The Toxpod

5 in 30 (Blood spots, pesticides, and post-mortem redistribution)

January 09, 2019 Season 1 Episode 8
The Toxpod
5 in 30 (Blood spots, pesticides, and post-mortem redistribution)
Chapters
The Toxpod
5 in 30 (Blood spots, pesticides, and post-mortem redistribution)
Jan 09, 2019 Season 1 Episode 8
Tim Scott & Peter Stockham
We look at 5 recent publications in the field of toxicology
Show Notes Transcript

In the final episode of season 1, we look at five recent publications in the field of toxicology.

  1. Voelker, SE, et al. Semi-quantitative determination of designer steroids by high performance liquid chromatography with ultraviolet detection in the absence of reference material. (2018) Drug Test. Anal. 1-7
  2. Moretti, M. A liquid chromatography-tandem mass spectrometry method for the determination of cocaine and metabolites in blood and in dried blood spots collected from postmortem samples and evaluation of the stability over a 3-month period. (2018) Drug Test. Anal. 10: 1430-1437
  3. Oenning, AL. et al. An effective and high-throughput analytical methodology for pesticide screening in human urine by disposable pipette extraction and gas chromatography - mass spectrometry. (2018) J Chrom B. 1092:459-465
  4. Bertol, E. et al. Proactive drugs in DFSA cases: Toxicological findings in an eight-years study. (2018) For. Sci. Int. 291: 207-215
  5. Brockbals, L. et al. Time-dependent postmortem redistribution of opioids in blood and alternative matrices. (2018) J. Anal. Toxicol. 42: 365-374


Contact us at thetoxpod@sa.gov.au


The Toxpod is a production of Forensic Science SA and the South Australian Attorney General's Department. The opinions expressed by the hosts are their own and do not necessarily reflect the views of their employer. 

Tim:
0:11
Hello and welcome to The Toxpod. I'm Tim Scott.
Peter:
0:12
And I'm Peter Stockham. So Tim, what's up with you? You're looking pretty happy with yourself, bouncing around the lab?
Tim:
0:22
Yeah, I am pretty happy actually Pete. I've just received an email from the Mathews open access journal of nutrition and dietetics asking me to be on their editorial board.
Peter:
0:32
That sounds prestigious.
Tim:
0:33
It does sound prestigious, doesn't it? I haven't been on an editorial board before.
Peter:
0:37
Do you know much about nutrition and dietetics?
Tim:
0:40
Well, I try and eat healthy.
Peter:
0:42
Oh, fair enough.
Tim:
0:43
Uh, I think I'll just learn on the job probably.
Peter:
0:47
Sounds fair.
Tim:
0:47
Yeah, so that's a real honor.
Peter:
0:48
So is that your first editorial board?
Tim:
0:50
It is.
Peter:
0:50
I'm already on the Analytical Journal of Toxicology.
Tim:
0:53
You mean Journal of Analytical Toxicology?
Peter:
0:55
No, no...
Tim:
0:56
A different one.
Peter:
0:57
Yeah.
Tim:
0:58
Not JAT?
Peter:
1:02
No, AJT. Or TAJ?
Tim:
1:02
Interesting. How'd you get on that one?
Peter:
1:03
They just sent me an email and I sent in 1,000 dollars.
Tim:
1:06
1000 dollars, that's a bargain to get on an editorial board!
Peter:
1:08
Speaking of which, if you wanted to get on the list of the top 100 scientists, all you have to do is send in your name and your address, your qualifications and I think it's about 800 US dollars and they send you a certificate.
Tim:
1:18
Do I google that or what?
Peter:
1:19
Yeah, just give it a google, see what happens.
Tim:
1:21
Alright. I'm definitely gonna do it.
Peter:
1:22
They invited me last year. And I haven't paid my money yet, but...
Tim:
1:25
You should get onto that.
Peter:
1:27
I might.
Tim:
1:27
And speaking of journals, today we're going to do another 5 in 30 episode, where we're looking at five recent articles in the field of toxicology and trying to draw out some of the interesting applications and some of the broader issues in toxicology from these papers. So the first paper we're looking at today is from Drug Testing and Analysis and it's by Voelker and colleagues. It's title is the Semi-quantitative determination of designer steroids by high performance liquid chromatography with ultraviolet detection in the absence of reference material.
Peter:
2:01
So it's not necessarily the typical toxicology paper that we might look at, but it does have some relevance in that often these materials, illicit material is coming out of the blue. It's very hard to get reference material. So these guys are looking at an alternative way to try and quantify some of these materials in capsules.
Tim:
2:18
Yeah. I think even though they're just focusing on steroids here, there are some wider implications in the sense that they, they're trying to do this in the absence of a reference material, which is a real problem for any kind of designer drugs. Here they're talking about designer steroids, but any kind of new psychoactive substances as well, getting reference materials is really hard to do sometimes or there's a long lag time, and so what they're trying to do here is sort of roughly quantify the amount of compound that they've got and then hopefully down the track, once these things do get synthesized, then they have a good idea of what kind of doses they've been seeing, even from this point.
Peter:
2:55
Yeah, it seems a reasonable sort of approach.
Tim:
2:57
Because drug users are constantly trying to evade legislation and standards and things by using chemically modified compounds so you have a bunch of compounds which are in the legislation and all they have to do is change one little functional group and now it's not covered by that legislation anymore and so it's not illegal to take it. Or it can result in a negative test because maybe laboratories have their methods set up to detect all the compounds of interest, but now you throw in a new one, they're not specifically looking for it and so they're not necessarily going to see it, so the test will come up as negative.
Peter:
3:28
And also the problem is there's obviously no knowledge on the pharmacological effects of these things, so that's quite worrying.
Tim:
3:34
Yeah, that's very worrying. People are just experimenting with their own bodies and their own lives basically taking some of this stuff. No one really knows what effects it's going to have.
Peter:
3:42
And in this paper, they are looking at quite large amounts they're detecting in these capsules, I understand. So if at a later time, if they've got an idea of how much is present in these capsules, at a later time when they have more information on what the health implications of these materials are, we can, they at least have got some data on how much people are taking.
Tim:
4:01
Yeah, but it takes quite a while sometimes for the health effects of these kinds of compounds to be properly studied. I mean even some of the new psychoactive compounds which are of interest in forensic toxicology, some of them have been around for years. We still don't really know what the pharmacological effects of them are beyond sort of a basic understanding.
Peter:
4:19
It used to be like there was maybe one or two a year of these new NPS would come out, but now there's literally dozens and dozens of them coming out and there's just not enough time to do that.
Tim:
4:27
Yeah, that's right. So here they're using an alternative standard as a reference material. They're quantifying using a similar compound but not actually the compound that they are looking at.
Peter:
4:40
So they're starting out with capsules and they're extracting the samples, or first of all, they're analyzing the samples, using GC/MS to identify what's present and they're using LC/UV to quantify what's in them.
Tim:
4:51
So the steroids that they're looking at are 1-androsterone and 6-beta-chlorotestosterone, and so the surrogate standards that they're using for those are dehydroepiandrosterone...
Peter:
5:01
DHEA.
Tim:
5:03
And testosterone. So because they quantifying by LC/UV, they've chosen those surrogate compounds to be very similar in structure and also in UV spectra, although even though they're very similar, they are still correcting for molar absorptivity.
Peter:
5:19
Yeah, so some drugs will absorb a little bit more strongly than other drugs because of conjugated bonds, et cetera, is that what the story is?
Tim:
5:26
Yeah, so they're making a correction factor for that.
Peter:
5:28
Yep. So they obviously have an idea of what these drugs are to start out with. It's a little bit like the NPS scene where you have a GC/MS library which may be available and although you've got the spectra, you may not have the authentic material. So they've identified these compounds using GC/MS, now they want to work at how much is present in the tablets.
Tim:
5:45
Yeah, and they do say there's a, there is an alternative to using a surrogate standard. You could extract out the material from the sample itself or from maybe from a different sample containing the same drug and then use that as a reference material. But of course that's not a certified standard.
Peter:
6:01
No.
Tim:
6:01
So we talked about the use of model compounds in a previous episode for validation of screening methods. But I guess this is kind of like using model compounds for quantitation as well.
Peter:
6:12
It makes sense if it's the best that you can do.
Tim:
6:14
But it can be difficult to validate, as they say here, establishing all the normal validation parameters.
Peter:
6:19
The level of validation really depends on what you're going to use it for and if it's for information only rather than prosecution, then that should be fine, I think.
Tim:
6:27
Yeah. Although even in this paper they did calculate a measurement uncertainty so they, so they are getting some information on the validity of the method. It's just difficult to get all the information that you would normally get for that compound. I think it's quite a interesting and useful approach. It might be harder to translate it to say postmortem forensic toxicology or drugs in drivers or something like that. Because a lot of the screening for those kinds of applications, a lot of the quantitation is done using MS instruments, mainly LC/MS and so unlike LC/UV where the response is quite predictable, on MS instruments, it's not always as predictable, so it's harder to translate that to another instrument or even to predict what it's going to be, you know, from one drug to another. You can change one small part of a drug and get a quite different response on an LC/MS.
Peter:
7:17
Or a GC/MS as well.
Tim:
7:18
Or a GC/MS, and it might be more susceptible to inter-day variation in your instrument.
Peter:
7:24
Yeah, so LC/UV is much more predictable in terms of the molar response that you're going to get.
Tim:
7:30
Yeah.
Peter:
7:30
So the next article we're gonna talk about is about dried blood spots. So it's a paper by Matteo Moretti et al, and it's entitled A liquid chromatography-tandem mass spectrometry method for the determination of cocaine and metabolites in blood and in dried blood spots collected from postmortem samples and evaluation of their stability over 3-months. And that's in Drug Testing and Analysis.
Tim:
7:51
Dried blood spots have really taken off in terms of the amount of research that's being done on these as a potential sample that's useful in forensic testing.
Peter:
8:00
So for several decades it's been used for newborn screening techniques but not necessarily until recently in the toxicology sphere.
Tim:
8:06
So one of the major advantages of using dried blood spots is that it's a less invasive technique than sticking a syringe into someone's arm and taking quite a few mils of blood. Here you just have to do a little prick and put it on a piece of paper and that's it.
Peter:
8:20
And I can see why people are interested in it because as well as being noninvasive, it also can be done by anyone really who's got maybe a finger pricking device, you can quickly dab it onto a piece of paper, let it dry, put it in the post, send it off to the lab.
Tim:
8:32
Yeah, and it's easier for the lab to store it then. You've just got these dried bits of paper rather than storing large amounts of blood in a freezer.
Peter:
8:40
And if it's dry, there's presumably less opportunity for bacteria to invade and contaminate the sample. And there's also, I guess more likely to be less biological hazard involved.
Tim:
8:49
So in this case obviously they're not looking at living people though. They're actually doing it in postmortem samples, which is a bit of a change from some of the other papers around.
Peter:
8:58
There's been a paper very recently from Lars Ambach looking at similar drugs, cocaine and metabolites as well. That was a controlled study where they were administering cocaine to live people and taking fingerprick samples. This is a different study where we're looking at samples taken at autopsy and dabbed onto paper, then dried and stored and then compared directly to the same blood stored in tubes.
Tim:
9:20
Yeah, so different application, but some of the same issues, you know, once you get the blood onto the paper then it's pretty much the same. And one of the major issues is that although it's good for qualitative analysis, the quantitative analysis is a bit more problematic. You can get different amounts of diffusion of the blood onto the paper depending on the hematocrit levels. If you only take part of the blood spot, the concentration may not be homogenous across the whole blood spot.
Peter:
9:49
So it sort of partitions across the bloodspot as it spreads out and dries doesn't it.
Tim:
9:53
And then you've got to make sure obviously that you're getting good recovery from the paper, you're trying to get it then back off the paper which its bound to reasonably strongly so you can get incomplete recovery from the paper sometimes. So all of those issues are the same no matter what the application is. But it's interesting here they're applying it to postmortem cases and one of the major advantages here is really the stability of the drugs and also just the ease of storage for post mortem samples.
Peter:
10:15
So a good application might be where you have an isolated community where someone's died, maybe in conditions where they don't have refrigeration. It may be even possible to take valid toxicology samples without refrigeration at those sites, and send them into the lab. When it comes to the extraction part, I understand they're using about 85 microliters of blood to spot onto the paper and they're also using 85 microliters of blood which they put directly into a tube. And they're taking several samples of, onto the paper so that they can test it at different time periods to see how the concentration changes. And apart from the initial step where they're getting the blood out of the paper, using some buffer, the extraction's virtually identical for the blood spot sample and the actual blood sample where they're using an SPE method.
Tim:
10:54
They had a bit of trouble with the ecgonine methyl ester, because the retention time was so fast, less than one minute, they were getting really significant matrix effects, which you're always going to get really if you have something coming out so early. There's gonna be a lot of other things coming out early as well and possibly suppressing it.
Peter:
11:12
Yeah, it's a common problem, isn't it? High polarity compounds come out way too early sometimes.
Tim:
11:16
So for the ecgonine methyl ester, they were getting matrix effects of around 40 percent, they've reported here, which I guess means 60 percent suppression. I mean, what is an acceptable level of matrix effects do you think, Pete?
Peter:
11:30
So in terms of guidelines, I think the GTFCh, the German speaking toxicology society, they say a maximum of 25 percent suppression. But if it works and they can show that it works across a number of samples, I don't really see what the problem is. Some samples that you assess might have a matrix effect of 30 percent were another one might have a matrix effect of only 10 percent. So that means there's going to be a large difference in your results.
Tim:
11:53
That's why they in a deuterated ecgonine methyl ester, just to limit that variability in the quantification.
Peter:
12:01
They had about 18 postmortem cases containing cocaine and metabolites and directly compared the blood result and the dried blood result. And we've got positive and negative differences between the dried blood spot sample and the blood sample, which is a bit unusual.
Tim:
12:14
Yeah, in some the dried blood spot was higher, in some the whole blood sample was higher.
Peter:
12:18
For the same, essentially the same blood that was spiked. The only difference being it was spiked on to paper instead of into a tube.
Tim:
12:24
Yeah. But some of the drugs are better than others. I mean cocaine looks like it's got a very good correlation. Ecgonine methyl ester and Cocaethylene, still a correlation but not as tight.
Peter:
12:35
Benzoylecgonine quite good, but still the odd sample that's quite different.
Tim:
12:38
And so the other aspect they were looking at was the stability.
Peter:
12:41
They looked at the stability from 3 to 10 weeks and cocaine, benzoylecgonine and ecgonine methyl ester seem to be pretty stable. Interestingly, cocaethylene seemed to have a couple of anomalies where there was severe degradation or severe decrease in response after even three weeks at room temperature.
Tim:
13:00
Yeah, there was some samples which were quite stable...
Peter:
13:02
10 weeks at room temperature. Couldn't do that with blood.
Tim:
13:05
Yeah, but then another couple which really degraded, as you said. Same with cocaine, there, a lot of them were quite stable, there was eight bloods that they studied there, but then they had one in particular which decreased by nearly half over that time. So you know, this is sample dependent as well, this kind of degradation.
Peter:
13:22
So the upshot for human performance toxicology, it's the ease of taking a sample, but for postmortem toxicology, dried blood spots is really only for transport.
Tim:
13:31
And for storage, you can store it at room temperature, which means that you need to have less freezers and so on, just a bit logistically easier.
Peter:
13:38
Promising work.
Tim:
13:39
Okay. So the next paper is from Journal of Chromatography B, it's by Oenning and colleagues and it's titled An effective and high-throughput analytical methodology for pesticide screening in human urine by disposable pipette extraction and gas chromatography mass spectrometry.
Peter:
13:55
It's good to talk about pesticides, we are often talking about drugs. And uh, this is another important part of toxicology that we often have to deal with.
Tim:
14:02
Yeah. Pesticides is still a huge cause of fatalities. In fact, they mention in this paper that the World Health Organization reports that poisoning from pesticides is one of the most common methods of suicide worldwide, so it's important to have a good method to detect pesticides.
Peter:
14:17
So what is this in-pipette extraction business Tim?
Tim:
14:21
So it's essentially a solid phase extraction, but rather than having cartridges or 96 well plates which a lot of labs are using, it's done within a single pipette, so it's scaled down. It eliminates the need for large equipment, manifolds and things like that, and basically you just suck up the sample and dispense several times, get it absorbed onto the solid phase material and then do the same thing with some solvent to rinse off your analytes. It's as simple that really. Here they're looking at 11 different pesticides.
Peter:
15:00
They include things like carbofuran, malathion, terbufos, carbaryl, things like that.
Tim:
15:01
So a lot of what they're talking about in this paper is about the method optimization, which was an important part of the method development obviously, there's quite a few different steps and there's different reagents and so on, so trying to optimize each of those to get the best possible recoveries of all the 11 different pesticides. And they use the Dohlert matrix in their method development, which is basically just a way of doing the least possible experiments to produce the best outcome for all the variables that you've got. So you basically do some experiments changing each variable by a set amount and you construct a 3D model essentially, and then that allows you to choose the best number for each of those variables, like the number of cycles, the extraction times, the solvent and so on.
Peter:
15:46
So it's easier to visualize a sweet spot for the best parameters that you want to use.
Tim:
15:49
Yeah, exactly. And there's a number of different models you can use. This is just one. And I have seen this taken too literally, where you can end up with some just ridiculously impractical volumes and things like that. You know, you end up with a method that uses 7.6 mils of solvent or something like that, so you don't want to take it too far.
Peter:
16:09
No, especially when you're working with urine. I mean we're not looking for accurate quantitative levels here, it's mainly identification, but they are quantifying, I believe.
Tim:
16:15
Yeah, as long as it's fit for purpose, that's really the main thing. And so they're analyzing these by GC/MS and the quantification is in selected ion monitoring mode.
Peter:
16:25
And the sample volume is only 0.1 mils which is pretty good for GC/MS especially.
Tim:
16:30
One thing I liked that they mentioned in this paper is that because they've got these pipette tips that they're using and you can recycle these pipette tips because you basically just take out the filter at the top, put in your solid phase material, put the filter back in, and then that's how you use it. Then you aspirate into that, but then afterwards you can just wash those pipettes and then reuse them again, which they did during their research and development. They do say that you shouldn't really do that for actual case samples. You want to make sure you avoid any carry over and so on, but I think the fact that they're, they've thought about the environmental impacts is a really good thing. As analytical chemists, you know, we use a lot of disposable materials, disposable plastics. It's good to be thinking about how to develop methods in an environmentally responsible way as well.
Peter:
17:17
It's very easy to fall into the disposable plastic mentality, isn't it? Just get a new one every time. But sometimes we have to do that for casework, but for validation work it may not necessarily be as important.
Tim:
17:28
Yeah, so where we, where you can, you obviously don't want to compromise the quality of your case results, but there's, there's probably a lot of areas where every toxicology lab can improve in their recycling of materials, in their reuse of materials rather than just throwing everything out. Actually I saw the other day an ad for a portable test kit, I think it was an immunoassay test kit which was virtually plastic free and that was sort of one of the big selling points of this kit because most of these portable immunoassay kits are almost all plastic and they are just disposable. You just throw them out as soon as you're done with them.
Peter:
18:02
Yep, it's a waste and they're going to be around here for our great, great grandchildren, those bits of plastic aren't they.
Tim:
18:07
So the method that they came up with had five aspirations cycles of the sample and then five aspiration and elution cycles of ethyl acetate and then they're injecting one microliter finally into the GC.
Peter:
18:23
They've got pretty good recoveries and their spiking way down to 5 or 10 micrograms per liter, so that's more than enough for forensic postmortem toxicology.
Tim:
18:31
And it is much easier to use this kind of technique with urine than with whole blood.
Peter:
18:36
So if you wanted to apply this sort of thing to blood, you'd probably have to dilute it and centrifuge it a bit because of the viscosity and particulates present in particularly postmortem blood.
Tim:
18:45
Yeah. Sometimes you see, even on solid phase cartridges, blood can be difficult to force through there, some blood is just really...
Peter:
18:52
I hate those samples.
Tim:
18:54
Yeah, and you can't always tell either. It's, some bloods, they don't necessarily look so thick or clotty or anything like that, but they just are really hard to pass through a solid phase cartridge and so here where your aspirating it and then dispensing it several times. Yeah, that could be quite difficult with blood and if you're using, if you're doing postmortem toxicology and you're looking at solid tissue macerates, liver macerates, then it becomes even more difficult because you've got a bit of solid particulates in there as well, but it works fine here for urine, which is the application that they're applying it to. All right. Let's move to the next paper which is from Forensic Science International. It's by Elisabetta Bertol and colleagues. It's entitled Proactive drugs in DFSA cases (that's drug facilitated sexual assault): Toxicological findings in an eight-years study.
Peter:
19:42
Eight years is a fair chunk of time.
Tim:
19:43
So this is a longitudinal study over that period of eight years looking at the types of drugs that are found in sexual assaults and also a few other factors as well.
Peter:
19:52
It can be hard putting these studies together over that length of time because often your methodology changes over that time.
Tim:
19:57
So they've looked at over 250 cases over this nearly a decade timespan. They're mostly looking at blood and urine samples and they do mention that they also asked for voluntary hair samples three months after the event, although they haven't mentioned the results of the hair analysis in these cases. I'm not sure if they got any or not.
Peter:
20:17
I worked on a project once where we were looking at blood and urine samples and then asked for a corresponding hair sample for three months later, and perhaps understandably, it was very hard to get them to come back for the followup hair sample.
Tim:
20:27
Yeah, one of the difficulties with sexual assault cases is that there's often a time delay between the incident and the sample being taken. Sometimes quite a long time delay, I mean usually at least hours, sometimes a day or several days between when the incident happened and when they actually have the samples taken, and so during that time, obviously the drugs are being eliminated from their system constantly.
Peter:
20:50
In this case however, they got two thirds of the victims arrived at the sexual assault center within 24 hours.
Tim:
20:57
Yeah but even in 24 hours, some drugs will be gone. I mean alcohol obviously, GHB, which is another one that's sort of commonly associated with DFSA.
Peter:
21:06
Disappears quickly.
Tim:
21:07
Yeah, it's going to be gone in 24 hours.
Peter:
21:09
What sort of drugs were they looking for then?
Tim:
21:10
They had a range of different extraction methods looking at different classes of drugs, including the most common types of drugs associated with sexual assault like GHB, benzodiazepines, I guess they're the typical sedatives that you might associate with drug facilitated sexual assaults, but interestingly they note here that they found quite a wide variety of drugs and that drugs might be used for different reasons. It might not just be a sedative that's used in these kinds of cases. Obviously someone might use alcohol or GHB to sedate the victim in order to assault them, but other drugs might be used to impair memory, to reduce inhibitions, even to heighten sexual experience, so it's important to do broad screening in these types of cases.
Peter:
21:52
So alcohol was obviously pretty prevalent.
Tim:
21:54
Yeah, that's commonly found in these types of studies. Alcohol is obviously widely used, so that might be the reason why it's there a lot, but it's also a very easy drug to use to spike someone's drink rather than spiking it with a powder or something. I think there's a common view in the media and just in society in general that people are adding powders to people's drinks, but actually it's a lot easier to just add alcohol. It's a liquid, it's readily available.
Peter:
22:18
Yep, and that's one of the reasons why they think that GHB's commonly used because it's fairly tasteless and easy to administer into a drink, you don't have to dissolve it. We rarely find GHB in these sort of cases.
Tim:
22:27
Yeah, they only found it in one case in, out of all of these 250 cases.
Peter:
22:31
In that one case, they analyzed blood and urine and in the urine it was about 19 milligrams per liter and in the blood was around about 2. So 19 milligrams per liter in urine's above what you normally would say is endogenous, but the presence in the blood really backs that up because it's rarely found in antemortem blood.
Tim:
22:49
Yeah, postmortem blood you have huge issues with interpreting GHB because it's produced postmortem. You can find massive levels, much greater than 3 milligrams per liter in a postmortem blood, but in an ante mortem blood, if it's been stored correctly and so on, if it's been preserved, you normally don't find GHB there.
Peter:
23:06
So in this case, they collected samples eight hours after the incident.
Tim:
23:09
Yeah, so based on the half life of GHB, that might have been quite a high concentration at the time of the incident, but the most commonly found drugs here after alcohol were cannabis and cocaine, which is consistent with other European data.
Peter:
23:21
Common recreational drugs, so once again maybe just recreational use rather than drink spiking.
Tim:
23:26
Yeah, and they found benzos in a handful of cases and it can be hard to interpret these in these types of cases as well because these are prescribed, you know, a lot of people take these kinds of drugs for a variety of reasons, but I guess if someone's not prescribed them and there's no other reason for them to be there, that's when they become really significant in these types of cases.
Peter:
23:45
Exactly. You really need to know the context of the results before you can interpret them.
Tim:
23:49
Yeah, and having that close contact with the people who are collecting the samples or the people who are investigating is really important.
Peter:
23:57
Okay. This is my favorite one. This is from the Journal of Analytical Tox by Lana Brockbals, Time-dependent postmortem redistribution of opioids in blood and alternative matrices. So this follows on from some work that they presented a few years ago now I think where they're actually using a robot to do sampling in post mortem cases.
Tim:
24:15
Yeah, this is pretty cool, isn't it? This is like the future of sample collection.
Peter:
24:20
It's like it's in space or something!
Tim:
24:21
Yeah, using a CT scanner and a robot together to really pinpoint the exact locations where they're going to take the samples from.
Peter:
24:28
Yeah, so they're taking what we might term admission samples into the mortuary or admission samples into the facility where they can very accurately position the needle into the femoral vein, into the ventricle of the heart and many other organs including brain, kidney, liver, muscle and all precisely taken so that they can get reproducibility from case to case. And also at autopsy, they're able to see where the needle was when the original sampling happened so they can go back and take a sample very adjacent to where the original sample was taken in terms of the tissue samples I mean. I imagine the whole purpose of this robotic sampling is to do admission samples when they come into the mortuary and so in some cases, depending on the results of the virtual autopsy they do with the CT scanner, they might not need to do an autopsy so they can use these toxicology results that they get on admission.
Tim:
25:17
Yeah, that's, that's a really useful application of it, but here they're just looking at postmortem redistribution. So they're comparing what they get when the, when the body first comes in, the samples they take then using this robot, compared to the samples taken at autopsy, and seeing how they change over that time and there's been some other studies into this as well. Postmortem redistribution is a well known phenomena that occurs. It's not precisely understood how it occurs in every case. I mean, the main thing is for the forensic toxicologist in interpreting post-mortem drug results is to know that postmortem redistribution is a real factor. Drugs can increase or decrease several fold and there's been lots of factors that have been used to predict postmortem redistribution. There's lots and lots of papers about this. People have looked at things like volume of distribution, protein binding affinity, lipophilicity, and experimentally people often use cardiac to peripheral blood ratios because the idea is that you'll get less redistribution in the peripheral samples, so taken from, you know, the femoral vein..
Peter:
26:21
Further away from the central parts.
Tim:
26:22
Yeah. Rather than the central parts near the heart. Because one of the main causes of postmortem redistribution is leaching from areas of high concentration to low concentration, so that's usually from muscles and tissues into the blood.
Peter:
26:35
Yeah, and liver and lungs and...
Tim:
26:37
Yeah, and speaking of liver, that's another thing that's often been used to try and predict how different drugs are gonna redistribute, is that liver to peripheral blood concentration. But none of these parameters has really been able to predict in every circumstance which drugs are going to redistribute and which ones aren't. There's always some inconsistencies that happen.
Peter:
26:56
I don't think they'll ever really get a handle on it to be honest because every case is different and what about just the movement of the body when they, the bodies on the ground, whether they, when they're carrying it or moving it, just the movement of the muscles being forced to move and pump blood through your veins, that's going to change distribution through the body. That's going to vary from person to person. If there was CPR that's going to distribute or maybe change distribution of blood around the body.
Tim:
27:20
Yeah. Move blood from the central region out to the periphery. Yeah. It's a very complex situation and so there hasn't been a predictive model that's been discovered and maybe there won't be, to give a reliable estimate of postmortem redistribution in a given case, you can say maybe that some drugs are more prone to redistribution than others, but that doesn't mean that they're going to have redistributed in this particular case that you're looking at. One of the things you should not use this kind of data for is to use it as a sort of correction factor. You find a particular concentration of a drug and then you multiply it by the median postmortem redistribution in all cases that have been studied, that's not the way to use this data.
Peter:
27:57
That's quite dodgy. This is reminiscent of some work back in 2012 by Gerostamoulos et al where they did a very similar thing where they took samples at admission and then samples at autopsy, and found pretty similar results in terms of redistribution for these particular drugs, I think.
Tim:
28:11
Yeah. The thing about using the robot here is that because they're able to really pinpoint where it's coming from, otherwise you're sort of relying on doing a femoral stab where you just, you're basically, you're sticking a needle into the thigh and pulling up some blood. You're not sure if it's coming from the vein or the artery, but most labs can't afford a robot like this, so that's just out of the question for a lot of labs. If you want to take samples prior to autopsy, really, that's the only way you can do it.
Peter:
28:37
That's right.
Tim:
28:38
So here they are looking specifically at opioids and how opioids redistribute. I mean opioids are very hard to interpret in post mortem cases anyway, or in any kinds of cases just because of the issues of tolerance and so on. People are taking opioids for a long time, they build up a tolerance and then so the doses increase over time, so the level of an opioid, it doesn't always correlate to its therapeutic effects. And then you talk about in a postmortem case you've got all these other issues with postmortem redistribution and so on. It can be really difficult to interpret these levels.
Peter:
29:08
So if you can get a sample earlier on in the process, all the better.
Tim:
29:12
One of the drugs they were looking at here is methadone and they found that the methadone concentrations generally increased over time in the femoral blood, that is. Although one case did decrease, so that just highlights the difficulty again, in making generalizations from this data to specific cases.
Peter:
29:28
Yeah, once again, you can't predict postmortem redistribution.
Tim:
29:31
And they hypothesize here that perhaps this, that particular case where it decreased, that might've been at a different stage of decomposition prior to the, when the sample was taken and so that might affect the time profile.
Peter:
29:46
So there was a constant time between the admission and the autopsy, but there was a fair bit of time beforehand.
Tim:
29:51
Yeah, that one case had a long postmortem interval, 59 hours compared to the others which were, most of them were quite a lot shorter than that. Interestingly though, EDDP, which is the metabolite of methadone, didn't really show any clear trend in terms of postmortem redistribution. So again, the ratio of metabolite to parent compound in postmortem cases isn't always reliable because they might not be changing at the same rates.
Peter:
30:17
They had 4 fentanyl cases and in each case there was a significant amount of postmortem redistribution between the time of admission into the mortuary and the autopsy.
Tim:
30:25
Yeah. So that was similar to methadone, but then the other opioids didn't really show any particular trend.
Peter:
30:31
No, it's hard to say anything from those at all. So because they've taken liver samples, muscle samples, spleen, kidney samples as well at t1, admission time, and autopsy time, they've also shown how drugs are redistributed in those samples. And in general they're fairly, fairly flat aren't they? There's not a great deal of change. Except in a couple of cases where muscle has gone down quite a bit.
Tim:
30:52
Yeah, and that's interesting because one of the things that they suggest here is that for the methadone, for example, because it was increasing in the blood and because the concentrations found in the thigh muscle were a fair bit higher than the femoral blood, which is sort of in the same region of the body, that it might be passive diffusion from the muscle to the blood, so the muscle is decreasing while the blood's increasing, but it's impossible to say for sure. I mean this seems likely, but it could also be diffusion along the blood vessels. It's really difficult to test that because you would need to take samples of blood all the way up from the femoral vein up to the central region of the body. Right up that vein to see if there's a concentration gradient happening, but you can't really do it because even sampling the blood changes the environment, pulls blood from other places.
Peter:
31:42
They are only taking 100 microliters though aren't they, so they could put in 100 needles in between the femoral vein and the heart.
Tim:
31:51
[laughing] Yes, maybe they'd need some adjustments to their robot to do that.
Peter:
31:53
They'd probably need some ethical approval for that sort of work too, I'd think.
Tim:
31:56
So they do note in the section on limitations that they did only have a small number of cases per analyte and one of the most difficult things with accounting for postmortem redistribution is that you can't account for changes that happen prior to receiving the body.
Peter:
32:10
So even if you do, this sort of study is not going to tell you anything about that, is it?
Tim:
32:14
No, except I guess that one of the really important things is to get the body to the mortuary as quickly as possible and then sampling as quickly as possible once the body arrives. So really in postmortem toxicology, because of all of these issues with redistribution, detecting the presence of the drug is really the most important thing and then interpreting it in light of the circumstances of the case, and the concentration is important but you can't rely on the concentration as being the thing that's going to tell you whether or not someone died of an opioid overdose or whether they were just taking their medication as prescribed. Alright. That's it for this 5 in 30 episode, and actually this is the last episode of the first season of The Toxpod.
Peter:
32:57
It's been an interesting experience and hopefully you're enjoying it.
Tim:
33:00
Yeah. Thanks for all the feedback from listeners all around the world. We appreciate that. If you want to contact us, you can do so by emailing thetoxpod@sa.gov.au. We'll be back for another season sometime in 2019.
Peter:
33:16
I'm looking forward to it, should be fun.
Tim:
33:18
And don't forget the Forensic and Clinical Toxicology Association conference is on in June. You can check out all the details at facta2019.com.au.
Peter:
33:28
Thanks for listening. So are you really an editor for that dianetics journal?
Tim:
33:32
No, I get those kind of emails all the time.
Peter:
33:36
Yeah, so do I.
Tim:
33:36
They're rubbish.
Peter:
33:36
I've still got my certificate though.