Navigating Challenges Associated with Bioanalysis of Therapeutic and Endogenous Peptides | Part 4 of

Navigating Challenges Associated with Bioanalysis of Therapeutic and Endogenous Peptides | Part 4 of


Hi. I’m Kim Haynes, a
product marketing manager in the Chemistry
Technology Center at Waters Corporation. In Part 4 of this
series, we’d like to talk you through
the calculations we use to evaluate our cleanup
methods for recovery and matrix effects. It is important to understand
how to do these calculations and how they are
different from each other. Recovery calculations are
done in sample matrix, while matrix effects
calculations are done specifically to evaluate
the effect of sample matrix on the recovery of the analyte. To calculate recovery, there
are two different solutions that you need to make– the post-extracted spike sample
and the extracted sample. To make the post-extracted
spike sample, you need to take a blank
sample matrix with no analytes and then run that through
your solid phase extraction procedure. At the end, you then
spike your standards directly into that
extracted matrix. And that creates your
post-extracted spike sample. To create your
extracted sample, you need to take your sample
matrix once again, but actually spike
your standards directly into that sample matrix. Then you run that through your
solid phase extraction protocol and collect your eluate, which
is now your extracted sample that contains your analytes. To do the actual
calculation, you take the response of
the extracted sample with the analytes
and divide that by the response of the
post-extracted spike sample. Multiply that by 100. And that gives you
your percent recovery. To effectively calculate
matrix effects, we once again need two samples. Just like in recovery, we need
a post-extracted spike sample, which we create by
taking a blank sample matrix with no analytes. We run that through our solid
phase extraction protocol. And then we spike our standards
into that extracted matrix. This creates our
post-extracted spike sample. Now, since we are
evaluating matrix effects, it’s important that we compare
that post-extracted spike sample to a solution
without matrix. So the second solution
that we prepare is a standard solution that
we spike the analytes into. So for example, if our
post-extracted spike sample is in 75% acetonitrile
and water, we want to make sure that
our standard solution where we spike our analytes
into it is also 75%. acetonitrile and water. Now, to do the calculations,
to calculate matrix effects, we take the response of our
post-extracted spike sample and divide that by the response
of our standard solution. Then we subtract 1 and
multiply that result by 100. That gives us our
percent matrix effects. If we see a negative value for
the percent matrix effects, we’re seeing ion suppression. If we see a positive
value, that means we’re seeing ion enhancement. We really hope this
module helped talk you through the calculations we
use to evaluate our cleanup methods for recovery
and matrix effects. For more information about
this or other related topics, you can always visit
the Bioanalysis Bootcamp to learn more and increase
your peptide and protein quantification fitness. Thank you.

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