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The following message was posted to: PharmPK
Dear all,
We are having difficulty in achieving sufficient sensitivity in LC/MS/
MS analysis of Caco-2 transport samples.
Here is the description of what and how we are doing:
The buffer that we use for Caco-2 transport study is Hanks balanced
salt solution with HEPES (4-(2-Hydroxyethyl)piperazine-1-
ethanesulfonic acid), and the transport samples are combined with 0.5
part of organic solvent, either MeOH or Acetronitrile. Injection
volume is 10 -20 ul.
Compounds are typically eluted around 5 to 7 minutes with 9-minute
gradients with 0.8 ml/min as a flow rate (Water and MeOH containing
either ammonium hydroxide or formic acid). The first 4 - 5 mintue-
flow is diverted to waste, and we split the flow into half before the
probe with a tee.
ESI mode and MRM in Quattro Micro. (Before we got this MS a month
ago, we had used Quattro LC, and rarely had sensitivity issue with
Caco-2 samples). Prior to LC/MS/MS analysis, all compounds are tuned
for MS tune page (source and analyzer) with infusion.
The infusion sample test run works pretty well, but we are
experiencing app. 10 fold loss of sensitivity in Caco-2 transport
samples. The (desired) lowest concentration of the standard curve is
2.5 ng/ml.
It would be greatly appreciated if experts provide me with some insight.
Sincerely,
KJ
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The following message was posted to: PharmPK
Hi,
Did you make a comparison to dissolve your compounds
in your mobile phase (water and MeOH with formic acid,
or ammonium hydroxide) and in the buffer you have
used? If yes, and you saw a 10 fold loss, I would
guess somehow your buffer got into Mass spectrometer,
even you have mentioned you sent first 4 to 5min
liquid to the waste. Maybe your post column switching
valve did not function properly as you have set.
This is the first two steps I would like to check.
Xiaodong
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The following message was posted to: PharmPK
Hi,
My message may be not so clear to you. I mean first
you can check the solubility of your compounds in your
mobile phase and in Hanks buffer to see if you have
solubility issue. If that is not the problem, then
check if you switching valve functions properly.
Xiaodong
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