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The following message was posted to: PharmPK
We are working on the HPLC method development from plasma for the compound
S-2 (2-aminoethylamino) ethyl phenyl sulphide dihydrochloride
for the subsequent use in pharmacokinetic and disposition studies in
rats /rabbits. The compound has m.p of 192-1940C, M. Wt: 287. The compound
is freely soluble in water and soluble in methanol. The UV l max
is 253 in MeOH and 248 in water. The pKa of the compound is 6.3 and 9.4
for the two amino groups.
We have spiked different concentrations of the compound in 100 =B5l of
plasma and we have constructed the calibration graph. We have done this
using two-sample work up procedures. I am giving here the details. However,
in both the cases the mobile phase composition is same.
Sample work up I: A volume of 100 =B5l of plasma mixed with different
concentrations
of compound, mix, vortex with MeOH, centrifuge and evaporate the supernatant
to dryness and reconstitute with 200 =B5l of mobile phase.
Sample work up II: A volume of 100 =B5l of plasma mixed with different
concentrations of compound, adjust the pH to 8.0 with NaOH, mix, vortex
with MeOH, centrifuge and inject the supernatant directly into the HPLC
system.
Mobile phase: Water: ACN (95:5), pH 3.0 (adjusted with acetic acid),
flow rate 1ml/min and l max is 248 nm.
The problem is when we inject the compound of interest by intravenous
route to the rats; we are not able to detect the compound even in the
immediately collected blood samples. We have given the highest dose of
25 mg/kg to the rats but we could not detect the compound in the blood
samples. We tried both the methods and could not succeed.
Can any one in this group help me in this regard? I will be glad to know
from the members if they can point out where I went wrong. Can any one
suggest me any other alternate ways of doing this?
Deepadevi D
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[A few replies - db]
From: David Foster
Date: Fri, 12 Oct 2001 17:07:58 +0930
To: david.-a-.boomer.org
Subject: Re: PharmPK
The following message was posted to: PharmPK
Do you have a decent recovery of your standards, and a working standard
curve? I think that you do from the details. If so, then you obviously
need a much lower Limit of Quantification. Probably cant do much with the
sample work up with that small volume, and I assume that you dont want to
move to LC-MS, so heres my suggestion:
I could be wrong in this case (with your compound), but often the
absorbance for a compound is markedly greater at lower wavelengths (ie
~210nm) compared to the "lambda max". For example, methadone has a lambda
max of around 300nm, but when you look at the UV spectra you can see that
the absorbance at 210nm is several times greater (over 5-fold I'd guess)
than the value at 300nm.
You could probably use a much lower wavelength (~210nm) as still have clean
chromatograms with either of the 2 assay methods IMHO, and the low ACN
content should help too -make sure you are using "far-UV grade" ACN though.
The acetic acid could be a probelm if there is a lot of it in the mobile
phase -could you use ortho phosphoric acid instead?
Id try injecting a water stock solution at a series of wavelength settings
on the UV detector: 250, 240, 220, 210, and 200nm -see if the sensitivity
improves- or look up/perform a UV scan 200-300nm. Of course you would have
to check that the blank extracted biofluid samples are clean too at the new
wavelength.
Hope this helps,
David
David Foster, PhD
Department of Clinical and Experimental Pharmacology
Faculty of Health Sciences
Adelaide University
Adelaide, South Australia 5005
Tel: +61 08 8303 5985
Fax: +61 08 8224 0685
Email: david.foster.aaa.adelaide.edu.au
http://www.adelaide.edu.au/Pharm/index.htm
---
From: Gan Siew Hua
Date: Fri, 12 Oct 2001 13:23:58 +0100 (BST)
To: david.-at-.boomer.org
Subject: Re: PharmPK HPLC method for S-2 (2-aminoethylamino) ethyl
phenyl sulphide dihydrochloride
The following message was posted to: PharmPK
Dear Deepadevi D,
You have got your HPLC method, but did you actually
extract the drug out of plasma? If the drug
concentration in plasma is in the range of nanogram,
you definitely cannot see any peaks (there in the
chromatogram).
What I suggest is try first to extract, maybe by using
a simple liquid-liquid extraction of the drug by
basifying the plasma first to two units above the pka
value, viewing the basic pKas. You can use any organic
solvent combinations, e.g. 4ml ethylacetate:hexane
(1:4) or diethylther:hexane (2:3) combination, to be
vortexed & centrifuged. Then the trick lies here:
CONCENTRATE the preparation (after having dried the
organic solvent off) by reconstitution
in only 20 or 50 ul (depending on your sample loop) of
a solvent similar to your mobile phase & bravo! you
don't have to kill your rats by increasing the dose
further...
Good luck,
Ms Gan Siew Hua,
PhD candidate,
Pharmacology dep,
Universiti Sains Malaysia,
16150 Kubang Kerian, Kelantan,
MALAYSIA.
---
From: "bvatul"
Date: Fri, 12 Oct 2001 09:56:15 -0400
To: david.aaa.boomer.org
Subject: Re: PharmPK HPLC method for S-2 (2-aminoethylamino) ethyl
phenyl sulphide dihydrochloride
The following message was posted to: PharmPK
Hello
A couple of things:
1. If you are able to construct a calibration curve and able to see your
molecule in HPLC it appears that it is stable in plasma. However you could
check this.
2. Is the molecule stable in the formulation?
3. Also you should consider extensive binding of your drug to red blood
cells. Did you check this?
I hope that this will help you.
Best
Atul
Post-Doctoral Fellow
Dept. of Pharmaceutics
University of Florida
---
From: "Deepadevi D"
To: david.-a-.boomer.org
CC: "bvatul"
MIME-Version: 1.0
Dear Dr. Atul,
Thank you very much for ur reply posted to pharmPK.
Yes, it appears that the drug is stable in plasma as we have constructed
the calibration graph.
The molecule has not yet been made into formulation. It is a new drug
altogether. I have used the pure drug it self.
We found out that the drug is not binding to RBC. We have done this by
adding known qty of drug to blood and later separating the plasma and
estimating. we found that the drug is not binding to RBC.
Any other reason ....it is surprising that the drug when given by iv
is not even present in the immediately collected blood samples.
Many thanks for ur info.
Deepadevi D
deepadevi.-a-.onebox.com - email
(512) 531-5067 x1112 - voicemail/fax
---
From: "bvatul"
To: "Deepadevi D",
Subject: Re: PharmPK HPLC method for S-2 (2-aminoethylamino) ethyl
phenyl sulphide dihydrochloride
Date: Fri, 12 Oct 2001 12:24:20 -0400
MIME-Version: 1.0
X-Priority: 3
Hello
This is surprising. In which solvent did you dissolve the drug before giving
iv? I presume it must be water or saline since your drug is soluble. I have
seen some drugs which are unstable in saline too. How long before you
prepared your formulation? Did you check the content uniformity. I do agree
this might be too basic but might be helpful in removing some doubts. Also
did you check the metabolism of this substance in blood? Another reason
might be some sort of intermediary conformational change in the structure
and its solubility and it might then be forming some sort of conjugates in
the blood. But mostly you can rule this out if you study any metabolism in
blood (You need to add cofactors). Capacity of RBC to metabolise can be
huge. Can your drug precipitate at the site of injection (after the salt
form is liberated)?
I think these factors should help in solving your problem.
Best
Atul
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