Detection of these drugs, synthesized to mimic existing illegal substances, demonstrates an achievement in technology^[7]. Among these new psychoactive substances, “ synthetic cathinones” burst into the market at an explosive rate which shows no signs of slowing. Synthetic cathinones are β -keto phenethylamines, structural analogs of cathinone, a psychoactive stimulant found naturally in the khat plant (Catha edulis)^{[8, 9, 10, 11, 12, 13, 14, 15]}.

Additional information about the pharmacological action of several pyrrolidinophenones (pyrrolidinovalero-phenone, pyrovalerone, 3, 4-methylenedioxypyrovalerone, 3, 4-methy-lenedioxypyrrolidinopropiophenone, and 3, 4-methylenedioxypyrrolidino-butyrophenone) was recently published^{[16, 17]}, showing that all pyrrolidi-nophenones were potent norepinephrine and dopamine inhibitors.

A study of the street drug mephedrone seized in South Wales over two years showed a decrease in sample purity after it was banned in the UK, with the most common cutting agents being sodium monoglutamate, sucrose and creatine^{[18, 19]}. Studies on the composition of “ legal highs” marketed in Portugal revealed that products were usually mixtures of synthetic cathinones and several other ingredients such as glucose, lactose and caffeine ^{[20, 21]}. Justin et al^[22] used the complementary techniques of IR and NMR spectroscopy to initially identify the contents of “ Raving Dragon Novelty Bath Salts” , the so-called first generation of this type of drug, and the second generation, “ Raving Dragon Voodoo Dust” . The NMR-generated structures agreed with the IR data and allowed the confirmation of the first-generation product, the Raving Dragon Novelty Bath Salts as methylone and of the second-generation product, Raving Dragon Voodoo Dust, as pentedrone. Khaled et al^[23] demonstrated, for the first time, the combination of HPLC-UV with amperometric detection (HPLC-AD) for the qualitative and quantitative analysis of 4-methylmethcathinone (4-MMC, Mephedrone) and 4-methylethcathinone (4-MEC) (fully characterised by ¹H-NMR, ¹³C-NMR, given the purity> 99.5% in both cases) using either a commercially available impinging jet (LC-FC-A) or custom-made iCell channel (LC-FC-B) flow-cell system incorporating embedded graphite screen-printed macroelectrodes (Fig.3). The protocol offered a cost-effective, reproducible and reliable sensor platform for the simultaneous HPLC-UV and amperometric detection of the target analytes. The two systems had similar limits of detection, in terms of amperometric detection (LC-FC-A: 14.66 μ g/mL and 9.35 μ g/mL, LC-FC-B: 57.92 μ g/mL and 26.91 μ g/mL), to the previously reported oxidative electrochemical protocol (39.8 μ g/mL and 84.2 μ g/mL), for the two synthetic cathinones, prevalent on the recreational drugs market.

Fig.3

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	Fig.3 FC-A impinging jet flow cell (a: closed; b: open) and FC-B iCell channel flow cell (c: closed; d:open), with (e) the flow diagram of HPLC-AD systems (LC-FC-A and LC-FC-B)[23]

In 2015, the Australian Border Force (ABF) seized a kind of viscous, light-red liquid^[24]. By a combination of NMR, GC-MS and infrared spectroscopy, the unknown substance was identified as N-tert.-butoxycarbonyl-MDMA (t-BOC-MDMA). Through exposure of the t-BOC-MDMA to a simulated gastric juice (pH 1.5) to validate the existence of a pro-drug and formation of MDMA, the -O-CH₂-O- resonance corresponding to t-BOC-MDMA was shown by NMR to decrease in size while the -O-CH₂-O- resonance from methylenedioxyregion of the proton NMR spectrum of the MDMA product increased during the same period. At t=305 min under pH 1.5 and T=37℃, the majority of the t-BOC-MDMA had converted to MDMA. Given this biochemical state does not naturally occur in a human stomach during physiological digestion, it seems conceivable that ingestion of the t-BOC-MDMA might result in a slow release of MDMA formation in vivo. It is believed that this is the first time in the world that MDMA, concealed as the tertiary-butoxycarbonyl derivative, has ever been detected by law enforcement agencies. Folker et al^[25] reported a seizure of the same compound in 2016 by police in North-Rhine Westphalia, Germany.

Jacek et al^[26] identified three novel hydrochloride salts of cathinones: 2-(pyrrolidin-1-yl)-1-(5, 6, 7, 8-tetrahydro-naphthalen-2-yl)pentan-1-one (TH-PVP), 2-(methyl-amino)-1-(2-methylphenyl)-1-pro-panone (2-MMC) and 1-(4-chlorophenyl)-2-(methylamino)propan-1-one (4-CMC). Their properties were examined through combinations of GC-MS, IR, NMR, electronic absorption spectroscopy and single crystal X-ray diffraction. In addition, characteristic reactions for the identification of these compounds were devised to focus on the carbonyl group, the most distinct part of cathinones in comparison with amphetamine-based compounds. Tetraphosphorus-decasulfide (P₄S₁₀), Davy, Heimgartner or Lawesson’ s reagents (LR) have been reported to convert carbonyls into thiocarbonyl compounds. However, this type of transformation has only been found to have limited applications for the synthesis of simple thioketones because they were relatively unstable at room temperature. So, P₄S₁₀ and LR were selected by the authors for their thionation reactions due to the low cost of the starting materials and simplicity of the synthesis. Complex mixtures of products were obtained, resulting from the reaction of both the carbonyl and amine groups with the thionation reagents. Their reactivity was evidenced by the complicated ³¹P NMR spectra with four intense signals at 93.9, 90.9, 83.5 and 81.4 ppm, which were characteristic for compounds owning N-P(=S)S₂fragments^[27]. Cemal et al^[28] reported similar type of reactivity leading to N-phosphorylated products. Shima et al^[29] detected 1-phenyl-2-(pyrrolidin-1-yl)octan-1-one (PV9) and 16 of its metabolites, including diastereomers and conjugates in PV9 users’ urine using newly synthesized authentic standards and/or based on their mass spectra and ¹H-NMR. Interestingly, it was suggested that the main metabolic pathways of α -pyrrolidinophenones depend on alkyl chain length. The authors concluded that their fi ndings will contribute to the establishment of a reliable analytical procedure for proving the intake of newly encountered designer drugs, as well as to predict their metabolic pathways. Similar research has been done with α -pyrrolidino-butiophenone (α -PBP)^{[30, 31, 32]}. Koutaro et al^{[33, 34]} exhibited a fatal case of PV9 poisoning where a PV9 metabolite (via reduction of keto group) was identifi ed based on LC-MS/MS data. Kayoko et al^[35] also narrated detections of 1-Phenyl-2-(pyrrolidin-1-yl)octan-1-ol and an oxidized metabolite of PV9 in human blood.

Many other newly distributed designer drugs have been reported^{[36, 37, 38, 39, 40, 41, 42, 43]}.

The composition of synthetic cannabinoids is ever-changing^[44]. New generations of these so-called “ Spice” products are constantly being released into the illicit drugs market, continuing to cause harm. As a result, it has become urgent for forensic laboratories to be able to promptly detect and identify synthetic cannabinoids in their original powder form and other consumable products, with minimal steps of sample preparation and cleanup. Most newly emerging synthetic cannabinoids exhibit an aromatic heterocyclic skeleton, for example indole, indazole, imidazole, or pyrazole rings, which can be substituted by diverse types of alkyl, aryl, acyl, ester, carbonyl, or carboxamide groups and lead to a large variety of new derivatives continuing to emerge on the synthetic drug market^{[45, 46, 47, 48, 49, 50]}.

In China, 116 narcotic drugs and psychotropic substances for non-medical use were placed under control on 1st October 2015, including 40 synthetic cannabinoids in addition to the 12 substances first regulated in 2013. Among the samples seized from a clandestine laboratory uncovered in Hubei province, seven synthetic cannabinoids and five substituted phenethylamine derivatives have been identified^{[51, 52, 53]}. NMR has been extensively used to derive the structures of purified synthetic cannabinoids. With NMR confirmation, the position of the substitution group on an aromatic ring can be determined (when compared to standard mass spectrum) as each proton in a unique chemical environment produces a unique signal that remains the same even in a herbal mixture (Fig.4). Slightly modified chemicals like JWH-073 flooded into the newly-born market for this kind of drug with a myriad of similar plant-based second-generation ‘ ‘ Spice-like’ ’ products misleadingly sold as incenses, meditation potpourris, bath additives and related others. Frequently, a compound had hardly been banned by the authorities when new structures/products were introduced even faster. NMR spectroscopy has long been used in forensic laboratories for elucidating the structures of novel compounds and is now much prized for the quantification of drugs and adulterants in potentially illegal products of abuse^{[54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87]}.

Fig.4

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	Fig.4 NMR strategies capable of disentangling overlapped 1D NMR peaks to yield accurate individual structures

Jankovics et al^[88] identified and characterized two new potential cannabimimetic molecules by several analytical techniques. The substances were seized in the form of pure powders by the customs office in Hungary. LC-MS/MS and UV spectra of JWH-018 and JWH-122 were used for their structure elucidation, which was confirmed by ¹H-NMR and ¹³C-NMR. Taking into account the circumstances of their seizure, both powder samples might have been intended to be used as designer drugs in their pure form or as ingredients of ‘ ‘ smart products’ ’ . Langer et al^[89] identified eight different synthetic cannabinoids by GC-MS: THJ-018, THJ-2201, MAB-CHMINACA, 5F-ADB, 5Cl-AKB48 (syn.:5C-AKB48), 4-pentenyl-AKB48, MDMB-CHMICA and 5F-AB-PINACA using JWH-018 as the GC-MS internal standard and the corresponding response factors to calculate the total amount of all synthetic cannabinoids in the commercial smoking mixtures. NMR and other techniques were used to fully characterize the compounds. The content of synthetic cannabinoids in the investigated products ranged from 23 to 120 mg/g (average: 57 mg/g), while the individual compounds ranging from 1 to 120 mg/g. Langer also carried out a full iterative band-shape analysis of the indazole and naphthalene proton signals. The metabolism of THJ-018 and THJ-2201 in human hepatocytes has already been examined^[90] and compared with the benzimidazole analogue of AM-2201 (=FUBIMINA)^{[91, 92]}. THJ-2201, an indazole analogue of AM-2201, was isolated from herbal mixture and was fully described in the literature. In an analysis of 104 chemical- and herbal- type products, THJ-2201 was one of eight newly distributed synthetic cannabinoids^[91]. MAB-CHMINACA, detected in herbal blend, is also known as ADB-CHMINACA whose mass spectra were first described by Wurita et al^[82] after it had been encountered in herbal mixtures. NMR data of 5F-ADB was obtained from a CD₃OD solution^[50]. The ¹³C chemical shifts in CD₃OD were generally higher than the literature values in DMSO-d₆ by 1.5 ppm with a standard deviation of 0.5 ppm. Synthetic cannabinoids with a 5-chloropentyl chain moiety have been isolated from herbal blends before, albeit generally only in minor amounts compared to the 5-fluorinated main product^{[89, 93]} among which the synthetic cannabinoid was found as the major ingredient at a concentration of 76 mg/g accompanied only by a hitherto unknown trace. The 5-fluoro analogue 5F-AKB48 showed a high affinity to the CB1 receptor with a Ki of 0.87 nM^[94]. MDMB-CHMICA, characterized and quantified in one herbal mixture, was reported in 2015 and cases of intoxication and death attributed to its consumption have been published^{[95, 96]}. 5F-AB-PINACA was fully characterized in 2015 when its metabolic profile in human hepatocytes^[97] and pharmacological characterization in rats^[98] were reported.

A sample of “ 4-methylpentedrone(4-MPD)” was obtained, along with information including incorrect structures and nonexistent CAS numbers, and analyzed by GC-MS, NMR, and liquid chromatography-electrospray ionization (LC-EIS)^[99]. The molecular mass obtained from GC-MS indicated that this vendor sample, labeled 4-MPD, might contain an additional -CH₂. Hence, the ¹H, ¹³C and, ¹³C DEPT NMR spectra for 2-(ethylamino)-1-(4-methylphenyl)pentan-1-one (4-MEAP), the above vendor sample and 1-(4-methylphenyl)-2-(propyl-amino)butan-1-one (4-MPB) were stacked to make a comparison of the NMR data along with the unique fragmentation patterns produced from these molecules. The spectra for the vendor sample and 4-MEAP were nearly identical, with the ppm shift values differing by < 0.2 ppm overall (Fig.5). Thus, the sample was finally identified as 2-(ethylamino)-1-(4-methylphenyl)-1-pentanone (4-MEAP), not 4-MPD or 4-MPB.

Fig.5

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	Fig.5 The 1H (left), 13C (center) and DEPT 13C NMR spectra (right) for the 4-MEAP, the vendor sample and 4-MPB[99]

Karen et al^[100] first reported an aryl sulphonamide cannabimimetic in seized plant materials in Western Australia. The identification of 8-quinolinyl-4-methyl-3-(1-piperidinylsulfonyl)benzoate (QMPSB) in methanol extracts of seized plant materials was shown to be hampered by almost complete transesterification to its methyl ester methyl 4-methyl-3-(1-piperidinylsulfonyl) benzoate (MMPSB), even at ambient temperatures. The presence of hydroxyquinoline in a seized plant material extract is an indicator that transesterification of a quinolinyl ester may have occurred. Edmunds et al^[101] described the isolation and characterization of a new synthetic cannabinoid. The mass spectrum highlighted structural similarities to JWH-081, though the spectrum showed no match in available libraries. Finally, the use of NMR spectroscopy plus high resolution LC/MS allowed for structural elucidation and identification of the unknown as (1-(cyclohexylmethyl)-1H-indol-3-yl) (4-methoxynap-thalen-1-yl)methanone according to the key signals in the ¹H NMR spectrum (Fig.6) to include a downfield doublet at 3.91 ppm due to the N-bound methylene protons.

Fig.6

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	Fig.6 1H NMR spectrum for (1-(cyclohexylmethyl)- 1H-indol-3-yl)(4-methoxynaphthalen-1-yl)- methanone[101]

Analytical challenges still remain in the detection of these synthetic cannabinoids due to the difficulty in obtaining all possible standards and in deconvoluting the interference signals from the background matrix. Dunne and co-workers^[102] developed an extraction procedure compatible with direct NMR quantification of synthetic cannabinoids in herbal smoking blends. Extraction media, time and efficiency were tested for different carrier materials containing representative synthetic cannabinoids. The developed protocol utilizes a 30 min extraction in d₄-methanol in the presence of an internal standard, allowing direct quantitation of the extract by NMR. The accuracy of the developed method was tested with in-house prepared herbal smoking blends. The results showed deviations less than 0.2 % from the actual content, proving that the method is sufficiently accurate for the relevant quantifications. Obtained results showed a variation in cannabinoid contents from 1.5 % (w/w) for mixtures containing MDMB-CHMICA to over 5 % (w/w) for mixtures containing 5F-AKB-48. This is an extremely effective advantage, since reference materials of new synthetic cannabinoids are often difficult to obtain when the substance is first introduced into the illicit drug market. Marino et al^[103] designed their NMR sample preparation as a simplified protocol to dramatically reduce both the time and sample size that were needed to positively identify cannabinoids in herbal products. The combination of rapid direct analysis in real time-mass spectrometry (DART-MS) and NMR can provide concrete cannabinoid structural information with no ambiguity, permitting this strategy to be a useful alternative, or complement, to the conventional GC-MS and LC-MS choices. According to their study, the four-minute 32-NMR scans generated a S/N of 4 to 1 for as little as 50 μ g (slightly above LOD) of a cannabinoid sample with successful identification. Fowler et al^[104] directly extracted synthetic cannabinoids with NMR solvent without any conventional lengthy isolation, purifi cation or chromatographic separations. ¹H NMR^[105] and proton correlation spectroscopy (COSY, HMQC and HMBC)^[106] were successfully employed to generate molecular fi ngerprints in herbal extracts, taking advantage of the spectroscopic separation power of NMR spectroscopy. The identification and quantification steps can be completed within an hour from sample-in to answers-out (Fig.7).

Fig.7

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	Fig.7 Analytical workflow illustrating the quantitative method capable of determining absolute metabolite concentrations[106]

New synthetic opioids and the sedative present a serious problem for public health due to their potency and risk of fatal intoxication^{[107, 108, 109, 110]}. “ Krokodil” , a homemade injectable mixture being used as a cheap substitute for heroin, is a light brown liquid that may cause the skin to become iridescent and discolored, and further lead to desquamation if injected without previous purification. Repeated or regular use may turn the skin around the injection site scaly and rough, like crocodile skin. Neves et al^[111] devised a thin-layer chromatography (TLC) profile, UV/Vis, ¹H-NMR and FTIR spectrum methods to screen and quantify desomorphine and codeine in ‘ ‘ krokodil’ ’ samples to show further information about the contaminants present in “ krokodil” . The synthesis was carried out as described in a previous paper^[112], where crude “ krokodil” appeared as a yellow to light brown solution due to the presence of iodine^[113]. Treatment of crude “ krokodil” with a strong base assured the presence of morphinans as free bases, which were subsequently extracted with ethyl acetate, an organic solvent of intermediate polarity. The qualitative analysis of the samples also showed the presence of other two morphinans (i.e. dihydromorphine-3, 6-dideoxy and morphinan- 4, 5-epoxy-3-ol) due to the highly reductive environment. The authors contacted users to understand the synthesis process and to know all the social and legal issues that lead a person to abuse “ krokodil” . A new synthetic analogue of fentanyl, N-phenyl-N-[1-(2-phenethyl)piperidin-4-yl]prop-2-enamide (acrylfentanyl), was identified in powder from a seized capsule found at a forensic psychiatric ward in Denmark^[114]. The proportion of deaths due to illicit drug overdose involving fentanyls has grown to exceed 50% in some parts of Canada^[115] during the recent few years. Since 2012, 12 deaths in Russia^[116] and more than 50 deaths in the US have been associated with acetylfentanyl^{[117, 118, 119]}. Other fentanyl analogues, such as butyrfentanyl, 3-methylfentanyl and furanylfentanyl, have also been linked to serious adverse events^{[120, 121, 122]}.