Myths in Forensic Toxicology

We're back and we're looking to bust some myths in forensic toxicology. No conventional idea is safe!


Contact us at toxpod@tiaft.org

Find out more about TIAFT at www.tiaft.org

The Toxpod is a production of The International Association of Forensic Toxicologists. The opinions expressed by the hosts are their own and do not necessarily reflect the views of TIAFT.

Chapters

• 0:42 Do we know what's happening inside our instruments?
• 5:31 Are matrix effects only a problem in LC/MS?
• 8:59 Does validating a method guarantee its performance?
• 13:48 Do you have to use low pH for basic compounds in LC/MS?
• 18:50 Is Baselt the final word in drug interpretation?
• 22:58 Are liver drug concentrations useful?
• 27:21 Am I the only one that doesn't understand this paper?

Transcript

Tim 0:00

Hello and welcome to the first episode of the second season of The Toxpod. I'm Tim Scott.

Peter 0:17

And I'm Peter Stockham. Thanks for joining us again. U h, last season was quite a success I think. What do you reckon, T im?

Tim 0:24

Yeah, we had a lot of fun. Quite a few listeners all around the world, which is great.

Peter 0:27

We'll keep going then shall we?

Tim 0:29

Well, today in our first episode we're going to look at some myths in forensic toxicology. So these are some ideas that need to die, right Pete?

Peter 0:38

There are quite a few myths aren't there?

Do we know what's happening inside our instruments?

Tim 0:39

I think there's a lot of myths in forensic tox. What about the idea that we know what's happening to compounds in our instruments?

Peter 0:48

We know what's happening to our... so for example, we're talking about ionisation process?

Tim 0:53

Yeah, I mean...

Peter 0:54

For liquid chromatography, mass spec, no one really knows how the ionisation process works, do they?

Tim 0:57

So we inject some liquid at the start of an instrument, solvent with some various things in there. At the other end, we get some kind of data coming out- response- and we're making a lot of assumptions about everything that's happening in between. We think we know everything that's happening. But time and time again, you know, people doing research, find out, oh actually this thing that we thought was happening doesn't quite happen that way. Or there's, there's nuances to the way that we thought these compounds are behaving inside our instruments.

Peter 1:27

So for example, you might think that when you inject on a GC/MS or LC/MS, everything that you inject gets ionized and goes through to the mass spec. But in reality, only a very small percentage of the compound actually gets ionized. The rest of it just goes into the vacuum pump.

Tim 1:41

Yeah. And even the stuff that does get ionized doesn't always get ionized into what we think it's going to get ionized into. Sometimes you get dimers forming and you get all sorts of crazy things happening. Sometimes you get doubly charged ions, which we don't really take into account very much in forensic toxicology.

Peter 1:59

Well unless you've got a TOF or a full scanning instrument, you won't see them, so...

Tim 2:03

That's true.

Peter 2:04

Well going on with targeted analyses, targeted instruments, like triple quadrupoles, you're only looking for specific analytes. There could be any number of compounds co-eluting with them at the same time. And this can be, they can cause matrix effects; ion suppression. You can't really validate for that, can you?

Tim 2:20

No, I think there's, we can probably improve the way that we validate for those things, but can you ever really properly validate to take everything into account? No, not really.

Peter 2:31

So one thing I still can't get my head around is why sometimes curves go quadratic.

Tim 2:35

Well that's another myth. The curves aren't actually quadratic. We call them quadratic. They're nonlinear curves. They look quadratic in the region that we're looking at. But a quadratic curve has a point of inflection and then goes back down again, which doesn't actually happen. They just sort of...

Peter 2:50

Level off.

Tim 2:51

Peter out, and yeah... that was just an aside.

Peter 2:53

Thanks. Yeah, because this even occurs if you've got deuterated internal standards, so why would you get a lower response for your parent analyte and not your internal standard? I'm pretty sure they don't ionize differently.

Tim 3:07

Don't they ionize differently? Why wouldn't they? If they behave differently in, on the column, you get slight separation between them. Wouldn't there be the possibility that they'd ionize differently?

Peter 3:19

Well, why do you have the slight separation?

Tim 3:21

Because the interaction of the deuterium is, it's interacting differently with the phase on the column. I assume.

Peter 3:29

I was told once that deuterium's got a smaller diameter, so the actual physical size of the molecule is smaller, so maybe that affects it.

Tim 3:36

Okay. Maybe, I mean they do tend to come out earlier in reverse phase chromatography.

Peter 3:42

But you wouldn't expect, say a basic drug where the nitrogen is the part that's protonated. Why would the detection of that be affected or the ionization of that part of the molecule be affected when the deuteriums are a long way away from that.

Tim 3:54

Yeah. I, ages ago, I started an investigation into looking at amphetamine which was deuterated on the ring versus amphetamine which was deuterated on the, the other part of the molecule around the nitrogen...

Peter 4:07

Yep, d3 versus d5?

4:09

Yeah, so presumably if it's going to have an effect, the one where it's closest to the nitrogen would have more of an effect. Unfortunately, it was a bit inconclusive, that study and uh, never really finished it. Like a lot of other things I've done.

Peter 4:22

Yeah. I guess it's not that important really in the scheme of getting casework out.

Tim 4:25

Yeah. It's fun science though. I saw a meme the other day on Twitter. Uh, the reason why I can't get my scientific research program done is because I keep getting distracted by science.

Peter 4:37

Yeah. I can relate to that.

Tim 4:39

Yeah that happens to me a lot.

Peter 4:39

What about saturation of your detector or saturation of your ionization? Do you know, can you tell the difference between those two?

Tim 4:45

I mean, I guess you can tell the difference if you try and compensate somehow using another isotopologue for example, that might confirm that it's saturation at the detector. Uh, but it's hard to tell sometimes.

Peter 4:58

So going back to the quadratic issue, is that quadratic thing happening at the detector or is that quadratic thing happening at the ionization stage, using LC/MS as I always do.

Tim 5:08

Could be either.

Peter 5:10

Mmm. So, we don't know what's going on basically inside the instruments. Is that what your saying?

Tim 5:13

Well that's what makes what we do so much fun because this is, this is the area that I really love to explore in research is trying to find out what's happening inside of our instruments.

Peter 5:23

They're the annoying bits Tim.

Tim 5:25

Yeah the annoying bits that lead to fun science!

Are matrix effects only a problem in LC/MS?

Peter 5:28

Yeah, okay. Fixing them up would be good I suppose. So the next myth I want to talk about was matrix effects are really only a concern in LC/MS.

Tim 5:36

Yeah. Matrix effects have really come to the front of the conversation in, you know, the last decade or so, which happens to coincide with when LC/MS has been really taking off. And so a lot of the talk has been about Matrix effects in LC/,MS which they definitely are an issue in LC/MS, but maybe we've ignored a little bit that matrix effects can also happen in other instruments as well.

Peter 5:59

Yeah. Well most of my experience now is using LC/MS but it used to be entirely GC/MS, and in retrospect now I think there was a lot of issues that were happening in GCs, which we would now term as matrix effects. For example with the, if you had a sample which was particularly dirty, then the next sample may be, have a poor response.

Tim 6:18

Yeah. So it can affect your GC column for example.

Peter 6:20

Or maybe even it's the insert. Maybe it's the injector liner, cause there's high temperatures in there. There's lots of active sites and some drugs are susceptible to these active sites and the active sites can be, can be made worse by a dirty sample.

Tim 6:36

So is it the endogenous compounds in the sample then, are you saying, that's causing those changes in the instrument performance?

Peter 6:45

Oh it can be, I think. So, a particularly putrefactive postmortem sample that obviously has a lot more rubbish in it, which can affect the liner, the GC liner.

Tim 6:55

I know sometimes when we, we used to use GC a lot and you'd get different results for different types of matrices spiked at the same concentration just because of the impact of the Matrix on the...

Peter 7:08

Yeah, there's a lot of chemistry happening in that liner at that high temperature. And add a few active sites and endogenous compounds and things were happening.

Tim 7:16

Actually I remember we used to do, we used to analyze benzodiazepines by electron capture detector, GC/ECD, and there was one particular benzodiazepine, I can't remember which one it is now, where if we analyzed it in a neat reference standard, we'd get a certain result, but if we analyzed the same concentration in a blood sample, we'd get a vastly different result.

Peter 7:41

An increased result.

Tim 7:41

Yeah. Now what was the cause of that? I mean it's clearly matrix effects. What is the actual fundamental cause of it? I don't know. Was there something coming out at that time, some endogenous compound, which you couldn't necessarily see from the electron capture detector, but which was somehow interfering with the detection?

Peter 8:00

So something could be chromatographing and interfering with the detection or it could be at the injector port where the compound is taking up the active sites.

Tim 8:08

Yeah.

Peter 8:08

And so then the drug's not getting stuck to the active sites and then it just comes through with a higher response.

Tim 8:14

But we didn't really have a name for it back then, like matrix effects, I mean it obviously was that, but it just wasn't as well known as it is now.

Peter 8:22

And when you have derivatives like pentafluoropropyl... is that right? I havent used it for so long, I've forgotten.

Tim 8:27

Anyhydride.

Peter 8:27

PFPA, those sort of derivatives, they're often esters, which can easily be hydrolyzed. And if you've got active sites in your injector port, such as on the glass wall and it just hasn't been silanized properly, then that can actually cause a decrease in response. And again, I've seen it with, when you inject a neat standard, you get a lower response than you do with when you would inject a spiked sample.

Tim 8:54

Yeah, it comes back to not knowing what's going on in our instruments.

Peter 8:57

Yes, you're starting to concern me now.

Tim 8:59

So here's another myth, Pete. This one's to do with validation. If you validated your method, it will work properly forever.

Peter 9:07

Course it will!

Tim 9:07

Why not? That's what validation is for, isn't it? To ensure your method's going to work always in every circumstance.

Peter 9:12

Every circumstance is taken care of. Of course, you can't take into consideration everything that can go wrong in a method. So there's a number of different angles. Is it, samples can never be replicated in validation. Experiments or instrumentation might change over time.

Tim 9:28

Yep. The measurements that you get from your validation are really just an estimate of the way that the method is going to perform. They're not a guarantee that the method is going to perform that way. If you get a certain accuracy in your validation, it doesn't mean that the accuracy is always going to be that or the precision is always going to be that.

Peter 9:45

Yeah. I guess that's why we have proficiency testing, inter lab proficiency testing programs. I guess that sort of can account for that and keep a, so you can keep an eye on your method.

Tim 9:57

Even things like the LOD, which you establish in validation, the limit of detection, but that can change as well. Depending on instrument performance, it goes up on down.

Peter 10:05

So you mean uh on a particular day your instrument might not be functioning as well as it should be or...

Tim 10:10

Yeah, it would be great if our instruments performed optimally all the time, but they just don't, you can't ever have that situation.

Peter 10:18

What about linear response as well? I've noticed that occasionally you might have a method where all of a sudden it starts going, let's say the word, quadratic. I know it's not quadratic. It starts going non linear.

Tim 10:29

Yeah, I won't yell at you.

Peter 10:30

Again.

Tim 10:31

Yeah, you can validate a method and it's linear up to a certain amount, but if you don't, if you're not continually monitoring that through having calibrators that go up that high, et cetera, there's no guarantee that it's still linear up to there.

Peter 10:44

And so what about sample matrix as well? I mean, that's gotta be the biggest variable doesn't it? I mean you try to take into account every different sort of matrix you can get by doing matrix effect experiments, pretty intensive one during the validation stage, but you can always get a sample which has got something in it that does affect your analytes.

Tim 11:01

Yeah. I think this is particularly true in forensic toxicology where you're dealing with the worst of the worst kind of biological samples, often decomposed or...

Peter 11:11

Oily.

Tim 11:12

Yeah. Yeah.

Peter 11:13

So dirty postmortems samples are sometimes not worried about so much because often laboratories will report the results as approximate, but in an analytical laboratory you really should be not worrying about what type of sample it is. You should be worrying about the result that you get out of that.

Tim 11:30

Yeah. You want to make sure that your result is, you're guaranteeing that your result is correct to some extent, even in a a really poor sample, but unfortunately, like you mentioned proficiency tests before, you'll never get a proficiency test, which is in a putrefactive blood sample. They're pretty pristine actually compared to the kinds of samples that you might come across in forensic tox, so they're not completely representing the sample types that you get, but you can't. That's the problem. So because of that, do you think that validation should be repeated to some extent from time to time? You validate a method and then you start using it and you might use it for many years after that. Do you think there's ever a need to go back and sort of revalidate just to check everything's performing the same way?

Peter 12:15

I think that'd be a good idea in an ideal world, but I don't think you can really. Validation's an exhaustive process, but you do have uh ruggedness as a parameter that you should be validating as well, which sort of tells you how robust your method is, so perhaps it's covered a little bit by that.

Tim 12:32

Yeah. Well, I think ruggedness though is one of those things that in articles where they're publishing validated papers, the ruggedness section doesn't seem to get as much attention as other sections of other validation parameters, which is, it's true in my case as well, when I've validated methods, I mean it's the ruggedness bit is hard to assess because it just takes time and different analysts and different instrument performance and different sample types.

Peter 12:59

There's an infinite number of different parameters you can change, you can have your extraction solvent composition slightly changed because someone else made it up. You should be evaluating that sort of thing. But yeah, the pH of your buffers, if it's out by a certain amount, will it make that much different to an extraction? That sort of validation is very onerous.

Tim 13:17

Yeah. And so some guidelines will say something like, um, you know, an expert scientist can usually judge which are the most significant factors which will affect the ruggedness of the method and can maybe test those ones but don't necessarily have to test every possible factor.

Peter 13:35

So you sort of make it fit for purpose or suitable for your application is what they often...

Tim 13:40

Yeah, but I guess the, the main thing is that validation is not a guarantee that your method is going to work that well under any circumstance.

Peter 13:48

What about pH in LC/MS Tim? You must use low pH for basic drugs because it gives you better ionization.

Tim 13:56

Yeah well that's, that's conventional thought. So you're saying you don't need to?

Peter 14:00

No it's been published quite a bit and I think it's quite well established that you can use high pH for basic drugs. Even though we're always thinking about ESI working by protonating the molecule so that it's in the ionized form even before it gets to the mass spectrometer. And so therefore it's easily ionized. But there's a lot of stuff happening, a lot of chemistry happening at that nebulizer tip in those tiny droplets and at that really highly charged environment where there's very high voltages and so you can get, um, even better sensitivity sometimes for basic drugs in high pH solvents.

Tim 14:32

Yeah it's interesting, isn't it? The sort of conventional rule of thumb with APCI versus ESI is that in APCI the ionization is happening in the gas phase. In ESI, the ionization is happening in the liquid phase before it gets to that gas phase, but it's actually more complex than that.

Peter 14:50

Much more complex. No one, again, we don't really know what's going on inside the instruments. We've got to stop saying that I think because people are going to start worrying about forensic tox, but that low pH for basic drugs does seem to make sense on paper. But in reality when you have, you still get just as good ionization for most drugs at high pH as you do at low pH.

Tim 15:10

But what about the chromatography though? That's another story, right?

Peter 15:13

That's the benefit of it because often you get better chromatography for basic drugs because they're less, once they get to, once they're on the column, they're less ionized so they're more neutral so they don't, so they have less interaction with active sites and they end up with nicer peak shape. And some drugs will be retained for longer on the column. So what's an example, say for morphine for example, which often comes out very early in a chromatogram on a normal phase, uh reverse phase column at low pH where there's lots of other stuff coming out as well and you're in a high aqueous environment and so you may get less effective, um, droplet formation and ionization down there, but on high pH gets retained on the column a lot longer. So it comes a bit later. You might even get nicer peak shape. And by the time it comes out in the chromatogram there's a different mix of aqueous and organic coming out. So you get better ionization too. So just give it a go and you'll be surprised that as long as you've got a column that can accept that sort of, um, difference in pH or a column that can go from a low pH to a high pH. It can be a very good way to try alternative chromatography as well. So you may have a method where you're getting some interference or lots of matrix effects for your analyte that you're looking at. Try a different pH and see what happens. So that saves a lot of money in buying different columns as well. Just try a different pH. There's a paper out in around the early 2000s I think and it was called uh what's going on in ESI, wrong way around ESI or something like that, and that talked about it well. It talked about caffeine and they talked about infusing a solution of caffeine at neutral pH and based on its pKa, there was no way that a mass spectrometer should have been able to detect caffeine because there should have been so few ionized molecules in solution and then they changed the pH and it didn't make much difference. That was a long time ago I read that. I've got one! It's LC/MS again, I love LC/MS. You have to inject your analytes in a solution that's the same as the starting composition of your analytical run.

Tim 17:17

Say that again.

Peter 17:17

So if you've got a method that starts at 90% aqueous and 10% organic, then you have to have your analytes in a similar sort of composition.

Tim 17:27

Ah I see, yeah.

Peter 17:30

So you can, you might not get the best chromatography, but i t w ould still work.

Tim 17:34

Yeah. And a lot of times you might not want to reconstitute your analytes in whatever those starting conditions are because you might not get the uptake of the compounds back into the...

Peter 17:44

Yeah or they may not be soluble enough. So they end up sticking to the glass or to the plastic insert. Like THC, I think is a good example where it's much more stable in a, in an organic solvent like methanol or ethanol than it is in large aqueous.

Tim 18:00

Yeah. So you want to have it in a ethanolic, methanolic solution, but you're obviously going to be starting your chromatography with mostly buffer.

Peter 18:08

Yep. So that'll mean that your chromatography will be affected unless you use some sort of process to mix up the sample before you inject it.

Tim 18:16

Like an injector program?

Peter 18:17

Yeah. You can use an injector program to sort of dilute that high organic content extract that you've got enough so that when it hits the column, it's not affected so much. That sort of undermines all the perfect chromatography that a lot of manufacturers talk about where they have, they insist you use these conditions, but in reality, in a, in a real laboratory you can't have, we're not injecting standards, we're injecting samples and we're injecting... not looking for one drug, you're looking for hundreds of drugs and there's never one solution or one... there's always a compromise is what I'm trying to say. So we should be flexible.

Tim 18:50

Alright changing topics slightly. Pete, here's another myth. There is a textbook which everyone calls Baselt in the field.

Peter 18:58

Baselt, I like Baselt.

Tim 18:59

Yes, I like Baselt too, but here is a myth common in forensic toxicology, especially among maybe less experienced people that Baselt is the last and final word on drug interpretation.

Peter 19:12

Yes. Well the information in Baselt's only taken from the literature and that, the person who summarized that literature is, you have to rely on them reading every single publication that's out there correctly and in its entirety.

Tim 19:25

And I think the author, Baselt himself, would be the first one to admit that this is not a comprehensive summary of all data on all drugs. It's kind of a snapshot. It's a useful guide. It's a good starting place.

Peter 19:39

But you can be tempted to use that as your only reference if you're in a hurry in a busy laboratory. But really you must look at those references just to make sure that what is being said In those paragraphs is correct.

Tim 19:51

And just that it's, it's the complete picture because it's, well it never is the complete picture. I guess that that's the thing about it, it's just a useful springboard to then go and have a look at some of those references and I mean any compilation of any kind of data is always going to be like that.

Peter 20:09

Drugs and their interactions are very complex. It's obviously very difficult to summarize 10 or 15 or 20 papers within a couple of paragraphs, which is what we really want. But in reality it's pretty difficult to do accurately and I'm pretty sure the author never intended that to be used as a sole source of your information that you're going to use, you have to use other references.

Tim 20:29

Exactly. And I mean we should say how indebted the field of forensic toxicology is to a compilation like this because it's an excellent place to go and and find some quick information about drugs. In fact, I feel like every time we say the name Randall Baselt, we should say something like, may he live forever afterwards, or something. May his textbooks never cease.

Peter 20:50

Yes.

Tim 20:50

One of the problems with compilations like this is that it obviously mentions some therapeutic levels that get found in clinical studies and things and then also some lethal levels that have been found in cases where people have died. Often there's a huge gap between those two things because the levels that get reported in fatalities in the literature, they might not be the minimum level that's required to kill someone. It could be that someone's just taken a massive overdose of something and so the concentrations in their blood are enormous. You could still die from a concentration that's much less than that.

Peter 21:25

Exactly. Or a lower concentration in conjunction with another drug. So interactions may not always be included in the monologues.

Tim 21:33

Yeah. And in drug fatalities they often are from polydrug use, so it's very difficult to give any firm postmortem ranges, which is why actually there's, there's other compilations around as well, like there's a recent paper from uh some guys in Helsinki, it's kind of an update on their paper from a few years ago, just summarizing all of the postmortem concentrations of drugs that they found in thousands and thousands of autopsies over the last however many years. And those kinds of compilations are good too, but it's still important to just look more broadly and take into account all of the available data rather than just looking at one compilation.

Peter 22:13

And these some guys are Raimo Ketola and Ilkka Ojanpera.

Tim 22:17

Yeah, sorry. That's my Australiana coming out, we call everyone some guys. By the way, we'd like to apologize for butchering people's names on this podcast.

Peter 22:25

And the English language in general.

Tim 22:27

Yeah

Peter 22:27

Those compilations are very good as well because they give an indication of what the therapeutic level is in post-mortem samples. So it gives you a little bit more of a snapshot of the sort of concentration you might expect in pm samples.

Are liver drug concentrations useful?

Tim 22:39

Yeah, but again, it's only, just like with Baselt, it's just the kinds of concentrations you might expect, but it's so complicated to assess drug effects. It's, it's not just based on the concentration of the drug especially in postmortem samples. So it's really difficult to have any kind of firm ranges. Let's talk about liver concentrations.

Peter 23:00

Ah, okay. Here we go.

Tim 23:01

So yeah, this is, I'm going to get out my soap box.

Peter 23:04

I thought you looked a bit taller.

Tim 23:05

Yep. Is it a myth, Pete, that liver concentrations can provide useful information in a postmortem case?

Peter 23:11

Of course they can provide useful information.

Tim 23:13

Look, I think they're, obviously they can provide useful information in some circumstances, but I think liver concentrations can be over relied on for how much information they actually do provide in cases because the concentrations that you find in liver, I mean if you think concentrations in postmortem blood are hard to interpret, try doing it in liver. The ranges, the overlap of the ranges that you find between nonfatal and fatal concentrations for many drugs, they seem to almost completely overlap. It might be at the very extremes, you can get some useful information. So if the drug's very low in the liver or very high in the liver, but I have never been convinced of a clear correlation between blood and liver concentrations.

Peter 23:58

So there also should be some clarification here. So you're talking about comparing liver results that you may get in your laboratory comparing to literature values.

Tim 24:08

Yeah, well that's the only way you can compare it isn't it?

Peter 24:10

Well you can have, you can have a large internal database I guess if you've done thousands of samples over the years of liver and blood and you've got a good comparison.

Tim 24:17

I suppose you can if you're using the same method.

Peter 24:18

Because they're all standardized methods within that laboratory. But I think the problem I've got with liver is a little, it's exactly like hair, you can't spike drugs into a matrix. And on top of that, it's...

Tim 24:28

Into a solid matrix.

Peter 24:30

Yeah.

Tim 24:30

Yeah and in many cases there is no post-mortem liver that you can use for your validation studies. People use other kinds of liver, animal liver sometimes. That doesn't behave exactly the same way as human liver.

Peter 24:43

And you can't, u m, unless y ou're using exactly the same method, you're going to extract the drug to a different extent. So if you use liquid liquid extraction after you've homogenized it, some laboratories w ill, homogenize it to different degrees than other laboratories, they might use a different solvent t o homogenize it in. So I think comparing l iver levels in one laboratory to another l aboratories or other published literature results can be a little bit risky, much more risky than comparing your own laboratories results.

Tim 25:09

Especially when you look at some of the methods that are published for liver analysis, or which maybe they, they're designed for blood, but they're also used for liver. And some of them I just don't think are appropriate. And so the concentrations that you get from them are, I would be very skeptical of the, the reproducibility of those concentrations. Like if you're using a protein precipitation method for liver, I mean if you're, even if you're macerating that liver in, let's say water or buffer or something beforehand, what you've got then is kind of a suspension of tiny particles of liver floating around in that water and then you're trying to extract all the drugs out of those particles just using some acetonitrile or acetone, I don't think you're going to be able to reproducibly pull all those drugs out of those solid particles using that kind of solvent.

Peter 25:56

Yeah, fair enough. And maybe extracting with a solvent when you're rolling it for a considerable time, like maybe better.

Tim 26:01

Yeah, maybe better. I think so. But because of that, it's really difficult to get any kind of compilation of the amount of data that you need. Like we're talking about Baselt before and some of these other compilations, if you want to be able to have any kind of firm grounding, you need lots and lots of data points. The number, the sheer number of data points that you need to generate is hard to get from any lab. Especially because most labs don't analyze liver routinely. There are some labs that do, but most labs will only do it when required to. And so there are times in certain cases where you might want to analyze, you know, as many specimens as you can from that case cause you're trying to work out the sort of total distribution of the drug throughout the body. So in some circumstances liver will play a part of that and can be useful. But I think the idea that if you have an equivocal blood result, which is hard to interpret and you're not sure, is this a fatal level, is it not? Analyzing the liver in those cases to provide a bit more surety, in most cases will just confuse the issue more. It's not going to add any more surety to that blood result.

Peter 27:07

The other problem is there's no therapeutic range of liver concentration, so...

Tim 27:11

Yeah, it's hard to get ethical approval to take living people's liver to analyze the drug concentrations.

Peter 27:17

Nonfatal concentrations are difficult to come by as well. So interpretation of that's hard.

Tim 27:21

So what about this Pete for, let's finish up here. One final myth. Every other toxicologist understands what they read in publications, but I don't.

Peter 27:29

What do you mean?

Tim 27:30

What I mean is that I think especially for people who are just starting out in the field, reading publications, there is a lot of information that's in each of them and the people who've written them are probably experts in that niche field that they're talking about. And so in every publication that you read, there's probably going to be things you don't understand. I mean, I feel like that to this day, there's publications I read and I might understand a lot of it and there's a few things in there that I don't really know that much about, but I think it's quite intimidating to people who are starting off reading the literature, and there's just so much they don't understand and they think they really should be understanding everything that they're reading and everyone else understands everything that I'm reading here and I don't,

Peter 28:07

And they're always written in a very authoritative way as well. So that sort of can be intimidating in that manner too. If you're reading several publications in a row, it's like a wall of knowledge to you because you have this vast amount of information and your brain can sometimes be tricked into thinking that this is all written by one person perhaps. And, but no, it's written by individual people and no one knows everything. And you shouldn't be discouraged by complex articles.

Tim 28:31

Yeah. Becoming an expert in any field is a process. It's a journey. It's not something that you achieve, becoming an expert. It's, it's a lifelong learning experience. And so...

Peter 28:43

And sometimes, um, because they're written in that way, being so authoritative that you don't question it at all.

Tim 28:50

Yeah. Which is why it's so great going to conferences and things like that where you can actually get to talk to people. And because I mean, I've found in my experience, people in our, in our field, I'll just talk about our field, are very accommodating and very willing to share information, you know, warts and all, of their method and, or the work that they're doing and say, look, we tried this, this didn't work very well and stuff. But that stuff doesn't go into papers obviously, because you can't discuss all that in a paper.

Peter 29:19

Exactly. It's a great place to be a scientist, the forensic community, I reckon.

Tim 29:22

Yeah. All right. I think that's enough myths busted for today, Pete.

Peter 29:26

Yep. I hope that your faith in toxicology hasn't been ruined.

Tim 29:30

We'll restore it as we go on in the second season. So stay tuned.

Peter 29:34

Thanks for listening.

Tim 29:34

See you next time.