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The following message was posted to: PharmPK
Dear Colleagues,
I am working on the analysis of angiotensin (Ang1-7) peptide in
plasma using LC/MS/MS. I got a good response on LC/MS/MS using pure
standards, but as soon as I switch to plasma samples and the sample
extraction using SPE, my analysis gets unreliable mostly due to the
low recovery only at 50%. I suspect that precipitation of general
proteins in plasma is affecting angiotensin. I have noticed that
even the sequence of standard additions to prepare the samples for
SPE is affecting the recovery. Any comments or advices for
angiotensin sample extraction would be appreciated.
Haejung
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Dear Haejung
Before commenting on your problem-unreliable analysis, I would like
to ask you one question. How did you arrive to 50% recovery?
If it is with respect to equivalent aqueous standard then it could be
a matrix effect from endogenous substances present in plasma. You can
change the chromatography keeping extraction procedure same to avoid
junk to elute at same retention time.
But as you mentioned, you observed this with your sample preparation
procedure also, then I would advice you to optimize sequentially
cartridge conditioning procedure, plasma treatment before loading and
elution procedure.
Hope this will help you.
Jignesh Kotecha
Torrent Research Centre
Gandhinagar, India
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Angiotensin sticks to glass unless it is siliconised. Also being a
peptide it is susceptible to enzymatic breakdown, so enzyme
inhibitors are also required. There may be more "modern" choices
these days, but in the seventies a mixture of 3 was commonly used:
2,3 mercaptopropanol (final concentration 1.53mM); 8-hydroxyquinoline
(3.23mM) and NaEDTA (2.56mM).
Hope this helps
Ian Smith
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The following message was posted to: PharmPK
Dear Haejung
You can try pre-fractionation of plasma with
acetonitrile or methanol precipitation prior to
loading to SPE.
I do not have experience with angiotensin, however
this is a recommended techniques for better recovery
of biomarkers (peptides). Give a try.
Jagdish
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