The key role of mass spectrometry in comprehensive research on new psychoactive substances

New psychoactive substances (NPS) are a wide group of compounds that try to mimic the effects produced by the ‘classical’ illicit drugs, including cannabis (synthetic cannabinoids), cocaine and amphetamines (synthetic cathinones) or heroin (synthetic opioids), and which health effects are still unknown for most of them. Nowadays, more than 700 compounds are being monitored by official organisms, some of which have been recently identified in seizures and/or intoxication cases. Toxicological analysis plays a pivotal role in NPS research. A comprehensive investigation on NPS, from the first identification of a novel substance until its detection in drug users to help in diagnostics and medical treatment, requires the use of a wide variety of instruments and analytical strategies. This paper illustrates the key role of mass spectrometry (MS) along a comprehensive investigation on NPS. The synthetic cannabinoid XLR‐11 and the synthetic cathinone 5‐PPDi have been chosen as representative substances of the most consumed NPS families. Moreover, both compounds have been investigated at our laboratory in different stages of the three‐step strategy considered in this article. The initial identification and characterisation of the compound in consumption products, the first reported metabolic pathway and the development of analytical methodologies for its determination (and/or their metabolites) in different toxicological samples are described. The analytical strategies and MS instruments are briefly discussed to show the reader the possibilities that MS instrumentation offer to analytical scientists. This publication aims to be a starting point for those interested on the NPS research field from an analytical chemistry point of view

Analytical key issues and challenges in the LC-MS/MS determination of antibiotics in wastewater

The research on antibiotics occurrence in the aquatic environment has become a hot topic in the last years due to their potential negative effects, associated to possible bacterial antibiotic-resistance, after continuous exposure to these compounds. Most of antibiotic residues are not completely removed in the wastewater treatment plants (WWTPs) and end up in the aquatic environment through treated wastewater (WW). The development of reliable analytical methodologies for the determination of antibiotics in influent (IWW) and effluent wastewater (EWW) is needed with different purposes, among others: monitoring their occurrence in the aquatic environment, performing environmental risk assessment, estimating removal efficiencies of WWTPs, or estimating the consumption of these compounds. In this paper, we perform an in-depth investigation on analytical key issues that pose difficulties in the determination of antibiotics in complex matrices, such as WW, and we identify challenges to be properly addressed for successful analysis. The analytical technique selected was liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), as it is the most powerful and widely applied at present for antibiotic residues determination. The mass spectrometric behavior of 18 selected antibiotics, the chromatographic performance, ion ratio variations associated to the sample matrix when using different precursor ions or protomers, and the macrolides adsorption to glass vial, were some of the issues studied in this work. On the basis of the detailed study performed, an analytical LC-MS/MS method based on sample direct injection has been developed for quantification of 18 antibiotics in IWW and EWW, allowing their determination at low ng L−1 levels.Funding for open access charge: CRUE-Universitat Jaume IDavid Fabregat-Safont acknowledges Ministerio de Universidades in Spain for his Margarita Salas postdoctoral grant (Ref. MGS/2021/15). Authors acknowledge financial support from General Directorate of Water (Conselleria de Agricultura, Desarrollo Rural, Emergencia Climática y Transición Ecológica) and Generalitat Valenciana (research group of excellence PROMETEO 2019/040). Authors also acknowledge to FACSA (Castelló, Spain) for the wastewater sample collection and the Ayuntamiento of Castelló for the authorization to collect wastewater samples from the WWTP of Castelló. Authors are very grateful to Alberto Celma for his comments on fluoroquinolones protomers evaluation

Rapid and sensitive analytical method for the determination of amoxicillin and related compounds in water meeting the requirements of the European union watch list

The presence of antibiotics in the aquatic environment is becoming one of the main research focus of scientists and policy makers. Proof of that is the inclusion of four antibiotics, amongst which is amoxicillin, in the EU Watch List (WL) (Decision 2020/1161/EU)) of substances for water monitoring. The accurate quantification of amoxicillin in water at the sub-ppb levels required by the WL is troublesome due to its physicochemical properties. In this work, the analytical challenges related to the determination of amoxicillin, and six related penicillins (ampicillin, cloxacillin, dicloxacillin, penicillin G, penicillin V and oxacillin), have been carefully addressed, including sample treatment, sample stability, chromatographic analysis and mass spectrometric detection by triple quadrupole. Given the low recoveries obtained using different solid-phase extraction cartridges, we applied the direct injection of water samples using a reversed-phase chromatographic column that allowed working with 100% aqueous mobile phase. Matrix effects were evaluated and corrected using the isotopically labelled internal standard or correction factors based on signal suppression observed in the analysis of spiked samples. The methodology developed was satisfactorily validated at 50 and 500 ng L − 1 for the seven penicillins studied, and it was applied to different types of water matrices, revealing the presence of ampicillin in one surface water sample and cloxacillin in three effluent wastewater samples.Funding for open access charge: CRUE-Universitat Jaume

Characterization of a recently detected halogenated aminorex derivative: para-fluoro-4-methylaminorex (4′F-4-MAR)

Despite the fact that 33% of the new psychoactive substances seized in 2015 were synthetic cathinones, the number of these derivatives has been decreasing in the last years, probably as a consequence of the unfavourable effects reported by users. Thus, the list of possible cathinone analogues is expected to get shorter, and it is likely that the same moiety changes applied for the preparation of synthetic cathinones will be applied in the near future to other stimulants in the search for favourable alternatives to controlled substances. This is evidenced by the increase in newly reported substances belonging to stimulant classes other than cathinones. One of the possible candidates for a new backbone from which to base new stimulants is aminorex, which is classified as a Schedule I substance by the Drug Enforcement Administration. Three derivatives have been reported until now: 4-methylaminorex or 4-MAR (also categorized as a Schedule I substance), para-methyl-4-methylaminorex (4,4′-DMAR) and 3′,4′-methylenedioxy-4-methylaminorex (MDMAR). Recently, the new halogenated 4-MAR derivative, para-fluoro-4-methylaminorex, characterised in this work (and abbreviated as pF-4-methylaminorex or 4′F-4-MAR) was detected by the Slovenian police. In the present work, 4′F-4-MAR has been characterized by high resolution mass spectrometry and nuclear magnetic resonance in a sample obtained from an anonymous consumer. This research shows that the same modifications applied for the preparation of synthetic cathinones are being used to prepare new stimulants based on the aminorex backbone

Metabolic profiling of four synthetic stimulants, including the novel indanyl-cathinone 5-PPDi, after human hepatocyte incubation

Synthetic cathinones are new psychoactive substances that represent a health risk worldwide. For most of the 130 reported compounds, information about toxicology and/or metabolism is not available, which hampers their detection (and subsequent medical treatment) in intoxication cases. The principles of forensic analytical chemistry and the use of powerful analytical techniques are indispensable for stablishing the most appropriate biomarkers for these substances. Human metabolic fate of synthetic cathinones can be assessed by the analysis of urine and blood obtained from authentic consumers; however, this type of samples is limited and difficult to access. In this work, the metabolic behaviour of three synthetic cathinones (4-CEC, 4-CPrC and 5-PPDi) and one amphetamine (3-FEA) has been evaluated by incubation with pooled human hepatocytes and metabolite identification has been performed by high-resolution mass spectrometry. This in vitro approach has previously shown its feasibility for obtaining excretory human metabolites. 4-CEC and 3-FEA were not metabolised, and for 4-CPrC only two minor metabolites were obtained. On the contrary, for the recently reported 5-PPDi, twelve phase I metabolites were elucidated. Up to our knowledge, this is the first metabolic study of an indanyl-cathinone. Data reported in this paper will allow the detection of these synthetic stimulants in intoxication cases, and will facilitate future research on the metabolic behaviour of other indanyl-based cathinones

Efficient Validation Strategies in Environmental Analytical Chemistry: A Focus on Organic Micropollutants in Water Samples

This article critically reviews analytical method validation and quality control applied to the environmental chemistry field. The review focuses on the determination of organic micropollutants (OMPs), specifically emerging contaminants and pesticides, in the aquatic environment. The analytical technique considered is (gas and liquid) chromatography coupled to mass spectrometry (MS), including high-resolution MS for wide-scope screening purposes. An analysis of current research practices outlined in the literature has been performed, and key issues and analytical challenges are identified and critically discussed. It is worth emphasizing the lack of specific guidelines applied to environmental analytical chemistry and the minimal regulation of OMPs in waters, which greatly affect method development and performance, requirements for method validation, and the subsequent application to samples. Finally, a proposal is made for method validation and data reporting, which can be understood as starting points for further discussion with specialists in environmental analytical chemistry.D.F.-S. acknowledges the Ministerio de Universidades in Spain for his Margarita Salas postdoctoral grant (Ref. MGS/2021/15). The authors from University Jaume I acknowledge financial support from Generalitat Valenciana (Research Group of Excellence Prometeo 2019/040) and Ministerio de Ciencia e Innovación (Ref. PID2021-127451OB-I00). J.B.Q. acknowledges funding provided by Xunta de Galicia (Ref. ED431C 2021/06), the Spanish Agencia Estatal de Investigación – MCIN/AEI/10.13039/501100011033 (Ref. PID2020-117686RB-C32).S

Variation in the pattern of synthetic cannabinoid use by a female patient during 2018

El consumo de cannabinoides sintéticos es un problema creciente. En España, el número de incautaciones e intoxicaciones relacionadas con estas nuevas sustancias psicoactivas (NPS) es menor que en el resto de Europa. No obstante, el análisis de mezclas de hierbas vendidas en smart-shops y páginas web refleja que estos productos sí están llegando a la población española. En este trabajo, se estudia el patrón de consumo de cannabinoides sintéticos en la ciudad de Valencia (España), a través del análisis de preparados de hierbas para fumar, cigarros y muestras de orina de potenciales consumidores recogidas en diferentes Unidades de Conductas Adictivas (UCAs) de la ciudad. Se analizaron diferentes productos disponibles en una smart-shop de la localidad, así como hierbas recogidas en las UCAs. Los análisis por espectrometría de masas revelaron la presencia de los cannabinoides sintéticos XLR-11 y UR-144. Se analizó también la orina de una paciente de la UCA, atendida por supuesto consumo de cannabis, que mostraba signos de consumo, aunque los análisis rutinarios daban resultados negativos. Se encontraron metabolitos de estos cannabinoides, previamente reportados en bibliografía. Tras el verano de 2018, se incautaron nuevas muestras de hierbas y cigarros, identificando el peligroso cannabinoide 5F-ADB. En las muestras de orina también se detectaron los principales metabolitos reportados para este cannabinoide sintético, y también se identificó este compuesto en una nueva hierba de la smart-shop. Es interesante resaltar que este cambio de consumo coincide con la prohibición en España del XLR-11 en el mes de julio de 2018, ilustrando cómo la legislación de estos compuestos afecta a su disponibilidad y, por tanto, a su patrón de consumo.The consumption of synthetic cannabinoids is a growing problem. In Spain, the number of seizures and poisonings related to these new psychoactive substances (NPS) is less than in the rest of Europe. However, the analysis of herbal mixtures sold in smart shops and web pages reflects that these products are indeed reaching the Spanish population. In this work, the consumption pattern of synthetic cannabinoids in the city of Valencia (Spain) is studied, through the analysis of herbal preparations for smoking, cigarettes and urine samples of potential consumers collected in different Addictive Behavior Units (ABUs) ) from the city. Different products available at a local smart-shop were analyzed, as well as herbs collected in the UCAs. Mass spectrometric analysis revealed the presence of the synthetic cannabinoids XLR-11 and UR-144. The urine of a patient from the ABU, treated for alleged cannabis use, who showed signs of use, was also analysed, although routine analysis gave negative results. Metabolites of these cannabinoids were found, previously reported in the literature. Following the summer of 2018, new herb and cigar samples were seized, identifying the dangerous cannabinoid 5F-ADB. The main metabolites reported for this synthetic cannabinoid were also detected in the urine samples, and this compound was also identified in a new herb from the smart shop. It is interesting to note that this change in consumption coincides with the ban on XLR-11 in Spain in July 2018, illustrating how the legislation on these compounds affects their availability and, therefore, their consumption pattern

Characterization of the recently detected cathinone N-cyclohexyl butylone: From structure elucidation to in silico supported pharmacological/ toxicological considerations

One of the most widely consumed new psychoactive substances (NPS) families in Europe are synthetic cathinones. Cathinone structure can be easily modified resulting in new derivatives that rapidly reach drug markets. In this work, the recently detected synthetic cathinone N-cyclohexyl butylone has been characterized by gas chromatography coupled to mass spectrometry (GC–MS), liquid chromatography coupled to high-resolution mass spectrometry (HRMS), nuclear magnetic resonance (NMR) and Fourier-transformed infrared (FTIR) spectroscopy, using research chemicals samples collected by the drug analysis service Energy Control from anonymous users. Compound identification was performed by the combination of HRMS and NMR data. The elemental composition and putative moieties of the compound were determined based on the accurate-mass ions observed by HRMS. Then, different NMR experiments, including bidimensional, allowed the establishment of the chemical structure and confirmation of compound identity. Furthermore, FTIR spectrum was also acquired in order to provide a complete analytical characterization of the novel cathinone. Finally, pharmacological/toxicological characterization was attempted using in silico methods. Based on these predictions, N-cyclohexyl butylone probably has similar effects to stimulants like MDMA

Proposal of 5-methoxy-N-methyl-N-isopropyltryptamine consumption biomarkers through identification of in vivo metabolites from mice

New psychoactive substances (NPS) are a new breed of synthetically produced substances designed to mimic the effects of traditional illegal drugs. Synthetic cannabinoids and synthetic cathinones are the two most common groups, which try to mimic the effects of the natural compounds 9Δ-tetrahydrocannabinol and cathinone, respectively. Similarly, synthetic tryptamines are designer compounds which are based on the compounds psilocin, N,N-dimethyltryptamine and 5-methoxy-N,N-dimethyltryptamine found in some mushrooms. One of the most important tryptamine compounds found in seizures is 5-methoxy-N,N-diisopropyltryptamine, which has been placed as controlled substance in USA and some European countries. The control of this compound has promoted the rising of another tryptamine, the 5-methoxy-N-methyl-N-isopropyltryptamine, which at the time of writing this article has not been banned yet. So, it is undeniable that this new substance should be monitored. 5-methoxy-N-methyl-N-isopropyltryptamine has been reported by the Spanish Early Warning System and detected in our laboratory in two pill samples purchased in a local smart shop. This has promoted the need of stablishing consumption markers for this compound in consumers’ urine. In the present work, the metabolism and pharmacokinetic of 5-methoxy-N-methyl-N-isopropyltryptamine has been studied by an in vivo approach, using adult male mice of the inbred strain C57BLJ/6. The use of ultra-high performance liquid chromatography coupled to high resolution mass spectrometry allowed the identification of four metabolites. After the pharmacokinetic study in serum and urine, the O-demethylated metabolite and the non-metabolised parent compound are proposed as consumption markers in hydrolysed urine. Data reported in this work will help hospitals and forensic laboratories to monitor the consumption and potential intoxication cases related to this tryptamine

Direct and Fast Screening of New Psychoactive Substances Using Medical Swabs and Atmospheric Solids Analysis Probe Triple Quadrupole with Data-Dependent Acquisition

New psychoactive substances (NPS) have become a serious public health problem, as they are continuously changing their structures and modifying their potency and effects on humans, and therefore, novel compounds are unceasingly appearing. One of the major challenges in forensic analysis, particularly related to the problem of NPS, is the development of fast screening methodologies that allow the detection of a wide variety of compounds in a single analysis. In this study, a novel application of the atmospheric solids analysis probe (ASAP) using medical swabs has been developed. The swab–ASAP was coupled to a triple quadrupole mass analyzer working under a data-dependent acquisition mode in order to perform a suspect screening of NPS in different types of samples as well as on surfaces. The compounds were automatically identified based on the observed fragmentation spectra using an in-house built MS/MS spectra library. The developed methodology was applied for the identification of psychoactive substances in research chemicals and herbal blends. The sensitivity of the method, as well as its applicability for surface analysis, was also assessed by identifying down to 1 μg of compound impregnated onto a laboratory table. Another remarkable application was the identification of cathinones and synthetic cannabinoids on the fingers of potential consumers. Interestingly, our data showed that NPS could be identified on the fingers after being in contact with the product and even after cleaning their hands by shaking off with a cloth. The methodology proposed in this paper can be applied for routine analyses of NPS in different matrix samples without the need to establish a list of target compounds prior to analysis