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We recently hosted a webinar focused on streamlining forensic toxicology workflows, featuring expert speakers Maria Sarkisian from the San Francisco Office of the Chief Medical Examiner (SFOCME) and Dr. Dick Paul Kloos from the Netherlands Forensic Institute (NFI). The webinar explored innovative LC-MS/MS strategies that help forensic labs improve efficiency. In this blog, we share highlights from the Q&A session, where our speakers addressed the audience’s questions and shared actionable insights for forensic laboratory professionals.
How is your method validated?
Dr. Dick Paul Kloos:
For us, the targeted screening was validated by the ASB Standard 36, as for the standard practices for method validation in forensic toxicology. Sometimes we also look at the European Medical Agency validation guidelines. These two are the most common for us.
Maria Sarkisian:
We used ASB Standard 36 as well.
Can your new methods meet the needs for sensitivity and specificity, especially for NPS (Novel Psychoactive Substances)?
Dr. Dick Paul Kloos:
Yes of course, this is a good question. There are many isomers or stereo isomers or maybe even chiral isomers to take into account. So, for us, we try to put in as many stereo isomers as possible. For example, 2-MMC, 3-MMC, and 4-MMC to see if you can separate them. In some cases, we know we cannot because they are too close. So for that, during the validation, we also have the selectivity. We automated these compounds in the reporting, so that there will be a mark indicating that it could possibly be another isomer.
And if it’s really necessary for the case, then we do a follow-up investigation, like a custom method, to make a chiral separation. So we basically put asterisks next to these compounds when we report them, if there is a chance that there is an isomer based on the structure.
Maria Sarkisian:
During method validation, when we’re doing selectivity studies, there are a lot of isomers that we are able to separate. But of course, there are some that we can’t like meta, ortho and para fluorofentanyl, for example. So what we do is choose a representative. Usually, that’s kind of maybe in the center in terms of retention time, and we spike that as a representative of LOD and QC’s. And in our actual components list, it’s listed as a group. But we can also identify it as a group.
What substances are included in the QC samples?
Dr. Dick Paul Kloos:
In the 571 method, it’s all 500. We basically have two levels of QCs, a lower and a higher QC, with all of them. It’s not one sample. It’s 13 per level because we have different working solutions, because they don’t all fit in one working solution. So, we have 13 QC lows and 13 QC highs for everything.
Maria Sarkisian:
I don’t know the exact number because of the groups, but it’s about 800, almost 900, that are spiked. We separated them into a mix A and a mix B to also be able to separate isomers that look close to each other. We have them in separate mixes in our QCs as well.
Dr. Dick Paul Kloos:
That’s a good point. That is not the best idea to put isomers in the same mix. So we also try to separate that and the metabolites of certain compounds. So, for example, cocaine and benzoylecgonine, we don’t put them in the same mix.
How do you deal with the identification of isomer pairs on the LC-QTOF? What were the requirements for RT and/or MS differences?
Maria Sarkisian:
In our method, we’re able to separate a larger group, like 3,4 something para-methyl acetyl fentanyl and fentanyl right after. They’re in separate mixes and separable by retention time. We came up with seven categories for grouping, where they are grouped if they are within 0.1 retention time in minutes, have the same precursor, and the same library fragmentation.
How is the compound library of 900+ analytes curated and maintained? And how frequently is it updated?
Maria Sarkisian:
We base it on recommended standards and guidelines, and also our already established in-scope Triple Quad methods and NPS alerts. We update them on a quarterly basis if there is an update to be made, usually based on NPS alerts or specific requests from medical examiners.
How do the results from your HRMS suspect screening and targeted semi-quant screening compare?
Dr. Dick Paul Kloos:
We look at the HRMS data as a suspect. We do this cautiously because there are no reference standards injected along with it. We really take the QTRAP method with the standards to confirm the results of the HRMS. If the HRMS data is still not in the QTRAP database, we would preferably like to confirm it with a reference standard as well.
This Q&A session showcased the importance of validation and adaptability in forensic toxicology workflows. Both speakers emphasized the need for robust, flexible methods to keep up with the evolving landscape of the recreational drug market and the challenges of high-throughput screening.
Want to learn more?
Watch the full webinar recording for further insights into advanced forensic toxicology workflows.
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