Drug consumption of suspected drug-influenced drivers in Hungary (2016-2018).

The hazard caused by driving under the influence of drugs (DUID) is determined by the time of consumption, dose and biological effects of a substance, as well as by synergistic drug interactions after multi-drug use. The aim of this work was to investigate the prevalence and pattern of psychoactive substance use of suspected DUID drivers and to present the advantages and disadvantages of the system currently used for determination of impairment in Hungary. Blood and urine samples, collected between 2016 and 2018, were taken from 2369 drivers with a positivity rate of 95% for at least one substance. Classical illicit drugs were detected in 76-87%, prescription medications in 9-15%, stimulant New Psychoactive Substances (sNPS) in 3-8%, and synthetic cannabinoids (SCs) in 20-22% of the positive samples. The most frequent substances according to substance groups were: classical illicit drugs: cannabis (n = 1240), amphetamine and methamphetamine (AM/MA) (n = 753), MDMA (n = 196), and cocaine (n = 180), medicines: alprazolam (n = 188) and clonazepam (n = 83), sNPS: N-ethyl-hexedrone (n = 115), SCs: 5 F-MDMB-PINACA (n = 267), AMB-FUBINACA (n = 92) and ADB-FUBINACA (n = 90). The median age of classical illicit drugs users was 29 years, prescription medicine users were 33 years old, sNPS users were 28 years, and SC users were 26 years old. Compared to the previous two years, we found pronounced changes in the ratio of sNPS (14% decrease) and SC users (10% increase), and in the pattern of NPS consumption. The ratio of multi-drug use varied between 38% and 50%. 69% of drivers tested positive were deemed impaired. Impairment was determined according to impairment limits (80-82%), multi-drug use (12-13%), and the result of medical investigation when a single active substance with no set impairment limit was detected in the blood (6-8%). The results of medical investigations may be uncertain due to the long time delay between arrest and clinical examination and to the structure of medical investigations created for determination of alcoholic impairment. In conclusion, a revision of the current medical investigation protocol is warranted to standardize clinical symptom scores that better correlate with driving impairment.

The Novel Psychoactive Substance Cumyl-CH-MEGACLONE: Human Phase-I Metabolism, Basic Pharmacological Characterization and Comparison to Other Synthetic Cannabinoid Receptor Agonists with a γ-Carboline-1-One Core.

Synthetic cannabinoids (SC) remain one of the largest groups of new psychoactive substances on the European drug market. In December 2018, Cumyl-CH-MEGACLONE, a novel SC based on a γ-carboline-1-one core structure, was firstly identified in Hungary and later also other European countries. This work aims to reveal the pharmacological characteristics and phase-I metabolism of Cumyl-CH-MEGACLONE and compare the data to its analogs Cumyl-PEGACLONE and 5F-Cumyl-PEGACLONE. The purified substance was characterized by means of gas chromatography-mass spectrometry (GC-MS), liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QToF-MS), attenuated total reflection infrared spectroscopy (ATR-FTIR) and nuclear magnetic resonance spectroscopy. Phase-I metabolites were identified by LC-QToF-MS analysis combined with a scheduled precursor ion list of authentic urine samples and confirmed by comparison with metabolites built in vitro by pooled human liver microsome assays. Pharmacological data were obtained in a competitive ligand binding assay and a receptor activation assay at the human cannabinoid receptor 1 (hCB1). The structure of 5-cyclohexylmethyl-2-(2-phenylpropan-2-yl)-2,5-dihydro-1H-pyrido[4,3-b]indol-1-one (semisystematic name: Cumyl-CH-MEGACLONE) was identified in a herbal blend as the main active ingredient. Investigation of phase-I biotransformation of Cumyl-CH-MEGACLONE led to three monohydroxylated metabolites (M08, M10 and M13) as reliable urinary markers for proof of consumption. At the hCB1, Cumyl-CH-MEGACLONE shows high binding affinity with Ki = 1.01 nM (2.5-fold higher than JWH-018), an EC50 of 1.22 nM and high efficacy with EMAX = 143.4% above constitutive activity of the receptor (1.13-fold higher than JWH-018). Comparison to the analogs 5F-Cumyl-PEGACLONE and Cumyl-PEGACLONE (both are hCB1 full agonists carrying a 5-fluoropentyl or pentyl chain instead of the cyclohexylmethyl moiety) suggests that Cumyl-CH-MEGACLONE is more likely to resemble the pharmacologic profile of the latter one.

The role of illicit, licit, and designer drugs in the traffic in Hungary.

The aim of this study was to investigate the prevalence and pattern of psychoactive substances among suspected DUID (Driving Under the Influence of Drugs) drivers in Hungary in 2014 and 2015. Blood and/or urine samples of 1252 suspected drivers (600 in 2014 and 652 in 2015) were analyzed for classical illicit and licit drugs, stimulant designer drugs (SDDs), and for synthetic cannabinoids, with 78.3% and 79.6% positive cases for at least one substance in 2014, and 2015, respectively. Impairment was proven in 39.2% (2014) and 35.7% (2015) of all drivers tested, based on the legal criteria of Hungary. Classical illicit drugs were found to be present in blood or urine of 89-61%, drivers tested. Drivers also tested positive for legal medications in 20-22%, SDDs in 21-28%, and synthetic cannabinoids in 15-19% of all cases. This indicates a drop in prevalence for classical illicit drugs and a slight but statistically non-significant increase for the other three substance groups. The distribution of drug types in each category were: [1] classical illicit drugs: cannabis (432), amphetamine (321), and cocaine (79); [2] medicines: alprazolam (94) and clonazepam (36); [3] SDDs: pentedrone (137) and α-PVP (33); [4] synthetic cannabinoids: AB-CHMINACA (46) and MDMB-CHMICA (30). The average age of illicit drug and SDD users was 30 years, while legal medications users were 36 years old on average, and the mean age of synthetic cannabinoid users was 26.5 years. The presence of both alcohol and at least one drug in samples was found in about 10% of the cases, both years. The ratio of multi-drug use was 33.0% in 2014 and 41.3% in 2015. Compared to former years the number of drivers who tested positive for drugs doubled in Hungary, but it is still low compared to alcohol positive cases. The relatively low detected rate of DUID can be explained by (1) combined alcohol consumption masking drug symptoms, (2) the absence of road-side tests for illicit and designer drugs and, (3) police officers not adequately trained to recognize milder symptoms of impairment. Targeted education of police officers, prompt medical examination and the use of a symptom-focused on-site survey, could improve the efficacy of DUID investigations. Our findings are not comparable with drug consumption habits of the general driving population. The last roadside survey (DRUID EU-6 Project) was performed in Hungary in 2008-2009, prior to the mass spreading of designer drugs. As their appearance has drastically changed the pattern of drug consumption of the population, a new roadside survey, targeting general drivers, would be necessary.

Structure-related related new approach in the gas chromatography/mass spectrometry analysis of cathinone type synthetic drugs.

A novel, structure-related derivatization principle has been developed in order to quantify cathinone-type synthetic drugs (CTSDs), focusing on the most common pentedrone (PENT), including also 4-fluoromethcathinone (4-FMC), methcathinone (MCTN), 4-methylethcathinone (4-MEC), 3,4-dimethylmethcathinone (3,4-DMMC), and 4-ethylmethcathinone (4-EMC). Firstly, oximated and, secondly, trimethylsilylated CTSD derivatives were characterized by mass fragmentation patterns using GC/MS that led to the development of a harmonized, quantitative, two-steps derivatization methodology. The two-step process involved i) oximation with hydroxylamine hydrochloride; and ii) trimethylsilylation with N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA). Next, the oximated-trimethylsilylated species were characterized by retention and mass fragmentation properties. Due to α-cleavage decomposition at their C1C2 bonds without exception, CTSDs uniformely exhibited a structure-related fragmentation pattern. The practical utility of this newly recognized mechanism was validated in urine samples employing an extraction-free and time-, work-, cost- and solvent-effective protocol in accordance with Green Chemistry. The centrifuged urines (10-40µL) were evaporated to dryness, followed by derivatization. The analytical performance of the methodology was characterized by repeatability (RSD%, varying between 1.43% and 5.44%), limit of quantitation (LOQ, 15-24µg/mL), linearity (R2, 0.9976-0.9998) and recovery (97-99%) values. The new principle was tested on drug users' urine: one specimen provided 56.8µg/mL PENT (3.8 RSD%). Simple trimethylsilylation of CTSDs confirmed their special fragmentation patterns, not yet described. The instantenous combination of the primarily formed, characteristic fragments with the mass m/z 44, led to the special equilibrium of products: confirming that direct trimethylsilylation of CTSDs is not suitable for their quantitation.

Comparison of five derivatizing agents for the determination of amphetamine-type stimulants in human urine by extractive acylation and gas chromatography-mass spectrometry.

Five acylation reagents have been compared for use as derivatizing agents for the analysis of amphetamine-type stimulants (ATS) in urine by gas chromatography-mass spectrometry (GC-MS). The evaluated reagents were heptafluorobutyric anhydride, pentafluoropropionic anhydride, trifluoroacetic anhydride, acetic anhydride (AA) and N-methyl-bis(trifluoroacetamide). The ATS included amphetamine, methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyethylamphetamine (MDEA). A mixture of the ATS was added to urine (1 mL) followed by KOH solution and saturated NaHCO(3) solution. The sample was then extracted with dichloromethane and the derivatizing agent and 2 µL were injected into the GC-MS instrument. The derivatizing agents were compared with reference to the signal-to-noise (S/N) ratios, peak area values, relative standard deviations (RSDs), linearities, limits of detection (LODs) and selectivities. The acetic anhydride proved to be the best according to the S/N ratio and peak area results for amphetamine, MA, MDMA and MDEA. The best RSD values of peak areas and of S/N ratios at 3 µg/mL were also given by AA in cases of MDA, MDMA and MDEA. At 20 µg/mL, the lowest RSD values of peak areas for MDA and the lowest RSD values of S/N ratios for MA, MDA, MDMA and MDEA were again given by AA. Additionally, the highest correlation coefficients for MA, MDA, MDMA and MDEA and the lowest LOD results for MA, MDMA and MDEA were produced by AA.

GC-MS determination of amphetamines in serum using on-line trifluoroacetylation.

An extraction and determination method of most important amphetamine derivatives in serum has been developed. The procedure comprises liquid-liquid extraction with tert-butyl methyl ether of the sample under basic conditions, centrifugation, formation of hydrochloric salts after the separation of organic phase, vacuum evaporation of the organic solvent at 60 degrees C, and trifluoroacetylation by on-line flash injection with MBTFA. GC analysis was performed by electron impact GC-MS in SIM mode. In this way satisfactory identification of 12 amphetamine derivatives could be obtained. Amphetamine, methamphetamine, MDA, MDMA and MDEA could be analyzed by using pentadeuterated analogs as internal standards. Low limits of detection 2.5-6.9 ng/mL could be reached. The assay was linear within the 5-100 ng/mL range with a regression coefficient greater than 0.999 for each compound. Our derivatization method is of low cost since only 1 microL of MBTFA is used for each flash trifluoroacetylation.