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.

Exploring the effect of microdosing psychedelics on creativity in an open-label natural setting

“…Analytical standards of psilocin, psilocybin, norbaeocystin, and baeocystin were synthesized in-house (purity ≥ 95%) at UCT. The evaluation of the developed analytical method (Hajkova et al 2016) encompassed the determination of Limit of Quantitation (LOQ) and the determination of the applicable concentration range for each compound studied. LOQ was determined as 10 times the ratio of signal to noise.…”

Synthesis and identification of deschloroketamine metabolites in rats' urine and a quantification method for deschloroketamine and metabolites in rats' serum and brain tissue using liquid chromatography tandem mass spectrometry

“…All identified metabolites were compared with the metabolites and fragments of MXE and ketamine found in the literature. For comparison with MXE, we also used data from our previous work, where we synthesized, confirmed, and quantified MXE, O ‐desmethylmethoxetamine, normethoxetamine, dihydromethoxetamine, O ‐desmethyl‐normethoxetamine, and dihydro‐ O ‐desmethylmethoxetamine in Wistar rat serum, brain, lungs, and liver …”

“…Because of the structural similarity of DXE with MXE, a slightly modified salting‐out assisted liquid–liquid extraction (SALLE) sample preparation procedure based on our previous work was applied. The sample preparation protocol was as follows: (a) 100 mg of brain tissue with IS added (2 μL of 2 μg/mL solution), 200 mg of ZY beads, and a mixture of 200 μL of 10 mmol/L ammonium bicarbonate and 100 μL of acetonitrile was homogenized in the BulletBlender homogenizer at speed 6 for 5 min; (b) the homogenate was cooled for 10 min at −20°C; (c) 300 μL of acetonitrile was added and the mixture was homogenized in the BulletBlender homogenizer at speed 6 for next 5 min; (d) then 20 mg of NaCl and 100 mg of Na 2 SO 4 were added and the mixture was homogenized in the BulletBlender homogenizer at speed 6 for 5 min; (e) 250 μL of supernatant was evaporated to dryness after the centrifugation at 18,700 × g for 5 min at 25°C; (f) 1 μL of the resulting supernatant was injected into LC–MS after the reconstitution in 250 μL of 10 mmol/L ammonium acetate/methanol (9/1, v/v).…”

Structure determination of butylone as a new psychoactive substance using chiroptical and vibrational spectroscopies

“…1 Because of the complicated legislative regulation (large increase of new substances occurring during the year) and consequent availability (Internet sales), there is a significant increase in the prevalence of these substances within the population. 5 Raman spectroscopy and high-performance liquid chromatography with mass detection (HPLC-MS) are the most commonly used techniques for the identification of pure substances. See the first articles for this special issue previously published in Volume 30:4.…”