Speaker 1: Imagine you have a severe, like a life threatening infection.
Speaker 1: You go to the pharmacy, you get your prescription, and
Speaker 1: you swallow this perfectly manufactured, chemically pure antibiotic.
Speaker 2: Right, you think you're safe exactly.
Speaker 1: But instead of curing you, it does absolutely nothing. I mean,
Speaker 1: the chemistry in the pill was completely flawless, but the
Speaker 1: biology failed. It's a it's a terrifying scenario.
Speaker 2: Oh absolutely. It completely shatters that illusion of binary perfection
Speaker 2: we all kind of walk around with. We tend to
Speaker 2: think a pill is just a pill. You know, it's
Speaker 2: either medicine or it's not.
Speaker 1: Yeah, it's black and white, right, But step.
Speaker 2: Into the hidden world behind that plastic bottle, into the
Speaker 2: actual rigorous landscape of pharmaceutical drug analysis, and you realize
Speaker 2: that a chemical compound actually becoming a save, life saving medicine.
Speaker 2: It's a modern miracle.
Speaker 1: It is the absolute definition of a high stakes microscopic
Speaker 1: obstacle course. And well, that is exactly our mission for
Speaker 1: this deep dive.
Speaker 2: Today.
Speaker 1: We have in front of us the textbook Pharmaceutical Drug Analysis,
Speaker 1: the revised second edition by Ashutoshkar.
Speaker 2: A fantastic comprehensive techt.
Speaker 1: It really is so whether you are a pharmacy student
Speaker 1: prepping for a career in the lab, a young professional,
Speaker 1: or just a learner who is insanely curious about how
Speaker 1: the modern world actually functions. Under a microscope, we are
Speaker 1: going to decode this whole process for you. We're covering
Speaker 1: everything from the underlying philosophy of drug creation all the
Speaker 1: way to the cutting edge analytical tools that guarantee purity.
Speaker 2: And we really have to start with that underlying philosophy, right,
Speaker 2: which medicinal chemistry today basically defines as the pursuit of
Speaker 2: better drugs for a better world.
Speaker 1: Yeah, we aren't just mixing standard chemicals and a beaker
Speaker 1: and hoping for the best anymore.
Speaker 2: Not at all. The sheer technological leaps we've made, they
Speaker 2: necessitate a level of scrutiny that would have been completely
Speaker 2: unimaginable just a few decades ago.
Speaker 1: Okay, let's untack this because to understand how we test
Speaker 1: these drugs, we really have to look at the tools
Speaker 1: we use to invent them in the first place. The
Speaker 1: source material highlights things like CATM right, computer aided drug modeling.
Speaker 2: Yes, KDM is huge.
Speaker 1: We aren't discovering drugs by accident anymore. We are literally
Speaker 1: using supercomputers to physically design a molecule on a screen
Speaker 1: like a three D puzzle piece meant to fit one
Speaker 1: specific disease receptor perfectly exactly.
Speaker 2: And we're pairing that with combinatorial chemistry, which basically means
Speaker 2: we use computer controlled robotics to rapidly generate thousands of
Speaker 2: slightly different molecular variations just to find the absolute most
Speaker 2: potent one.
Speaker 1: Right, and then delving deep into biotechnology to engineer these
Speaker 1: incredibly specific molecules designed to be highly toxic to a disease,
Speaker 1: but you know, virtually harmless to a human.
Speaker 2: Being, which is an incredible feed of engineering.
Speaker 1: It makes me think of engineering a highly advanced spacecraft.
Speaker 1: You don't just bolt the titanium together, fill it with fuel,
Speaker 1: and hit the launch button. You definitely shouldn't, right, Modern
Speaker 1: drug development is completely defined by its pre flight inspections,
Speaker 1: because when you are dealing with a microscopic chemical that
Speaker 1: is potent enough to alter human biology, a single microscopic
Speaker 1: flaw in the manufacturing process isn't just a simple error.
Speaker 1: It's a catastrophe.
Speaker 2: It is. And if we connect this to the bigger picture,
Speaker 2: you start to see why the regulations are so astronomically strict.
Speaker 2: Because these new drug entities are so active and so specific,
Speaker 2: the risk benefit ratio has to be pegged to an
Speaker 2: absolute bare minimum.
Speaker 1: There's no room for air zero.
Speaker 2: We are looking at strict, legally binding enforcement by official compendia,
Speaker 2: So the USP, the British pharmacopia, the European pharmacopia.
Speaker 1: And we hear these acronyms all the time, right, like
Speaker 1: cGMP or PAIS. But these aren't just bureaucratic red tape.
Speaker 2: So far from it. Current Good Manufacturing Practices or cGMP
Speaker 2: is a relentless legal framework. Yeah. I mean it mandates
Speaker 2: that every single piece of equipment, every scale, every thermometer
Speaker 2: in a lab is calibrated and validated continuously.
Speaker 1: Pais the pre approval inspections right.
Speaker 2: That means the FDA or the relevant regulatory body physically
Speaker 2: sends inspectors into the manufacturing plant. They audit everything before
Speaker 2: a drug is even allowed to hit the market. They
Speaker 2: demand that our analytical methods evolve just as fast as
Speaker 2: the drugs themselves.
Speaker 1: Okay, wait, I need to stop you there. This brings
Speaker 1: us back to that terrifying scenario I mentioned at the
Speaker 1: very start.
Speaker 2: The pure pill that fails.
Speaker 1: Yes, if a lab follows all these strict cGMP rules
Speaker 1: and they produce a pill that is one hundred percent
Speaker 1: chemically pure, how can it fail? Isn't the act of
Speaker 1: ingreding exactly what it says on the bottle?
Speaker 2: Well, it might be perfectly pure in the glass vial,
Speaker 2: but glass viles don't have stomachs, they don't have liver
Speaker 2: ensigns or intestinal walls. This is the critical transition from
Speaker 2: the highly controlled realm of laboratory chemistry into the chaotic,
Speaker 2: wet environment of human biology. We really have to talk
Speaker 2: about bioavailability.
Speaker 1: Bioavailability the textbook defines this as the rate and extent
Speaker 1: to which the active ingredient is absorbed from the drug
Speaker 1: product into the body or to the specific site.
Speaker 2: Of action, exactly which analysts measure by tracking things like blood,
Speaker 2: serum plasma levels, or even urinary excretion data. Over time,
Speaker 2: medical scientists rely on blood level attainment studies as the
Speaker 2: ultimate indicator of therapeutic equivalents.
Speaker 1: Because if it's not in the blood, it's not working.
Speaker 2: Precisely. Clinical efficacy entirely depends on how well that drug
Speaker 2: actually enters your bloodstream, and there are three major factors
Speaker 2: governing this the onset of therapeutic activity, the intensity of
Speaker 2: the therapeutic effect, and the duration of that effect.
Speaker 1: Wait, I want to dig into how a chemically perfect
Speaker 1: pill fails those three factors because the source material points
Speaker 1: to formulation variables, right, especially for drugs with low solubility.
Speaker 2: Yeah, think about the physical characteristics of the powder inside
Speaker 2: that capsule. What is the particle size, what's the crystalline structure,
Speaker 2: because a molecule can arrange itself into different crystal shapes,
Speaker 2: which you call polymorphs.
Speaker 1: Okay, and why does that matter?
Speaker 2: Well, one shape might dissolve in water in five minutes
Speaker 2: while the other takes five hours. What binding agents or
Speaker 2: excipients were used to pack the powder into a tablet.
Speaker 2: All of these things dictate how fast that tablet actually
Speaker 2: breaks apart and dissolves in gastrointestinal fluids.
Speaker 1: It's like it's like having the exact, perfectly cut key
Speaker 1: to unlock a door, but the key is encased in
Speaker 1: this solid block of ice.
Speaker 2: Oh that's a great analogy.
Speaker 1: Yeah, the chemistry the shape of the key is flawless.
Speaker 1: But if the ice won't melt in time for you
Speaker 1: to actually use it before the body naturally eliminates it.
Speaker 1: The delivery completely fails. The biology the onset is delayed,
Speaker 1: the intensity never peaks, and the duration is just zero.
Speaker 2: That is a brilliant way to picture it. If the
Speaker 2: drug is eliminated before the ice melts, it is medically useless.
Speaker 1: So knowing how incredibly crucial both this chemical purity and
Speaker 1: this biological delivery are, how do scientists actually measure them?
Speaker 1: How do they prove to an FDA inspector that the
Speaker 1: key isn't trapped in ice and that the key itself
Speaker 1: is the right shape?
Speaker 2: They rely on the analyst's toolbox. It starts with establishing
Speaker 2: very specific physical constants and then backing them up with
Speaker 2: rigorous statistical validation. These constants are essentially the unique measurable
Speaker 2: fingerprints of a pharmaceutical chemical.
Speaker 1: Here's where it gets really interesting to me. We aren't
Speaker 1: just talking about, you know, putting a powder on a
Speaker 1: digital scale. One of the constants is specific optical rotation.
Speaker 1: We are literally talking about measuring the exact angle that
Speaker 1: light rotates when it passes through a molecule.
Speaker 2: Yeah. We use a device called a polarimeter for this.
Speaker 2: Because pharmacological activity is intimately related to the three dimensional
Speaker 2: configuration of a drug molecule. Many complex drugs are chiral, meaning.
Speaker 1: They have a left handed version and a right handed version.
Speaker 2: Exactly chemically they have the exact same atoms, but biologically
Speaker 2: they are mirror images.
Speaker 1: Which matters because the receptors in your body are also
Speaker 1: three D. If the drug is the right handed version,
Speaker 1: it might cure your disease perfectly, but if it's the
Speaker 1: left handed version, it might not fit the receptor at all,
Speaker 1: or worse, it can cause terrible side effects.
Speaker 2: Right, and shining polarized light through the solution tells you
Speaker 2: exactly what shape you have. The textbook uses ergometrine malleot
Speaker 2: as an example here to prove its optically pure and
Speaker 2: safe to use, and analyst shines polarized light through a
Speaker 2: one percent solution of it in water.
Speaker 1: And it has to hit a specific angle.
Speaker 2: It is legally required to rotate exactly between plus fifty
Speaker 2: degrees and plus fifty six degrees. If it rotates ay
Speaker 2: forty five degrees, the physical shape of the molecule in
Speaker 2: that batch is wrong. The batch is rejected.
Speaker 1: Wow, and the specificity extends to everything, doesn't it? Like weight?
Speaker 1: Permeliator is used to differentiate liquids that might look completely
Speaker 1: identical to the naked eye. The booknoes Araqi's oil must
Speaker 1: weigh between point nine zero eight and point nine two
Speaker 1: zero grams per mili leader.
Speaker 2: While benzal alcohol is significantly heavier.
Speaker 1: Right, it has to weigh between one point zero four
Speaker 1: zero and one point zero five zero grams.
Speaker 2: We also look at loss on drying, which is basically
Speaker 2: a highly regulated test to measure moisture and volatile impurities.
Speaker 1: I love how strict this particular test is. You don't
Speaker 1: just like leave the powder out in the sun. If
Speaker 1: you want to test aspirin, you have to put it
Speaker 1: under reduced pressure, specifically over silica jel, which absorbs moisture
Speaker 1: for exactly five hours.
Speaker 2: Yes, the conditions are incredibly exact.
Speaker 1: And the compendia dictates it can lose not more than
Speaker 1: or nmt zero point five percent of its weight. Any
Speaker 1: more than that and your aspirin is contaminated with too
Speaker 1: much water or some volatile solvent.
Speaker 2: What's fascinating here is how analysts manage the inevitable errors
Speaker 2: that occur when measuring all these constants. Because in any
Speaker 2: laboratory operating under the laws of physics errors will happen.
Speaker 2: The textbook makes a vital distinction here between determinate errors
Speaker 2: and indeterminate errors.
Speaker 1: Determined errors are systematic, right. They are things you can
Speaker 1: actually pinpoint and fix. So if your analytical balance is
Speaker 1: calibrated wrong, or you're using a solvent that has a
Speaker 1: known impurity in it, that's a systematic error, right.
Speaker 2: And you minimize those by running parallel control tests, calibrating
Speaker 2: your apparat as constantly, or wenning a blank test with
Speaker 2: just the solvent.
Speaker 1: But indeterminate errors are random. They are the slight, uncontrollable
Speaker 1: variations in the temperature of the room, or microscopic fluctuations
Speaker 1: in the electrical current running the polarimeter, or just simple
Speaker 1: human observational limits.
Speaker 2: You can't eliminate them physically. You have to handle them mathematically.
Speaker 1: Which means we are bringing statistics into the chemistry lab.
Speaker 2: Absolutely, you use statistical treatments of finite samples. Analysts look
Speaker 2: at the distribution of random errors, and they use complex
Speaker 2: tools like the method of least squares.
Speaker 1: Let's make sure we clarify that conceptually, the method of
Speaker 1: lead squares is a way to find the truth hidden
Speaker 1: inside noisy data. So if you plot a bunch of
Speaker 1: test results on a graph, and they look like a
Speaker 1: scattered cloud of dots because of those random errors.
Speaker 2: The method of lease squares is a mathematical formula that
Speaker 2: draws the absolute best fitting straight line through that cloud. Right.
Speaker 1: It literally minimizes the square of the distance between every
Speaker 1: single dot and the line, giving you the most accurate
Speaker 1: posts average of your data.
Speaker 2: Which is critical because if you are testing a batch
Speaker 2: of powerful heart medication, you can't just guess if a
Speaker 2: weird test result was a random statistical fluke or a
Speaker 2: sign of a highly toxic impurity. You need strict mathematical
Speaker 2: criteria for rejecting an observation. That is what ensures the
Speaker 2: data you are using to release that drug to the
Speaker 2: public is scientifically sound.
Speaker 1: Okay, so we've got the basics down. We're drying out
Speaker 1: aspirin over silica jel, we're shining light through ergometrin. We're
Speaker 1: drawing statistical lines of bentunefit. But as we discussed earlier,
Speaker 1: molecules are getting vastly more complex, very much so. A
Speaker 1: simple physical constant weight or optical rotation is great for
Speaker 1: a relatively simple substance in a beaker, But what happens
Speaker 1: when you have a massive, highly complex biological protein.
Speaker 2: That is exactly where the basic toolbox hits its limit.
Speaker 2: When we move into the realm of complex organic medicinal compounds,
Speaker 2: biotechnological proteins, or trace level hormones, we have to rely
Speaker 2: on it advance separation and detection technologies.
Speaker 1: We are talking about high performance liquid chromatography HPLC.
Speaker 2: HPLC is arguably the cornerstone of modern pharmaceutical analysis. It's
Speaker 2: used for isolating natural active compounds, essaying cephalosporins, which is
Speaker 2: a major class of antibiotics, and controlling microbiological processes.
Speaker 1: Let's explain how this actually works for everyone, because it
Speaker 1: sounds intimidating. Chromatography at its core is kind of like
Speaker 1: a racetrack. You have a stationary phase, which is the
Speaker 1: track itself, often packed inside a metal column.
Speaker 2: Okay, I like this analogy, and you have a.
Speaker 1: Mobile phase, which is a liquid solvent pumped through the
Speaker 1: column under incredibly high pressure. You inject your drug sample
Speaker 1: into this liquid flow. Now, as the mixture travels down
Speaker 1: the column, different molecules stick to the track differently based
Speaker 1: on their specific chemical properties.
Speaker 2: So some move fast, some move slow.
Speaker 1: Exactly some molecules breeze right through and others lag behind.
Speaker 1: By the time they reach the detector at the finish line,
Speaker 1: they have separated completely.
Speaker 2: In the level of separation we can achieve with this
Speaker 2: racetrack is just staggering. The textbook highlights this with insulin,
Speaker 2: specifically differentiating human insulin from poor sign or pig insulin.
Speaker 1: This is a huge deal because for a long time,
Speaker 1: diabetics had to use animal insulin, which could cause bad
Speaker 1: immune reactions. Human insulin is much safer, but the two
Speaker 1: proteins are incredibly similar. Telling them apart is notoriously.
Speaker 2: Difficult, very difficult to do it. Animals have to use
Speaker 2: a very specific HPLC setup. They use a vite ac
Speaker 2: C eighteen column, which is a specific type of stationary track.
Speaker 2: They maintain the column at exactly forty degrees celsius just
Speaker 2: to control the viscosity of the flow. Everything has to
Speaker 2: be perfect everything. They use a highly specialized liquid mobile
Speaker 2: phase and the detection at the finish line happens by
Speaker 2: shining ultraviolet light at a wavelength of exactly two hundred
Speaker 2: and fourteen nanimeters.
Speaker 1: And even with all of that, the peaks on the
Speaker 1: read out basically the visual representation of the molecules crossing
Speaker 1: the finish line. They are incredibly close together. The compendious
Speaker 1: states that if the resolution between those two peaks isn't
Speaker 1: at least one point three, the test is invalid. You
Speaker 1: cannot legally certify the batch as pure human insulin.
Speaker 2: It's a remarkable level of precision. But HPLC is designed
Speaker 2: for analyzing the drug itself, usually during manufacturing, and this
Speaker 2: raises an important question. What do we do when the
Speaker 2: drug is no longer in a clean factory environment.
Speaker 1: Oh, like when it's in a person?
Speaker 2: Right? How do we detect drugs at absolute trace levels
Speaker 2: like picograms floating around in a chaotic complex human fluid
Speaker 2: like blood plasma? Right?
Speaker 1: Yeah, you can't just pump a patient's blood through an
Speaker 1: HPLC racetrack to find a microscopic trace of a barbiturate.
Speaker 1: I mean, it would completely clog the machine.
Speaker 2: It would ruin it. This is where the textbook introduces
Speaker 2: radioimmunoassay or RIA RIA.
Speaker 1: The book says it's a technique based on haptin determinants
Speaker 1: and competitive antibody binding using isotopically labeled compounds.
Speaker 2: Let's translate that a bit lese. A haptin is just
Speaker 2: a small molecule that can't trigger an immune response on
Speaker 2: its own, but it can if you attach it to
Speaker 2: a larger carrier protein. By doing that in a lab,
Speaker 2: scientists can essentially force an animal to create antibodies that
Speaker 2: are perfectly designed to target one specific drug molecule.
Speaker 1: Okay, So once you have those highly specific antibodies, ARIA
Speaker 1: is widely used for analyzing drugs like morphine, hydromorphone, clonazepam,
Speaker 1: and flunosolide directly in human plasma.
Speaker 2: Yes, when I.
Speaker 1: Was trying to visualize the actual mechanism of how this
Speaker 1: test is performed, I realized Ria is basically a microscopic,
Speaker 1: high stakes game of musical chairs.
Speaker 2: It absolutely is. Let's break that down. How does that work?
Speaker 1: So think about a test tube. The antibodies that the
Speaker 1: analysts put in the tube are the chairs. There is
Speaker 1: a limited, strictly fixed number of them. Now, the analysts
Speaker 1: introduce the players to the game.
Speaker 2: Right.
Speaker 1: The players are a mixture of two things, radioactive artificially
Speaker 1: labeled drug molecules that the analysts add themselves and the
Speaker 1: actual unlabeled drug molecules from the patient's blood sample.
Speaker 2: And all those molecules are the exact same shape, so
Speaker 2: they're all fighting for those limited antibodied chairs exactly.
Speaker 1: It's competitive binding. The radioactive molecules and the patient's drug
Speaker 1: molecules compete to sit down. When the reaction reaches equilibrium.
Speaker 1: When the music stops, you literally wash away any molecules
Speaker 1: that didn't get a seat.
Speaker 2: You're out of the game right now.
Speaker 1: Because you know exactly how many radioactive molecules you started with.
Speaker 1: You simply measure the radioactivity of the ones that manage
Speaker 1: to get a chair.
Speaker 2: And the math here is just beautifully simple in its logic.
Speaker 2: If the radioactivity left in the tube is really low,
Speaker 2: it means the patient's blood was absolutely flooded with the
Speaker 2: actual drug, and the actual drug stole all the seats
Speaker 2: from the radioactive ones.
Speaker 1: Yeah. By measuring the radioactive losers, you can calculate the
Speaker 1: exact concentration of the drug in the patient's plasma.
Speaker 2: It is an ingenious mechanism, really is. And the reason
Speaker 2: RIA is so incredibly powerful for clinical monitoring is because
Speaker 2: of its combined isotope dilution and stereospecificity. The antibodies we
Speaker 2: use as chairs are so specific they will only accept
Speaker 2: that exact molecular shape, completely ignoring the thousands of other
Speaker 2: proteins and chemicals naturally floating in a patient's blood. It's amazing.
Speaker 2: It allows us to monitor the pharmacokinetics, basically, how the
Speaker 2: drug is moving through a patient's body in real time,
Speaker 2: at concentration levels that were completely invisible to science thirty
Speaker 2: years ago.
Speaker 1: So what does this all mean for us? We've journeyed
Speaker 1: from the high minded philosophy of medicinal chemistry, you know,
Speaker 1: using supercomputers to build better drugs for a better world,
Speaker 1: right down to the invisible, statistical and radioactive battles happening
Speaker 1: in test tubes to guarantee those drugs actually work in
Speaker 1: a human stomach.
Speaker 2: It is a true testament to the rigorous, often completely
Speaker 2: unappreciated work of the pharmaceutical analyst. They are the gatekeepers.
Speaker 2: They are the only thing standing between a theoretical chemical
Speaker 2: breakthrough and a practical, safe cure.
Speaker 1: Adapting our specific focus as educational content creators for you
Speaker 1: the listener, whether you are prepping for a lab, practical
Speaker 1: or you're just fascinated by the science of medicine, we
Speaker 1: want to do a quick mental exercise to reinforce what
Speaker 1: we've unpacked today. Are you ready?
Speaker 2: That's good?
Speaker 1: Think back to the transition from the lab to human biology.
Speaker 1: The key in the block of ice. Can you name
Speaker 1: the three major factors that govern a drug's efficacy through
Speaker 1: its bioavailability? Take a second, think about it, got it?
Speaker 1: It's the onset of therapeutic activity, the intensity of the
Speaker 1: therapeutic effect, and the duration of that effect. If those
Speaker 1: three don't align perfectly in the body, driven by formulation
Speaker 1: variables like particle size, the chemical purity in the lab
Speaker 1: simply doesn't matter.
Speaker 2: And that reality brings me to a quote from the
Speaker 2: source material that perfectly captures this endless, relentless pursuit of precision.
Speaker 2: It's some zick Meister and Chalmooky who wrote, every scientific
Speaker 2: advance is an advance in method. The invention of a
Speaker 2: new specialized laboratory procedure brings about rapid conquests and new
Speaker 2: fields of science and technology. Finally, it exhausts itself and
Speaker 2: is replaced by still more practical method.
Speaker 1: We build the polarimeter, we build the HPLC column, and
Speaker 1: eventually we replace them with something even better, always pushing
Speaker 1: the boundary of what we can see, which.
Speaker 2: Leads us with a profound question to ponder as we
Speaker 2: wrap up. As our analytical methods become so incredibly sensitive,
Speaker 2: like advanced HPLC and ria that they could detect a
Speaker 2: single solitary rogue molecule in a batch of millions, will
Speaker 2: our very definition of a pure drug eventually have to
Speaker 2: change entirely? At what point does a microscopic trace impurity
Speaker 2: simply cease to matter to human biology? And how will
Speaker 2: the analytical gatekeepers of the future draw that line.
Speaker 1: That is a fascinating thought to leave on, because right now,
Speaker 1: when we open that medicine cabinet, we expect absolute binary perfection,
Speaker 1: but ensuring that perfection is a constant, rigorous fight against
Speaker 1: the messy realities of chemistry, statistics, and biology. Thanks for
Speaker 1: joining us on this deep dive. Keep questioning the things
Speaker 1: you assume are simple, and we'll catch you on the
Speaker 1: next one.