Dear Mr Kasiram,Back to the Top
I\0xEDve read the discussion about IV precipitation and I am interested in
the analytical reason of incubating the drug with blood.
I am a chemist (not a physician), and in my work I develop and
validate methods for determination of drugs in biological fluids by
HPLC for Bioequivalence Studies. Now our team has to develop a method
for determination of a drug which is extensively bounded to red blood
cells. By this reason we will determine the drug in whole blood of
healthy volunteers (not in a plasma - blood/plasma ratio is~6:1). We
extract the blood (control and calibration samples) by liquid-liquid
extraction and recovery is high and reproducible. Our team for the
present is validating the analytical method and we have not analyzed
the blood samples of the volunteers yet.
May I ask you if we have to incubate the calibration, control and
blank samples in this case? What is the aim of incubating the drug with
blood - I think that it is for simulating the in-vivo binding of the
drug with RBC? As we don\0xEDt incubate the blood samples in our recent
method, will we have to expect differences between extent of extraction
(recovery) of calibration /control samples and blood samples of
volunteers. If it is so, should we incubate the calibration samples and
then sonicate (before proceeding to extraction) both the calibration
samples and blood samples of volunteers? What is the recommended time
for such incubation?
Thank you very much in advance.
Denitsa
Dear Denitsa,Back to the Top
To answer your questions one by one-
"May I ask you if we have to incubate the calibration, control and
blank samples in this case?"
Generally not required if extraction recovery of your drug is close to
>80 or 90%. However, you can check whether or not it is required (in your case) by extracting spiked blood with (say for about an hour) and without incubation. If there is significant difference you can include sonication step before extraction and repeat the experiment. If you still find difference in recovery, then you can consider introducing incubation step for your calibration, control and blank samples.
"What is the aim of incubating the drug with blood - I think that it is for simulating the in-vivo binding of the drug with RBC?"
That is obviously one of the reasons. But, the main reason for
suggesting that step to Mr.Sivakumar is to check time dependent degradation of his drug as it is known that blood contains enzymes such as esterases and amidases that hydrolyse ester and amide containing drugs.
"As we don\0xEDt incubate the blood samples in our recent
method, will we have to expect differences between extent of extraction
(recovery) of calibration /control samples and blood samples of
volunteers. If it is so, should we incubate the calibration samples and
then sonicate (before proceeding to extraction) both the calibration
samples and blood samples of volunteers? What is the recommended time
for such incubation?"
As mentioned above, you can check if it is necessary to include the
incubation and/or sonication step by analysing drug spiked blood samples - with and without incubation.
Please note that sonication step is to lyse the RBC for easy recovery of drug thereby reducing vortexing time.
Hope this helps.
Kasiram.
Dear Dr. Denitsa,Back to the Top
You may find the difference if you do not incubate your drug with whole
blood for some time at room temperature especially for a drug binds to
RBCs and lipoprotein. Although we did not do the equilibration time for
cyclosporine but an hour time should be sufficient (as we did not see
difference for longer time). Cyclosporine is an example of drug that is
extensively bound to RBCs and some lipoproteins in blood.
Afterincubation you can simply freeze (-20 degree Celsiusfor 30
min)your whole blood sample to rupture RBCs. Generally sonication at
high frequency generates heat and lot of extraneous material from the
breakdown of matrix to trouble your HPLC assay.
Hope this helps.
Jagdish K.Jaiswal, Ph.D.
Research Associate
Drug Discovery (Pharmacology)
Torrent Research Centre
Gandhinagar 382428, Gujarat
India
Phone: 91-79-2396100 Ext.237/238
Fax:91-79-23969135
Dear Mr Kasiram and Mr Jagdish,Back to the Top
thank you for the quick response of my questions. Could you explain me
how to
perform an incubation procedure of a drug in blood?
Mr Jagdish mentioned that the RBCs are ruptured under freezing the blood
samples at - 20 oC. In our Lab we use blank blood for calibration curve
and
QC's which we keep in a fridge at -20 oC. The blood samples of the
volunteers
in BE studies are also carried to our laboratory in a frozen state. Our
team
thaw all samples at room temperature before analytical procedure.
Does it mean that the RBCs in our samples are already ruptured and all
drug
molecules are free? And if the blank blood has been frozen once, will I
be
able to perform the incubating or the RBCs are totally destroyed? I
need to
have the same recovery for the calibration and unknown samples. Is the
incubating necessary in this case?
Thanks.
Denitsa
Dear Denitsa,Back to the Top
"Could you explain me how to perform an incubation procedure of a drug
in blood?
Mr Jagdish mentioned that the RBCs are ruptured under freezing the blood
samples at - 20 oC. In our Lab we use blank blood for calibration curve
and QC's which we keep in a fridge at -20 oC. The blood samples of the
volunteers in BE studies are also carried to our laboratory in a frozen
state. Our
team thaw all samples at room temperature before analytical procedure."
Yes, freezing does lyse RBC's. In order to carry out incubation
experiments for the method validation purposes or drug stability testing purposes, you need to use freshly collected blood only. Once you rule out the extraction issue/stability issue, you might use stored blood.
"Does it mean that the RBCs in our samples are already ruptured and all
drug molecules are free?"
If RBC get ruptured at freezing conditions that doesnt mean that your
drug is free. Drug would still be bound to proteins and membranes but access to it by extraction solvent gets easier. Thats why I suggested to use brief sonication step. As Mr.Jagadish pointed out sonication might lead to additional extraneous peaks in your chromatogram, you have to practiacally check whether you need a sonication step and if so how long.
"And if the blank blood has been frozen once, will I be
able to perform the incubating or the RBCs are totally destroyed? I
need to have the same recovery for the calibration and unknown samples.
Is the incubating necessary in this case?"
You may not get correct picture on whether incubation leads to
differences in extraction recovery of your drug if you use stored blood. Therefore, as mentioned above, you better use fresh blood if at all you want to check the recovery differences with and without incubation. Once confirming that there is no difference or by using correct extraction procedure, you can very well use frozen blood for your calibration and QC samples.
Hope this helps.
Kasiram.
Dear all,Back to the Top
i incubated the drug in blood at 37 C and analysed the
presence of drug in blood as well as plasma. we are
not able to find the presence the drug.
But, when the drug was put in blood and incubated at
20+2 C, room temperature, the drug is seen in blood as
well as in plasma. what are the possible reasons for
the conversion or degradation of the drug.Blood/plasma
contains esterases and amidases. But we couldnot any
hydrolysed product of the drug also appreciably
products on incubation at 37C. what happened to the
drug?
What is the reason for these observations.
In invivo, on intravenous injection of this drug also,
we could get the presence of drug only on immediately
centrifuging the collected blood and analyse it. But
the drug concentration falls very rapidly and we are
not able to get any PK parameters. In contrast, the IV
study, when the blood was centrifuged after some time
and analysed the plasma, the drug was not detected at
any time points from 3 minutes also. The drug has poor
aqueous solubility (1ug/ml) and log P is also very
high. The drug is also having highly protein binding
property.
* The reason for the presence/absence of drug in 37
and room temperature is not understood.
* In IV study, the drug is not seen in blood when
centrifuged later.
Is the blood constitutents degrades the drug or the
stability of drug in blood or plasma is major factor.
I need some opinion on this
regards
sivakumar
Dear Sivakumar,Back to the Top
Your observations clearly indicate that your compound is getting
degraded in
blood/plasma over time and that process seems to be rapid at 37oC.
I think its time for you to talk to your medicinal chemistry people and
learn more about the structural features of the molecule and know the
liable sites for possible hydrolysis by amidases/esterases.
Also, if a molecule contains reactive groups (such as sulfhydryl) could
readily react with endogenous compounds such as glutathione, cysteine
etc and form conjugates. Also they can form dimers of its own. So, to know
what could actually be happening its important to understand the chemistry of
your molecule.
If the degraded products are hydrophilic, you may not see any peak in
your chromatogram as there is every chance for the degraded products to get
eluted in solvent front (as the mobile phase was intended for the
parent, it would be too strong for degraded products). Therefore, delaying your
elution or using a gradient might help.
Hope this helps.
Kasiram.
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