Webinar Highlights: Insights into a Forensic Lab – Mass Spectrometry Beyond CSI
Using LC-MS/MS to tackle toxicological screening
5 Aug 2015
Dr. Jürgen Kempf, Freiburg Institute of Forensic Medicine, Germany
Screening for xenobiotics is a crucial part of forensic toxicological analysis. Furthermore, comprehensive and reliable screening of human body fluids for the presence of drugs, toxicants, poisons and their metabolites is an ever increasingly challenging task. In this exclusive webinar, Dr. Jürgen Kempf, from the Freiburg Institute of Forensic Medicine, describes the use of two complementary LC-MS/MS technologies to address this issue. Learn how LC-MS/MS makes high sensitivity, high confidence forensic drug screening a reality, with LC-MSn ion trap technology (Toxtyper™) and accurate mass Quadrupole Time of Flight Mass Spectrometry (ToxScreener). Watch the webinar on-demand here.
The Toxtyper looks like a very convenient solution for routine analysis, but how can it cope with the changing landscape of new psychoactive substances that are constantly emerging?
Although the Toxtyper itself is a closed system, there is an open access version where you can add data specific to your application, such as your analyte and its spectra. Methods for recording spectra in positive or negative mode are included, so you only need to add the mass/charge ratio and retention time to the scheduled precursor list. Depending on the kind of analytes you want to screen for – for example, synthetic cannabinoids or psychotropic medical drugs – it might be a good idea to set up specific screening methods. It depends on the number and chemical properties of the compounds you are screening for.
For target screening of compounds that are in a database, which technology (Toxtyper MSn or QTOF accurate mass) gives you the highest confidence answer?
That depends on how you describe highest confidence − both methods surpass identification criteria laid out in European guidelines. The Toxtyper gives you nominal MS/MS and MS/MS/MS spectra combined with the retention time, while the QTOF system will give you accurate mass data comprising of, precursor mass, isotopic pattern and MS/MS fragments. Therefore, both methods will accurately identify substances that are in there. However, if you are only looking for compounds in a database, for example during forensic screening, the Toxtyper is an easier way to get an answer.
How do you go about finding reference materials, especially for the newly emergent designer drugs?
A lot of these compounds are available from standard reference compound vendors. In addition, we work closely with police and customs agencies throughout Europe and we are part of a European monitoring program that actively searches online retailers for chemicals. It is an ongoing process, a cat and mouse game.
What would you say the main advantages are for using a QTOF for screening over other LC-MS technologies?
The main advantage is the full scan/broad band CID approach that allows the detection of all ions coming from the ion source or collision cell. Every compound that enters the mass spectrometer and can be ionized will be detected, so there's no need to update acquisition methods, as one method fits all. All available data is recorded then you can choose what you want to look for afterwards. Even months after the original analysis, you can go back to the data and look for new compounds. This is especially interesting in the case of these rapidly emerging new psychotropics, as you can go back and see if they were present last month or two months ago.
How much resolution do you really need for unknown screening? Is more always better?
If you have the money, and the time, then of course more is better. For every additional decimal place, you need more expensive instruments, better climate systems and so on. For the compounds that we are dealing with, the resolution of the QTOF system is sufficient, but it is the ability to simultaneously perform MS/MS analysis at this level of resolution that delivers the high confidence in the end result.
You presented a lot of compelling information supporting the use of QTOFs for in-depth target and unknown screening, but there was only a brief mention of this aspect: How compatible is the ToxScreener QTOF for undertaking accurate quantitative analysis?
Triple quadrupole MS is probably the gold standard for quantitative analysis of xenobiotics in body fluids, so if targeted quantitation is your main task you should use such a system. Nevertheless, we have shown that you can easily quantify with a QTOF system − we reanalyzed some batches of samples with the QTOF after routine quantitation on a triple quadrupole instrument and the results were comparable.
The use of a qual-quant approach may be limited by factors such as sample handling and correct use of internal standard, for example. After a large batch of samples is analyzed, the data must be evaluated, analyte calibration samples must be prepared, and you must consider internal standards. Therefore, it's not so much a technical problem as a problem of sample preparation. You may also need to re-run the samples, which may be a problem depending on the stability of the analytes.
In general, for a ToxScreener analysis followed by calibrations standards, you can get some semi-quantitative results. If you want to have accurate quantitation, you will have to run the appropriate calibration and QC samples, within the batch as on a triple quadrupole system, but then you would probably get the same results.
To achieve quantitation of a drug, did you use one internal standard, or multiple deuterated standards?
For semi-quantitated results, or quantitating whether or not the levels of the analyte are at or below therapeutic levels, you may only require one to three internal standards combined with a three-point calibration that is analyzed after the sample. For accurate quantitation, however, you should use the appropriate deuterated version of your analyte, although that is more a problem of the ESI process than of the QTOF itself.
Where you may run into a problem is if you're analyzing 1,000 compounds; which internal standard should you use? It can be very hard to find the right analytical deuterated standard. In this case, the best thing to do would be to use four to five deuterated standards across the whole run time and try for semi-quantitative results. If you really want to get accurate quantitation, you should know what you're looking for before you do the run.
You mentioned amphetamine derivatives. How popular are these derivatives?
Unfortunately I can't tell you how popular or prevalent these substances are − the data you would need would be from the criminal police labs in Germany. Body fluids are analyzed by the forensic institutes, but the materials and the drugs themselves are analyzed in those labs.
Which analytical techniques, or any variants thereof, would you suggest for analyzing the foreign constituents of fingerprints residues, such as drugs of abuse, and/or their metabolites?
As far as I know, the general approach would be MALDI perhaps, but I don't know if it can be done with the DESI source; I haven't tried that yet. It's really a question of the limit of detection – I don't know how many ng or pg of material you would find in fingerprints. I know that analysis using a DESI source has been done in the UK, but everything else I have seen uses MALDI, using special ambient ionization. You can combine the Toxtyper and the QTOF with the DESI source without a problem, but as I have no experience with it, I don't know how well or if it would work.