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Dear All,
Please excuse the ignorance of a novice but I am not by any means a
kineticist. need to generate a calibration curve of a prostaglandin
in order to quantify the contained amounts in plasma samples. I
thought one would just make up a known concentration from a standard
and then inject decreasing volumes into the lc/ms, limit the TIC
chromatogram to show the ion of interest, and integrate the peak to
get the area. This would then give me a graph of amount vs. area
which would then serve as my calibration curve provided that my
plasma samples give integrated area values within this range. What I
am finding is that the difference in area between a 20 ul injection
and a 10 ul injection is basically the same which leaves me
dumbfounded and at a loss. Am I doing this wrong? I am using
MassLynx 4.1 and the integration is done with its default settings
(told you I am a novice...).
Thanks in advance.
Tai
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The following message was posted to: PharmPK
Tai,
Biofluid calibration and quality control samples are formulated by
fixing the volume of biofluid (usually 100 uL to 1 mL, but held
constant throughout a particular analysis method), removing an amount
of biofluid from each calibration or QC "sample," and adding back the
same amount of formulated standard solution. The standard solution is
usually formulated in two steps: (1) create a aqueous or lightly
buffered standard solution at about a 10x concentration to the
highest desired biofluid sample calibrant; (2) dilute this "spiking
solution" in biofluid so that you can create a full calibration curve
and the QC that fit within the curve. At this point, you are
replacing the removed biofluid with a primarily (a little buffer,
perhaps a little organic) biofluid solution - this keeps the biofluid
from becoming markedly different than the patient samples you will
receive from the clinical site. You then treat the resulting
calibrants and QC to the sample preparation methodology you have
developed and validated - this may be any number of different
methods, from "dilute and shoot" to solid phase extraction to a
classic liquid-liquid extraction.
With prostaglandins, you will probably require an antioxidant in your
biofluid collection tubes and you will probably need to control pH or
antioxidant concentration throughout the sample collection, calibrant
and QC formulation, and sample processing steps, otherwise you will
have instability in your samples, calibrants, and QC. Sample and
standard instability will result in an unreliable methods
development, validation, and implementation process that will
manifest as high variability in known concentrations that should be
constant, accurate, and precise. If these parameters are not
established, your will have an unstable calibration curve, your QC
will fail imprecisely, and the concentrations you determine for
patient samples will be unreliable.
All of this is beyond the scope of an e-mail and should result in
conversations with seasoned bioanalytical scientists. Doing this
stuff incorrectly results in undue scrutiny of clinical trial results
- and no one wants to hear the assay was faulty!
Ian Davis
Director, Operations
Strategic Consulting Services
Pharsight Corporation
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The following message was posted to: PharmPK
There are so many practical factors to consider that your approach is
never going to work.
Your approach is based on the assumption that there is perfect linear
and amount-proportional response on the LC/MS with the injected
amount of the analyte. This is almost never the case. A variety of
factors influence your instrument performance,, such as instrument
conditions, instrument performance degradation, ionization efficiency
change related to the sample composition, mobile phase composition, etc.
In addition to your mass spectrometry part, many other aspects of
your procedure affect the reliability of your analytical results,
such as sample processing, recovery of your analytes during sample
extraction, reliability of your autosampler, to name just a few.
It is imperative to set up an LC/MS/MS based quantitaton assay with a
reliable internal standard. Any publication on an LC/MS based
quantitation assay should give you the general aspects involved:
selection of an internal standard, sample extraction/clean up, HPLC
conditions, instrument optimization, etc.
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The following message was posted to: PharmPK
Dear Tai,
If you want a very quick way to generate a calibration line then the
approach you have suggested is viable. However this is not the best
scientific practice since it does not take into account matrix effects
(supression of signal in the ion source) or the % recovery from your
plasma extracts - refer to the e-mail of Ian Davis. But if this is a
very quick and dirty experiment that doesn't require absolute
quantitation then it should work. There are several reasons why you may
not see a difference between a 10uL and a 20uL injection. Firstly check
that the peak actually relates to your compound, inject a blank and make
sure you get a flat baseline to start with. Secondly check that your
sample is not saturating the detector. If its too concentrated then a
10uL and a 20uL injection would look the same.
I hope that helps.
David
David Neville, BSc MRSC
Discovery Team Leader
HFL Ltd
Newmarket
UK
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Copyright 1995-2010 David W. A. Bourne (david@boomer.org)