title: Salting-out-assisted liquid-liquid extraction as a suitable approach for determination of methoxetamine in large sets of tissue samples.
abstract: A new designer drug, a dissociative anesthetic, and a putative N-methyl-D-aspartate receptor antagonist, methoxetamine (MXE) noted by the EU Early Warning System has been already identified as a cause of several fatalities worldwide. The primary objective of this work was to develop a suitable sample preparation method allowing for isolation of MXE and its main metabolites in high yields from rat brain, liver, and lungs. For the purpose of the project, MXE and five metabolites were synthesized in-house, specifically O-desmethyl-normethoxetamine, O-desmethylmethoxetamine, dihydro-O-desmethylmethoxetamine, normethoxetamine, and dihydromethoxetamine. A sample preparation procedure consisted in the homogenization of the tissue applying salting-out-assisted liquid-liquid extraction (SALLE). A subsequent liquid chromatography-mass spectrometry (LC-MS) analysis was based on reversed-phased chromatography hyphenated with a triple quad MS system in a positive electrospray mode. Multiple reaction monitoring (MRM) was used for qualification and quantification of the analytes. The quantification was based on the application of an isotopically labeled internal standard, normethoxetamine-d3. The matrix-matched calibrations were prepared for each type of matrix with regression coefficients 0.9943-1.0000. The calibration curves were linear in the concentration range of 2.5-250 ng g(-1). Limits of quantification (LOQs) were estimated as 2.5 and 5 ng g(-1), respectively. Recovery (80-117%) and matrix effect (94-110%) at 100 ng g(-1) and intra- and inter-day accuracy and precision at low (2.5 ng g(-1)), middle (25 ng g(-1)), and upper (250 ng g(-1)) concentration levels for all the analytes in all three types of tissues were also determined. The developed analytical method was applied to a set of real samples gathered in toxicological trials on rats and MXE, and its metabolites were determined successfully.
authors.affiliation: Forensic Laboratory of Biologically Active Substances, University of Chemistry and Technology Prague, Technická 5, 166 28, Prague 6, Dejvice, Czech Republic.|Department of Analytical Chemistry, University of Chemistry and Technology Prague, Technická 5, 166 28, Prague 6, Dejvice, Czech Republic.|National Institute of Mental Health, Topolová 748, 250 67, Klecany, Czech Republic.
authors.forename: Katerina
authors.initials: K
authors.lastname: Hajkova
authors.affiliation: Forensic Laboratory of Biologically Active Substances, University of Chemistry and Technology Prague, Technická 5, 166 28, Prague 6, Dejvice, Czech Republic.
authors.forename: Bronislav
authors.initials: B
authors.lastname: Jurasek
authors.affiliation: Department of Analytical Chemistry, University of Chemistry and Technology Prague, Technická 5, 166 28, Prague 6, Dejvice, Czech Republic. david.sykora@vscht.cz.
authors.forename: David
authors.initials: D
authors.lastname: Sykora
authors.affiliation: National Institute of Mental Health, Topolová 748, 250 67, Klecany, Czech Republic.
authors.forename: Tomas
authors.initials: T
authors.lastname: Palenicek
authors.affiliation: Forensic Laboratory of Biologically Active Substances, University of Chemistry and Technology Prague, Technická 5, 166 28, Prague 6, Dejvice, Czech Republic.|National Institute of Mental Health, Topolová 748, 250 67, Klecany, Czech Republic.
authors.forename: Petra
authors.initials: P
authors.lastname: Miksatkova
authors.affiliation: Forensic Laboratory of Biologically Active Substances, University of Chemistry and Technology Prague, Technická 5, 166 28, Prague 6, Dejvice, Czech Republic.|National Institute of Mental Health, Topolová 748, 250 67, Klecany, Czech Republic.
authors.forename: Martin
authors.initials: M
authors.lastname: Kuchar
mesh_terms: D000818:Animals; D001923:Brain Chemistry; D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods; D003512:Cyclohexanones / Q000032:analysis* / Q000378:metabolism / Q000493:pharmacokinetics; D003514:Cyclohexylamines / Q000032:analysis* / Q000378:metabolism / Q000493:pharmacokinetics; D015198:Designer Drugs / Q000032:analysis; D057230:Limit of Detection; D059625:Liquid-Liquid Extraction / Q000379:methods*; D008099:Liver / Q000737:chemistry; D008168:Lung / Q000737:chemistry; D051381:Rats; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D003512:Cyclohexanones; D003514:Cyclohexylamines; D015198:Designer Drugs; C569113:2-(3-methoxyphenyl)-2-(ethylamino)cyclohexanone
keywords: LC-MS/MS; Methoxetamine; New psychoactive substances; SALLE; Tissues