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Dear all,
My queries are specific for all, who have an experience of analysis
of buffer samples.
We were working in a project in which we have to determine four
analytes simultaneously in Caco-2 buffer sing LC-MS/MS. The problem
was tedious because the analytes are of different physicochemical
properties and the matrix is complex buffer ( HBSS buffer composed of
electrolyte and glucose). Because of ion suppressing effect of the
buffer, analytes couldn't be analyzed by direct injection of samples
in to the MASS. Even dilution up to 30 times could not solve the
problem.
Subsequently, we tried sample extraction using LLE and SPE, but it
didn't work as the solubility of the analytes were not similar.
However, we observed that using a diverter for first 3 min to avoid
flow of buffer into the MASS, at a flow of 600 uL/mL using a C18
column (50 X 4.6) and 90% aqueous mobile phase, all analytes were
eluted within 10 min using slight higher organic mobile phase from
3-7 min in a 12 min run time. The method was having good
reproducibility too and only 2 uL sample was used after diluting 2
times. I assumed that all buffer components will be washed out within
3 min as I didn't found any ion suppression ( i.e., 100% recovery).
I discussed the same with my colleagues, who are expert in
bioanalysis using MASS and they were not very happy with my approach.
They rather suggested me not to use buffer just after diluting twice
because direct injection (without sample extraction) could harm the
MASS. I encouraged the suggestion and did stop my efforts using
diverter. Now, I am confused .. What exactly to be used?... as my
efforts are claimed as non scientific.
So, my queries are....
1. Should I use the same method?
2. Is sample preparation needed in analysis of caco-2 buffer
samples, if we are using a diverter for 3 min?
3. As I didn't observe any ion suppression, should I assume that
no buffer is entering into the MASS?
4. What should be the approach while handling a complex buffer
system?
I would like to mention again here that, the analytes are having
different physicochemical properties (4 compounds from four BCS class).
Sincere regards,
Bhagwat Prasad
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Dear Bhagwat Prasad,
I've been using a similar method you mentioned to analyze samples
from a permeability experiment.
I was using a Ringer buffer with glucose and mannitol in EasyMount
(TM) diffusion chambers, separated with
a patch of live duodenal mucosa.
For sample preparation, I only used addition of equal volume of
acetonitrile with internal standard to stop all
enzymatic activity and to precipitate the proteins. I presume some
phosphates might have precipitated as well.
After a brief centrifugation (eg. 10000 g, 5 min), the supernatants
were diluted 10 times with mobile phase
and injected directly into LC-MS/MS. I used a flow diverter valve and
a gradient elution like you
and collected only a small part of efluent (between 4 and 6.5 min).
At all other times, the flow was
diverted to waste. The make up flow of 50 % acetonitrile at 0,05 mL/
min was used to prevent
the spray needle from running dry. This prevented the contamination
of ESI spray chamber and any
ion suppression / enhancement as well.
I would advise you however, that you should use your guard column and
change it more frequently
when injecting samples without a prior extraction into lc/ms.
Best regards,
Jurij Trontelj,
Faculty of Pharmacy
University in Ljubljana
Slovenia
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Dear Bhagwat,
Using diverter is a very useful method. Even while using SPE and LLE,
keeping the diverter program running will help in maintaining source
clean.
Vinayak Nadiger
Vinayak Nadiger
Manager , Bioanalytical Chemistry
11 Biopolis Way, Helios #08-05
Singapore 138667
E Mail: vnadiger.at.combinatorx.com
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The following message was posted to: PharmPK
using a diverter is good practice, specially when you do have an
interferant that is not retained. You could think of it as somewhat of
an on off technique where you are simply diverting the materials not
retained by the column. If you have an internal standard, you could
enhance the system by getting the material to stick to the column, elute
the non-retained material, then elute witth an organic more compatible
with the detection, then recondition, and recycle for the next sample.
Ed O'Connor, PhD
Technical Director, Immunoanalytical
Tandem Laboratories
115 Silvia Street
West Trenton, New Jersey
609-228-0243
ed.oconnor.aaa.tandemlabs.com
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