- So, Jonathan do we have anything in the email today?
- I dunno.
You usually check.
- Ugh.
- I'll check, I'll check, no problem.
- 'Kay.
- Whoa, wow, holy smokes, we got another one on SPE.
- Oh!
- Yeah, one about, is protein precipitation
good enough when dealing with biological samples?
- Oh, that is a fantastic question.
I'm excited to tackle this one.
- [Jonathan] Yeah, yeah, we know that
biologics have things in there like salts,
fats, proteins, even these things, phospholipids.
- Yep, but we have to think about
how those things are going to affect our sample.
Because they can cause things like matrix effects,
cause ion suppression, they can cause
chromatography to not be reproducible.
They can have contaminants build up on our system,
and cause us all kinds of problems.
So, - Okay.
- this definitely one I'm excited to look at.
- Alright, well let's take a look at it.
- [Kim] Okay.
- Aright, Kim, I'm excited to test this myth.
How we gonna do it?
- Sure.
So this one can be straightforward.
I mean, what we'll wanna test is the
ability to just use protein precipitation
and see if we can get reproducible
quantification and chromatography.
Right? - Okay, right.
- [Kim] So, what we can do is
do a traditional protein precipitation.
We'll do a one to three protein crash
We'll use plasma.
- [Jonathan] That's good.
- [Kim] And then we'll just keep making injections
of the sample over and over and over again
with a typical gradient that you would use in bioanalysis.
- Alright. - So we'll go to
a high percentage of acetonitrile
to potentially wash off the column.
And then we'll just monitor the phospholipids,
since they're the major cause of, you know, matrix effects.
- Okay. - Can cause
issues with quantification.
- Excellent. - And then we can just
see what that looks like after,
when we take a look at the actual trace.
- So we can look at something like the area count,
for example, and we'll just
monitor that and see if that fluctuates.
- Absolutely.
- It's a good idea.
- Yep, let's set it up.
- Alright, let's do it.
(cheery music)
So, Kim, we talked about one of the major problems
when doing these biological samples is phospholipids.
So that's something that I think
we definitely are gonna have to monitor.
- [Kim] Absolutely.
- So for those that don't know
what a phospholipid is, it's this long
kind of molecule that has a polar head group
up front, and this long hydrophobic
chains that tail off at the end.
It's those hydrophobic chains that can kinda
stick or bond to a most reversed-phase column.
In this experiment, we're gonna monitor
the transition, that 184, and we'll
take a look at the data over some replicate injections.
- [Kim] Awesome.
- So, here we've done some multiple injections.
Just one after the other, just kinda monitoring
how these phospholipids kinda build up on the column.
And you can see really early on, say up to
maybe injection five or six, the phospholipid count
coming off the column isn't that much.
But over time, boy you really can see how
they're really starting to coat the column,
come off the column, and not come off at the same rate.
So you get a lot of irregularity, irreproducibility,
which causes a lot of problems for the chromatography.
- [Kim] Yep, you're exactly right, JT.
And if we look at the chromatography
to see how these phospholipids would actually
impact the analytical results,
we can see a really interesting story.
If we look at the top chromatogram here,
we can see that the first protein precipitation injection,
and here we're just monitoring the phospholipids,
but you can see that they've just started
to build up on the column, but they're really
not causing us any kind of a problem.
- [Jonathan] Right.
- [Kim] But if we look at the bottom chromatogram,
we can see that by the last injection,
now those phospholipids have really built up,
and they're starting to coelute with our
risperidone and our clopidogrel,
and they could be causing things like
matrix effects, causing higher column backpressure,
but most importantly, causing us problems
with being able to get reproducible quantification.
- [Jonathan] Yeah, so protein precipitation just
isn't good enough to clean up these biological samples.
- [Kim] Unfortunately, it's not.
It's easy, and, you know, sometimes
you know, we scientists like to do
the easiest thing. - Yep.
- [Kim] But, you know, in this case,
it's really not getting us where we need to go.
- Okay.
So Kim, how do you wanna call this one?
We kinda did our experiments.
What do you think?
- I know.
Well, I think we have to call it busted
because protein precipitation really isn't enough
to make sure that you have good reproducible quantification.
- Yeah, I agree, but I don't feel real good about it.
We are kinda leaving the customer hanging.
- I know, I feel like we're not giving them the best advice.
So, maybe we can just go a little bit
further and extend this experiment.
- Yeah, this calls for bonus time.
- I think bonus time.
- [Both] Let's do it.
- Alright I got the perfect sample,
Kim, for this experiment.
Synthetic cannabinoids, there's 22 species
in this sample mix, and the sample's whole blood.
So a real challenging, complex mixture
and a really tough matrix.
- [Kim] Oh, definitely.
So since we're gonna do whole blood,
I think let's really go after cleaning up
this sample and show our scientists what we can do.
- [Jonathan] Sure.
- [Kim] Let's do solid phase extraction,
and let's use maybe four different sorbents.
- Okay. - To see how
they all compare in performance.
But let's see specifically how they do
with cleaning up what we just looked at, phospholipids.
- [Jonathan] Alright, let's do it.
- [Kim] Okay.
(cheery music)
- [Jonathan] So here's the chromatogram
from those 22 synthetic cannabinoids.
The method is really complex.
We don't wanna be tinkering around with it too much.
You see we got everything mostly separated.
Those two isobaric compounds, peaks nine and ten,
we're able to resolve those chromatographically.
So we don't wanna mess around with that at all.
- [Kim] Yeah, sure, a lot of times
people will try to resolve matrix interferences
away from their compounds of interest.
But when you've got a complex chromatogram like this,
it's better just to take a step to remove them.
- [Jonathan] Okay.
- [Kim] So since we talked a lot about phospholipids
and matrix effects specifically, let's take a look
at those four different SPE sorbents that we used,
and also the impact of our cleanup
in terms of matrix effects, which is
the most important thing for a quantification.
So we can see here that for most of the compounds,
everything looks pretty good, but we definitely
have a problem with compound JWH-203.
We can see that there's a lot of ion suppression going on.
- [Jonathan] Yeah, what's going on with that?
- [Kim] Yeah, what exactly is happening?
- [Jonathan] What did you do?
- [Kim] (laughs loudly) So if we take a closer look,
we can see that phospholipid 524 is actually
coeluting exactly with our compound of interest, JWH-203.
So, this is definitely going to cause
a problem with our matrix effects.
So if we take a closer look at the impact
of the phospholipids, we can see that there's
a direct correlation between the presence
of the phospholipid in our final sample,
and the matrix effects.
So if we take a look at the left-hand column,
we see that sample cleanup.
And you can see the presence of the phospholipid
that we see on the left-hand side,
directly correlates to the amount
of matrix effects that we see on the right-hand side.
So you can see with the three bottom examples,
we have a lot of matrix effects.
With the top example, where the phospholipid
has effectively been removed,
we see very little matrix effects.
- Oh, Kim, way to put in a little
extra overtime there today.
- I know. - You know, I was just
trying to help out the customers.
- But it was worth it, it was.
- So let's get back to the original myth.
You know, is protein precipitation good enough?
- (sighs) I think we have to say
that one was busted,
- Yep. - like we did earlier.
But, there are things you can do
to take care of the problem.
- Exactly right.
Using some sort of SPE.
- Yep, that works.
- Yep, to get rid of those pesky little phospholipids.
- I know they're sticky.
- Yeah, so I'll write back to the customer.
I'll let them know, you know. - Okay.
- Yep.
- Yep, let them know there are options.
If you'd like your question to be answered
on a future episode, please feel free
to email us at trustyourscience@waters.com.
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