WEBINAR | Toxicology Screening Using High Resolution Mass Spectrometry for Forensic Cases

WEBINAR | Toxicology Screening Using High Resolution Mass Spectrometry for Forensic Cases

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Hello, everyone. Welcome to today's live broadcast, Toxicology Screening Using High Resolution Mass Spectrometry for Forensic Cases. I'm Laura Bush, the editorial director of LCGC. And I'll be your moderator for today's event. We're pleased to bring you this webcast, presented by LCGC and sponsored by Waters Corporation. From the everyday consumer to scientist in the laboratory, we all rely on accurate information to make critical decisions.

Waters Corporation is a world leading specialty measurement company focused on improving human health and well being through the application of high value analytical technologies and industry leading scientific expertise. For forensic drug chemistry and forensic toxicology laboratories, Waters provides a wide range of easy to use solutions, including compact, low cost mass detectors, high performance tandem quadrupoles and high resolution time of flight mass spectrometers. Today's webinar will focus on the advantages of high resolution mass spectrometry for forensic toxicology laboratories. We have a few housekeeping announcements before we get started.

First, the webcast is designed to be interactive. So we encourage you to ask questions. You can submit questions at any time by typing them in the Q&A box.

And you can find that on the right-hand side of your screen. You can enlarge the slide window by clicking on a small square icon in the upper right-hand corner of the slide window, or by hovering your mouse over the lower right-hand corner and dragging the window to the desired size. The slides will advance automatically during the webcast. And if you have any technical problems viewing or hearing the presentation, please click on the question mark Help widget in the dock at the bottom of your window. Before I introduce today's speaker, I would like to ask everyone in the audience to participate in two brief polling questions. In each case, please click directly on the screen to enter your answers.

So here's our first question now on your screen. "Is the analysis of novel psychoactive substances or designer drugs a challenge in your laboratory?" Yes or no? So again, you can click directly on the screen to enter your answers. "Is the analysis of novel psychoactive substances or designer drugs a challenge in your laboratory?" Yes or no? I'd now like ask our audience to participate in our second polling question. Please click directly on the screen to enter your answers. So here's that question.

"With the current COVID-19 pandemic, as a percent, how would you characterize the volume of your laboratory test operation at this point in time?" 0% to 20% of normal, 30% to 40% of normal, 50% to 60% of normal, 70% to 80%, 90% to 100%? So once again, you can click directly on your screen to enter your answers. And that question is, "With the current COVID-19 pandemic, as a percent, how would you characterize the volume of your laboratory test operations at this point in time? 0% to 20% of normal, 30% to 40% of normal, 50% to 60% of normal, 70% to 80% of normal, 90% to 100% of normal? Thank you very much for participating in our second poll. I would now like to introduce today's speaker. We are very pleased to be joined today by Vincent Di Fazio. Vincent Di Fazio currently works as a judicial expert in toxicology for the National Institute of Criminalistics and Criminology in Brussels, Belgium.

The Institute's major activities include post-mortem cases and cases involving living persons, such as drug facilitated sexual assault or driving under the influence of drugs. In his current position as a forensic expert, Vincent Di Fazio gives lectures regularly to judicial magistrates to inform them of the possibilities and limitations of toxicological investigation. He's a biomedical scientist from the Université Catholique de Louvain, or UCL, in Brussels, and was awarded the Paul Meiu prize in 2005 for the best end study work of the Biomedical Sciences School at UCL. Vincent Di Fazio is a member of the BLT, SRMLB, TIAFT, SOHT, and SFTA.

In addition, he is currently listed in the Belgian National Register of Judicial Experts and also a European registered toxicologist certified by EUROTOX since 2013. Vincent, thank you for joining us today. Please go ahead and get us started. Good morning, good afternoon, or good evening, depending on where you are.

My presentation will focus on the impact of high resolution mass spectrometry in the daily work of forensic toxicologists. I will try to show you with some real cases the benefit of this technology in a tox lab. First, a quick preview of our Institute. The NICC, the National Institute for Criminalistics and Criminology, is part of the Ministry of Justice in Belgium. We have different fields of expertise, like toxicology, DNA, ballistics, et cetera.

And we are working only after a specific demand from the judicial authorities. In our toxicology department, our daily work consists of a mix of forensic cases. We can have driving under the influence of drugs cases, drug facilitated sexual assault cases, or cases involving living persons and also post-mortem cases.

Then and luckily, we are not only doing reports but also research projects on different topics, like alternative matrices or new psychoactive substances. We are also often requested by the government to give them scientific advice. And finally, we also play a role of educator by giving lectures to judicial authorities about the possibilities of toxicology analysis in a forensic context.

Depending on the case we have to deal with, we apply different lab strategies. For example, in our DUID cases, where we receive a blood sample for confirmation of drugs of abuse, after a positive, on site oral fluid screening test. The Belgian law has blood cutoff values for THC, amphetamine, MDMA, cocaine, and benzoylecgonine. For these cases, we are only performing a confirmation and blood quantification by UPLC MS/MS of the on site positive screened drugs abused.

But for the other cases, like DFSA or post-mortem cases, we never know what has been consumed. So the strategy includes GC headspace, FID method for alcohols, but also target methods by LC MS/MS for illicit or prescribed drugs. And of course, a comprehensive drug screening, which in our lab before the acquisition of the Tof combines two different techniques. GC /MS without and after derivatization and UPLC DAD.

In Belgium, except for blood alcohol quantification, there is no legal requirement to use a specific analytical technique in a forensic context. So there are big differences between labs. For example, one uses UV detection and the other one MS/MS to quantify benzodiazepines. Of course, as detection windows will be totally different for the two techniques, the use of a specific technique will have a significant impact on the conclusions of the expertise report. In some types of cases, this issue may not be of importance.

But for DFSA cases, where sensitivity is key, these differences are of crucial importance for judges to know and to consider. And here you can see the importance of sensitivity that LC MS/MS can provide by extending the urine detection window for bromazepam from 24 hours to over five days. This can be important for DFSA cases, where the victim may not report the crime immediately. So the screening method on Xevo G2-XS QTof starts with a protein precipitation using 100 microliters of sample and 400 microliters of ice cold acetonitrile. The extracted example is injected first in negative electrospray ionization mode.

And then the next day, we keep the same column. We change to mobile phase and we re-inject the same samples in positive electrospray ionization according to the method developed by Waters in Manchester. The same sample preparation is used for all sample types, a lot of post-mortem blood, urine, or other post-mortem matrices. The data acquisition is non-targeted, which means we do not exclude any compounds as you would if you were using a tandem quadrupole in MRM mode. All ions are entering the collision cell and data are collected at both low energy and high energy, which enables the collection of exact mass data on precursor ions and fragment ions. Both fragment ion prescuror ion information combined with retention time is used to identify compounds by comparing against a library of over 1,600 compounds.

The library was produced under the same acquisition conditions as the samples are run. Data is recorded from 50 to 1,000 Daltons. Once the data is acquired, it is automatically processed by UNIFI software system. A typical workflow would be to compare the acquired data against the library of approximately 1,600 compounds in which the UNIFI software will identify compounds by using retention time data and exact mass data of precursor and fragment ions. The data can also be investigated using other workflows to investigate compounds that are present but not in the library.

One advantage of non-targeted data acquisition is that the data is always available to investigate at a later date when or if more information becomes available. For example, on a new NPS. Here is an example of a UNIFI targeted workflow, where the compound is identified using the UNIFI scientific library. Actually, in our run, we are using a homemade QC. It's a blood sample spiked with a mixture of five different prescription drugs to be identified in positive electrospray mode. Our criteria to accept a batch are the correct identification of our five compounds, a mass error lower than five ppm, isotope match error mass lower than 20%.

Then we check the retention time observed and retention time expected. We also need a minimum of one fragment found versus expected fragments. And a response higher than 10,000. Here you have the example of bromazepam with low energy and high energy spectra with the different fragments and their associated mass error. The first case concerns an eight-month-old girl who was admitted in a hospital for surgery.

After that, she stayed one week brain dead and finally died of cardiac arrest. An autopsy was performed. Question of the magistrate was, as the origin of the cardiac arrest is unknown, exclude a possible intoxication during surgery. The autopsy samples were, therefore, not relevant.

But we received approximately 350 microliters of surgical blood sample. With this low sample volume you can forget your screening by UPLC, DAD or GC /MS. So we decided to use 100 microliter to screen the blood sample with the QTof. Here are the results.

With the Tof, we found morphine. Of course, everybody knows the substance. Midazolam, this is a benzodiazepine used especially in a hospital environment for sedation.

And levetiracetam, an anti- epileptic drug. The next step is the confirmation and quantification of these compounds to verify if we are in a toxic range or not. So we used tandem mass spectrometry, and more specially, Xevo TQ-S system with, as you can see, quite limited sample volumes. And finally, when we compare our results with therapeutic ranges of these compounds, we can answer the question of the judge and exclude any possible overdose.

In post-mortem cases, it can be possible that no blood sample is available. This can be problematic for the final interpretation. Other autopsy samples, like urine, liver, bile will give an overview of the relatively recent use of substances. But it will not help us to say this substance may be the cause of the death.

So in this context, there is an interest to investigate vitreous humor for several reasons. Vitreous humor is an isolated metric from the decomposed processes and also from possible post-mortem redistribution phenomena. The alcohol ratio between blood and vitreous humor is around one, and scientifically proven. Detection windows of drugs in vitreous humor will be close to blood and so more related to the moment of the death in comparison with other matrices. Vitreous humor is used for the post-mortem quantification of glucose and electrolytes. And vitreous humor is also a very clean matrix due to its water composition, which is an interesting advantage for pre-analytical steps.

Finally, vitreous humor can be taken during an external examination of the deceased person. But unfortunately, we have also some disadvantages. The first one is a limited sample volume and a limited sample availability because you don't find any more vitreous humor approximately five days after death.

And until today, we do not have therapeutic concentrations of drug in vitreous humor. So is qualitative screening of vitreous humor by Tof possible? The answer is yes. of course. For this particular case, we can determine a recent use of codeine and paracetamol separately, or the recent use of pharmaceutical specialty, combining the two substances.

But we cannot say if this administration could be implicated in the death of the victim. Maybe another comment. We have also tried the Tof to analyze other post-mortem matrices, like liver homogenate. And it also works. Next case is concerning a 17-year-old girl who declared that she had been sexually abused. Blood and urine samples were collected 12 hours later.

According to her declarations, she only drank one glass of an alcohol drink. And she supposed to not consume drugs of abuse or any prescribed drugs. Before looking at the results, you probably know that there is a huge variety of drugs that can be used in cases of DFSA.

Furthermore, some products can be used in very low dose. And this can of course complicate their detection in biological samples. On this slide, you can see the results obtained with the three different chromatographic screening methods.

So aripiprazole is a neuroleptic compound, as risperidone. 9-hyrdroxy risperidone is an active metabolite of risperidone. Trazadone is an antidepressant. And MCPP is its metabolite. If you combine GC /MS with DAD, you will find two of the three compounds present in the blood and urine samples. When you look at concentration found by MS/MS techniques, you can see that the sensitivity of the Tof is quite interesting.

And this is of major interest for DFSA cases. So let's come back to the interpretation of this case. As the victim declared no comsumption of prescribed drugs, we have to propose two different hypotheses.

First, she takes these compounds as medication. And the other one, one or more compounds could have been used in low dose to impair the victim. Next case is a DUID case concerning a 22-year-old driver arrested by the police following a significant increase speed and a particular driving style.

The driver shows a noticeable waking impression despite the early hour. He says that he is coming from a party. The police officer observed a clearly agitated behavior and pupils are not dilated.

The oral fluid on site screening test was performed but revealed no recent consumption of classical drugs, namely cannabis, cocaine, amphetamines, and heroin. And in Belgium, if a negative on site test result is obtained, usually the driver is free to drive on. But if impairment is evident, the police officer via agreement of the magistrate can decide to take a blood or urine sample for further toxicological investigation. That's what happened in this case.

And blood and urine samples of the driver were taken. What about the results? Screening in blood by GC /MS and UPLC DAD was negative. In urine, we found MDMA, or ecstasy, and it's metabolite MDA.

These two compounds are confirmed by MS/MS techniques. We targeted MRM techniques. We also found small amounts of THC COOH in blood and urine. With this result, you can demonstrate a non-recent consumption of ecstasy and cannabis. But you cannot explain the impairment observed by the police officer.

Finally, when we applied the Tof method, we found naphyrone in blood and in urine samples. And of course, THC COOH and MDMA in urine. Naphyrone is an NPS belonging to the cathinone chemical class. It's a stimulant and is sold as an alternative of mephedrone. This slide shows you the results of the driver's urine simple.

All our identification criteria, as for example, retention time, precursor, ion mass, at least one fragment mass, and a signal above 10,000 were satisfied for MDMA MDA and naphyrone The following case is a post-mortem case of 2013. So this concerns a 21-year-old man found dead in his tent during a summer music festival. There was an overdose suspicion.

The comprehensive drug screening and also target methods realized in 2013 did not demonstrate any evidence of overdose. In 2018, at the parents' request, further investigations were requested. And especially due to recent analytical developments, a new toxicological expertise.

So we decided to re-analyze the blood and urine samples by GC MS and Tof. Even with updated MS libraries, results obtained by GC /MS were always negative. But with the Tof, things were a bit different. According to our identification criteria, we found by ToF in the urine sample a suspicion of 25B NBOMe. These molecules are NPS belonging to the phenylethylamine chemical class.

It is frequently sold as LSD for its hallucinogenic effects. Here you have the results of the Tof urine screening. You can see that 25B NBOMe is identified by the library with a retention time of eight minutes. And you have two fragments found for two fragments expected. So it was the first NPS identified in our lab. And so we decided to inject reference standards of 25B NBOMe but also 25C and 25I to see how the method deals with these compounds.

25C and 25I NBOMe have similar structures. With the bromine atom replaced by other halogens, chlorine and iodine. This slide shows you the injection of the reference standard of 25B on the left side and 25C on the right side.

Of course, exact masses are different, but also retention time. 25B and 25C were correctly identified by the Waters library. And this was exactly the same observed for the injection of the reference standard of 25I. This little experiment, and especially in terms of retention time, exact mass, identified fragments, has contributed to confirm the 25B NBOMe consumption in this post-mortem case.

In conclusion, we all agree in our lab that this instrument is essential for our daily toxicological work. And the acquisition methods from Waters were easily applied to our instrument. We only optimized a pre-analytical step. A protein precipitation with ice cold acetonitrite. So you can have your screening results combining positive and negative electrospray mode in approximately 24 hours.

And this is a great advantage for giving a quick and relevant qualitative screening in an urgent post-mortem context, for example. You also have seen that the sensitivity is good, allowing us to cover the presence in very low concentrations of a large amount of compounds and their metabolites. A necessary tool when you analyze DFSA cases or when you are confronted with very low sample volumes. Up to date libraries are important because number and types of NPS are in continuous development. We have decided to use this instrument only for qualitative evaluation, even if you can quantify.

But our strategy is to confirm and quantify, depending on the case context, by MS/MS technique what we have identified by the QTof. The UNIFI software allows us to save a lot of time. By establishing your identification criteria, you have a nice, quick overview of what's inside your sample. We decided to inject all our samples with two different injection volumes in a run to avoid missing a correct identification of a compound present in a high concentration in the sample. Another interesting point is the easy instrument start up, including different critical steps, like LockSpray, positive and negative calibrations.

Everything's automated but you have to check each parameter before starting your run. So LockSpray is a reference solution set up prior to any analysis and which is constantly infused during the analysis, acting as a lock mass during all the experiment. You also can classify none identified candidates in terms of response intensity, for example, and only decide to elucidate the more intense ones.

The Elucidation Tool in UNIFI uses accurate mass helps via ChemSpider. Concerning unknowns, this is something interesting to check quickly if you have missed something in your sample. This is a Mirror Plot showing at the top the base peak ion chromatogram at low energy and down the identification compounds.

If something present in the BPI chromatogram is not identified, then you can, with its own retention time, decide to elucidate this candidate. So we have unfortunately not yet finished with this method. We only have a homemade QC sample in positive mode. We do not have any control over batch in negative mode.

So we are currently working to make a homemade QC for the negative mode. Another important thing is that for the moment, we do not use internal standards. This is not really correct, as we are now using an internal standard for the positive mode.

But we do not still have any internal standard for the negative mode. So this is something to develop in order to be more confident of our sample's integrity. I would like to thank you very much for your attention. Thank you Mr. Di Fazio for that informative presentation.

It's now time for the question and answer period. So I would just like to remind everyone in the audience that if you'd like to submit a question, you can do that by typing it in the Q&A box. And you can find that on the right-hand side of your window. OK. So starting with one, how do you find operating the high resolution MS system compared to a tandem quadrupole MS or GC /MS system? Well, we bought the Tof instrument in the beginning of 2018. And I have to admit that we were a bit worried, as our lab uses GC FID, GS /MS, and LC MS/MS as techniques.

But we had no experience with high resolution mass spectrometry. It took approximately 10 months since the installation of the system to be fully trained and operational. In addition to training of our team on site, a more specific training dedicated to tox screening with UNIFI by specialist at Waters MS headquarters in Wilmslow-- sorry-- was really effective. We did not really have problems to learn how to use the system. It was not any more complicated than for other systems.

And I don't think it requires a high skilled operator to use the system. For example, in our lab, analysts using the Tof and processing data using the in-house made workflows combined with the library generated by Waters. Of course, after that, experts are in charge to control and validate the obtained data.

And moreover, to decide, depending on the case, to go further with the investigation. For example, using the Elucidation Tool for the identification of unknowns. Interpretation of data in comparison with classical MS/MS systems does take more time with the Tof. Thank you. Here's another one.

If a standard for a new NPS is not available, can you still report it as a positive identification based on the HRMS data? This is a very good question. If the NPS is also identified by GC /MS, I will report it. If it is not the case, it's more complicated.

If you had to ask me this question two years ago, I would have said definitely no. But now with the experience of the system, I would say it depends. I think we have several levels of certainty in the identification workflow. You have the exact mass error on the precursor ion and fragments; the number of fragments found compared to the number of fragments expected in the library; retention time found compared to the retention time of the library; response; and finally, the isotope match intensity RMS percent, and the isotope match and that RMS ppm.

These two criteria indicates the degree of matching between the theoretical isotopic pattern and the observed pattern for the precursor ion plus term. So if all criteria are satisfied, and if I can confirm its presence by detecting also a metabolite of this compound-- sorry-- I will report it even if I do not have the reference standard. If it is not the case, I would suggest to say it is suspicious but not confirmed by a reference standard. Why do you think naphyrone was not detected by GC /MS? I would expect it would have been easily detected by GC /MS. Yes. In this particular case, maybe it's a problem of concentration because we did not quantify the level of naphyrone in blood.

So maybe you will find very, very low concentrations of naphyrone. And that's maybe why we have detected by the Tof, but not by GC /MS. In what percent of samples do you find an NPS? As I mentioned during the presentation, for DUID cases, it is difficult to know the NPS prevalence, as we are not looking for them.

The Belgium DUID legislation uses its legal cutoff for classical drugs of abuse. So for these cases, we are not using the Tof but target LC MS/MS method for confirmation and quantification of the required drugs of abuse. However, our research team looked at this problem. And we made a first study published in DTA a couple of years ago, in which we analyzed 556 blood samples collected after a positive on site oral fluid screening test.

This study showed 7% of NPS in this DUID population already positive for classical drugs. In a second study from drivers that had signs of recent drug use, but were screened negative for classical drugs of abuse, the NPS prevalence was around 10%. These two studies were very important for us because even if you could think that NPS prevalence is quite low. It is also clear that some drivers are using NPS sometimes in combination with classical illicit drugs and would present a real danger in terms of road safety.

For other cases, like post-mortem or DFSA cases, I do not have concrete results. But in my opinion and my experience, the NPS prevalence for these cases is lower than the prevalence observed in the DUID population. Are you doing the HRMS QTof analysis for all the samples in your lab or just for some special cases? If so, how do you decide which samples will need more investigation? Yes.

As I told you, for example, for DUID cases, we are not using the Tof because this is a very target substances we have to detect and to quantify. But for all other investigations, then DUID, we are using the Tof, of course, because we never know what's inside the samples. So we have to make a comprehensive drug screening because it is very important to go further than for DUID cases.

Do you encounter false positives when you confirm with LC MS/MS? Not really. Not for the moment. No because every substance we detect by the Tof we confirm with MRM methods. Until now, we haven't had some false positive result. What are the identification criteria that you use for drugs detected in case samples? Oh, yes. So I mentioned, we have some criteria concerning the exact mass error on the precursor ion.

We have also a minimum of one fragment that has to be found versus fragments expected. We check also the retention time compared to the library. We need also a response higher than 10,000 to accept the identification. And for the isotope match intensity RMS percent, we have to be lower than 20% to accept the compound identified by the library. How do you handle the data processing of a batch when faced with several hundred drugs? And how long does it take to process one batch of samples? Oh, yes. So, the processing of the data is really automatically done.

So then we are only looking on the screen just to see that for the compounds identified our criteria are all respected. So depending on the case we have to deal with, if we see some peaks that are not identified by the library then we go to the next step and to the Elucidation Tool to check if we have candidates. And to see with other libraries available on the net to see if we can find another new NPS, or something like that. But, this is the job of the expert. This is not the job of the analyst in the lab.

This is something that we are doing in your office calmly to check new possible candidates. But it takes time, of course, of course. Because this is not automated so you have to investigate. Do you plan to use the QTof for quantification, as well? This is something that we are thinking about and that we will maybe develop it in the future for some specific cases. First, to quantify some classical drugs of abuse or therapeutic drugs that we often detect in our blood samples.

In this case, we could provide in a short period of time a preliminary semi-quantitative result. And this could be interesting for an urgent forensic case. Secondly, to quantify some compounds like NPS, for example, that we do not detect on a regular basis in our samples.

In this way, we do not need to add to the compounds to our routine quantitative methods, reducing costs, less standards to buy, and diminishing possible errors in preparing stock solution with enormous amount of compounds. This could be a first step. And we will see how it works and if it can be extended to other things. Is there a reason you run negative mode first and then positive mode? No. I have no explanation for that. You can also check the positive first and next data, the negative mode.

I think the most important thing is that in the negative mode, you have less compounds to search because you have probably all the acidic compounds. But, the run time is around eight minutes for a negative mode. In the positive mode, the runtime is around 15 or 16 minutes. So it takes more time, the investigation of positive mode. So if you want to inject your batch in negative mode maybe at the end of the day, you can re-inject all the batch in positive mode and you have all your data.

the next day. That's the only reason why we suggest to inject in negative mode the first day. Does Tof have more downtime compared to GC or triple quadrupole instruments? I don't know, really, if it takes-- the sample prep is maybe more quick with the Tof because we only perform protein precipitation. Thus, it is faster with the Tof than for GC /MS because you have the liquid liquid extraction then the step of derivatization.

So in terms of sample prep, the Tof is very quick. But maybe you have to take more time in processing and in reporting with the Tof then for GC /MS. What ppm error do you think is acceptable for confirmation purposes? I think it's 2 ppm, 2 ppm on mass error. In terms of detection limits, are QTof limits sufficient to meet DUID screening criteria for low concentration blood drugs around 5 to 10 nanograms per milliliter? Though it's difficult because for the moment, we haven't checked each limit of detection of every compound you can find in the library with the Tof. But in our experience, we see that the sensitivity of the Tof is very powerful. For example, detect some levels of ketamine that were under our limit of quantification by MS/MS methods.

So generally, we are really well surprised with the sensitivity of the Tof. So I don't think it is a real problem with the sensitivity. But of course, we did not investigate each compound. But generally, in what we observe, it is very powerful. Does chilled acetonitrile work significantly better than room temperature, acetonitrile? Yeah, I think it's better for the precipitation.

I will recommend to use ice cold acetonitrile for the precipitation. Yeah, it works very well. Can you walk us through how long it takes for the processing of the results? Is it automated or do technicians or professionals have to review each peak? I just talked about that. So the processing takes maybe, if you look at all your batch, it takes maybe several minutes, maybe a half an hour approximately, depending on the number of samples you have to process. But in our lab, the analysts are processing the data, then they are reporting the results. And we as experts, we are just checking and looking at each compound identified to check all the criteria on the screen.

This takes, of course, some time. But this is necessary because you have also to consider if you have to check candidates. So yeah, of course it takes time. But I think this is very important. And when you try this method, I think you don't want to come back with another classical screening method.

But I think just try it to see how it works. Have you looked at hair analysis for drugs of abuse? No. I think for our lab, it was not really interesting because we have a lot of MRM target methods for the quantification of drugs of abuse or classical drugs for hair.

So it was not important for us to develop a screening method by the Tof. And we do not have also so many hair samples to deal with in our lab. So it was not a priority for us to detect. But you can, of course, try to do that.

But we have not done that. Can you briefly outline how you validated the Tof method for screening post-mortem samples? Yeah. We did not perform a validation of our screening method.

I think it's important to consider that the Tof method in our lab is not the only one method we apply in our strategy of investigation. And besides this method, we classically detect and quantify with target MS/MS methods classical drugs of abuse and therapeutic drugs, such as benzodiazepine, antidepressants, and neuroleptics. As I mentioned before, we worked several months with three different screening methods. But finally, it is very difficult to compare the results because each technique has limitations in terms of sensitivity. And there is no one reference screening method.

We think the best way to perform a validation of the Tof method would be to make a cross-validation with another tox lab. But this is something still to investigate. Our strategy for the moment is to confirm by MS/MS technique each compound of interest detected with the ToF to avoid a false positive result. But I have to mention that it never happened until now. How many samples do you run with the QTof per year? Oh, this is a good question. I have not made the exact number.

But I would say, approximately 500 samples. But, well, this is very, very approximate. Using only protein precipitation, how frequently are you changing the UHPLC column? In other words, after how many injections? Oh, I have to ask to my colleague in the lab maybe because I don't know exactly.

But I have the feedback. And I don't think there are some problems. You have to change relatively frequently the column.

No, I think you have to take care about the injection volume. This is very important. But, we have no problem to change more than frequently the column of the mobile phase.

But, you have to consider also that with protein based precipitation, you can investigate glucuronides, compounds like that, so the metabolites. And this is very important because if you are doing liquid- liquid extraction you will not detect them. So I think, putting protein precipitations a good compromise when you have to make a comprehensive drug screening. Do you think there will be a time when high resolution MS systems will completely replace tandem quadrupole MS systems in forensic labs, assuming HRMS can also perform quantitative analysis comparable to a triple quadrupole? Well, I don't think that high resolution will completely replace tandem quadrupole MS systems in the future.

I know that you can, of course, also perform quantitative analysis with the QTof as you can do with any triple quad. I see more these two techniques as complementary. However, I think that high resolution mass spectrometry has revolutionized and improved efficiency of the toxicology screening in forensic laboratories. Fantastic. Well, with that, I think we should wrap up. Thank you so much, Vincent Di Fazio for your excellent talk today and for answering all those questions.

Thank you. You too. I'd also like to thank everyone in the audience for attending and participating in our event today. And I'd like to thank our sponsor, Waters Corporation, for making today's webcast possible. You'll receive an email alerting you when this webcast is available for replay.

We invite you to forward that announcement to your colleagues who might have missed today's live event. We hope to see you all next time. Goodbye.

2021-02-17 05:04

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