Analysis of fragmentation pathways of fentanyl derivatives by electrospray ionisation high-resolution mass spectrometry.

RATIONALE: During the last decade there has been a large increase in the availability of new synthetic opioids on the European drug market. Fentanyl analogues accounted for a significant proportion of these compounds. When there is a rapid introduction of new compounds from a given chemical class onto the market, a computer-assisted library search is not recommended and the spectra have to be interpreted individually. Therefore, the knowledge of how different groups of new psychoactive substances are fragmented can be very helpful in identifying new compounds. METHODS: In this study, the fragmentation patterns of 33 fentanyl derivatives were investigated using electrospray ionisation (ESI). The analyses were conducted using liquid chromatography quadrupole time-of-flight mass spectrometry (LC/QTOFMS). Based on measurements carried out under various conditions, the fragmentation pathways of the tested compounds that were divided into groups due to their chemical structure were established. RESULTS: The performed study allowed for the determination of characteristic ions that were formed during the fragmentation of fentanyl derivatives using ESI. Due to the high mass accuracy of the LC/ESI-QTOFMS technique, it was proved that the cleavage of the tested molecules occurred mostly on the bonds adjacent to the nitrogen atoms. Based on the proposed fragmentation scheme, the general structure for fentanyls, and the presence of some characteristic ions, it is possible, after applying simple mathematical operations, to calculate the masses of individual substituents in the formulas of the new fentanyl analogues, which may appear on the drug market. Furthermore, based on the exact masses, it is possible to determine the formulas of these substituents. CONCLUSIONS: Knowledge of the specific fragments generated under ESI conditions can be used in forensic laboratories to determine the structures of novel compounds from the group of fentanyl derivatives.

The Presence of Stimulant Drugs in Wastewater from Krakow (Poland): A Snapshot.

An analysis of wastewater from Krakow (Poland) for the presence of controlled and uncontrolled stimulant drugs of abuse was performed. Samples were collected from the Plaszow wastewater treatment plant, Krakow, Poland, and prepared by solid phase extraction. The LC-QTOFMS method was applied for identification and quantification of popular stimulants: MDMA, mephedrone, 4-MEC, MDPV and mCPP. Environmental loads of illicit drugs were calculated; the WWTP discharged loads ranging from 3.6 to 6.7 mg day(-1) 1000 inhabitants(-1) of MDMA, 3.6 to 7.1 mg day(-1) 1000 inhabitants(-1) of mephedrone and 4.8 to 5.8 mg day(-1) 1000 inhabitants(-1) of 4-MEC. The results confirmed the growing popularity of new psychoactive substances in Poland.

Structural elucidation and identification of a new derivative of phenethylamine using quadrupole time-of-flight mass spectrometry.

RATIONALE: In recent years, the phenomenon of uncontrolled distribution of new psychoactive substances that were marketed without prior toxicological studies has been observed. Because many designer drugs are related in chemical structure, the potential for misidentifying them is an important problem. It is therefore essential to develop an analytical procedure for unequivocal elucidation of the structures of these compounds. The issue has been discussed in the context of 25I-NBMD [2-(4-iodo-2,5-dimethoxyphenyl)-N-[(2,3-methylenedioxyphenyl)methyl]ethanamine], a psychoactive substance first discovered on the drug market in 2012. METHODS: The substance was extracted from blotter papers with methanol. Separation was achieved via liquid chromatography. Analysis was conducted by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QTOFMS). Identification of the psychoactive component was supported by electron impact gas chromatography/mass spectrometry (GC/EI-MS). RESULTS: The high accuracy of the LC/ESI-QTOFMS method allowed the molecular mass of the investigated substance (M(exp) = 441.0438 Da; mass error, ∆m = 0.2 ppm) and the formulae of ions formed during fragmentation to be determined. The main ions were recorded at m/z = 135.0440, 290.9876 and 305.9981. Structures of the obtained ions were elucidated in the tandem mass spectrometry (MS/MS) experiments by comparing them to mass spectra of previously detected derivatives of phenethylamine. CONCLUSIONS: The performed study indicated the potential for using LC/QTOFMS method to identify new designer drugs. This technique can be used supplementary to standard GC/MS. Prior knowledge of the fragmentation mechanisms of phenethylamines allowed to predict the mass spectra of the novel substance--25I-NBMD.

Comparison of "herbal highs" composition.

Popularity of new psychoactive substances, known as legal highs or herbal highs, is continuously growing. These products are typically sold via internet and in so-called head shops. The aim of this study was to identify active ingredients of herbal highs and to compare their chemical composition. Twenty-nine various products seized by the police in one of the "head shops" were analysed. Herbal mixtures (0.2 g) were prepared by ultrasonic-assisted extraction with 2.0 ml of ethanol for 2 h. The extracts were analysed by gas chromatography coupled to mass spectrometry (GC/MS). The main active compounds of the herbal mixtures were synthetic cannabinoids: JWH-018, JWH-073 and cannabicyclohexanol (CP-47,497-C8-homolog). Their content differed between the products; some contained only one cannabinoid whereas the others contained two or more. Cluster analysis and principal component analysis revealed that chemical composition of many products was very similar. The similarity was connected with their flavour and not the common name. This statement was true for the synthetic cannabinoids, other potential agonists of cannabinoid receptors (amides of fatty acids) and ingredients of natural origin and confirms that herbal highs are a threat to human health because the purchaser has no information on their real composition.

Profiling of 3,4-methylenedioxymethamphetamine by means of high-performance liquid chromatography.

An impurity-profiling method for 3,4-methylenedioxymethamphetamine (MDMA) is presented. The impurities of interest were extracted by solid-phase extraction (SPE) on Bakerbond C18 spe columns from a weakly alkaline solution (pH 8.5). Development of the extraction conditions covered selection of the buffer for dissolution of the sample and the volume of the eluent used to elute the impurities. An important part of the studies was to optimise the separation conditions, and the simplex method was used for this purpose. Cluster analysis was applied for comparison of samples and its grouping. The developed method was based on the areas of 33 selected peaks corresponding to MDMA impurities. All examined samples were correctly classified into clusters corresponding to the synthetic route.

Analysis of Fragmentation Pathways of New-Type Synthetic Cannabinoids Using Electrospray Ionization.

Recently, dozens of new psychoactive substances have appeared on the European drug market every year. The most abundant group of these compounds is synthetic cannabinoids. In the first few years of the "legal highs" phenomenon, JWH (John W. Huffman) compounds were especially popular among drug users. However, the group of synthetic cannabinoids is constantly expanding, as new compounds are created by replacing known structural elements with different chemical groups. The problem with the identification of novel substances in forensic laboratories results from the structural similarity of the compounds and the rapid introduction of newer designer drugs on the black market. In this study, the fragmentation patterns of 29 new-type synthetic cannabinoids using electrospray ionization were investigated. The analysis was performed using quadrupole time-of-flight mass spectrometry. Based on measurements carried out under various conditions, the way of fragmentation of the tested compounds that were divided into groups due to their chemical structure was established. The study showed that the bond between the carbon atom of the carbonyl group and the ring or NH group attached to the ring was mainly cleaved. This mechanism was adequate for the fragmentation of first-generation synthetic cannabinoids. This paper presents characteristic ions formed by synthetic cannabinoids (i.e., ions originating from an indole/indazole ring and an adamanyl/naphthalene/quinoline ring) using electrospray ionization. Knowledge of these specific fragments can be used in forensic laboratories to determine the structure of novel compounds from the group of synthetic cannabinoids. Graphical Abstract ᅟ.

Alpha-PVP as an active component of herbal highs in Poland between 2013 and 2015.

Alpha-PVP (alpha-pyrrolidinovalerophenone, α-PVP) is a synthetic derivative of cathinone. It has been one of the most frequently detected new psychoactive substances (NPS) available on the drug market in recent years in Poland. The usual routes of administration of the drug include oral, insufflation, and injection. Unexpectedly, we dealt with a great number of herbal samples that turned out to contain α-PVP. A total number of 352 herbal samples from 19 cases in which we detected synthetic cathinones, were investigated in the Institute of Forensic Research (IFR) from 2013 to 2015. The seized products that were received by our laboratory were first screened by gas chromatography coupled to mass spectrometry (GC-MS). Quantification of α-PVP and other cathinones was performed by ultra-performance liquid chromatography with photodiode array detection (UPLC-PDA). Of the samples, 84% contained only α-PVP. Other groups of products were those containing only α-PVT, α-PVP and α-PVT, α-PVP and synthetic cannabinoid A-834, 735, and α-PVP and cannabis. In one herbal sample, α-PVP was detected along with caffeine and tadalafil. The herbal products present on the market containing only α-PVP usually had a mass of 0.3 to 0.6 g, and concentration range in this group of samples was 3.0-44.0% (content: 13.0-222.0 mg per package). The amount of α-PVP in samples below 0.30 g was in a range 9-18 mg whiles in samples above 0.60 g it was in the range 30-716 mg. There were also products containing a mixture of α-PVP and α-PVT. In those samples, α-PVP concentrations were: 3.0-6.0% (amount: 15.0-34.0 mg). Copyright © 2016 John Wiley & Sons, Ltd.

Fatal intoxication with methoxetamine.

Methoxetamine (MXE) is a new synthetic drug of abuse structurally related to ketamine and phencyclidine. A case of a 29-year-old male with acute toxicity related to the analytically confirmed use of MXE is reported. The man was found dead at his residence. Biological material was analyzed using liquid chromatography-tandem mass spectrometry. The concentration of MXE in urine of the deceased was 85 µg/mL. Despite the vial containing the blood sample being destroyed during transportation and the blood leaking out into the cardboard packaging, the blood level of MXE was estimated. After determination of the cardboard grammage (approx. 400 g/m(3) ) and the mean mass of the blood obtained after drying (0.1785 ± 0.0173 g per 1 mL), the estimated blood concentration of MXE was found to be 5.8 µg/mL. The high concentration of MXE in blood and urine and the circumstances of the case indicate an unintentional, fatal intoxication with this substance.

Optimization of solid-phase microextraction conditions for gas chromatographic determination of ethanol and other volatile compounds in blood.

A procedure for the determination of acetaldehyde, acetone, methanol, ethanol, 1-propanol and 2-propanol in blood was developed. Separation of analytes was carried out on DB-wax capillary column (l = 30 m, I.D. = 0.32 mm, dF = 0.5 microm) at 40 degrees C, hydrogen was used as a carrier gas (at 30 kPa) and FID as a detector. Quantification was performed with the use of 2-butanol as an internal standard. Headspace solid-phase microextraction was applied as the sample preparation technique. The usefulness of most commercially available fiber coatings was checked and 65 microm Carbowax/DVB proved most effective. Microextraction was carried out from the headspace at 60 degrees C for 10 min. The sample was stirred at 750 rpm. In order to improve the extraction efficiency of analytes, salting-out agents were also applied. Potassium carbonate turned out to be the most efficient. A 1.0-g amount of this salt and 0.1 ml of I.S. were added to 0.5 ml of sample. Validation of the worked-out method was performed. For each analyte, the limits of detection and quantification, linearity, working range, accuracy and precision were determined or tested.

Concentration of ethanol and other volatile compounds in the blood of acutely poisoned alcoholics.

The presence of volatile compounds, such as acetone, acetaldehyde, methanol, ethanol, isopropanol, and n-propanol, in the blood of 169 acutely poisoned alcoholics was determined. The clinical diagnosis of addiction was made on the basis of a patient interview as well as physical, psychological, and psychiatric examination. At the time of the patients' admission to the clinic, the mean concentration of ethanol in blood was 3.14 +/- 1.10 g/l and its elimination rate in the studied group was 0.27 +/- 0.08 g/kg/hr, an elimination rate significantly higher (P <.001) than that of social drinkers, which averages to 0.014 +/- 0.04 g/kg/h. The presence of other volatile compounds in the blood of alcohol-addicted patients is common. The calculated elimination rate constant of methanol was about 0.2 h(-1). This rate seems to indicate that, in heavy drinkers, the elimination of methanol may be relatively fast even if the ethanol concentration is above 1 g/l. The elimination of other volatile compounds can be accelerated by large doses of ethanol, although it is not correlated with actual blood ethanol level. Moreover, in most of the blood samples with a methanol concentration below 10 mg/l, the measured concentration of acetone was below 7 mg/l and that of isopropanol was below 2 mg/l.

Fatal intoxication with 3-methyl-N-methylcathinone (3-MMC) and 5-(2-aminopropyl)benzofuran (5-APB).

The emergence of a large number of new psychoactive substances (NPSs) in recent years poses a serious problem to clinical and forensic toxicologists. Here we report a patient who administrated ca. 500mg of 3-MMC (3-methyl-N-methylcathinone) and 400mg of 5-APB (5-(2-aminopropyl)benzofuran) in combination with 80g of ethyl alcohol. The clinical manifestations included agitation, seizures, hypertension, tachycardia, hyperthermia and bradycardia. The patient did not recover and died around 4h after the use of drugs. The cause of death was acute cardiovascular collapse that occurred following mixed intoxication with NPSs and alcohol. Toxicological analysis of post-mortem blood revealed 3-MMC and 5-APB in concentrations of 1.6µg/mL and 5.6µg/mL, respectively. Moreover, the serum alcohol concentration was 1.4g/L in ante-mortem sample collected 1h after admission to the hospital. This is the first report on blood concentration of 3-MMC and 5-APB in fatal intoxication.

Identification and characterization of 2,5-dimethoxy-3,4-dimethyl-ß-phenethylamine (2C-G)--a new designer drug.

This study presents and discusses the mass spectrometric, infrared spectroscopic and nuclear magnetic resonance spectroscopic data of 2,5-dimethoxy-3,4-dimethyl-ß-phenethylamine (2C-G), a new designer drug. A powder sample containing 2C-G was seized in Poland in 2011. The paper focuses on a comparison of the analytical features of 2C-G and other members of the 2C-series, in order to assess the possibility of unequivocal identification. The occurrence of intense peak at m/z = 178 and different intensities of the ions at m/z = 165 and 180 in the gas chromatography-electron impact-mass spectrometry (GC-EI/MS) spectrum of 2C-G made it possible to distinguish it from 2C-E. Differences in relative intensities of the ions at m/z = 192, 179 and 177 were observed for GC-EI/MS spectra of TFAA derivatives of 2C-G and 2C-E. An identical set of ions was recorded for these substances using the liquid chromatography-electrospray ionization/quadrupole time of flight mass spectrometry (LC-ESI/QTOFMS) method in both MS and tandem mass spectrometry (MS/MS) mode, but the distinction was possible based on differences in the ion intensities at m/z = 193.1223 and 178.0988. The Fourier transform infrared (FTIR) spectrum of 2C-G was significantly different from other members of the 2C-series, with a characteristic doublets at 993-1014 cm(-1) and 1099-1124 cm(-1) , and the ratio of bands at higher wavenumbers. Final elucidation of the structure of 2C-G was carried out by (1) H and (13) C NMR spectroscopy. The study indicated that the marketing of analogues of controlled substances poses a real analytical challenge for forensic laboratories, and the application of sophisticated methods is often required for unequivocal identification of a new substance.

Analytical characterization of three hallucinogenic N-(2-methoxy)benzyl derivatives of the 2C-series of phenethylamine drugs.

This publication reports analytical properties of three new hallucinogenic substances identified in blotter papers seized from the drug market, namely 25D-NBOMe [2-(2,5-dimethoxy-4-methylphenyl)-N-(2-methoxybenzyl)ethanamine], 25E-NBOMe [2-(4-ethyl-2,5-dimethoxyphenyl)-N-(2-methoxybenzyl)ethanamine] and 25G-NBOMe [2-(2,5-dimethoxy-3,4-dimethylphenyl)-N-(2-methoxybenzyl)ethanamine]. These substances are N-(2-methoxy)benzyl derivatives of the 2C-series of phenethylamine drugs. The applied procedure covered a variety of analytical methods, including gas chromatography with electron impact mass spectrometry (GC-EI-MS; without derivatization and after derivatization with trifluoroacetic anhydride (TFAA)), liquid chromatography-electrospray ionization-quadrupole time of flight mass spectrometry (LC-ESI-QTOF-MS), Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR), which made it possible to identify the active components unequivocally. The GC-MS spectra of analyzed compounds were very similar, with dominant ions observed at m/z = 150, 121, and 91. The remaining ions were analogous to those observed for parent substances, namely 2C-D, 2C-E, 2C-G, but their intensities were low. Derivatization allowed determination of molecular masses of the investigated substances. Their exact masses and chemical formulas were confirmed by LC-QTOF-MS experiments and the fragmentation patterns of these compounds following ESI were determined. The tandem mass spectrometry (MS/MS) experiments confirmed that the studied substances were N-(2-methoxy)benzyl derivatives of the 2C-series compounds. Final elucidation of the structures was performed by NMR spectroscopy. The substances were also characterized by FTIR spectroscopy to corroborate the identity of the compounds.

Prevalence and co-existence of active components of 'legal highs'.

The results of a study performed on samples of 'legal highs' seized in head shops by law enforcement and health services in Poland between mid-2008 and mid-2011 are presented. In total, 449 preparations which differed in labelling, net masses, forms of distribution, etc., were analyzed. A variety of sophisticated analytical methods, including gas chromatography-mass spectrometry (GC-MS), liquid chromatography-quadropole time-of-flight mass spectrometry (LC-QTOF-MS), high performance liquid chromatography (HPLC), and nuclear magnetic resonance (NMR) were applied for component identification and quantification. The most common ingredients of legal highs were (in descending order): MPDV, caffeine, butylone, TFMPP, lidocaine, 4-MEC, mephedrone, pFPP, BZP, and MDPBP. The scatter of substances changed over time, and piperazines were often ousted by cathinones. Most of the preparations were composed of two or more ingredients. Cathinones and piperazines were mixed mainly within the chemical classes (77.6% and 56.1% of dual links, respectively), caffeine was mixed both with piperazines (24 products) and cathinones (22 products), whereas lidocaine only with the latter class (47 products). A great inconsistency in the qualitative and quantitative composition of products with identical labelling was shown in an example of Coco products seized after August 2010; we found 10 different single component or mixture preparations, and the content of individual ingredients varied from several to hundreds of mgs. This paper summarizes potential dangers connected with the uncontrolled sale of psychoactive substances, and indicates important issues concerning the analysis of legal highs.

Analysis of UR-144 and its pyrolysis product in blood and their metabolites in urine.

UR-144 [(1-pentyl-1H-indol-3-yl)(2,2,3,3-tetramethylcyclopropyl)methanone] is a synthetic cannabinoid, which has been detected in many herbal blends, resinous samples and powders seized from the Polish drug market since the beginning of 2012. This paper presents the case of intoxication by this substance. A complete picture of the symptoms observed by a witness, paramedics and medical doctors are given. In the analysis of powder residues from the plastic bag seized from the intoxicated person by gas chromatography-mass spectrometry (GC-MS), UR-144 and its major pyrolysis product [1-(1-pentyl-1H-indol-3-yl)-3-methyl-2-(propan-2-yl)but-3-en-1-one] were detected. Both substances were also identified in a blood sample collected on admission of the patient to hospital using liquid chromatography-triple quadrupole tandem mass spectrometry (LC-QqQ-MS). Blood concentration of UR-144 was 6.1 ng/mL. A urine sample collected at the same time was analyzed by liquid chromatography-quadruple time-of-flight tandem mass spectrometry (LC-QTOF-MS). The parent substance and its pyrolysis products were not detected in urine, while their five metabolites were found. The experiments allowed the location of derivative groups to be established, and thus elucidate rough structures of the metabolites; a dihydroxylated metabolite of UR-144 and mono-, dihydroxylated and carboxylated metabolites of its pyrolysis product were identified.

Detection of buphedrone in biological and non-biological material--two case reports.

Buphedrone (2-(methylamino)-1-phenylbutan-1-one, α-methylamino-butyrophenone, MABP) is a positional isomer of mephedrone. In Poland, it was marketed in the second half of 2010 after the banning of mephedrone. Buphedrone is a stimulant that is snorted, smoked or taken orally. This substance was identified in 15 products seized by law enforcement after August 2010 and analysed in the Institute of Forensic Research (IFR). Buphedrone was the sole psychoactive substance in only 5 products. It was mixed mainly with 4-MEC and MDPV. This paper presents two cases in which both biological and non-biological materials were delivered to the IFR for toxicological analysis. In the first case, a passenger car crashed into a truck. The car driver suffered severe injuries resulting in his death. During external inspection of the deceased the police discovered several packages containing a white powder. In the second case, a man was arrested for possession of illicit drugs. Analysis of powders was carried out using gas chromatography-mass spectrometry (GC-MS) and high-pressure liquid chromatography with diode array detection (HPLC-DAD). The purity of buphedrone found in powder samples was in the range of 58-68%. Analyses of blood were carried out using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Buphedrone was found in the blood of the deceased at a concentration of 127 ng/mL and of the drug user/seller at 3 ng/mL.

25C-NBOMe--new potent hallucinogenic substance identified on the drug market.

This publication reports analytical properties of a new hallucinogenic substance identified in blotter papers seized from the drug market, namely 25C-NBOMe [2-(4-chloro-2,5-dimethoxyphenyl)-N-(2-methoxybenzyl)ethanamine]. The identification was based on results of comprehensive study including several analytical methods, i.e., GC-EI-MS (without derivatization and after derivatization with TFAA), LC-ESI-QTOF-MS, FTIR and NMR. The GC-MS spectrum of 25C-NBOMe was similar to those obtained for other representatives of the 25-NBOMe series, with dominant ions observed at m/z=150, 121 and 91. Fragment ions analogic to those in 2C-C (4-chloro-2,5-dimethoxy-ß-phenylethanamine) were also observed, but their intensities were low. Derivatization allowed the determination of molecular mass of the investigated substance. The exact molecular mass and chemical formula were confirmed by LC-QTOF-MS experiments and fragmentation pattern under electrospray ionization was determined. The MS/MS experiments confirmed that the investigated substance was N-(2-methoxy)benzyl derivative of 2C-C. The substance was also characterized by FTIR spectroscopy to corroborate its identity. Final elucidation of the structure was performed by NMR spectroscopy.

Alpha- and beta-asarone in herbal medicinal products. A case study.

The composition of pellets and tablets, which were sold as the preparations of Chinese natural medicine, was studied. The drugs were seized by the police due to the intoxication of a young woman. Analyses were performed by GC-MS (HP-5 ms column) and by HPLC (RP-18e Chromolith(®) monolithic column). Diverse content of asarone isomers was found in the tested material. The dose of alpha-asarone ranged from 0.49 to 42.5 µg per pellet, while its average concentration in the tablets was 51.4 µg. Beta-asarone was not detected in the pellets with low content of alpha isomer (below 1 µg), while its content in the remaining pellets ranged from 142 to 645 µg. The tablets contained higher doses of beta-asarone (1526 µg in average). The total content of asarones in most of the examined pellets and tablets exceeded 115 µg, which is the maximum acceptable daily intake. Taking into account the dosage written on packages that ranged from several to more than ten pellets/tablets a day, one can assume that the intake of such doses of asarone might be health-threatening.

Identification and characterization of 2,5-dimethoxy-4-nitro-ß-phenethylamine (2C-N)--a new member of 2C-series of designer drug.

The online sale of psychoactive substances, including hallucinogens, is becoming a serious problem in many countries. This paper presents and discusses the mass spectrometric, infrared spectroscopic and nuclear magnetic resonance spectroscopic data of 2,5-dimethoxy-4-nitro-ß-phenethylamine (2C-N), which was identified in a powder sample seized by the authorities in 2011 in Poland. The molecular mass of 2C-N (226.0954 amu) was confirmed in the LC/ESI-QTOFMS experiment. The molecular ion was also observed in the GC-EI/MS spectrum. A characteristic set of ions for the parent substance was found using both chromatographic methods, and when derivatization with trifuluoroacetic anhydride (TFAA) was applied. Two broad dominant bands at 1520 cm(-1) and 1342/1322 cm(-1), observed in the FTIR spectrum of 2C-N, originated from the nitro group. NMR spectroscopy helped unequivocal elucidation of the structure. The applied identification procedure proved to be a powerful tool to determine the structure of a new designer drug.

Identification of naphthoylindoles acting on cannabinoid receptors based on their fragmentation patterns under ESI-QTOFMS.

'Herbal highs' have been advertised as legal and natural substitutes to cannabis, but a detailed examination of these products has revealed that the herbal matrix is laced with synthetic substances that mimic the effects of marijuana. Producers select the ingredients based on the results of scientific studies on the affinities of different chemicals to cannabinoid receptors. Naphthoylindoles have turned out to be the most popular class of substances identified in the products. Legal actions taken in order to tackle the problem of uncontrolled access to one substance have usually resulted in the marketing of derivatives or analogues. In the study, the mass spectral behavior of twelve synthetic cannabinoids from the naphthoylindole family under electrospray ionization (ESI) was investigated. LC-QTOFMS experiments were performed in three modes (low fragmentor voltage, high fragmentor voltage with/without collision energy), and they enabled the identification of protonated molecules and main ions. A general fragmentation pattern under this ionization method was proposed, and mechanisms of ion formation were discussed. The developed procedure allowed the determination of substituent groups of the core naphthoylindole structure and distinction between positional isomers. The obtained results were used for the prediction of the ESI-MS spectra for many naphthoylindoles with a high affinity to cannabinoid receptors. Similarities and differences between ESI-MS and electron impact-MS spectra of naphthoylindoles were discussed. The developed identification process was presented on an example of an analysis of an unknown herbal material, in which JWH-007 was finally identified. Knowledge of the fragmentation mechanisms of naphthoylindoles could also be used by other researchers for identification of unknown substances in this chemical family.