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I am trying to analyze a discovery compound in Krebs buffer from a heart
perfusate experiment. The analyte behaves very well by LC/MS/MS when
standards are prepared in plasma, serum, or brain homogenate, with
linearity
in the range of 5-5000 ng/mL. However, when the standard curve is
prepared
in the buffer, I get no signal at all until almost 500 ng/mL. I am
guessing
that this is due to ion suppression. As a rule we run "fast gradients"
with
runtimes of <2 minutes. I tried extending the gradient to ~5 minutes,
but
with no success.
Does anyone have hints, in their own labs or in the literature, on
analysis
of similar types of samples. My next step was to do some de-salting by
solid phase extraction of the perfusate, and/or liquid/liquid
extraction.
Best Regards,
Jim Schmidt
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We have lot of experience in this phenomena. We think what happened
is that because of the high ionic strength of the buffer, most of the
test article was lost to the absorption to the container well before it
was analyzed.
Ta Kung
Pre-Clinical Development Department
Neurocrine Biosciences, Inc.
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Two suggestions: dilute the sample with water and use a divert valve to
avoid the front elution.
Best regards,
Alvaro Cardenas
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Jim,
Yes, suppression of ionization by the high concentration of salts might
have reduced your LC/MS/MS assay sensitivity if you introduce the
buffer directly into LC/MS and your LC/MS ion flow and the spraying
flow are the same. To test it, you can try column-switching to switch
the eluent of the first few minutes to the waste since mostly buffer
salts will be eluted first if you are running reversed phase. Then
introduce the rest of eluent to the Mass spectrometry and see the
response.
Hope this helps,
Shirley
Xiaowei Teng (Shirley), PhD
Faculty of Pharmaceutical Sciences
University of British Columbia
Vancouver, BC, V6T 1Z3
Canada
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Copyright 1995-2010 David W. A. Bourne (david@boomer.org)