Martin Kuchař

Chiral separation of new designer drugs (Cathinones) on chiral ion-exchange type stationary phases

We present the enantioseparation of new designer drugs from the cathinone family on structurally different chiral ion-exchange type stationary phases. A novel strong cation-exchange type chiral stationary phase was synthesized and its performance compared with previously reported ion-exchange type chiral stationary phases. The influence of structural elements of the chiral selectors on their chromatographic performance was studied and the possibilities of tuning chromatographic parameters by varying the polarity of the employed mobile phases were determined. Evidence is provided that a change in mobile phase composition strongly influences the solvation shell of the polarized and polarizable units of the selectors and analytes, as well as ionizable mobile phase additives. Furthermore, the structural features of the selectors (e.g. the size of aromatic units and their substitution pattern) are shown to play a key role in the effective formation of diastereomeric complexes with analytes. Thus, we have achieved the enantioseparation of all test analytes with a mass spectrometry-compatible mobile phase with a chiral strong cation-exchange type stationary phase.

Emerging toxicity of 5,6-methylenedioxy-2-aminoindane (MDAI): Pharmacokinetics, behaviour, thermoregulation and LD50 in rats.

MDAI (5,6-Methylenedioxy-2-aminoindane) has a reputation as a non-neurotoxic ecstasy replacement amongst recreational users, however the drug has been implicated in some severe and lethal intoxications. Due to this, and the fact that the drug is almost unexplored scientifically we investigated a broad range of effects of acute MDAI administration: pharmacokinetics (in sera, brain, liver and lung); behaviour (open field; prepulse inhibition, PPI); acute effects on thermoregulation (in group-/individually-housed rats); and systemic toxicity (median lethal dose, LD50) in Wistar rats. Pharmacokinetics of MDAI was rapid, maximum median concentration in serum and brain was attained 30min and almost returned to zero 6h after subcutaneous (sc.) administration of 10mg/kg MDAI; brain/serum ratio was ~4. MDAI particularly accumulated in lung tissue. In the open field, MDAI (5, 10, 20 and 40mg/kg sc.) increased exploratory activity, induced signs of behavioural serotonin syndrome and reduced locomotor habituation, although by 60min some effects had diminished. All doses of MDAI significantly disrupted PPI and the effect was present during the onset of its action as well as 60min after treatment. Unexpectedly, 40mg/kg MDAI killed 90% of animals in the first behavioural test, hence LD50 tests were conducted which yielded 28.33mg/kg sc. and 35mg/kg intravenous but was not established up to 40mg/kg after gastric administration. Disseminated intravascular coagulopathy (DIC) with brain oedema was concluded as a direct cause of death in sc. treated animals. Finally, MDAI (10, 20mg/kg sc.) caused hyperthermia and perspiration in group-housed rats. In conclusion, the drug had fast pharmacokinetics and accumulated in lipohilic tissues. Behavioural findings were consistent with mild, transient stimulation with anxiolysis and disruption of sensorimotor processing. Together with hyperthermia, the drug had a similar profile to related entactogens, especially 3,4-metyhlenedioxymethamphetamine (MDMA, ecstasy) and paramethoxymethamphetamine (PMMA). Surprisingly subcutaneous MDAI appears to be more lethal than previously thought and its serotonergic toxicity is likely exacerbated by group housing conditions. MDAI therefore poses greater risks to physical and mental health than recognised hitherto.

Pharmacokinetic, Ambulatory, and Hyperthermic Effects of 3,4-Methylenedioxy-N-Methylcathinone (Methylone) in Rats

Methylone (3,4-methylenedioxy-N-methylcathinone) is a synthetic cathinone analog of the recreational drug ecstasy. Although it is marketed to recreational users as relatively safe, fatalities due to hyperthermia, serotonin syndrome, and multi-organ system failure have been reported. Since psychopharmacological data remain scarce, we have focused our research on pharmacokinetics, and on a detailed evaluation of temporal effects of methylone and its metabolite nor-methylone on behavior and body temperature in rats. Methylone [5, 10, 20, and 40 mg/kg subcutaneously (s.c.)] and nor-methylone (10 mg/kg s.c.) were used in adolescent male Wistar rats across three behavioral/physiological procedures and in two temporal windows from administration (15 and 60 min) in order to test: locomotor effects in the open field, sensorimotor gating in the test of prepulse inhibition (PPI), and effects on rectal temperature in individually and group-housed rats. Serum and brain pharmacokinetics after 10 mg/kg s.c. over 8 h were analyzed using liquid chromatography mass spectrometry. Serum and brain levels of methylone and nor-methylone peaked at 30 min after administration, both drugs readily penetrated the brain with serum: brain ratio 1:7.97. Methylone dose-dependently increased overall locomotion. It also decrease the amount of time spent in the center of open field arena in dose 20 mg/kg and additionally this dose induced stereotyped circling around the arena walls. The maximum of effects corresponded to the peak of its brain concentrations. Nor-methylone had approximately the same behavioral potency. Methylone also has weak potency to disturb PPI. Behavioral testing was not performed with 40 mg/kg, because it was surprisingly lethal to some animals. Methylone 10 and 20 mg/kg s.c. induced hyperthermic reaction which was more pronounced in group-housed condition relative to individually housed rats. To conclude, methylone increased exploration and/or decreased anxiety in the open field arena and with nor-methylone had short duration of action with effects typical for mixed indirect dopamine–serotonin agonists such as 3,4-metyhlenedioxymethamphetamine (MDMA) or amphetamine. Given the fact that the toxicity was even higher than the known for MDMA and that it can cause hyperthermia it possess a threat to users with the risk for serotonin syndrome especially when used in crowded conditions.

Pharmacokinetic and behavioural profile of THC, CBD, and THC+CBD combination after pulmonary, oral, and subcutaneous administration in rats and confirmation of conversion in vivo of CBD to THC

Metabolic and behavioural effects of, and interactions between Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) are influenced by dose and administration route. Therefore we investigated, in Wistar rats, effects of pulmonary, oral and subcutaneous (sc.) THC, CBD and THC+CBD. Concentrations of THC, its metabolites 11-OH-THC and THC-COOH, and CBD in serum and brain were determined over 24h, locomotor activity (open field) and sensorimotor gating (prepulse inhibition, PPI) were also evaluated. In line with recent knowledge we expected metabolic and behavioural interactions between THC and CBD. While cannabinoid serum and brain levels rapidly peaked and diminished after pulmonary administration, sc. and oral administration produced long-lasting levels of cannabinoids with oral reaching the highest brain levels. Except pulmonary administration, CBD inhibited THC metabolism resulting in higher serum/brain levels of THC. Importantly, following sc. and oral CBD alone treatments, THC was also detected in serum and brain. S.c. cannabinoids caused hypolocomotion, oral treatments containing THC almost complete immobility. In contrast, oral CBD produced mild hyperlocomotion. CBD disrupted, and THC tended to disrupt PPI, however their combination did not. In conclusion, oral administration yielded the most pronounced behavioural effects which corresponded to the highest brain levels of cannabinoids. Even though CBD potently inhibited THC metabolism after oral and sc. administration, unexpectedly it had minimal impact on THC-induced behaviour. Of central importance was the novel finding that THC can be detected in serum and brain after administration of CBD alone which, if confirmed in humans and given the increasing medical use of CBD-only products, might have important legal and forensic ramifications.

Enantioseparation of Chiral Sulfoxides on Amylose-Based Columns: Comparison of Normal Phase Liquid Chromatography and Supercritical Fluid Chromatography

We present enantioseparation of a series of racemic sulfoxides on three different amylose-based polysaccharide columns. Two of them have the amylose units modified with dimethylphenyl carbamoyl groups (Chiralpak AD-H and Chiralpak IA), while the third one possesses a carbamoyl moiety with an additional chiral centre (Chiralpak AS-H). The enantioseparation of selected analytes was achieved in high-performance liquid chromatography (HPLC) and the full analyte set was enantiomerically resolved using supercritical fluid chromatography (SFC). Comparing the results obtained in both modes, we show that enantioseparation under SFC conditions is superior to HPLC mode in terms of speed, while retaining excellent enantioselectivity and resolution. Faster elution of analytes was observed on increasing the polarity of the co-solvent (modifier) in the mobile phase. This trend is apparent in both chromatographic modes. Documenting the important role of the additional chiral centre, Chiralpak AS-H provided the best chromatographic parameters resulting in the enantioseparation of all analytes.Graphical Abstract

Mephedrone (4-Methylmethcathinone): Acute Behavioral Effects, Hyperthermic, and Pharmacokinetic Profile in Rats

Mephedrone (MEPH) is a synthetic cathinone derivative with effects that mimic MDMA and/or cocaine. Our study in male Wistar rats provides detailed investigations of MEPH’s and its primary metabolite nor-mephedrone’s (nor-MEPH) pharmacokinetics and bio-distribution to four different substrates (serum, brain, lungs, and liver), as well as comparative analysis of their effects on locomotion [open field test (OFT)] and sensorimotor gating [prepulse inhibition of acoustic startle reaction (PPI ASR)]. Furthermore, in order to mimic the crowded condition where MEPH is typically taken (e.g., clubs), the acute effect of MEPH on thermoregulation in singly- and group-housed rats was evaluated. Pharmacokinetics of MEPH and nor-MEPH after MEPH (5 mg/kg, sc.) were analyzed over 8 h using liquid chromatography with mass spectrometry. MEPH (2.5, 5, or 20 mg/kg, sc.) and nor-MEPH (5 mg/kg, sc.) were administered 5 or 40 min before the behavioral testing in the OFT and PPI ASR; locomotion and its spatial distribution, ASR, habituation and PPI itself were quantified. The effect of MEPH on rectal temperature was measured after 5 and 20 mg/kg, sc. Both MEPH and nor-MEPH were detected in all substrates, with the highest levels detected in lungs. Mean brain: serum ratios were 1:1.19 (MEPH) and 1:1.91 (nor-MEPH), maximum concentrations were observed at 30 min; at 2 and 4 h after administration, nor-MEPH concentrations were higher compared to the parent drug. While neither of the drugs disrupted PPI, both increased locomotion and affected its spatial distribution. The effects of MEPH were dose dependent, rapid, and short-lasting, and the intensity of locomotor stimulant effects was comparable between MEPH and nor-MEPH. Despite the disappearance of behavioral effects within 40 min after administration, MEPH induced rectal temperature elevations that persisted for 3 h even in singly housed rats. To conclude, we observed a robust, short-lasting, and most likely synergistic stimulatory effect of both drugs which corresponded to brain pharmacokinetics. The dissociation between the duration of behavioral and hyperthermic effects is indicative of the possible contribution of nor-MEPH or other biologically active metabolites. This temporal dissociation may be related to the risk of prolonged somatic toxicity when stimulatory effects are no longer present.

Highly sensitive avidin-biotin ELISA for detection of nandrolone and testosterone in dietary supplements

Avidin-biotin technology was used for the implementation of an enzyme-linked immunosorbent assay (AB-ELISA) as a sensitive method for the detection of anabolic androgenic steroids (AAS) present in dietary supplements. Using click chemistry, novel haptens (linker-optimized biotinylated nandrolone (NT) and testosterone (T) at positions C-3 and C-17, respectively) were designed and synthesized to be then applied as four different immobilized competitors in a proposed set of four indirect competitive AB-ELISAs. Four rabbit polyclonal antibodies of various specificities were prepared using four different immunogens synthesized from C-3 and C-17 carboxymethyloxime and hemisuccinate derivatives of NT and T, respectively. Assembled AB-ELISAs were characterized to establish method parameters such as a half-maximum inhibition concentration (0.18-12.99 ng/mL), limit of detection (0.004-0.032 ng/mL) and linear working range (the best with 0.02-1.38 ng/mL). The stability of the set simulating storage in different conditions was demonstrated. Cross reactivity (CR) was tested for 59 steroids including both endogenous and synthetic analogues in four assembled AB-systems. The focus was placed on the practical use of the method in detection of various AAS in 49 samples of counterfeit dietary supplements. The concordance between ultra high performance liquid chromatography-mass spectrometry (UHPLC-MS) and the CR corrected data from AB-ELISA indicated the potential of this method even to quantification of T propionate, NT phenyl propionate, and NT decanoate in such a complex matter. Copyright

Lateral flow immunoassay and enzyme linked immunosorbent assay as effective immunomethods for the detection of synthetic cannabinoid JWH-200 based on the newly synthesized hapten

Graphical abstract

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

Recently, there has been a worldwide substantial increase in the consumption of new psychoactive substances (NPS), compounds that mimic the structure of illicit drugs, such as amphetamines or ecstasy. The producers try to avoid the law by a slight modification of illicit structures, thereby developing dozens of temporarily legal NPS every year. The current trends in the detection and monitoring of such substances demand a fast and reliable analysis. Molecular spectroscopy represents a highly effective tool for the identification of NPS and chiroptical methods can provide further information on their 3D structure, which is the key for the determination of their biological activity. We present the first systematic study of NPS, specifically butylone, combining chiroptical and vibrational spectroscopies with ab initio calculations. According to density functional theory calculations, 6 stable lowest energy conformers of butylone were found and their molecular structure was described. For each conformer, the relative abundance based on the Boltzmann distribution was estimated, their population weighted spectra predicted and compared to the experimental results. Very good agreement between the experimental and the simulated spectra was achieved, which allowed not only the assignment of the absolute configuration, but also a precise description of the molecular structure.

Use of novel haptens in the production of antibodies for the detection of tryptamines

Tryptamines are a group of hallucinogenic drugs whose detection in body fluids could be simplified by immunochemical assay kits. Antibodies for these assays are obtained by the immunization of laboratory animals with conjugates of a hapten similar to the target analyte and a suitable protein. Therefore we synthesized novel haptens derived from tryptamine-based drugs, with N,N-dimethyltryptamine (DMT), 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) and N,N-diisopropyltryptamine (DiPT) selected as the target analytes. Their structures were modified with a short linker ended with a carboxylic group. The haptens were conjugated with bovine serum albumin (BSA) and rabbits were immunized with the conjugates. The obtained polyclonal antibodies showed good reactivity and the LOD of the constructed ELISAs was in the range 0.006–0.254 ng mL−1. Thus, they are suitable for the development of immunochemical assay kits.