Non-uniform sampling to enhance the performance of compact NMR for characterizing new psychoactive substances.

Efficient and robust analytical methods are needed to improve the identification and subsequent regulation of new psychoactive substances (NPS). NMR spectroscopy is a unique method able to determine the structure of small molecules such as NPS even in mixtures. However, high-field NMR analysis is associated with expensive purchase and maintenance costs. For more than a decade, compact NMR spectrometers have changed this paradigm. It was recently shown that a dedicated analytical workflow combining compact NMR and databases could identify the molecular structure of NPS, in spite of the lower spectral dispersion and sensitivity of compact spectrometers. This approach relies on 1H-13C HSQC to both recognize NPS and elucidate the structure of unknown substances. Still, its performance is limited by the need to compromise between resolution and experiment time. Here, we show that this strategy can be significantly improved by implementing non-uniform sampling (NUS) to improve spectral resolution in the 13C dimension of HSQC at no cost in terms of experiment time. Gains in the range of 3 to 4 in resolution are achieved for pure NPS and for a mixture. Finally, 2D HSQC with NUS was applied to improve the identification of NPS with the assistance of databases. The resulting method appears as a useful tool for the characterization of NPS in mixtures, which is essential for forensic laboratories.

Characterization of new psychoactive substances by integrating benchtop NMR to multi-technique databases.

New psychoactive substances (NPS) have become a serious threat for public health due to their ability to be sold in the street or on internet. NPS are either derived from commercial drugs which are misused (recreational rather than medical use) or whose structure is slightly modified. To regulate NPS, it is essential to accurately characterize them, either to recognize molecules that were previously identified or to quickly elucidate the structure of unknown ones. Most approaches rely on the determination of the exact mass obtained by high-resolution mass spectrometry requiring expensive equipment. This motivated us to develop a workflow in which the elucidation is assisted with databases and does not need the exact mass. This workflow combines 1D and 2D NMR measurements performed on a benchtop spectrometer with IR spectroscopy, for creating a multi-technique database to characterize pure and mixed NPS. The experimental database was created with 57 entries mostly coming from seizures, mainly cathinones, cannabinoids, amphetamines, arylcyclohexylamines, and fentanyl. A blind validation of the workflow was carried out on a set of six unknown seizures. In the first three cases, AF, AB-FUBINACA, and a mixture of 2C-I and 2C-E could be straightforwardly identified with the help of their reference spectra in the database. The two next samples were elucidated for the first time with the help of the database to reveal NEK and MPHP substances. Finally, a precise quantification of each characterized NPS was obtained in order to track NPS trafficking networks.

High-field and benchtop NMR spectroscopy for the characterization of new psychoactive substances.

New psychoactive substances (NPS) have become a serious threat to public health in Europe due to their ability to be sold in the street or on the darknet. Regulating NPS is an urgent priority but comes with a number of analytical challenges since they are structurally similar to legal products. A number of analytical techniques can be used for identifying NPS, among which NMR spectroscopy is a gold standard. High field NMR is typically used for structural elucidation in combination with others techniques like GC-MS, Infrared spectroscopy, together with databases. In addition to their strong ability to elucidate molecular structures, high field NMR techniques are the gold standard for quantification without any physical isolation procedure and with a single internal standard. However, high field NMR remains expensive and emerging "benchtop" NMR apparatus which are cheaper and transportable can be considered as valuable alternatives to high field NMR. Indeed, benchtop NMR, which emerged about ten years ago, makes it possible to carry out structural elucidation and quantification of NPS despite the gap in resolution and sensitivity as compared to high field NMR. This review describes recent advances in the field of NMR applied to the characterization of NPS. High-field NMR methods are first described in view of their complementarity with other analytical methods, focusing on both structural and quantitative aspects. The second part of the review highlights how emerging benchtop NMR approaches could act as a game changer in the field of forensics.

Forensic application of position-specific isotopic analysis of trinitrotoluene (TNT) by NMR to determine 13C and 15N intramolecular isotopic profiles.

2,4,6-trinitrotoluene (TNT) is a molecule which is easily identified with current instrumental techniques but it is generally impossible to distinguish between sources of the same substance (TNT). To overcome this difficulty, we present a multi stable isotope approach using isotope ratio monitoring by mass spectrometry (irm-MS) and Nuclear Magnetic Resonance spectrometry (irm-NMR). In the one hand, irm-MS provides bulk isotopic composition at natural abundance in 13C and 15N. The range of variation between samples is rather small particularly for 13C. In the other hand, irm-13C NMR and irm-15N NMR enable the determination of positional intramolecular 13C/12C ratios (δ13Ci) and 15N/14N ratios (δ15Ni) with high precision that lead to larger variation between samples. The present work reports an application of the recent methodology using irm-15N NMR to determine position-specific 15N isotope content of TNT. The interest of this methodology is compared to irm-13C NMR and irm-MS (13C and 15N) in terms of TNT samples discrimination. Thanks to the use of irm-NMR the results show a unique isotopic fingerprint for each TNT which enable origin discrimination between the samples without ambiguity.

Isotopomics by isotope ratio monitoring by 13 C nuclear magnetic resonance spectrometry on cutting agents in heroin: A new approach for illicit drugs trafficking route elucidation.

In the battle against the illicit drugs market, methodologies have been developed by forensic laboratories to address the determination of the origin and dismantlement of the trafficking route for various target molecules such as heroin and cocaine. These drug profiling methods are not straightforward, especially when the target molecules are synthetic and very pure, resulting in poorly informative impurity profiles, e.g. new psychoactive substances and cutting agents. A tool based on the determination of intramolecular isotopic profiles has been developed to provide origin discrimination with a new way to profile seized cutting agents and heroin samples. Whereas stable isotope analyses by mass spectrometry give the bulk isotopic composition, nuclear magnetic resonance gives direct access to the position-specific isotope content at natural abundance. This report shows how both 13 C NMR spectrometry and 13 C, 15 N MS might provide complementary and valuable information to link seized caffeine and paracetamol to their origin. Here, isotopic ratio monitoring by 13 C NMR (irm-13 C NMR) offers additional benefits over irm-MS in its capability to determine a detailed isotopic profile, leading to a better method to distinguish different caffeine and paracetamol batches.

Position-specific 15 N isotope analysis in organic molecules: A high-precision 15 N NMR method to determine the intramolecular 15 N isotope composition and fractionation at natural abundance.

The position-specific 15 N isotope content in organic molecules, at natural abundance, is for the first time determined by using a quantitative methodology based on 15 N Nuclear Magnetic Resonance (NMR) spectrometry. 15 N NMR spectra are obtained by using an adiabatic "Full-Spectrum" INEPT sequence in order to make possible 15 N NMR experiments with a high signal-to-noise ratio (>500), to reach a precision with a standard deviation below 1‰ (0.1%). This level of precision is required for observing small changes in 15 N content associated to 15 N isotope effects. As an illustration, the measurement of an isotopic enrichment factor ε for each 15 N isotopomer is presented for 1-methylimidazole induced during a separation process on a silica column. The precision expressed as the long-term repeatability of the methodology is good enough to evaluate small changes in the 15 N isotope contents for a given isotopomer. As observed for 13 C, inverse and normal 15 N isotope effects occur concomitantly, giving access to new information on the origin of the 15 N isotope effects, not detectable by other techniques such as isotope ratio measured by Mass Spectrometry for which bulk (average) values are obtained.

Full Spectrum Isotopic 13C NMR Using Polarization Transfer for Position-Specific Isotope Analysis.

For the last ten years, quantitative isotope ratio monitoring 13C NMR (irm-13C NMR) has been successfully tested and proven as an efficient tool for the determination of position-specific 13C/12C ratios. Several applications in different domains have shown the interest in this technique. In the context of origin assignment, the possibility to track the distribution network of illicit drugs or cutting agents is of prime importance. However irm-13C NMR still suffers from a relative lack of sensitivity limiting its dissemination among control laboratories. Improvements were proposed to reduce experiment time by using the INEPT sequence ("Insensitive Nuclei Enhanced by Polarization Transfer") based on polarization transfer from highly sensitive 1H to less sensitive 13C. Several applications based on the use of the one bond scalar coupling between 1H and 13C (1 JCH) have shown the potential of this methodology in terms of short experimental duration. However, the isotopic information given by quaternary carbons was lost. The aim of this study is to extend this approach by using short- and long-range coupling (1 JCH, 2 JCH, and 3 JCH) in order to have access to all 13C/12C position-specific ratios, i.e., acquisition of the full spectrum (FS-INEPT). It is shown that this innovative tool provides both sensitivity gain-thanks to the long-range polarization transfer-and appropriate repeatability. The relative isotopic profiles allowed the classification of two cutting agents, caffeine and paracetamol (acetaminophen), according to their origin, as it was previously observed with "classical" irm-13C NMR but consuming much less sample and/or reducing the experimental time.

A study of cannabis potency in France over a 25 years period (1992-2016).

Cannabis contains a unique class of compounds known as the cannabinoids. Pharmacologically, the principal psychoactive constituent is Δ9-tetrahydrocannabinol (THC). The amount of THC in conjunction with selected additional cannabinoid compounds (cannabidiol/CBD, cannabinol/CBN), determines the strength or potency of the cannabis product. Recently, reports have speculated over the change in the quality of cannabis products, from nearly a decade, specifically concerning the increase in cannabinoid content. This article exploits the analytical data of cannabis samples analyzed in the five French forensic police laboratories over 25 years. The increase potency of both herbal and resin cannabis in France is proved through the monitoring of THC content. For cannabis resin, it has slowly risen from 1992 to 2009, before a considerable increase in the last four years (mean THC content in mid-2016 is 23% compared to 10% in 2009). For herbal cannabis, it has known three main stages of growth (mean THC content is 13% in 2015 and mid-2016 compared to 7% in 2009 and 2% in 1995). The calculation of THC/CBD ratios in both herbal and resin samples confirms the recent change in chemotypes in favor of high potency categories. Finally, the CBN/THC ratios in marijuana samples were measured in order to evaluate the freshness of French seized hemp.

IRMS to study a common cocaine cutting agent: phenacetin.

Phenacetin is a pharmaceutical closely related to acetaminophen that has been banned in France for a long time due to its nephritic and carcinogenic adverse effects. It frequently appears in cocaine seizures as a cutting agent. Following both sanitary and intelligence motivations, this molecule was chosen for this study, and stable isotopes seemed to be the most appropriate tool. A total of 228 seized samples were collected over a 6-year period, and 8 standards of known origin were purchased. They were submitted to gas chromatography (GC) or elemental analysis - isotope ratio mass spectrometry (EA-IRMS) measurements, depending on their complexity. Stable isotope ratios of carbon, hydrogen, and nitrogen for a part of the sample set, were acquired. The isotopic values of phenacetin standards acquired from various providers located worldwide are quite spread, which indicates that stable isotopes could be used to discriminate manufacturers. However, the measured values of most of the seized samples are concentrated in a narrow range, tending to demonstrate that phenacetin is smuggled from a single source or similar ones. Consequently, stable isotopes could only be used to exclude that several samples come from a common source. Copyright © 2016 John Wiley & Sons, Ltd.

Headspace profiling of cocaine samples for intelligence purposes.

A method for determination of residual solvents in illicit hydrochloride cocaine samples using static headspace-gas chromatography (HS-GC) associated with a storage computerized procedure is described for the profiling and comparison of seizures. The system involves a gas chromatographic separation of 18 occluded solvents followed by fully automatic data analysis and transfer to a PHP/MySQL database. First, a fractional factorial design was used to evaluate the main effects of some critical method parameters (salt choice, vial agitation intensity, oven temperature, pressurization and loop equilibration) on the results with a minimum of experiments. The method was then validated for tactical intelligence purposes (batch comparison) via several studies: selection of solvents and mathematical comparison tool, reproducibility and "cutting" influence studies. The decision threshold to determine the similarity of two samples was set and false positives and negatives evaluated. Finally, application of the method to distinguish geographical origins is discussed.

Drug intelligence based on MDMA tablets data: 2. Physical characteristics profiling.

One of the tasks of the European project entitled "Collaborative Harmonisation of Methods for Profiling of Amphetamine Type Stimulants" (CHAMP) funded by the sixth framework programme of the European Commission was to develop a harmonised methodology for MDMA profiling and the creation of a common database in a drug intelligence perspective. Part I was dedicated to the analysis of organic impurities formed during synthesis in order to investigate traffic tendencies and highlight potential links between samples, whereas this part focuses on physical characteristics of the MDMA tablets. Diameter, thickness, weight and score were demonstrated to be reliable and relevant features in this drug intelligence perspective. Distributions of samples coming from the same post-tabletting batch (post-TB) and samples coming from different post-TB were very well discriminated by using the squared Euclidean or the Manhattan distance on standardised data. Our findings demonstrated the possibility to discriminate between MDMA samples issued from different post-TB and to find out links between samples coming from a same post-TB. Furthermore, the hypothesis that most of the MDMA samples found on the international market come from the same countries was supported.

A quick and automated method for profiling heroin samples for tactical intelligence purposes.

A computerized procedure is presented for the profiling and comparison of illicit heroin seizures. The system involves the derivation and gas chromatographic separation of five major constituents followed by a fully automatic data analysis and transfer to a PHP/MySQL database. Comparisons via the square cosine function between a single chromatographic profile and the whole database (several hundred of samples) are performed in a few seconds. Advantages of this profiling method compared to the classical minor constituents one are discussed.

Optimization of extraction parameters for the chemical profiling of 3,4-methylenedioxymethamphetamine (MDMA) tablets.

The extraction of impurities from illegally produced 3,4-methylenedioxymethamphetamine (MDMA) has been studied in order to optimize the parameters. Two different MDMA samples were used. Particular attention was paid to the influence of the pH, the evaporation step, and the sample storage. The method used was an extraction of impurities by diethyl ether from a buffer solution at pH 11.5, followed by gas chromatography (GC) mass spectrometric (MS) analyses after a dryness concentration under monitored conditions of the ethereal extract. Repeat extractions of the same sample gave an average relative standard deviation (RSD) of less than 8.5% within day and less than 10.5% between days.

A contribution to the chemical profiling of 3,4-methylenedioxymethamphetamine (MDMA) tablets.

A profiling method for the identification of impurities found in seized 3,4-methylenedioxymethamphetamine (MDMA) tablets is presented. Impurities of interest are extracted from an alkaline solution (pH 12.8) by diethyl ether and submitted to gas chromatography (GC)-mass spectrometry (MS) analyses. Identification of impurities is performed by electron impact ionization (Ei) mass spectrometry and confirmation by positive chemical ionization (Ci+) MS or, when possible, MS/MS (MS(2)). Repeat extractions of the same sample give an average relative standard deviation (R.S.D.) of less than 8% within the same day and 15% between days (results were obtained after normalization by the sum of peak areas, each one being acquired by selected ion monitoring (SIM)). Possible application toward batch comparison of samples is discussed. Chromatographic profiles are compared using the cosine function for evaluating similarity and/or dissimilarity among exhibits.