Dear Laurian,Back to the Top
I feel the information given by you is very important if any body wants
to analyze the drug in plasma. But i would like to know about the
volumes of plasma and the solvents being used for extraction of drug
from plasma, in case of drugs which are highly protein bound, e.g.
NSAIDS. Is the volume of different solvents necessarily sufficient to
extract the whole amount of drug from plasma? What if one uses a 1: 10
or 1: 50 ( plasma: organic solvent) to extract the drug and then
evaporating and reconstituting the residue before injecting into HPLC?
As my knowledge, the nature of organic solvent used for LLE does notBack to the Top
depend on degree of drug bounded to plasma proteins.You must to choose
an organic solvent depending on selectivity and recovery of extraction.
In oder to prepare plasma samples by LLE for HPLC analisys, could be
followed the next steps:
1. 1 ml water + 0.1 ml solution containing drug, adjust pH, extract
with 5-6 ml solvent, evaporate or reextract at opposite pH, inject.
Calculate recovery. If it is > %50, that solvent is a possible
candidate for final method.
2. 1 mlplasma + ....idem....... . First look at the SELECTIVITY in
HPLC.
If it is OK, calculate recovery.If it is poor, try 1 ml plasma +8 ml
solvent or 1 ml plasma +5 ml solvent twice. More than 8 ml sovent or
than twice steps in extraction needed: think about changing solvent
(think about chemical structure - local and global polarity), pH
(sometimes even the drug is a week base, due the problems in stability,
pH can be changed---example tamoxifen extracted at pH 7 with 85%
recovery)or change to SPE or protein precipitation (example:
ciprofloxacin, norfloxacin, atenolol works better in protein
precipitation than in LLE)
If selectivity is NOT OK, resolve first chromatographic problem and
then go to (2.)
!! Sometimes the recovery is OK but is very difficult to resolve
endogenous componds from plasma and you must to choose another solvent,
with a lowerrecovery but with a cleaner chromatogram.
You can try a solvent screening (using diethylether, dichlormethane,
hexane or, very useful, combinations). Think about changing polarity
using 1.5% ACN or isoamilic alcohol in hexane for some basic compounds
(fluoxetine recovery in hexane 55% and 80% in hexane containing 1.5%
alc. isoamilic). you can use also heptane or MTBE
I have some experience with NSAIDS, I used diethylether for extraction
but NSAIDS work better with protein precipitation.
About 1:50 ratio, I think is very non-economical method, time consuming
and think to change solvent or extraction conditions.
About "extraction the whole amount of drug from plasma" it is not
necessary as time you have a constant recovery and you validate the
method (...and do not think about 100% recovery by LLE!!!)
Hope this helps
Vlase Laurian
Vlase Laurian Univ. Med. & Pharmacy "I. Hatieganu" Dept. of
Pharmaceutical Technology and Biopharmaceutics Cluj-Napoca Romania
...."Dear Laurian,Back to the Top
I feel the information given by you is very
important if any body wants
to analyze the drug in plasma. But i would like to
know about the
volumes of plasma and the solvents being used for
extraction of drug
from plasma, in case of drugs which are highly
protein bound, e.g.
NSAIDS. Is the volume of different solvents
necessarily sufficient to
extract the whole amount of drug from plasma? What
if one uses a 1: 10
or 1: 50 ( plasma: organic solvent) to extract the
drug and then
evaporating and reconstituting the residue before
injecting into HPLC?"....
As my knowledge, the nature of organic solvent used
for LLE does not depend on degree of drug bounded to
plasma proteins.You must to choose an organic
solvent depending on selectivity and recovery of
extraction.
In oder to prepare plasma samples by LLE for HPLC
analisys, could be followed the next steps:
1. 1 ml water + 0.1 ml solution containing drug,
adjust pH, extract with 5-6 ml solvent, evaporate or
reextract at opposite pH, inject. Calculate
recovery. If it is > %50, that solvent is a possible
candidate for final method.
2. 1 ml plasma + ....idem....... . First look at the
SELECTIVITY in HPLC.
If it is OK, calculate recovery. If it is poor, try
1 ml plasma +8 ml solvent or 1 ml plasma +5 ml
solvent twice. More than 8 ml sovent or than twice
steps in extraction needed: think about changing
solvent (think about chemical structure - local and
global polarity), pH (sometimes even the drug is a
week base, due the problems in stability, pH can be
changed---example tamoxifen extracted at pH 7 with
85% recovery) or change to SPE or protein
precipitation (example: ciprofloxacin, norfloxacin,
atenolol works better in protein precipitation than
in LLE)
If selectivity is NOT OK, resolve first
chromatographic problem and then go to (2.)
!! Sometimes the recovery is OK but is very
difficult to resolve endogenous componds from plasma
and you must to choose another solvent, with a lower
recovery but with a cleaner chromatogram.
You can try a solvent screening (using diethylether,
dichlormethane, hexane or, very useful,
combinations). Think about changing polarity using
1.5% ACN or isoamilic alcohol in hexane for some
basic compounds (fluoxetine recovery in hexane 55%
and 80% in hexane containing 1.5% alc. isoamilic).
you can use also heptane or MTBE
I have some experience with NSAIDS, I used
diethylether for extraction but NSAIDS work better
with protein precipitation.
About 1:50 ratio, I think is very non-economical
method, time consuming and think to change solvent
or extraction conditions.
About "extraction the whole amount of drug from
plasma" it is not necessary as time you have a
constant recovery and you validate the method
(...and do not think about 100% recovery by LLE!!!)
Hope this helps
Vlase Laurian
Vlase Laurian
University of Medicine & Pharmacy "I. Hatieganu"
Dept. of Pharmaceutical Technology & Biopharmaceutics
13, Emil Isac
Cluj-Napoca 3400
Cluj
Romania
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