Identification of Novel Psychoactive Substances Using Hyphenated Mass Spectrometric Techniques

March 1, 2012

Special Issues

Volume 0, Issue 0

Gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–mass spectrometry (LC–MS) techniques offer advantages in separating and confirming the identity of constituents in novel psychoactive substances.

The abuse of novel psychoactive substances (NPS) has been increasing over the last few years especially as these products have become so readily available through the Internet. These products are emerging mostly as analogues or precursors of well-known drugs of abuse, such as amphetamine. However, a major problem that is more complicated than their abusive potential is that these products may not contain what is on the label. In fact, they have been shown to contain a mixture of drugs and excipients that may be toxic or even lethal. The number of deaths caused by NPS products has been increasing over the last year. This increase stimulates the need for sensitive techniques to identify these substances and detect any organic impurity in their products. Hyphenated mass spectrometric techniques, such as gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–mass spectrometry (LC–MS) offer some advantages. These techniques can both separate the constituents as well as confirm the chemical identity of the individual constituents in the product.

Novel psychoactive substances (NPS), also called "legal highs," represent a major threat to society, particularly to the younger generation. The threat caused by these drugs ranges from chronic psychological or physical illnesses to death. These drugs emerge as analogues or precursors of well-known drugs of abuse to bypass regulations, and the popularity of abusing them is increasing worldwide (1–4). The European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) witnessed the emergence of 41 NPS in 2010, which was high compared to 24 substances in 2009 and 13 substances in 2008 (1). This was the largest number of substances reportedly introduced in a single year and included the following derivatives: 15 cathinones, 11 cannabinoids, a synthetic derivative of the plant based substance arecoline, five phenethylamines, one tryptamine, one piperazine, one cocaine, one ketamine, one phencyclidine, one aminoindan, one benzofuran, one aliphatic amine, and a substance that was described as designer medicine (1). The largest threat of these drugs is "amphetamine type" stimulants whose use, according to the United Nations report, has exceeded that of heroin and cocaine (3).

Several factors contribute to the emergence of these new drug derivatives, such as the drop in the purity of ecstasy, heroin, and cocaine over the last few years (3); the change in the sociological and behavioral context (5); the change in the drug phenomena from life-threatening treatment to lifestyle drugs; and the emergence of unregulated Internet websites. These drugs are marketed in an attractive way and can be found under the names "Legal," "Speed," "Research Chemical," "Plant Food," "Bath Salts," and "Not for human consumption" (6–8). They also are sold at a cheap price. A survey by Schmidt and colleagues showed that an average price of a designer drug in the UK was 9.69 GBP (9). In addition, a survey conducted by Carhart-Harris and colleagues showed that the popularity of common NPS was related to their legal status (2,10). The issue associated with the emergence of NPS is not simply related to increased use, but in the chemical diversity of the products available (1). The EMCDDA warns that the situation could become even more complex if the future witnesses a growing trend in the synthesis of new illicit drugs based on pharmaceutical products (1).

The toxicity and death case reports caused by these drugs have been increasing over the last few years (1,2,11). According to the United States Poison Control Center, 2237 calls were received from January to May 2011 relating to "bath salts" from 47 states; whereas, the number of calls in 2010 relating to these drugs was 303 (3,12). The cases from the last few years include paranoia and psychotic illnesses (13), acute liver failure (14,15), myocardial inflammation (16), acute sympathomimetic toxicity, hallucinations (1,17–24), and even death (1,20,25–29). The toxic effects associated with these products are not only related to the NPS pharmacological effect and dose, but also to the impurities and adulterants found in them. Often, these products do not only contain what is stated on the product label, or "label claim," but also contain a mixture of drugs and impurities. These additional substances are both deliberate (for example, to counteract a side effect) and accidental (such as, resulting from a poor quality synthesis). They range from organic to inorganic materials and include illicit drugs, pharmaceutical active ingredients, chemical precursors, and common excipients (30). For instance, analysis of products from patients admitted to the emergency department showed that out of 33 products analyzed 19% of nonliquid samples did not contain any drug and 23% contained a pharmaceutical active ingredient (31). The remaining samples contained both classic and novel recreational drugs, including methamphetamine, ketamine, chlorophenylpiperazine, and 1-benzylpiperazine. This highlights a significant issue of confirming the identity of these substances and ultimately the quantity in which they are present in a product.

Hyphenated mass spectrometric techniques, such as gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–mass spectrometry (LC–MS), offer the advantage of selectivity and sensitivity for the analysis of NPS. These techniques can separate and potentially identify NPS of similar chemical structure, in contrast to rapid colorimetric tests that are often unable to discriminate between chemical analogues and isomers. The objective of this review is to highlight the MS methods used in the literature to date for the analysis of NPS. The review focuses on NPS products purchased from the Internet as well as product seizures. Most of the references in the literature cited the use of GC–MS; whereas only one reference utilized the application of LC–MS.


GC–MS has been the mass spectrometric method of choice for NPS analysis because GC can often separate structural isomers and MS provides fragmentation that is essential in identifying different analogues of a substance. Also, data collected by electron ionization (EI) is easily checked against fragmentation libraries. This is beneficial when analyzing NPS products that are often contaminated with additional analogues, isomers, synthesis precursors, and other active ingredients or organic impurities.

Figure 1: Chemical structures of (a) amphetamine and (b) cathinone.

GC–MS has been used to identify drug analogues in NPS products seized (32,33) and purchased from the Internet (34). For example, the cathinone "mephedrone" was identified in two products initially believed to be "amphetamine" (Figure 1) (32). Italian police seized these products in the form of an off-white powder and a white tablet, labeled with a dolphin logo (Table I), which initially tested positive for amphetamine using a standard colorimetric test. The products were further analyzed using GC–MS, yet the data did not confirm the presence of amphetamine. Examination of the mass spectrum showed the presence of mephedrone (MW 177) and its major fragment at m/z 58, which corresponded to the immonium ion (Table II). The drug had retention times of 6.03 min and 11.06 min before and after derivatization with 2,2,2-trichloroethyl chloroformate. Derivatization of mephedrone increased the major fragment from m/z 58 to m/z 232, 234, and 236, showing the three chlorine atoms with their clustering pattern. Another case was observed for two samples obtained during a large product seizure submitted by the police department in Australia (33). The GC–MS analysis showed that the samples had very similar mass spectra with the same major ion peak of m/z 58 corresponding to cathinone fragmentation, yet the chromatograms showed peaks at different retention times, 5.54 and 6.96 min. The two drugs were identified as the 3-fluoromethcathinone analogues 3,5-difluoromethcathinone and 3,5-dichloromethcathinone, respectively. Another case identified a mixture of cathinone analogues in capsules purchased from the Internet that claimed to contain only the active ingredient mephedrone (34). The products purchased were Spirit, Sub Coca 2, Neo Dove, and Sub Coca. The products all were different colors. However, the results showed that only the first product (Spirit) contained mephedrone (retention time = 4.36 min), the second product (Sub Coca 2) contained a mixture of α-phthalimidopropiophenone (retention time = 6.45 min) and 2-fluoroamphetamine (retention time = 2.56 min), and the third (Neo Dove) and fourth (Sub Coca) contained a mixture of caffeine, mephedrone, N-ethylcathinone, and α-phthalimidopropiophenone (Table I). The MS spectra of the four substances showed the same fragmentation pattern as the previous cathinones.

Table I: Details of the substances found in novel psychoactive substances products

Because the synthesis of NPS is often done in uncertified laboratories, there is little effort made to control or purify isomeric forms. The incidence of analogues present as isomers, yet still different to that on the label claim of an NPS product, was much more prominent in the literature. These included isomers of fluoromethcathinone (35), methylmethcathinone (36), N-ethylethcathinone (37), methylenedioxycathinone (38), isocathinone (39), and naphyrone (40).

A number of studies developed methods using isomer standards to identify a single isomer present in a sample. A method to distinguish the 2-, 3-, and 4-isomers of fluoromethcathinone was developed using GC–EI-MS (35). The chromatogram of the 4′-isomer (9.28 min) was clearly distinguished from the 2′-isomer (9.15 min) and the 3′-isomer (9.23 min). However, the three isomers were not stable so acetylation was needed to improve their stability. In this respect, the retention time of the acetylated derivatives of 4′-, 3′-, and 2′-isomer increased to 11.44, 11.46, and 11.66 min, respectively. The fragmentation of the three isomers was the same as the cathinone derivatives with a major ion peak corresponding to the immonium ion at m/z 58. The presence of the 4′-isomer of fluoromethcathinone was then confirmed in three capsule products labeled as Sub Coca Dragon, High Spirit, and Neo Dove 2. However, in this case GC–MS could not detect caffeine and methylamine salt, which were present in the capsules and were confirmed using nuclear magnetic resonance (NMR) and infrared (IR) spectroscopy. A method to distinguish 2-, 3-, and 4-isomers of methylmethcathinone in NPS products also was developed (36–38). All isomers showed the same MS fragmentation pattern with the major ion peak at m/z 58 (36), yet they were successfully separated 2<3<4 by GC between 6 and 9 min, respectively. The seized product analyzed was found to contain the 4- methylmethcathinone (mephedrone) isomer as well as benzocaine and showed GC retention times of 8 and 12 min, respectively.

Table II: Ions observed from the analysis of novel psychoactive substances using chemical ionization (CI) and electron ionization (EI) mass spectrometry

In some cases, samples contained a substance and an isomer of the substance, demonstrating the typically poor quality of the product obtained. Isomers of mephedrone and ethcathinone were detected in two seized products using GC–EI-MS (37). The isomers were detected at different retention times than mephedrone (8.2 min) and ethcathinone (6.5 min) and were seen at 6.4 min for isomephedrone and 5.4 min for isoethcathinone. Both isomers had a major ion peak at m/z 134 corresponding to isocathinone instead of m/z 58, which corresponds to cathinone (Table II). Benzocaine also was found in these products. Methods to distinguish 3,4-methylenedioxymethcathinone and butylone isomers found in two seized products using GC–EI-MS also was developed (38). The chromatogram of the first sample showed the separation of 3,4-methylone and 3,4-MDPV from two other impurities, benzocaine and phenacetin, and were between 12 and 16 min (Table I). On the other hand, the second sample lacked these two impurities and the GC data showed the separated isobutylone and 3,4-butylone between 13 and 15 min. The mass spectra showed base peaks for methylone, butylone, and MDPV at m/z 58, 72, and 126, respectively (Table II). Isomers of pentedrone and pentylone, were seen in two NPS product seizures: white powder seized from the customs department at the airport in Austria and green capsules brought to the police from a head shop in Germany (39). For the white powder, the mass spectrum in chemical ionization (CI) mode for both isomers showed the same [M+H]+ at m/z 192, but the mass spectrum in EI mode showed a difference in the major ion peak of the characteristic immonium ion that was m/z 86 for pentedrone and m/z 120 for isopentedrone (Figure 2 and Table I). Similarly, the capsules showed to contain pentylone and iso-pentylone had similar results with [M+H]+ at m/z 236 in CI mode and showed different major ion peaks in EI mode of the characteristic immonium ion that was at m/z 86 and 164 for pentylone and isopentylone (Figure 2), respectively. A recent study using GC–ion trap-EI-CI-MS to distinguish α- and β-isomers of naphyrone also was done (40). In this case, both isomers showed similar mass spectra with a major ion peak at m/z 126 corresponding to the formation of the iminium ion in EI mode, and [M+H]+ at m/z 282 in CI mode. However, both isomers had different retention times at 12.41 and 12.86 min corresponding to α and β-naphyrone respectively. The β-isomer was detected in one powder sample from the Internet, whereas both isomers were found in a second powder sample (Table I).

Figure 2: Chemical structures of (a) pentedrone, (b) isopentedrone, (c) pentylone, and (d) isopentylone.

In many cases, NPS products contain mixtures of isomers, analogues, and organic and inorganic impurities. This was observed with the analysis of 24 NPS products purchased from the Internet (41). Most of these products were labeled as naphyrone (NRG-1) but contained one or a binary mixture of other cathinone analogues or isomers, an anesthetic, a stimulant, or other substances with no psychoactive effect. The cathinones found included 4-methyl-N-ethylcathinone, butylone, flephedrone, MDPV, mephedrone, and naphyrone (Table I). The substances with no psychoactive effects included benzocaine, caffeine, lidocaine, procaine, and inorganic materials (Table I). A GC–MS method was developed to separate all substances found in the purchased products. Thus, the retention times for the cathinone derivatives were 7.97, 9.54, 6.73, 11.53, 7.64, and 12.41 min, corresponding to 4-methyl-N-ethylcathinone, butylone, flephedrone, MDPV, mephedrone, and naphyrone, respectively. The mass spectra for these cathinones showed major fragmentations corresponding to the iminium ion at m/z 58, 58, 126, 72, 126, and 72, respectively (Table II). On the other hand, benzocaine, caffeine, lidocaine, and procaine showed retention times of 8.36, 9.9, 10.05, and 10.92 min, respectively. Whereas all these products contained a single substance or binary mixtures of two substances, further binary mixtures and more complicated quaternary mixtures of cathinones were detected in an additional three products. Two of the products were labeled as mephedrone and one was labeled as naphyrone (42). Five different cathinones were identified in these samples (Table I) and had retention times of 6.73, 9.95, 11.12, 11.53, and 9.39 min, corresponding to 4-fluoromethcathinone, pentylone, MDPBP, MDPV, and MPPP, respectively. The mass spectra in EI mode showed a major ion peak corresponding to these cathinones at m/z 58, 86, 112, 126, 98, and 164, respectively (Table II). In addition, the [M+H]+ values detected for these cathinones in CI mode were observed at m/z 182, 236, 262, 276, and 218, respectively.

A number of studies used GC–MS to identify substances chemically unrelated to the label claim. In some cases, NPS products contained neither an isomer nor an analogue of their label claim, but instead contained a completely different substance. This was shown with seven products purchased from the Internet and labeled as MDAI (three products), Benzofury, 5-IAI, NRG-3, and E2. Apart from one MDAI sample, the GC–MS analysis of the products showed that they contained completely different ingredients to their label claim (43). These products contained a mixture of BZP, 3-TFMPP, and caffeine, except E2, which contained only caffeine (Table I). The chromatography method was able to separate the compounds present that appeared at 5, 5.3, 5.7, and 7 min corresponding to BZP, 3-TFMPP, MDAI, and caffeine, respectively. The major ion peaks observed in the mass spectra were m/z 91, 188, 160, and 194, respectively. In comparison, products claiming to contain legal substances including amino acids, salts, ketones, and minerals contained a NPS (44), usually a cathinone derivative (Table I). Additional molecular components found in theses products included piperazines, ephedrine, and caffeine.


LC–MS also has been used to develop methods to separate known NPS. Five seized powder samples were obtained from the Hungarian customs and finance guard (45). The five samples contained the cathinones mephedrone, butylone, MDPV, flephedrone, and 4´-methylethcathinone (Table I). The method was developed using these samples and reference standards of methylone and methedrone. The LC method was able to separate the seven substances in less than 8 min. In addition, the mass spectra (in ESI mode) showed characteristic ions [M+H]+ at m/z 178, 222, 276, 182, 208, 194, and 192, respectively.


Products of novel psychoactive substances represent a major threat ranging from chronic illness to death. Unlike traditional pharmaceuticals, these products are often made in uncertified laboratories where the synthesis and manufacturing quality is not properly controlled. Hyphenated MS techniques offer rapid and sensitive procedures for identifying a range of molecular constituents found in these products including NPS analogues, NPS isomers, and common anesthetics. Analysis of novel psychoactive substance products showed that more than 60% of products contained a mixture of novel psychoactive substances in addition to their label claim, and more than 25% contained an anesthetic or caffeine.

Suzanne Fergus is a senior lecturer in pharmaceutical chemistry in the School of Pharmacy at the University of Hertfordshire in Hertfordshire, UK.

Jacqueline L. Stair is a senior lecturer in analytical science in the School of Pharmacy at the University of Hertfordshire in Hertfordshire, UK.

Sulaf Assi is a postdoctoral research fellow in the School of Pharmacy at the University of Hertfordshire in Hertfordshire, UK. Please direct correspondence to:


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