The last few years have witnessed an exponential increase in the use of novel psychoactive substances (NPS). These drugs, also known as “legal highs”, are commonly sold as bath salts, plant food, or herbal incenses, and labeled as “not for human consumption”.
As reported by the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA), over the past 20 years more than 700 NPS have appeared on Europe’s drug market. They contain sedatives, stimulants, and hallucinogens, such as tramadol, oxycodone, benzodiazepines, and cannabinoids, to name a few.
NPS pose serious challenges
With new drugs appearing on the market almost every week it is difficult to keep up with the determination of their composition and their psychoactive effects. Moreover, NPS stays often ahead of legislation since subtle modifications in the chemical formulas make new products available almost as fast as existing ones are banned.
Screening for these drugs is problematic since analysts need to have a good idea of the compounds that are likely to be present in a sample before analysis and often no reference standards are available. Therefore, laboratories rely on the creations of accurate and up-to-date libraries to identify NPS.
Common screening methods for NPS
The most prevalent screening tool for NPS identification is gas chromatography-mass spectrometry (GC-MS), a widely available analytical platform in most laboratories. For quantification purposes instead, liquid chromatography-tandem mass spectrometry (LC-MS/MS) is more commonly used.
The identification is based on libraries of reference standards, which are not always readily available for unknown NPS. Also, mass spectra and/or retention times of closely-related drugs are very similar, compromising a reliable identification.
Researchers are constantly working on alternative methods for the analysis of NPS. In this regard, a promising technique is liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS), which can detect the accurate mass of compounds in biological samples to four decimal digits, providing a high degree of specificity.
By combining the measured accurate mass of the compound with the accurate mass of its fragments, as well as the isotopic pattern and retention time, it is possible to perform a broad screening of NPS in plasma to accurately identify a drug by comparing these parameters with a reference library.
A study using LC-Q-Orbitrap HRMS in targeted-MS/MS mode was able to identify and quantify 25 NPS in plasma, particularly cathinones and synthetic cannabinoids. The method used calibration with standard references and provided high sensitivities with a low limit of detection (LOD 10-2–10-3 ng/ml).
ATR-IR spectroscopy for the fast, preliminary investigation of NPS
A potential technique that can overcome some of the limitations of GC-MS and LC-MS is attenuated total reflectance infrared (ATR-IR) spectroscopy. The analysis requires very little amount of sample and no pre-treatment, enabling the fast, preliminary investigation of NPS.
The ATR-IR analysis of 45 samples led to the identification of 31 different NPS, including synthetic cathinones, cannabinoids, phenethylamines, and opioids. The identification of each drug was based on the comparison between the registered spectrum and reference spectra reported in libraries.
ATR-IR also allows for the identification of compounds with very similar chemical structures. For instance, cathinones such as 4-chlor-omethcathinone (4-CMC), 4-chloroethcathinone (4-CEC), and 4-chlorobutylcathinone (4-CBC) differ for the number of methylene groups, therefore their spectra are very similar.
Nevertheless, ATR-IR can discriminate against these compounds thanks to subtle differences especially in the 3500–2000 cm-1 range. The technique can also distinguish between structural isomers which in GC-MS or LC-MS analysis would have the same retention time. For example, the spectra of 3-methyl methcathinone (3-MMC) and 4-MMC show differences in the 1500-1700 cm-1 and can therefore be discriminated against.
Automated NPS analysis using low field 1H NMR spectroscopy
Nuclear Magnetic Resonance (NMR) is a powerful spectroscopic technique for the identification of organic compounds. Yet, so far NMR has not found extensive use in drug analysis, due to the complexity of the measurement and the elevated costs associated with the need for superconducting magnets and related technology costs.
The recent rise in benchtop spectrometers can however make NMR a more appealing and cost-effective technique. A new approach using an automated NMR benchtop spectrometer, which acquires, processes, and interprets the spectral data in a short time with minimum user knowledge, can be used for the identification of NPS.
In a recent study, researchers developed an algorithm that analyzes 1H NMR spectra acquired with a low-field spectrometer (59.7 MHz) by matching them with a library of known substances. The recognition was based on two portions of the spectra; the “class” and the “fingerprint” regions.
In particular, the region between 0.46 and 1.54 ppm is characteristic of the class of a compound, while the fingerprint region (3.90–12.50 ppm) allows for the discrimination of different compounds within a class.
The method was validated by performing 130 analyses, with a 99% success rate for the class identification and a 95% success rate for the compound identification.
The 1H NMR analysis of 416 seized samples revealed that the results were in agreement with the GC-MS analysis in 93% of the cases, proving the process can be a viable alternative to current methods. Most of the drugs identified were MDMA, cocaine, and ketamine, but also benzocaine and diazepam.
Future work will be focused on developing a quantification method to extend the applicability of the qualitative approach described and to improve the ability to handle complex mixtures with multiple components.
The rise in NPS production and consumption is pushing society and science to a race against time. Most of the efforts are focused on the generation of up-to-date libraries for the accurate screening of these new drugs.
Although there are advanced tools, they are often only accessible to a limited number of facilities. Therefore, there is also a need to develop new methods and techniques to make NPS analysis available in most laboratories.
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- Rab, E. & Martin, S. 2019. Novel Psychoactive substances: a toxicological challenge [Online]. The Royal College of Pathologists. Available: https://www.rcpath.org/profession/publications/college-bulletin/july-2019/novel-psychoactive-substances-a-toxicological-challenge.html [Accessed 8 August 2020].
- Montesano, C., Vannutelli, G., Gregori, A., Ripani, L., Compagnone, D., Curini, R. & Sergi, M. (2016). Broad Screening and Identification of Novel Psychoactive Substances in Plasma by High-Performance Liquid Chromatography-High-Resolution Mass Spectrometry and Post-run Library Matching. J Anal Toxicol, 40, 519-28.10.1093/jat/bkw043
- Piorunska-Sedlak, K. & Stypulkowska, K. (2020). Strategy for identification of new psychoactive substances in illicit samples using attenuated total reflectance infrared spectroscopy. Forensic Sci Int, 312, 110262.10.1016/j.forsciint.2020.110262
- Antonides, L. H., Brignall, R. M., Costello, A., Ellison, J., Firth, S. E., Gilbert, N., Groom, B. J., Hudson, S. J., Hulme, M. C., Marron, J., Pullen, Z. A., Robertson, T. B. R., Schofield, C. J., Williamson, D. C., Kemsley, E. K., Sutcliffe, O. B. & Mewis, R. E. (2019). Rapid Identification of Novel Psychoactive and Other Controlled Substances Using Low-Field (1)H NMR Spectroscopy. ACS Omega, 4, 7103-7112.10.1021/acsomega.9b00302
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