Anal Bioanal Chem. 2016 Feb;408(4):1171-1181.

Salting-out-assisted liquid-liquid extraction as a suitable approach for determination of methoxetamine in large sets of tissue samples

Katerina Hajkova1,2,3, Bronislav Jurasek1,4, David Sykora*2, Tomas Palenicek3, Petra Miksatkova1,3,4, Martin Kuchar1,3,4

1Forensic Laboratory of Biologically Active Substances, University of Chemistry and Technology Prague, Technicka 5, 166 28 Prague 6, Dejvice, Czech Republic

2Department of Analytical Chemistry, University of Chemistry and Technology Prague, Technicka 5, 166 28 Prague 6, Dejvice, Czech Republic

3National Institute of Mental Health, Topolova 748, 25067 Klecany, Czech Republic

4Department of Chemistry of Natural Compounds, University of Chemistry and Technology Prague, Technicka 5, 166 28 Prague 6, Dejvice, Czech Republic

PMID:26661068; DOI: 10.1007/s00216-015-9221-1



Introduction: New psychoactive substances (NPS) are global phenomenon which causes health and socio-pathological problems. These substances are rapidly worldwide spread via Internet and are highly popular among people as a “legal” substitution of illicit drugs. One NPS class consists of arylcyclohexylamines that belonging to dissociative drugs. The main representatives of this class are phencyclidine (PCP), ketamine and methoxetamine (MXE). MXE is the latest member of the group and it seems as dangerous as its forerunners. This N-methyl-d-aspartate (NMDA) antagonist shares dissociative properties of PCP and ketamine and it could be less harmful than these compounds. However, gathered data are not in accordance with this assumption. The first MXE notification to the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) came from the UK in 2010. Until now, EMCDDA has recorded 110 intoxications and 20 fatalities in the EU.


Fig. 1 MXE and its main metabolites of phase I


Aims: The aim of this study was to develop a suitable and a cost-effective sample preparation technique followed by liquid chromatography–mass spectrometry (LC-MS) method for the determination of MXE and its main metabolites (see Fig. 1) in tissue samples brain, liver, and lung. The overall procedure had to be validated according to bioanalytical guidelines and applied to sets of real samples from animal study.


Methods: MXE was subcutaneously administered to Wistar male rats to evaluate its impact on their behaviour and also to estimate distribution of MXE and its main metabolites to serum, brain, liver and lung tissue. Groups of eight animals were decapitated at several time points; 0.5; 1; 2; 4 and 6 h, respectively. Samples were stored at -40 °C. (Samples of urea and serum were also collected within this study and gathered data are a part of the behavioural study reported in the article of Horsley R.R. et al. (2016)).

First, several frequently used sample preparation protocols were tested; for instance, a protein precipitation and/or a solid phase extraction (SPE). As the results were not satisfactory, we decided to ground the procedure on a salting-out liquid-liquid extraction (SALLE) and QuEChERS approach. The final sample preparation protocol is summarized in the following scheme (see Fig. 2).


Fig. 2 Scheme of the sample preparation procedure applied for tissues with a picture of a real sample


For quantitation a calibration curve was constructed and validation parameters, limit of quantification (LOQ), limit of detection (LOD), accuracy, precision, and repeatability, evaluated. Fig. 3 shows an example of a typical LC-MS chromatogram obtained analysing fortified tissue samples during calibration procedure.


Fig. 3 Example of the MRM chromatogram of a calibration sample in real matrix (50 ng/mL, liver tissue)


Results: The results from the analysis were plotted to illustrate time distribution of analytes in each tissue, thus, you can see curves for MXE, O-desmethylmethoxetamine, and normethoxetamine in one plot for each tissue (see Fig.4). The error bars as a standard deviation of average value are included. There are also the examples of LC-MS chromatograms of real samples per each tissue (see Fig.4, the right side of the plots). The main analyte detected in all tissues was MXE. Other analytes found in relatively significant concentration were O-desmethylmethoxetamine and normethoxetamine. For illustration, kinetic profiles and example chromatograms for each tissue are included (see Fig.4).



Fig. 4 Plots describe a distribution of MXE, normethoxetamine, and O-desmethylmethoxetamine in a) brain, b) liver and c) lung and examples of real chromatograms for each tissue (the right side of the plots).


Conclusions: In this study a suitable, reliable and cost-effective sample preparation technique was developed, tested and applied to real tissue samples in animal study. The procedure was based on SALLE principle. The aim of our work was to gain reliable data on MXE and its metabolites in brain, liver, and lung tissues. This was successfully realized by combining SALLE with LC-MS. The evaluation of the data from psychiatrists’ point of view is provided in the article of Horsley R.R. et al. (2016).



This study was funded by a specific university research grant (project MSMT No 20/2017) and by the Ministry of Interior of the Czech Republic (project VI20172020056, New synthetic drugs – complex interdisciplinary research centre).



Horsley, R. R.; Lhotkova, E.; Hajkova, K.; Jurasek, B.; Kuchar, M.; Palenicek, T. Detailed pharmacological evaluation of methoxetamine (MXE), a novel psychoactive ketamine analogue—Behavioural, pharmacokinetic and metabolic studies in the Wistar rat. Brain Res Bull 2016.



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