Samuel Mesihää (Presenter)
University of Helsinki
Bio: I received my MSc in biochemistry from University of Oulu (Finland) in 2015. In my MSc thesis I applied mass spectrometry for discovery of potential urinary tumor markers. Currently I'm a PhD student at University of Helsinki, Department of Forensic Medicine.
Authorship: Samuel Mesihää (1), Raimo Ketola (2), Ilpo Rasanen (2), Anna Pelander (2), Ilkka Ojanperä (1,2)
(1) Department of Forensic Medicine, Faculty of Medicine, University of Helsinki, (2) National Institute for Health and Welfare, Forensic Toxicology Unit, Helsinki, Finland
Lack of authentic reference standards is a common problem in quantitative mass spectrometric analysis of new psychoactive substances (NPS) and drug metabolites. A new instrument platform was utilised for concurrent identification and quantification, by dividing the GC flow between a high resolution QTOFMS instrument and a nitrogen chemiluminescence detector. The high mass accuracy of QTOFMS combined to APCI ionisation enabled tentative substance identification relying on the accurate mass of protonated molecular ion. Quantification was based on the equimolar response of drugs to nitrogen. This concept was applied to study the concentrations of α-PVP and its metabolites in post-mortem urine samples.
According to the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA), nearly one hundred new psychoactive substances (NPS) are entering the illicit drug market annually (1). NPS are produced mainly in the Far East to avoid drug legislation and to experiment with new psychotropic effects. Many of the NPS share structural similarities with commonly abused psychotropic drugs such as amphetamine or lysergic acid diethylamide (LSD).
Accelerated availability of NPS in 2010s has been a challenge even for clinical toxicology laboratories because conventional analysis of drugs in biological matrices still rely on the use of reference standards. Continuous appearance and disappearance of NPS complicates the acquisition of authentic reference standards because the standards are expensive and difficult to obtain in a sufficiently short period of time. Urine metabolites of NPS are rarely commercially available, and thus expensive custom synthesis may be required.
Mass spectrometry is a method of choice in the analysis of drugs in biological matrices. High resolution mass spectrometer (HRMS) instruments with high mass resolution and mass accuracy can be used for tentative screening of NPS without reference standards, if the structure of the molecule is known (2). However, quantification still remains a challenge. Nitrogen chemiluminescence detection (NCD) shows equimolar response to nitrogen that could be exploited for single-calibrant quantification as approximately 90% of drugs contain nitrogen. The N-equimolar response of this detector allows quantification of both traditional illicit drugs and NPS using a single secondary nitrogen-containing reference standard. A considerably high signal sensitivity can be reached when NCD is hyphenated to gas chromatography (GC) rather than liquid chromatography (LC).
In this study, we apply a novel analytical platform consisting of a GC System coupled through a two-way flow-splitter to a quadrupole time-of-flight mass analyser (QTOFMS) and to a NCD. Atmospheric pressure chemical ionization (APCI) in the positive mode was used in the GC-QTOFMS system to enable accurate mass based identification based on the elemental composition of the protonated molecule as identification criterion. Unlike electron ionization in GC-MS, APCI is a soft ionization technique that usually preserves the protonated molecule. In our previous studies we proved that the MS/MS spectra produced by GC-APCI-QTOFMS were comparable with the commercially available LC-ESI-QTOFMS spectra, which allows the later confirmation of target compounds by using the more easily accessible in-house or commercial LC-ESI based MS/MS spectral libraries (3).
In this study we applied GC-NCD-APCI-QTOFMS to study urine concentrations of α-pyrrolidinovalerophenone (α-PVP), a common cathinone type NPS that is currently controlled in Finland, and three of its metabolites. Simultaneous identification and quantification of α-PVP and its metabolites was demonstrated by analysing post-mortem urine samples after liquid-liquid extraction with a mixture of ethyl acetate and butyl chloride at a basic pH.
α-PVP and three of its metabolites were studied in ten post-mortem urine samples with α-PVP concentration ranging from 0.05 to 5mg/L. In most cases, all metabolites were successfully detected in full scan mode along with two characteristic fragment ions that were found in the MS/MS mode. Tentative identification was confirmed by MS/MS using authentic reference standards.
The accuracy of single-calibrant NCD quantification for α-PVP and its metabolites in urine was generally better than 30%, which is comparable to mass spectrometric quantification with reference standards. These results represent the first GC-NCD quantifications of drug metabolites from authentic samples.
Conclusions & Discussion
This novel analytical platform has a great potential in the analysis of nitrogen-containing drugs with a unique advantage of simultaneous identification and quantification without using reference standards. Retrospective quantification can be carried out even if the NPS have escaped initial screening. In the future we plan to apply this approach to study the toxicity, prevalence and metabolism of a wider range of NPS in forensic blood and urine samples.
References & Acknowledgements:
1. EMCDDA (2016) European drug report 2016: Trends and developments. http://www.emcdda.europa.eu/system/files/publications/2637/TDAT16001ENN.pdf. Accessed 11 May 2017.
2. Ibánez M, Sancho JV, Bijlsma L, Nuijs AL, Covaci A & Hernandez F (2014) Comprehensive analytical strategies based on high-resolution time-of-flight mass spectrometry to identify new psychoactive substances. Trends in Analytical Chemistry 57:107-117.
3. Mesihää S, Ojanperä I, Pelander A, Rasanen I & Ketola R (2017) Development of a GC-APCI-QTOFMS library for new psychoactive substances and comparison to a commercial ESI library. Analytical and Bioanalytical Chemistry 409: 2007-2013.
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