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Dear members
In-vitro liver microsomal stability is a routine scenario in a drug =20
discovery program. Microsomal stability studies (along with =20
metabolite/possible metabolic liable sites identification) have =20
become an important screening tool for NCE=92s especially in early =20
stage of drug discovery. The samples generated from such studies are =20
subjected for HPLC-UV/fluorescence detection by isocratic/gradient =20
flow. I am quite comfortable with this methodology and no complaints.
But, I am apprehensive about subjecting such study =20
samples on LC-MS/MS. I understand that in-vitro metabolism is =20
performed in 0.1M KH2PO4 buffer (inorganic) and samples containing =20
inorganic buffer should be avoided for MS detection due to their non-=20
volatile nature as they accumulate in the ion source and cause ion =20
suppression. I have the following queries regarding this issue:
1. Is there any methodology or sample treatment which will avoid =20
KH2PO4 buffer present in the sample from reaching MS?
2. Is it like that the small amount of inorganic buffer in the =20
injection volume not going to affect MS?
3. Can I subject the deproteinized/reconstituted samples for MS? Do =20
they have negligible or no effect on MS?
4. It is stated that inorganic buffers should be avoided for ESI. Is =20
it an indication that I can use in APCI?
5. I am unaware of any microsomal stability studies which do not =20
employ KH2PO4 buffer. Do members have any alternative methodology for =20
performing the study when samples should be subjected to LC-MS/MS =20
analysis.
Expert opinions / literature citations from every member are equally =20
appreciated.
Thanks in advance
L.Chakradhar
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Dear Chakradhar,
When you are subjecting the microsomal stability samples for LC-MS/
MS, the samples most probably will be for qualitative purpose than
quantitative purpose, and also the concentration of the buffer in the
final samples will be very less to effect the results of your analysis.
Pavan
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Dear chakri
Your question is absolutely correct. In microsoaml stability studies
we are using only less than 50ul phos buffer, moreover finally the
analyte is extracted with organic solvents and then evporated, hence
there is no problem for ESI in LC MS MS analysis.
Hope this helps you .
A.Karthik
Best wishes
A.Karthik
Research scholar
MCOPS
Manipal
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The following message was posted to: PharmPK
A good way is to use a diverter valve (six port valve for example)
and to
divert the flow of the first minutes of your analytical run, before the
elution of your product.
You could also use an auxiliary pump to maintain the same elution
conditions
in the source during this time.
Inorganic buffers are not recommended in both apci and esi.
Your microsomal stability samples could (or should) be precipitated
with an
organic solvent and then centrifuged prior analysis.
Regards
Fabrice Guillet
Xenoblis-Biopredic
Saint Gregoire
France
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