Successful use of a novel lux® i-Amylose-1 chiral column for enantioseparation of "legal highs" by HPLC.

Bath salts, fumigations, cleaners and air fresheners, behind these terms substances are hidden, which count as "Legal Highs". These fancy names are used to pretend Legal Highs as harmless compounds, to circumvent legal regulations for marketing as well as to increase the sales. Besides classic illicit drugs of synthetic origin such as amphetamines, cocaine and MDMA, the trade of these compounds, also known as new psychoactive substances (NPS), is not uncommon today. In many countries, NPS are still not subject to drug control. Among them, there are stimulants such as new amphetamine derivatives or cathinones, which possess a chiral centre. Little is known about the fact that the two possible enantiomers may differ in their pharmacological effect. The aim of this study was to test a novel HPLC column for the enantioseparation of a set of 112 NPS coming from different chemical groups and collected by internet purchases during the years 2010-2018. The CSP, namely Lux® 5 µm i-Amylose-1, LC Column 250 x 4.6 mm, was run in normal phase mode under isocratic conditions, UV detection was performed at 245 nm and 230 nm, injection volume was 10 µl and flow rate was 1 ml/min. With a mobile phase consisting of n-hexane/isopropanol/diethylamine (90:10:0.1), herein, 79 NPS were resolved into their enantiomers successfully, for 37 of them baseline resolution was achieved. After increase of lipophily of the mobile phase to 99:1:0.1, another 27 compounds were baseline separated. It was found that all separated NPS are traded as racemic compounds.

Mix of new substances psychoactive, NPS, found in blotters sold in two Colombian cities.

The new psychoactive substances (NPS) in Colombia are detected by national authorities, in blotters strip, in different circumstances and places: airports, music concerts, discos and parks. Blotters are marketed as LSD and cause several cases of intoxication and death in some consumers: due to acute intoxication or when mixed with other drugs and may have different effects on the central nervous system (CNS). This study was conducted to research into and identify the chemical composition of the drugs impregnated in the blotters sold in two Colombian cities. This research provides the analysis of 70 doses coming from forensic cases of the Colombian Attorney General's Office in Bogota and from the Laboratory of Narcotics of the Colombian National Institute of Legal Medicine and Forensic Sciences (North Headquarter) in Barranquilla. Mixtures of drugs, such as DOB, 25I-NBOMe, MDMA and 25I-NBOMe imine were found within the blotters through gas chromatography coupled to mass spectrometry (CGMS); these drugs are classified by international authorities as NPS belonging to the phenylethylamines group. The results clearly warn about a growing public health problem in the country.

Rapid determination of designer benzodiazepines, benzodiazepines, and Z-hypnotics in whole blood using parallel artificial liquid membrane extraction and UHPLC-MS/MS.

Benzodiazepines (BZD) and Z-hypnotics are frequently analyzed in forensic laboratories, and in 2012, the designer benzodiazepines (DBZD) emerged on the illegal drug scene. DBZD represent a particular challenge demanding new analytical methods. In this work, parallel artificial liquid membrane extraction (PALME) is used for sample preparation of DBZD, BZD, and Z-hypnotics in whole blood prior to UHPLC-MS/MS analysis. PALME of BZD, DBZD, and Z-hypnotics was performed from whole blood samples, and the analytes were extracted across a supported liquid membrane (SLM) and into an acceptor solution of dimethyl sulfoxide and 200 mM formic acid (75:25, v/v). The method was validated according to EMA guidelines. The method was linear throughout the calibration range (R2 > 0.99). Intra- and inter-day accuracy and precision, as well as matrix effects, were within the guideline limit of ± 15%. LOD and LLOQ ranged from 0.10 to 5.0 ng mL-1 and 3.2 to 160 ng mL-1, respectively. Extraction recoveries were reproducible and above 52%. The method was specific, and the analytes were stable in the PALME extracts for 4 and 10 days at 10 and - 20 °C. No carry-over was observed within the calibration range. PALME and UHPLC-MS/MS for the determination of DBZD, BZD, and Z-hypnotics in whole blood are a green and low-cost alternative that provides high sample throughput (96-well format), extensive sample clean-up, good sensitivity, and high reproducibility. The presented method is also the first method incorporating analysis of DBZD, BZD, and Z-hypnotics in whole blood in one efficient analysis. Graphical abstract.

Changes in illicit, licit and stimulant designer drug use patterns in South-East Hungary between 2008 and 2015.

The aim of this work is to present the changes in classical illicit and licit drug, as well as stimulant designer drug (SDD) consumption of suspected drug users in South-East Hungary between 2008 and 2015. Urine and/or blood samples of 2976 subjects were analyzed for these groups of substances of which 1777 (59.7%) were tested positive. THC was the most frequent (32.2%) substance, followed by classical stimulants (amphetamine, metamphetamine, MDMA, cocain) (21.4%), SDDs (17.0%), benzodiazepines (15.5%), medical opiates (codeine without morphine, methadone, tramadol) (4.03%), and morphine with or without 6-acethyl-morphine (1.98%). The annual rate of cannabis consumption continuously decreased after 2010. The use of classical stimulants was constant, except for a significant increase in 2015. Benzodiazepine incidence increased and remained steady after 2011. Medical opiate and morphine frequency was variable. SDDs were found in the highest number in 2012-13, exceeding the frequency of classical stimulants. The most prevalent SDDs were as follows: 2010 - mephedrone, 2011 - 4-MEC, methylone, MDPV, 4-FMC, and 4-FA, and 2012-2015 - pentedrone. Beside pentedrone, 3-MMC, αPVP, αPHP, and 4-CMC were detected with a notable number in this period. Multi-drug use was found in 30-43% of suspects tested positive between 2008 and 2014, which elevated to 52% in 2015. The frequency of substances in the biological samples corresponded to their seizure rate. When SDDs were included on the NPS list, their frequency in biological samples and in seized materials slightly decreased or did not change. However, a marked decrease was observed following classification as illicit drugs.

A new challenge in forensic toxicology exemplified by a case of murder under the influence of a synthetic cannabinoid - AM-2201.

Among new psychoactive substances (NPS) available on the narcotic market, a significant number consists of synthetic cannabinoids commonly known as smokable herbal "spice" and "K2", and which are legally treated as a legal alternative to marijuana. The dearth of information on the pharmacology of these intoxicants as they are introduced into the market has created the urgent need among healthcare providers for case studies on the substances belonging to this group, both in terms of the consequences of using such intoxicants, and in methods of detection. The subject of the present report is a multi-parameter analysis of a criminal case of an 18-year-old male who was charged with murder of his female relative and attempted murder of two other victims by stabbing. The defendant pleaded guilty, but he claimed that he had been acting without volition, because he was under the influence of the synthetic cannabinoid AM-2201, which had been purchased from a dealer as a 10g package labelled "Mr Green - No bad trip". Analytical methods including gas chromatography - electron ionization - quadrupole ion trap mass spectrometry (GC-EI-QIT/MS) and liquid chromatography, electrospray ionization, tandem mass spectrometry (LC-ESI-MS-MS) were developed to determine the presence of AM-2201 in the Mr Green - No Bad Trip, and in the blood of the perpetrator, respectively. Toxicological findings are discussed in the context of psychoactive and adverse physical effects resulting from the presence of AM-2201 in the human body; the observations were also analyzed in conjunction with data from the literature.

Challenges in Modern Anti-Doping Analytical Science.

The challenges facing modern anti-doping analytical science are increasingly complex given the expansion of target drug substances, as the pharmaceutical industry introduces more novel therapeutic compounds and the internet offers designer drugs to improve performance. The technical challenges are manifold, including, for example, the need for advanced instrumentation for greater speed of analyses and increased sensitivity, specific techniques capable of distinguishing between endogenous and exogenous metabolites, or biological assays for the detection of peptide hormones or their markers, all of which require an important investment from the laboratories and recruitment of highly specialized scientific personnel. The consequences of introducing sophisticated and complex analytical procedures may result in the future in a change in the strategy applied by the Word Anti-Doping Agency in relation to the introduction and performance of new techniques by the network of accredited anti-doping laboratories.

The role of diode array ultraviolet detection for the identification of synthetic cathinones.

The utility of diode array ultraviolet (UV) detection for aiding in the identification of synthetic cathinones, including different sub-classes and positional isomers is presented. For 35 synthetic cathinones, unique UV spectra are obtained for seven sub-classes, including mostly beta ketones, where position and type of substitution on benzene rings give rise to differences in UV maxima and relative intensity of the spectral bands. This aspect is key to distinguishing positional isomers that contain differences in R substitution (mono and di) around the benzene ring, which provides complementary information to electron ionization mass spectrometry, where the latter technique cannot distinguish between these types of positional isomers. In addition, it is possible to ascertain the substitution position based on the UV spectra. For ten sets of positional isomers, it was possible to distinguish most of the positional isomers within a set. For ultra-high performance supercritical fluid chromatography (UHPSFC) versus reversed phase ultra-high performance liquid chromatography (UHPLC), there was at least a 10 nm blue shift in UV maximum (shift to shorter wavelengths). This highlights the importance of taking in account the effect of mobile phase on the UV maximum when performing method development in UHPSFC. Copyright © 2017 John Wiley & Sons, Ltd.

DART - LTQ ORBITRAP as an expedient tool for the identification of synthetic cannabinoids.

Synthetic cannabinoids as designer drugs constitute a major problem due to their rapid increase in number and the difficulties connected with their identification in complex mixtures. DART (Direct Analysis in Real Time) has emerged as an advantageous tool for the direct and rapid analysis of complex samples by mass spectrometry. Here we report on the identification of six synthetic cannabinoids originating from seized material in various matrices, employing the combination of ambient pressure ion source DART and hybrid ion trap - LTQ ORBITRAP mass spectrometer. This report also describes the sampling techniques for the provided herbal material containing the cannabinoids, either directly as plant parts or as an extract in methanol and their influence on the outcome of the analysis. The high resolution mass spectra supplied by the LTQ ORBITRAP instrument allowed for an unambiguous assignment of target compounds. The utilized instrumental coupling proved to be a convenient way for the identification of synthetic cannabinoids in real-world samples.

Chiral resolution and absolute configuration of the enantiomers of the psychoactive "designer drug" 3,4-methylenedioxypyrovalerone.

Illicit rac-MDPV (3,4-methylenedioxypyrovalerone), manufactured in clandestine labs, has become widely abused for its cocaine-like stimulant properties. It has recently been found as one of the toxic materials in the so-called "bath salts," producing, among other effects, psychosis and tachycardia in humans when introduced by any of the several routes of administration (e.g., intravenous, oral, etc.). The considerable toxicity of this "designer drug" probably resides in one of the enantiomers of the racemate. In order to obtain a sufficient amount of the enantiomers of rac-MDPV to determine their activity, we improved the known synthesis of rac-MDPV and found chemical resolving agents, (+)- and (-)-2'-bromotetranilic acid, that gave the MDPV enantiomers in >96% enantiomeric excess as determined by (1) H nuclear magnetic resonance and chiral high-performance liquid chromatography. The absolute stereochemistry of these enantiomers was determined by single-crystal X-ray diffraction studies.

Enantiomeric separation of new cathinone derivatives designer drugs by capillary electrochromatography using a chiral stationary phase, based on amylose tris(5-chloro-2-methylphenylcarbamate).

In this study, a chiral CEC method for the enantiomeric separation of ten cathinone derivatives, by means of a polysaccharide-based chiral stationary phase, has been developed. Capillary columns of 100 µm id packed with amylose tris(5-chloro-2-methylphenylcarbamate) coated on silica, also called Sepapak 3 or Lux Amylose-2, were used to achieve the enantioseparation of the studied designer drugs. Enantioresolution, chromatographic retention, and separation efficiency were evaluated in dependence of mobile-phase composition in terms of the content of the organic modifier, nature, and pH buffer. To obtain a sensitivity improvement, a field-amplified sample injection was evaluated optimizing the sample solvent composition and injection time. The LODs and LOQs values were in the range 25-100 and 50-150 ng/mL, respectively, for all the racemic compounds. Good results in terms of resolution (Rs ), separation efficiency (N/m), and short analysis times were obtained using a mixture of ACN/methanol/sodium acetate pH 9 (89/10/1, v/v/v). Applying a voltage of 10 kV and a temperature of 20°C, the analyzed cathinone derivatives were separated in their enantiomers in less than 10 min. A study, concerning the method precision, in terms of intra- and interday repeatability and column-to-column reproducibility was carried out in accordance with the analytical procedures for method validation. Intra- and interday repeatability provided RSD values in the ranges 1.1-1.7, 1.3-2.3% for retention time and 1.3-2.6, 2.1-3.4% for peak area, respectively.

Simple segmental hair analysis for α-pyrrolidinophenone-type designer drugs by MonoSpin extraction for evaluation of abuse history.

For detection of a history of drug abused, we developed a simple method for extracting pyrrolidinophenone-type designer drugs in human hair by using a MonoSpin(®) C18 column. Target drugs were extracted from a single alkaline-digested hair segment (length, 10mm; weight, ca 0.1mg). The analytes extracted were then analyzed by high-performance liquid chromatography-mass spectrometry without evaporation of the eluent after MonoSpin extraction. Linearity from 0.5 to 500ng/mg was observed for all the tested drugs using an internal standard method (correlation coefficients >0.998) and the limit of detection was 0.2ng/mg. The recoveries were between 0.7 and 11.1%. The coefficients for intraday and interday variations at 4, 40, 200, and 400ng/mg in hair were between 0.7 and 11.1%. This method was successfully applied to the identification of these designer drugs in segmented human hair from drug abusers and indicated their history of drug abuse. The results were consistent with the patients' statements, indicating that this rapid method can be used to detect a history of drug abuse.

Chemiluminescence detection of piperazine designer drugs and related compounds using tris(2,2'-bipyridine)ruthenium(III).

We present an exploration of the chemiluminescence from reactions of benzylpiperazines and phenylpiperazines with tris(2,2'-bipyridine)ruthenium(III). The selectivity of the reagent towards these compounds was found to be highly dependent upon the pH of the solution, and the relative emission intensity was strongly influenced by electron donating or withdrawing substituents on the phenyl or benzyl ring. In spite of previous investigations showing poor responses for aromatic-substituted amines (compared to their aliphatic amine counterparts), intense emissions were observed with phenylpiperazines under acidic conditions, particularly those with halogen or trifluoromethyl substituents on the aromatic ring. Buffered alkaline conditions provided much broader selectivity for the detection of both phenylpiperazine and benzylpiperazine compounds, which we have applied to a rapid HPLC procedure for the determination of piperazines of forensic interest in 'party pill' samples.

Identification of novel third-generation synthetic cannabinoids in products by ultra-performance liquid chromatography and time-of-flight mass spectrometry.

Synthetic cannabinoids are a group of compounds that are structurally diverse and are commonly found in various herbal incense and potpourri blends, which are sold in convenience stores, smoke shops and over the Internet. During the past few years, multiple state and federal legislations have been enacted controlling various subsets of these compounds that have been detected in compound categories generally considered the first and second product generations. As shown in previous studies, as compounds become controlled, new compounds emerge and become prevalent. We report on the emergence and prevalence of five different compounds (A796,260, MAM-2201, UR-144, URB597 and XLR-11) in the state of Indiana through their qualitative detection in solid-dosage herbal products via rapid solvent extraction and ultra-performance liquid chromatography with time-of-flight mass spectrometry (UPLC/ToF). We demonstrate the use of UPLC/ToF to be a suitable tool in the identification of these substances in a crime laboratory or forensic laboratory setting, which ultimately enables a laboratory to design assays for the detection of specific analytes in biological specimens in regard to regional trends and prevalence.

Sports doping: emerging designer and therapeutic ß2-agonists.

Beta2-adrenergic agonists, or ß2-agonists, are considered essential bronchodilator drugs in the treatment of bronchial asthma, both as symptom-relievers and, in combination with inhaled corticosteroids, as disease-controllers. The use of ß2-agonists is prohibited in sports by the World Anti-Doping Agency (WADA) due to claimed anabolic effects, and also, is prohibited as growth promoters in cattle fattening in the European Union. This paper reviews the last seven-year (2006-2012) literature concerning the development of novel ß2-agonists molecules either by modifying the molecule of known ß2-agonists or by introducing moieties producing indole-, adamantyl- or phenyl urea derivatives. New emerging ß2-agonists molecules for future therapeutic use are also presented, intending to emphasize their potential use for doping purposes or as growth promoters in the near future.

Chiral separation of new cathinone- and amphetamine-related designer drugs by gas chromatography-mass spectrometry using trifluoroacetyl-l-prolyl chloride as chiral derivatization reagent.

Since cathinone derivatives gained high popularity on the recreational drugs market within the past 5 years the development of analytical methods for the achiral and chiral determination of this substance class is of great interest. Not at least because it is obvious that the pharmacological potency differs between both enantiomers. Cathinones are structurally closely related to amphetamines, which have similar stimulating effects and are somehow better investigated. The goal of this research was to perform indirect enantioseparation of novel psychoactive cathinone and amphetamine derivatives. Trifluoroacetyl-l-prolyl chloride was served as chiral derivatization agent (CDA). Chromatographic separation was performed using a commercially available HP5-MS capillary column with a length of 30 m. Helium was used as carrier gas with a constant flow of 1.0 ml/min. Under optimum conditions 14 amphetamine derivatives were successfully resolved into their enantiomers and detected with the single quadrupol detector. Racemic methcathinone derivatives analyzed with the same method showed different peak areas for each of the produced diastereomeric isomers, even if they are structurally closely related to the amphetamines. Derivatization experiments with the single isomers of methcathinone led to both diastereomers whereas the S(-) enantiomer seemed to racemize more likely. Based on comparative experiments with R-(-)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride (MTPA) as CDA, racemization due to the keto-enol-tautomerism of the cathinone derivatives seemed to be responsible for this phenomenon. Nevertheless, 18 cathinone derivatives were successfully enantioseparated and an approach of quantitative evaluation is demonstrated.

Natural product 'legal highs'.

The last five years have seen a dramatic increase in the global popularity of 'Legal Highs', often referred to as Novel Psychoactive Substances (NPS). These materials are single chemicals, plant or fungal materials or their extracts, which may be purchased and possessed without legal restriction in certain countries. The single chemical entity drugs are often closely structurally related to existing controlled drugs of abuse from, for example, the amphetamine (phenethylamine), cannabinoid-mimetic or tryptamine classes, whereas the natural products are from plant or fungal materials that have a long history of pharmacognosy. These natural product legal highs are by their very nature highly chemically complex, and in the clear majority of cases, chemical studies were conducted some considerable time ago. Their pharmacology and toxicology generally focuses on the major active principles with few studies detailing the potentially highly complicated and multiple effects of their extracts. This complexity, coupled with the inherent natural product variability of plant and fungal species, adds a further dimension to the potential harms associated with natural product legal high use. This review encompasses the most popular current legal highs and describes their basic chemistry, usage and preparation, pharmacology, toxicology and discusses the issues surrounding the complexity of these materials, and how this can impact on the evaluation of their harms.

Molecularly imprinted solid-phase extraction for the selective determination of methamphetamine, amphetamine, and methylenedioxyphenylalkylamine designer drugs in human whole blood by gas chromatography-mass spectrometry.

A novel method is described for the extraction of methamphetamine, amphetamine, and methylenedioxyphenylalkylamine designer drugs, such as 3,4-methylenedioxy-methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxyethylamphetamine, N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine, and 3,4-(methylenedioxyphenyl)-2-butanamine, from human whole blood using molecularly imprinted solid-phase extraction as highly selective sample clean-up technique. Whole blood samples were diluted with 10 mmol/L ammonium acetate (pH 8.6) and applied to a SupelMIP-Amphetamine molecularly imprinted solid-phase extraction cartridge. The cartridge was then washed to eliminate interferences, and the amphetamines of interest were eluted with formic acid/methanol (1:100, v/v). After derivatization with trifluoroacetic anhydride, the analytes were quantified using gas chromatography-mass spectrometry. Recoveries of the seven amphetamines spiked into whole blood were 89.1-102%. The limits of quantification for each compound in 200 µL of whole blood were between 0.25 and 1.0 ng. The maximum intra- and inter-day coefficients of variation were 9.96 and 13.8%, respectively. The results show that methamphetamine, amphetamine, and methylenedioxyphenylalkyl-amine designer drugs can be efficiently extracted from crude biological samples such as whole blood by molecularly imprinted solid-phase extraction with good reproducibility. This extraction method will be useful for the pretreatment of human samples before gas chromatography-mass spectrometry.

The identification of a nitrosated prodrug of the PDE-5 inhibitor aildenafil in a dietary supplement: a Viagra with a pop.

A new unapproved analogue of sildenafil was detected in capsules of a herbal dietary supplement promoted as a libido enhancing product. Using LC-DAD-MS, MS-MS, HRMS, IR and NMR the analogue was shown to be a derivative of the PDE-5 inhibitor aildenafil with a nitrosamine moiety. A hydrolysis experiment showed that the new analogue was a prodrug of aildenafil and was therefore named nitroso-prodenafil. A capsule contained 108 mg of nitroso-prodenafil which is equivalent to 84 mg of aildenafil and 5.1 mg of nitrogen monoxide (NO). Although it is unknown how much NO can be usefully generated there is 3-fold more NO present than in a 10 mg isorbide nitrate tablet. Both PDE-5 inhibitors and nitrosamines cause vasodilatation by increasing levels of NO. To their coincidental use is warned against because it may cause a fatal drop in blood pressure. In addition, nitrosamines are known carcinogens. This is the first time a PDE-5 inhibitor and a potential NO donor were identified in one molecule. The findings indicate the dangerous level of advancement in medicinal chemistry by producers of unapproved drugs.