124 - Spectroscopy & Quality Control (S9E4)

Welcome to the deep dive. Today we're going to
be going pretty deep into the world of spectroscopy.
It's something that maybe a lot of folks don't
think about on a daily basis, but it's pretty
vital in making sure that all the medicines we
rely on are safe and effective. Absolutely. Think
of it kind of like a detective working behind
the scenes in the pharmaceutical world. Exactly.
Making sure everything is up to par. Making sure
that that pill that you take actually has what
it says it has in it. Right. And that it's pure
and it's going to work the way it's supposed
to. Absolutely. Yeah. And to really get into
this, we've got a pretty interesting stack of
sources. We've got everything from really in
-depth scientific articles that get into the
nuts. and bolts of each technique, all the way
to some pretty serious regulatory documents that
spell out what's required from a quality standpoint.
Absolutely. So it's going to be a pretty comprehensive
view. Our mission today is really to unpack these
techniques, understand how they're applied across
the entire lifespan of a drug, from its initial
development to its large scale manufacturing.
Yeah, from start to finish. Yeah. How do these
things ensure the quality of the final product
that ends up at your pharmacy or in your medicine
cabinet? Absolutely. Yeah. So first off, what
are these techniques that we keep mentioning?
So spectroscopy, in a nutshell, is the study
of how matter interacts with electromagnetic
radiation. OK. Now that might sound. a little
bit complicated, but think of it this way. By
analyzing the patterns of absorption or emission
of light, we can get a ton of information about
the composition and structure of a substance.
And we're going to focus on a few key methods
that are widely used in the pharmaceutical industry.
Got it. And they all work together to make sure
that the medicine you take is good. Let's start
with UVV spectroscopy. I think this one, a lot
of people, maybe they've heard the name. Sure.
But maybe they're not exactly sure what it is.
So UVV spectroscopy, or ultraviolet visible spectroscopy,
is based on a pretty simple principle. It looks
at how molecules absorb light in the ultraviolet
and the visible parts of the electromagnetic
spectrum. The amount and the pattern of that
absorption is unique to different compounds.
And it's related to the electronic structure.
So really what UVVs lets us do is get a really
reliable head count of the molecules that we're
interested in. So you can use it to identify
what's there. Absolutely. And how much is there.
Exactly. Kind of like using a, I don't know,
shining a light through a colored solution and
seeing how much of that light gets through. Absolutely.
And based on that, you know, you can get a feel
for the concentration. You got it. OK, so UVVs,
it's kind of like a molecular fingerprint based
on how it absorbs light. Precise. And it's used
to identify things and quantify, right? Yes.
You mentioned the regulatory aspects too. Yeah,
so this ties in directly with making sure that,
you know, pharmaceutical manufacturers are following
the rules. Things like 21 CFR211, which is a
regulation that dictates how drugs should be
manufactured. Right. And that ultimately affects
the quality of medicine that you receive. All
right. Yeah. So that's UV VICE. Yes. What about...
infrared or IR spectroscopy. Okay. I've heard
about that. So IR spectroscopy looks at a different
part of the electromagnetic spectrum. Okay. This
one tells us about how molecules vibrate. Vibrate.
Yeah. Right. So when a molecule absorbs infrared
light of a specific wavelength, it causes its
bonds, its chemical bonds, to start vibrating.
Huh. Right. And those different functional groups,
different parts of a molecule, they vibrate at
characteristic frequencies. Okay. So for example,
a carbonyl group or a hydroxyl group, these are
different parts of a molecule. They're going
to vibrate at different frequencies. Got it.
So think of it like IR spectroscopy is giving
each molecule a unique vibrational signature.
And that allows us to really pinpoint. what functional
groups are there. So you're looking at what wavelengths
of light are absorbed. Exactly. And from that,
you can infer the functional groups that are
present. You got it. Okay. So it's like identifying
the ingredients in a recipe based on, you know,
if you knew what each ingredient tasted like,
you could kind of tell by the overall flavor
what's in there. So IR is good for identifying
materials and figuring out the structure based
on these functional groups. Absolutely. It's
like identifying the specific building blocks
within a molecule. Okay. Which is really important
to make sure that the drug you take has the right
structure. Got it. Yeah. So we've got UVVs for
identifying compounds, quantifying them. We've
got IR for the functional groups. Yes. Now there's
this thing called Raman spectroscopy, which sounds
a little bit different. It is a little different.
So Raman spectroscopy, it actually looks at the
scattering of light. So not absorption, but scattering.
Right. And specifically, it looks at inelastic
scattering. Inelastic. Yeah. OK. So when light
interacts with a molecule, most of it's going
to scatter off with the same energy that it had
when it went in. Right. But a small fraction
of it will be scattered inelastically, meaning
it loses or gains a little bit of energy. OK.
So its wavelength changes slightly. Got it. And
we call that change in wavelength the Raman shift.
OK. And that gives us information about the molecules'
vibrational modes. Okay. Kind of similar to what
we were talking about with IR. Got it. But it's
a slightly different approach. So instead of
looking at what light is absorbed, you're looking
at how the light changes when it scatters off
the molecule. Exactly. Okay. Yeah, it's like
shining a light on something and seeing how it
wiggles the light back. Right. And those wiggles
tell us something about the molecule structure.
Got it. Okay, so you mentioned inelastic scatterings,
the light kind of, it loses or gains a little
bit of energy. Exactly. When it interacts with
the molecule. Yeah. And that's kind of the basis
of the technique. That's how we get the information.
OK. Yes. And you were saying that this technique
has some advantages over the others. It does
in some cases. So one of the cool things about
Raman is that it can often be used for non -destructive
analysis. OK. Meaning you don't have to destroy
your sample to analyze it. Right. So you can
imagine if you had a medicine, a sealed medicine,
you could actually analyze it through the packaging.
Wow. Without ever having to open it. That's pretty
impressive. Yeah. Okay, so non -destructive.
Non -destructive. What else? Another advantage
is that water doesn't scatter Raman light very
strongly. Okay. So this makes it really useful
for analyzing aqueous solutions, right? Right.
Things that are dissolved in water or biological
samples. Got it. Which are very common in the
pharmaceutical world. Right. Yeah. And didn't
you mention something about it being used in,
in like manufacturing? Yes. So Raman actually
has a really great potential for in -process
monitoring. Okay. So what that means is you can
actually use it while you're making a drug. Wow.
Wow. So for example, Raman spectroscopy can be
used to monitor blend uniformity. Okay. So in
continuous manufacturing, you're making a tablet,
right? You're mixing up all these powders, the
active ingredient, with other things. Right.
And you want to make sure that it's all mixed
evenly. Absolutely. So Raman can be used to check
that in real time. Okay. And if something goes
wrong, if it's not homogeneous, it can actually
trigger an alarm. Wow. And you can adjust the
process right away. So it's like a feedback loop.
Exactly. Core quality control. Real time quality
control. Right there in the manufacturing line.
Exactly. That's incredible. Yeah, it's a really
powerful technique. OK, so UV vis, IR, now we
got to talk about NMR. OK, yeah. Nuclear magnetic
resonance. That one, I always got to admit, that
one always sounded pretty complicated. It's definitely
the most intricate of the techniques that we've
talked about. OK. But it gives you just incredible
detail about the structure of molecules. OK.
So the way it works is you place a sample in
a really strong magnetic field. OK. And then
you expose it to radio frequency radiation. OK.
Now certain atomic nuclei like hydrogen 1 and
carbon 13, they can absorb this radiation. Right.
And then they emit it back out at specific frequencies.
OK. And those frequencies are sensitive to the
magnetic environment of that nucleus. Okay. So
basically you're listening to those nuclei as
they send out these signals. So it's like you're
tuning into a radio station, but the radio station
is a specific atom in the molecule. Exactly.
And it's telling you something about its environment.
Exactly. It's like a little radio transmitter
inside the molecule. Okay. Giving us information
about what's around it. Got it. Yeah. And from
that, you can kind of get a feel for the connectivity.
Like how are all these atoms connected? You can
figure out the bonds between the atoms. You can
figure out the three -dimensional shape of the
molecule. Wow. You get a lot of information from
NMR. So that one is definitely the most detailed.
Yes, absolutely. You were saying that's particularly
good for organic compounds? It is. It's really
the gold standard for identifying and characterizing
organic compounds. Got it. And in fact, our sources
talk about how it's used in drug development.
Okay. Right, so when they're doing organic processed
research and development, right, they're actually
using NMR to figure out, you know, are they making
the right molecule? Got it. And that helps ensure
that the drugs are safe and effective. So it's
like it's used across the board in a lot of...
It's a very versatile technique. Yeah. Yeah.
All right, so we've got these four techniques.
Yes. They all provide different information about
the molecule. Right. Now, how are these things
used in quality control, specifically in the
pharmaceutical world? So one of the primary roles
of these spectroscopic techniques is compound
identification. OK. So at every stage of the
drug making process, you need to confirm what
you have. Right, so you start with your raw materials,
you have your intermediates as you're making
the drug, and then of course the final drug product.
Got it. So you need to make sure that you have
the right thing at each step. Right. So we do
that by comparing the spectrum of an unknown
sample to a reference standard. So the reference
standard is basically a known pure sample of
the thing that you're trying to make. And so
if they match, you're good. Right. You know you
have what you're supposed to have. And that's
super important, you know, for making sure that...
It's absolutely critical. Yeah, the drug that
ends up on the shelf is actually what it's supposed
to be. Yeah, you don't want any mix -ups, right.
Right. And again, the regulations are really
clear about this. Things like 21 CFR 330, which
talks about the identity, strength, quality,
impurity of drug substances. Right. So you have
to be able to prove that you have what you say
you have. Got it. And spectroscopy is the way
to do that. So it's like having a whole library
of fingerprints. Right. But instead of fingerprints,
they're molecular fingerprints that you can use
to compare again. Exactly. It's like a molecular
database. OK. So that's compound identification.
Yes. What about purity? Purity is absolutely
critical as well. Right. And spectroscopic techniques
can also be used to detect and quantify impurities.
OK. So if you're looking at a spectrum and you
see some unexpected peaks or deviations from
the pure standard, that's a sign that something
else is there. Right. That something might be
a contaminant. OK. And that's important because
Impurities could affect the efficacy of the drug.
They can also have toxic effects. So you really
want to make sure that your drug substance is
as pure as possible. And our sources actually
talk about how much effort goes into this. So
there's a handbook on isolation and characterization
of impurities, which goes into detail about all
the different ways that you can try to isolate
and identify these impurities. And there are
even organizations like the PQRI, the Product
Quality Research Institute, which specifically
focuses on mutagenic impurities. So these are
impurities that could potentially cause mutations
in your DNA. Wow. So there's a lot of work that
goes into making sure that the drugs are free
from those kinds of impurities. So it's not just
a matter of like, oh, is it pure or not? It's
like, are there any specific impurities that
are going to be particularly problematic? Exactly,
yeah. And you need sensitive methods to detect
those. Got it. Yeah. All right. So we've talked
about compound identification, purity. Right.
Now what about the actual formulation itself?
So the final pill or the liquid that you would
take. How does spectroscopy play a role there?
Right, because it's not just about the active
ingredient. It's about how it's all put together
into the final product. So we can use spectroscopy
to characterize the formulation. OK. So for example,
we can use IR and Raymon to look at the solid
state of a drug. Solid state. Yeah, so things
like polymorphism. Polymorphism, now that's where
things get a little bit... Polymorphism is basically
the ability of a solid... to exist in different
crystal structures. So the same molecule can
pack together in different ways. And that can
affect things like solubility and bioavailability,
how well the drug is absorbed by your body. And
we actually have a whole book on polymorphism
in our sources that goes into a lot more detail
about that. So IR and Raman can tell us about
those different crystal structures. And then
NMR. It can even be used to study reaction conversion
during formulation. So how much of the starting
material has actually been converted into the
final product? Got it. And it can also be used
to look at the impact of different additives
on the formulation. Got it. So things like preservatives
or stabilizers that are added to the drug to
help it last longer. Right, all the inactive
ingredients. Exactly. OK. So it's really about
understanding Not just the active ingredient
itself, but how the whole formulation behaves.
Absolutely. How does it dissolve? How stable
is it? All these things are important. Okay,
so we've talked a lot about, you know, what each
of these techniques can do. Yes. Now every tool
has its limitations. Of course. So let's talk
about some of those. Okay. Like where does each
of these techniques fall short? So yeah, each
one has its advantages and disadvantages. Right.
So starting with UVVs, it's very sensitive. OK.
And it's really good for quantitative analysis.
Right. Especially for compounds that absorb UV
or visible light. We call those chromophores.
Right. So those are compounds that are good at
absorbing those specific wavelengths of light?
Exactly. OK. However, it can sometimes lack specificity.
OK. different compounds might have similar absorption
spectra. Right. So it can tell you that something's
there, but it can't always tell you exactly what
it is. So it's like, it's like if you're trying
to identify a fruit based on its color. Right.
You know, there's a lot of red fruit. Exactly.
So it narrows it down, but it doesn't give you
the definitive answer. Right. OK. So that's one
limitation of UVves. Got it. Now, IR spectroscopy.
that gives you a lot more structural information.
Because it's sensitive to those functional groups
we talked about. So it's better at identifying
compounds. However, sometimes you need to do
some sample preparation to get a good spectrum.
And certain materials, like water, can interfere
with the measurement. So that's something to
keep in mind with IR. What about Raman? So Raman,
one of the big advantages there, we talked about
it, is that it's non -destructive. you don't
have to mess up your sample. Right. You don't
have to dissolve it in something or crush it
up or anything like that. Exactly. You can analyze
it as is. OK. And you can analyze a variety of
sample types. Oh, right. Including aqueous solutions
and even through packaging. Right. So that's
a big plus. Yeah. However, the signals can be
kind of weak sometimes. Oh, OK. So you need a
more specialized instrument to get a good signal.
Right. And those instruments can be a little
more expensive. OK. So that's something to consider
with Ramon. And finally, NMR, which we said is
kind of the most powerful in terms of structural
information. Yeah, NMR can tell you so much about
the structure. Right. You know, how the atoms
are connected, the 3D arrangement, everything.
Right. But it is less sensitive than the other
techniques. Right. So you need more sample. OK.
And it takes longer to get the data. Right. So
it's not really ideal if you're dealing with
very low concentrations, or if you need to analyze
a lot of samples very quickly. Right, if you're
doing high throughput screening or something
like that. Exactly. Okay. And the instruments
themselves, the NMR instruments, they're quite
expensive. Okay. So that's another factor to
consider. So it sounds like these techniques,
they all have strengths and weaknesses. They
do. And they're kind of complementary in a way.
They're all part of the toolkit. Yeah. And you
choose the right tool depending on the job. Right.
Like if you need to quantify something very quickly,
UVV might be the way to go. Exactly. But if you
need to really get into the weeds of the structure,
NMR might be the answer. Exactly. Or if you have
a sample that you don't want to destroy, you
might go with Remon. Right. Right. Depends on
the situation. Exactly. OK. Now, to really bring
this to life, do we have any case studies? We
can infer some potential case studies based on
the information we've read. So let's imagine
a scenario. Right. Let's say a pharmaceutical
company receives a batch of an active pharmaceutical
ingredient. Right. And they use IR spectroscopy
to analyze it. OK. And they find that it's missing
a key functional group that should be there.
OK. That's a huge red flag. Right. It could mean
that it's counterfeit or that it's degraded somehow.
Right. And so they don't use it. Right. And that
prevents potentially dangerous product from reaching
patients. Right. So that's a case where IR spectroscopy
really helped protect public health. Absolutely.
Yeah. OK. How about an example with Raymon in
a manufacturing setting? OK. So let's say you're
making tablets in a continuous manufacturing
process. Right. And you're using Raymond spectroscopy
to monitor the drug content in the powder blend.
Okay. Right, as it's being mixed. Right. And
let's say the Raymond spectrum starts to show
some deviations. Okay. Meaning that the blend
is not homogenous. Right. Some parts of the mixture
have too much drug and some parts don't have
enough. Exactly. So the system can actually trigger
an alarm. Right. Tell the operators to stop and
adjust the process. Okay. And that prevents a
batch of tablets from being produced. Right.
that don't meet the specifications. So again,
it's kind of like a real -time quality check.
Exactly. That prevents problems. Right. It ensures
that every tablet has the right amount of drug
in it. OK. Yeah. What about NMR? So NMR is often
used to deal with impurities. Right. So let's
say in drug development. OK. They're making a
new drug. Right. And they detect an impurity.
OK. And they want to figure out what it is. Right.
So they use NMR to analyze the structure. And
they're able to figure out where this impurity
came from in the synthesis. And they can modify
the process to prevent it from forming. Got it.
So that's a really important application of NMR.
So it's not just identifying the impurity. It's
like using that information to change the process.
To improve the process. To make a better product.
Exactly. Got it. And finally, a lot of folks
are familiar with the idea of a pill or a liquid
having a certain concentration of the active
ingredient. How does UVV come into play there?
So UVVs is often used for just that, to determine
the concentration of a drug. So if you have a
bottle of cough syrup, it's going to say on the
label it has this much active ingredient per
milliliter. So UVVs is used to verify that, to
make sure that it actually has the right concentration.
a day -to -day quality control. type of test.
Absolutely. OK. And it's required by good manufacturing
practices or GMP. Right. Which is a set of regulations
that ensures the quality of pharmaceuticals.
Right. So all these techniques, we see them in
kind of every aspect of drug development and
manufacturing. They're involved at every stage
from the very beginning, you know, when they're
first discovering a new drug all the way to the
end. Right. When it's sitting on the shelf of
the pharmacy. Right. Yeah. So let's talk about
that a little bit. You know, where do we see
it? early on in development. So in early drug
development, spectroscopy is really important
for characterizing new chemical entities. So
when they first make a new molecule that they
think might have some therapeutic potential,
they need to figure out what its properties are.
How soluble is it? How stable is it? How does
it interact with other things? So the spectroscopy
can.