Navigating Challenges Associated with Bioanalysis of Therapeutic and Endogenous Peptides | Part 5 of


[MUSIC PLAYING] Hello. I’m Kim Haynes, a
product marketing manager in the Chemistry
Technology Center at Waters Corporation. Part 5 of this
series is a follow-up to our previous module about
calculating recovery and matrix effects. In addition to understanding
the calculations, it is important to understand
how to actually make the solutions and how
to set the experiment up on your 96-well solid
phase extraction plate or set of cartridges. I’d like to take a few minutes
to explain exactly how we make these solutions to
calculate recovery and matrix effects. In my conversations
with scientists, I often find that the
preparations of these solutions can be confusing, so I think
it’s helpful to explain them. So first let’s talk
about explaining the creation of the
post-extracted spike sample. First we take 500 microliters
of blank sample matrix and run that through our solid
phase extraction protocol. Our elution step requires that
we elute in 50 microliters. Then we take our post-spike
solvent and we add 50 microliters of a
100-nanogram-per-mil solution equaling a total solution
of 5 nanograms per mil. To create the extracted
sample, we once again take 500 microliters
of sample matrix, but this time, it’s been spiked
with 10 nanograms per mil of analyte. When we run that through our
solid phase extraction protocol and elute with 50
microliters, that gives us a solution of 50
microliters that contains 5 nanograms of analyte. So the result is that these
two vials both contain matrix components from 500
microliters of matrix that’s been eluted with 50
microliters of elution solvent. We’ve added 50 microliters
of post-spike solvent and theoretically
have an equivalence to 5 nanograms of
analyte in each solution. To calculate matrix
effects, we once again need the post-extracted
spike sample. And I just explained in
recovery how to create that. But here, we also need
a standard solution containing the analytes. To create our standard
solution, what we need to do is post-spike 50 microliters of our
100-nanogram-per-mil solution into the sample solvent that
matches the elution solution from our post-extracted
spike sample. So once again, if our
final elution solution is 75% acetonitrile,
25% water, once again, our standard solution
that we’re spiking into would be 75% acetonitrile
and 25% water. There is a special
consideration to keep in mind when you’re preparing
the post-spike solvent. Due to peptide solubility
issues or non-specific binding considerations, it
may be helpful to add 50 microliters of stock
solution that contains a carrier protein and the organic, such
as 80/20 acetonitrile in water with 1% formic acid
containing 0.05% rat plasma. So to calculate
the matrix effects, effectively, we have two vials. Both vials contain
5 nanograms per mil of analyte, which
means we’ve had 50 microliters elution
solvent and 50 microliters of the post-spike solvent. The post-spike sample also
contains the compounds extracted from
the sample matrix. The other part of calculating
recovery and matrix effects that I often find can be
confusing to scientists is actually just how to set
this up in a 96-well plate or in cartridges, so I’d like
to take just a few minutes to talk about that as well. Now, in the
calculations, we talked about creating your
post-extracted spike sample, your extracted sample, and also
your standard solution, so it’s important to know where to put
these on your 96-well plate and in what order so that
you can get accurate results. If you take a look at this
diagram of a 96-well plate, you can see how we
would set this up. For example, if we focus in
on the post-extracted spike sample, or PESS,
you can see that we would run our blank sample
matrix through the SPE process, and then actually spike
the standards directly into the collection plate
and those wells at the end. And here we do four replicates. If we look at the
extracted sample, you’ll see that we spike
our standards directly into that sample matrix
before the SPE process and just collect the final
eluate into those wells. To create the sample for
the standard solution, what we actually do is pipette
the final elution solution that’s used in the
SPE protocol directly into the wells of
the collection plate, and then we spike
in the standards. There’s no SPE actually
being performed at all into those wells. We really hope this module will
help prepare you to calculate recovery and matrix effects. For more information about
this or other related topics, you can always visit the
Bioanalysis Boot Camp to learn more and increase
your peptide and protein quantification fitness. Thank you.