficiently chaotropic to cause release of highly
protein
bound drugs. Increasing the volume of organic and adding additional
components as well as chilling (ice cold) or heating the matrix (56oC
for 30
minutes)prior to organic may help.
In any of these manipulations, the test article (or its metabolite(s)
must
be added to the matrix in order to define stability limits and actual
recoveries during method development.
As a caution The addition of salts or acids to assist in the
precipitation
and separation of analytes from protein will have effects on both the
chromatography and mass spectroscopy of the analyte.
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Yes, even I believe chilling with the stepwise addition of the
precipitant will increase the chromatography a lot!!
And it has worked for no. of compounds with the diverse chemical
nature and differential potein binding will release the drug readily
"In vitro and in vivo investigation of metabolic fate of rifampicin
using an optimized sample preparation approach and modern tools of
liquid chromatography-mass spectrometry". This one of the good examples.
Thanks a lot for the information.
Best Regards,
Amrit
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Dear Raffaella
You can give a try using LLE method. Check the extraction recovery of
your drug using different organic solvents. Select the best solvent
for LLE.
Hope this helps.
Regards
Khalid
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Be sure to include comparison of recoveries in PBS and plasma or whole
blood. Make sure you know the problem is with binding to plasma
proteins
and not to red cell/platelet binding, uptake or degradation in plasma.
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Dear Raffaella,
Q1. Proteins are considered as biological amphoteric molecules and
there is reversible interaction exists between drug and protein. It is
generally inferred that low protein binding drugs give good recovery
in protein precipitation (Ex. Ribavirin <5%, Levetiracetam <10%).
Nevertheless it all depends on the molecule binding to the type of
protein (Albumin, Alpha-1-acid glyco protein, Lipo proteins etc..) and
type of precipitant. If the precipitant able to disrupt protein drug
binding by any means (changing the salvation potential, removal of
hydration layer around protein, change of dielectric constant, change
of pH to bring the mixture to the isoelectric point), recovery can be
obtained for highly protein bound drugs also (Ex. Ziprasidon 99%,
Meloxicam 99.4% which gives >90% recovery in protein precipitation
methods).
In solid phase extraction sample is pretreated to disrupt the binding
to some extent by adding water or buffer. Preferably OPA is added to
disrupt the binding. However one can easily get some portion of
protein in eluent (may not interfere in your analysis because of
negligible quantity).
Q2. As per my knowledge type of anticoagulant does not significantly
modify the plasma protein binding.
Regards
KJ
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