Dear All.Back to the Top
We would like to hear your suggestion regarding the following points,
1. In the case of Quantification by LCMS, in the absence of drug (drug
may
degrade to metabolite)how can we detect the unknown metabolite without
knowing the Mass number?
2.As the metabolite Mass number is unknown, in LCMS we have to scan for
the
full scan, where the sensitivity is very less compared to MRM mode. In
that
case how can we detect the mass number of the metabolite present in
biological sample at ng level.
Advance thanks,
Thanks & Regards,
Dr.B.Sivakumar
Dear siva kumarBack to the Top
For the detection and determination of metabolites and there masses u
need to have a elaborate HPLC/uv (gradient) method for a good resolution
of parent drug and metabolites - In vivo/In vitro.
You need to do a full scan (amu range encompassing the mass of the
parent compound) with tuning parameters of parent compound/default
parameters and obtain the Total ion chromatogram (TIC). From the TIC and
the UV retention times of the metabolites and parent compound masses is
obtained by extracted ion chromatograms (XIC). (For this work LC/MS-MS
with in line uv detector is very usefull or otherwise Relative retention
times (RRT's) of parent compound and the metabolites on the TIC has to
be followed.
Then you need to obtain and match the fragmentation (MS-MS data) pattern
of the parent and metabolites. Later precursor ion scan has to be done
for a stable product ion of parent and the metabolites to confirm the
origin of metabolites.
To over come the smaller concentration of metabolites in In vivo/In
vitro samples, you need to work on extraction methods and pooling of
samples to achieve the requisite higher concentrations for LC-MS/MS
detection.
This is the usual basic procedure for the determination of masses of
metabolites. Alternative ways do exist which need to be explored. Hope
this helps you
Ansar Ali Khan, M.Sc.,
Research Scientist,
Drug Metabolism and Pharmacokinetics,
Glenmark Research Centre,
Plot No.-A-607, TTC, Industrial Area,
Mahape, Navi Mumbai-400 709,
India.
Phone No: 91-22-5590 2491/92 Extn.308
Fax No.: 91-22-5590 2318
Dear Sivakumar,Back to the Top
"1. In the case of Quantification by LCMS, in the absence of drug (drug
may degrade to metabolite)how can we detect the unknown metabolite
without
knowing the Mass number?
2.As the metabolite Mass number is unknown, in LCMS we have to scan for
the full scan, where the sensitivity is very less compared to MRM mode.
In
that case how can we detect the mass number of the metabolite present in
biological sample at ng level."
Develop an HPLC method that can separate metabolites and parent. If
concentration of metabolites
generated in-vivo is low, you can perform in-vitro metabolic incubations
using liver microsomes and
use these samples to characterise metabolites.
Step-1) To know mass of your metabolites - Run your in-vitro/in-vivo
samples
in Q1 mode using suitable hplc method.
Then get mass for each metabolite peak.
Step-2) To characterise your metabolite - you optimise conditions for
fragmentation for your parent in Q2 mode
and run the same samples which will give fragmentation for metabolites
(although not optimal).
Step-3) Quantification of your metabolites - using the precursor mass
and
the major fragment mass you can create MRM's for
all metabolites and run the same samples in Q3 mode (MRM mode). If you
have
metabolites with same MRM as that of parent
you got to separate them in your hplc part.
Step-4) To confirm the structures and accurately quantify your
metabolites
you got to synthesise the metabolites and use them.
Hope this helps.
Good luck,
Kasiram.
Dear Dr Sivakumar,Back to the Top
Firstly, to detect a metabolite without knowing the mass number you may
try
the folowing things :
- Screen for phase II metabolites (or for metabolites with a mass upper
from
the parent drug) using the precursor ion scan, you will find the
metabolites
that give the parent drug when fragmented with the same sensitivity of
the
MRM mode.
- A another good solution is to screen hypothetical metabolites (the
most
common phase I modification) using the MRM mode. In this case you have
to
study your drug structure and the fragmentation pattern (using a
product ion
scan) to find potential metabolite mass and fragments. You will have a
greater sensitivity. To my mind MS mode (Q3MS or Q1MS) won't work
because of
the noise ratio for ng level in biological sample.
- finally have you got any in vitro data ? searching a unknown
metabolite
in biological sample is hard, try after in vitro incubations on
microsomes
or cytosol, In this case you will have less noise.
I hope it will help you.
Nicolas Picard
Pharm D, PhD student
Laboratoire de Pharmacologie et Toxicologie - Equipe DEXO
CHU Dupuytren
87042 LIMOGES-FRANCE
Dear Sivakumar,Back to the Top
Detection of unknown metabolite is never a straightforward easy task.
But there are always starting points like Oxidation and demethylation etc.
You can get an XIC for these mass numbers based on parent drug mass. Once
you see a small peak corresponding to any of these masses, you can set a
daughter ion scan for these masses and make a fresh run. usually you
get a good MSMS spectra so that you can speculate the position of
transformation. As far as sensitivity is concerned, you can adapt various strategies as discussed by the group. New instruments have good sensitivity in ng range. But, incorporation of microbore and capillary HPLC as the front end will help to get better sensitivity.
Hope this is useful.
Best wishes,
Vinayak Nadiger
PharmPK Discussion List Archive Index page
Copyright 1995-2010 David W. A. Bourne (david@boomer.org)