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The following message was posted to: PharmPK
We are investigating the oxidative metabolism of a compound using Rat
liver microsomes (RLM).
Our observation is : 40 - 45% of the parent compound disappears after
incubation with RLM's.
However, we are not able to find any additional peaks appearing on our
Chromatogram using either a isocratic elution or a gradient elution
system ???
How do we account for the loss of parent drug??
Y.Chugh
Wockhardt Research Centre
Aurangabad, India.
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[A few good replies - db]
From: "Shirley Teng (Xiaowei Teng)"Date:
Thu, 17 Jan 2002 15:40:10 +1100
To: david.-at-.boomer.org
Subject: Re: PharmPK Metabolism with rat liver microsomes
The following message was posted to: PharmPK
Dear Y.Chugh,
Are you using uv detector? If yes, you need to consider if the
metablite(s) has uv absorption. [and any difference in molar absorbance
- db] 'The loss of parent drug' could be estimated from the
disappearence of the parent drug.
Shirley
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From: "atul"
Date: Wed, 16 Jan 2002 22:58:33 -0800
To: david.-a-.boomer.org
Subject: Re: PharmPK Metabolism with rat liver microsomes
The following message was posted to: PharmPK
Hello Dr Chugh
Based on your structural properties is it possible that the molecule
cleaves in a position that the chromophore in the structure is lost.
Then in that case you will hardly see anything in your chromatograms.
Also it depends on your extraction procedures and I am sure that you
must have used a photodiode array detector for your work. Doing this
study on LCMSMS might be a safe bet coupled with an appropriate
derivatization technique. A lot depends on the elution techniques too.
Cheers
Venkatesh Atul Bhattaram
Post-doctoral fellow
Dept of Pharmaceutics
University of Florida
Gainesville-32601
Ph:352-846-3257 (O)
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From: "PERSIANI Stefano"Date: Thu, 17 Jan
2002 09:06:16 +0100
To: david.aaa.boomer.org
Subject: R: PharmPK Metabolism with rat liver microsomes
The following message was posted to: PharmPK
If you are using the radilabelled derivative of your compound you might
want to check if the labelling is in a metabolically stable position.
Products labelled in metabolically unstable positions produce either
unlabelled metabilites or 14CO2.
If your product is labelled with 3H the loss of radioactivity might be
due to exchange with H2O (again metabolically unstable labelling).
If you are using the cold compound I suggest you to check the non
specific absorption onto the glassware used for the experiment (also the
SPE hardware, if any) by performing a symple recovery experiment without
microsomes.
Recovey experiments with the radiolabelled drug by measuring
radioactivity might also be useful.
I hope this help.
Stefano.
Stefano Persiani, PhD
Manager, Pharmacokinetics and Phase I
Rotta Research Laboratorium, S.p.A.
Via Valosa di Sopra, 7-9
20052 Monza (MI)
ITALY
e-mail stefano.persiani.-a-.rotta.com
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From: "Cardenas Armesto, Alvaro Jesus"Date: Thu, 17 Jan 2002 09:31:02 +0100
To: david.at.boomer.org
Subject: Re: PharmPK Metabolism with rat liver microsomes
The following message was posted to: PharmPK
Dear colleage,
I do not know which is your sample preparation procedure and the kind of
detection (UV, MS) that you are using. However, assuming that you are
running a blank of incubation (i.e. microsomes without NADPH) and
excluding problems of solubility, I think that at least there are two
possible explanations:
- Binding of the metabolites to microsomes - Changes in the UV spectra
(or a lack of ionisation in MS) of the metabolites
For the first case, previous to HPLC analysis I would try to pre-treat
the incubated sample with an organic solvent (i.e. dilution with ACN).
For the second case, if you use UV detection I would use a diode array
detector (or merely detect at 210 nm) and if you use MS detection I
would change from ESI to APCI.
Finally, in these cases I think is useful to run incubations at
different periods of time to confirm the results.
Alvaro Cardenas
AlmirallProdesfarma
acardena.at.almirallprodesfarma.com
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From: Stephen Day
Date: Thu, 17 Jan 2002 09:22:10 -0500 (EST) To: david.-at-.boomer.org
Subject: Re: PharmPK Metabolism with rat liver microsomes
The following message was posted to: PharmPK
Hi,
Here are a few possibilities regarding your low recovery:
1) Extensive covalent binding of reactive metabolite to protein.
2) Highly polar metabolite that elutes in the solvent front... but you
may have ruled this out.
3) Loss of chromaphore during metabolism (UV-VIS detection)... this is
rare in my experience.
Obviously, radiolabelled compound is a great tool in these situations.
Regards,
Steve
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The following message was posted to: PharmPK
I do not know which detector you are using to monitor the metabolites.
Photodiode array with 200 to 400 nm range should be able to pick up your
uv absorbing metabolites.
hope it helps
Dr. Jyoti Paliwal
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The following message was posted to: PharmPK
But only if 1) they are well resolved from the parent and 2) if their
spectra are significantly different
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