New Psychoactive Substances (Npss) Abuse in Romania: Analytical Strategies for Drug Screening in Biological Samples Using High Resolution Mass Spectrometry

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New psychoactive substances (NPSs) abuse in Romania: analytical strategies for drug screening in biological samples using high resolution mass spectrometry

Carmen Lidia Chiţescu1,2,*, Ana Doina Radu3, Florina Aciu3, Monica Moraru1,2, Iuliu Fulga1,2

______Abstract: New psychoactive substances (NPSs) are rapidly spreading among Romanian youth, and toxicology laboratories are requested to identify such compounds in biological samples as blood, urine, or gastric content. In this work, LC-HRMS/MS technique represented by Q Exactive -Orbitrap was applied in addition to GC-MS, for the identification of the NPSs. Different extraction methods such as liquid-liquid extraction, solid phase extraction were applied to samples from deceased or living persons. The results were compared and discussed. Although the reference standards were not always available, the identification of the NPSs was successfully achieved, via LC- HRMS in both full scan and targeted ion fragmentation (t-MS2) modes using a predictive approach. Key Words: forensic sciences, toxicology, new psychoactive substances, screening, HRMS, predictive approach.

INTRODUCTION The governmental measures wasn’t followed by a significant decline of consumption, as, according to fifth Due to its geographical location, Romania is ESPAD Survey 2015, about 5% of the Romanian adult part of the Balkan route of drugs traffic. If in the past population had experimented with NPSs (compared Romania was mainly a transit area, drug consumption to 2% at the fourth ESPAD Survey conducted in 2011) has more than doubled in the last years, according to the [1]. According to the last ANA report on drug situation, fifth general population survey conducted by European 20172, NPSs are the second consumed drugs after School Survey Project on Alcohol and Other Drugs cannabis [2]. In Romania, NPSs are currently being (ESPAD) in 2015 [1]. purchased online or on illegal market as drug of choice New psychoactive substances are designer drugs and their availability and use has been increased. that intend to mimic controlled substances and are not Therefore, there is a growing concern about the yet covered by international laws. Those “legal-highs” associated issues: drug-related intoxications, deaths, appeared on the Romanian drug market in 2008, and infectious disease or criminality. Regarding drug related were legally commercialized in “smart” or “dreams” emergency room visits in 2016, from 4518 cases, 6% were shops. In 2 years, in 2010, Romania was ranked fourth diagnosed as intoxication due to ingestions of an unknown in the EU, regarding NPSs use. As a response, by drug [2]. Regarding the NPSs drug related deaths, these government control laws in 2010, 36 new psychoactive were likely to be underestimated due to the difficulties of substances were placed under control and new drugs analytical confirmation of exposures, since only two cases commercialization was banned. are reported in 2015 and four cases in 2016 [2, 3].

1) Emergency Hospital of Galati, Department of Legal Medicine, Galati, Romania 2) “Dunarea de Jos” University of Galaţi, Faculty of Medicine and Pharmacy, Galaţi, Romania * Corresponding author: “Dunarea de Jos” University of Galaţi, Faculty of Medicine and Pharmacy, 35 Alexandru Ioan Cuza street, 80010, Galaţi, Romania E-mail: [email protected] 3) “Mina Minovici” National Institute of Legal Medicine, Bucharest, Romania

173 Chiţescu C.L. et al. New psychoactive substances (NPSs) abuse in Romania

This brings up the problem of drug testing diethyl ether. Formic acid (98%), Tris(hydroxymethyl) methods for NSPs. Only a few of toxicology laboratories aminomethane, acetic acid, ammonium acetate, are able to detect and confirm the drug consumption ammonia, ultrapure water (LC-MS grade) were purchased in Romania, most of them engaged in legal medicine from Merck Romania. As derivatization agents: acetic structures. Furthermore, forensic toxicology laboratories anhydride/pyridine, trifluoroacetic acid (TFA), and are required to identify NSPs in biological samples or pentafluoropropanol (PFPOH) were purchased seized materials without the availability of a reference from Sigma-Aldrich (Germany). β-Glucuronidase/ standard. Arylsulfatase from Helix Pomatia for urine hydrolysis, Gas chromatography coupled with mass was purchased from Sigma-Aldrich (Germany). spectrometry (GC–MS) has been the most used technique Solid phase extraction (SPE) Chromabond for general unknown screening in toxicological analysis Drug 200 mg/3 ml were purchased from Chromabond [4]. (Macherey-Nagel GmbH & Co., Germany). However this method is generally not suitable Analytical standards: diazepam, methadone, for NPSs the identification because it is based on library benzoylecgonine, morphine, codeine, THC, THC- spectra or reference standards and spectra information COOH, α-PVP and prolintane were purchased from on NPSs are not always available. Lipomed GmbH, Germany as solutions 1 mg ml-1 in From this perspective, high-resolution mass methanol. spectrometry (HRMS) analyses allowed for the accurate Analysed samples mass determination of the analytes and therefore of their Biological samples in 17 forensic cases were empirical formulas and molecular structures directing collected between January 2015 and December 2016, the investigation to one compound or to a limited due to suspicion of NPSs ingestion. Urine and blood number of compounds [5-7]. MS-MS methods applied specimens were provided by police department for in addition to HRMS screening can raise the reliability the persons involved in traffic events (2 cases) or two of screening at the level of confirmation analysis [7,8]. attempts of rape. Blood, urine, gastric content from Because the lack of reference standards, approaches patients presented to the emergency department were based on theoretical and predicted reference data proved provided by clinic laboratories in (2 cases). Post-mortem to be useful [9]. specimens (blood, urine, gastric content) were obtained The presented study was conducted in two during the autopsy for 11 cases of drug related death. All forensic toxicology laboratories of National Institute samples were stored frozen (-20°C) until were analysed. of Legal Medicine, Bucharest and Legal Medicine Blank urine and blood samples and spiked samples Service of Galati, Romania with the technical support for the available analytical standards were analysed as of the Laboratory of Chromatography and Imagistic of control samples. “Dunarea de Jos” University of Galati, Romania. GC-MS and HRMS techniques were both Methods applied. The manuscript presents analytical strategies, Sample preparation for HRMS analysis emphasizing the potential of the HRMS/MS approach, LL extraction for blood samples: blood samples which allows the identification of a large number of were extracted with an ethyl acetat/methylene chloride/ compounds including NPSs in one single analysis. isopropanol (3/1/1) mixture in both basic condition Different sample preparation methods as: liquid-liquid (pH 9, with Tris solution) and acidic condition (pH 3, extraction (LL), solid-phase extraction (SPE), enzymatic with acetic acid 4%). After centrifugation, the extract hydrolysis, and derivatization were applied to blood, was evaporated under a high purity nitrogen flow at urine, and gastric content. The results were compared 40°C (Thermo Scietific, Germany). The residue was and the key role of HRMS-MS analysis in identifying reconstituted in methanol and filtrated thru 0.2 µm NPSs was discussed. micro-filter. In the context of the currently poor official Urine enzymatic hydrolysis followed by LL reporting of drug abuse in Romania, the aim of the extraction: urine was adjusted to pH 5.5 with acetate buffer presented manuscript is to describe our experiences and incubated for 2.5 h at 37°C with β-glucuronidase. The regarding the analytical challenge of NPSs identification urine was then extracted by liquid-liquid extraction with in biological samples and to discuss some methodological diethyl ether/methylene chloride (1/1). After evaporation issues raising analysing new psychoactive substances. at 40°C under a high purity nitrogen flow, the residue was dissolved in methanol and filtrated (0.2 µm). Experimental Gastric content LL extraction: a volume of Chemicals, reagents and materials gastric content was filtered and extracted with methylene Organic solvents used were purchased from chloride/ diethyl ether (70/30). After evaporations of the Merck Romania: methanol, acetonitrile, methylene solvents at 40°C under high purity nitrogen flow, the chloride, ethyl acetate, isopropanol, ethyl acetate, residue was dissolved in methanol.

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Sample preparation for GC-MS analysis An ultra-performance Accucore U-HPLC SPE extraction for blood/serum/plasma sample: Column C18 (150 x 2.1 mm, 2.6 µm), (Thermo Scientific) 1 ml of sample was applied on Chromabond Drug was used. A flow rate of 0.4 ml min−1 was set for cartridge previously preconditioned with methanol and separation of the selected compounds in the U-HPLC water. The analytes were eluted with methylene chloride/ system. The mobile phase consisted of: water at pH 3.5 isopropanol/ammonia mixture (80/20/2). The eluate was with formic acid (A) and methanol (B). A 15 minutes concentrated by evaporation under flow of high purity gradient was used. The step gradient was follow: 0–1 min nitrogen, at 40°C. Derivatization step was done with TFA 100% A; 1–2.5 min linear increase to 40% B; 2.5–10 linear and PFPOH. After the evaporation to dryness the sample increased; to 100% B and hold 3 min; 13–13.2 decreasing was reconstituted in ethyl acetate. to 0% B; 13.2–15 min 100% A. Injection volume was set Urine LL extraction: urine was extracted at 20 μl. with ethyl acetate/ methylene chloride/ isopropanol HESI (Heated Electrospray) ion source was used (3/1/1) after enzymatic hydrolysis (β-glucuronidase, for the ionization. The HESI parameters were optimized pH 5.5). Evaporation of the solvent under flow of high as follow: sheath gas flow rate 40 unit; aux. gas unit flow purity nitrogen, at 40°C was followed by derivatization rate 10; capillary temperature 250°C; aux gas heater procedure with pyridine/ acetic anhydride mixture temperature 300°C; spray voltage 2800 V (-2800 V for (2/3). After the evaporation to dryness the sample was ESI-); S lens RF level 50. reconstituted in ethyl acetate. MS parameters Detection of compounds was performed using Instrumentation a Q-Exactive mass spectrometer. Full scan data in both GC-MS analysis positive and negative mode was acquired at a resolving A GC- MS configuration (GC6890N MS 5975B) power of 70 000 FWHM at m/z 200. For the compounds powered by Agilent Technologies with a 30 m x 25 of interest, a scan range of m/z 130–1000 was chosen; mm i.d. x 0:25 µm Hewlett Packard HP-5 MS capillary the automatic gain control (AGC) was set at 3e6 and column was used in the present work. Carrier gas was the injection time was set to 200 ms. Scan-rate was set helium (purity: 99.999%) and the flow rate was held at at 2 scan sec-1. External calibration was performed by constant velocity of 1 ml min-1. Ionization voltage was set calibration solution in positive and negative mode. at 70 eV. In addition to the full scan acquisition, a targeted The GC-MS was operated both in full scan and MS/MS analysis was performed using the mass inclusion SIM modes. A volume of 1 µl of sample was injected. In list and expected retention times of the target analytes, full scan analysis, the oven temperature was held at 100 with a 30 sec. time window. The Orbitrap spectrometer °C for 3 min. following injection, and then programmed was operated both in positive and negative mode at at 30 °C min-1 to 310 °C, which was held for 10 min for 17500 FWHM. The AGC target was set to 2e5, with the full scan analysis. A scan range 45-590 amu was used in maximum injection time of 20 ms. The precursor ions are full screening mode. In SIM mode a temperature gradient filtered by the quadrupole which operates at an isolation was used. window of m/z 2. Collision energy was set at 30, 35 and Data were evaluated by the Chemstation software 45 eV. using WileyY6, NIST05 and Pfleger-Maurer-Weber- Data was evaluated by the Quan/Qual Browser TOX3 mass spectra database. Xcalibur 2.3 (Thermo Fisher). The mass tolerance HR-MS-MS analysis window was set to 5 ppm for the two analysis modes. Q Exactive high-performance quadrupole- Detection was based on accurate mass Orbitrap LC–MS/MS was used to identify psychoactive measurements and pattern recognition of the product substances from biological samples in a post target ions compared to standards solution or with MS/MS approach. Screening of the samples for targeted data generated by HighChem Mass Frontier 7.0 software contaminants in full scan mode was followed by an in case of no standard available. MS/MS analysis as targeted ion fragmentation (t-MS2) achieving high sensitivity and selectivity and enabling Analytical approach confirmatory analysis. The analytical methodology included: LC parameters (a) Immunological testing of the A Thermo Scientific Dionex Ultimate 3000 urine samples using qualitative immunoassay Series RS pump coupled with a Thermo Scientific INSTALERT, (Innovacon, SUA) for: amphetamines, Dionex Ultimate 3000 Series TCC-3000RS column methamphetamines, barbiturates, benzodiazepines, compartments and a Thermo Fisher Scientific Ultimate cocaine (benzoylecgonine), cannabinoids, opiates, 3000 Series WPS-3000RS autosampler controlled by MDMA, TCA, methadone, phencyclidine. Chromeleon 7.2 Software (Thermo Fisher Scientific, (b) Routine analyses of the urine samples Germany) were used for analysis. by single quadrupole GC/MS in full scan mode

175 Chiţescu C.L. et al. New psychoactive substances (NPSs) abuse in Romania followed by comparison of the spectra against spectra ion at m/z 126 was the most abundant fragment. The peak library. For blood samples, analysis was done on SIM was identified by PMW-TOX 3 lybrary as the prolintane mode using characteristic ions for nine basic drugs: metabolite – OH-phenyl-prolintane with match factor of methamphetamine and amphetamine, MDA, MDMA, 70-80% (Fig. 1). NIST 03 library identified the compound MDA, benzoylecgonine, methadone, morphine, codeine. as pyrovalerone with a match factor of 75% in urine (c) Analyses of the samples by full scan screening samples in case no. 5. Although speculative, due to library with LC-HRMS in the positive/negative ESI mode. A matches, these findings suggested that NPSs should post-target approach was applied for a comprehensive list further be investigated. of 102 compounds, belonging to different drugs classes Analysis of the gastric lavage sample available for such as: illegal drugs (amphetamines, opioids, methadone sample no. 4, revealed the presence of the characteristic cannabinoids); NSPs: synthetic cathinones; synthetic ions at m/z 126, but library search didn’t identify the cannabinoids, tryptamines analogues; prescription drugs spectra. (benzodiazepine, barbiturates, opiates). The analysis disclosed the presence of opiates (d) Analyses of samples using LC-HRMS/MS in (methadone, morphine and codeine) in urine and blood the positive/negative ESI mode applying fragmentation samples in cases no. 8, 9, 12; diazepam and metabolite voltages as targeted ion fragmentation (t-MS2) to study were identified in cases no. 6 and 8; 11-nor-9-carboxy- the accurate masses of the fragment obtained. THC (THC-COOH) was identified in urine sample no. (e) Generating MS/MS data using HighChem 21. No other psychoactive substances were identified. Mass Frontier 7.0 software in order to pattern recognition Urine and blood sample for cases 1-13 were reanalysed of the productions. by high resolution mass spectrometry. (f) If available, spectra of pure standards were LC/HRMS analyses used to compare the responses of positive samples. Full Full scan screening by LC/HRMS analyses scan acquisition and targeted MS/MS analysis were of samples no. 1 to 17 was performed. In a post-target performed. As confirmation criteria the detection of screening approach, the presence of the protonated a precursor ion and at least four product ions in t-MS2 molecule in the samples was searched by processing mode was applied. method based on monitoring theoretical exact masses In those cases where pure standards were not using narrow mass windows (5Δppm) in combination available, the postulation of their molecular structures with the application manager XCalibur which permits was made by exact mass determination and significant mass error examination. Full-scan acquisition data were fragment interpretation using HRMS/MS technique processed using a home-made database which included compared to fragmentation patterns generated by 102 compounds: illegal drugs, prescription drugs together HighChem Mass Frontier 7.0 software. with designer drugs as synthetic cannabinoids, synthetic cathinones, phenylethylamines, piperazines, synthetic RESULTS tryptamines. Full scan HRMS analysis and examination of In eleven of seventeen cases, synthetic the accurate-mass spectra allowed for the accurate mass cathinones were identified in blood or urine samples identification of some monitored drugs and their main by HRMS analysis: α-pyrrolidinopentiophenone (α metabolites: – PVP), 3,4-methylenedioxypyrovalerone (MDPV), Alpha-pyrrolidinovalerophenone (α –PVP) and 3',4'-methylenedioxy-α-pyrrolidinobutyrophenone the main metabolites: OH-α-PVP, 2-oxo-α- PVP, N-N- (MDPBP), pyrovalerone; two urine samples were bis, dialkyl-α- PVP were identified in urine and blood positive for cannabinoids. Ephedrine was identified sample in six cases of drug abuse related deaths (no. 1-3, in the gastric lavage for a teenage girl, brought in the 5, 8, 10). emergency room after unknown drug ingestion. MDMA MDPV (3,4-methylenedioxypyrovalerone) and (3,4 - methylenedioxy - N - methylamphetamine) four metabolites were identified in two cases of suicide and metabolites were found in the urine sample of a (case 7 and 13). Pyrovalerone was identified in a case of driver. Other identified psychoactive substances were: death by drug overdose together with opiates (case no. opiates and methadone, benzodiazepines. No synthetic 12). MDPV and MDPBP were identified in both victim cannabinoid was founded in the analysed samples. The and perpetrator in a sexual assault with suicide tentative result of the analysis are summarised in the Table 1. (cases no 16, 17). GC-MS analysis THC-COOH and MDMA (methylendioximeth- The submitted blood in cases 1 to 13 was amphetamine) together to its active metabolites subjected to a GC-MS in SIM mode analysis for basic drug 4-hydroxy-3-methoxymethamphetamine (HMMA) and detection. If urine sample was available, a GC-MS full 3,4-methylenedioxyamphetamine (MDA) (10) were scan screening was performed using library approaches. identified in two drivers. In the urine samples no. 1, 2, 3, 5, and 10 the characteristic Ephedrine and nor-ephedrine were identified

176 Romanian Journal of Legal Medicine Vol. XXVI, No 2(2018) in blood and gastric lavage from a teenager brought A. Metabolites identification in the emergency room after the consumption of As we mentioned in the result section in urine “ethnobotanical” drugs, probable herbal stimulants (case samples for the cases no. 1 - 3 and 10, GC-MS analysis no. 4). in full scan mode indicate the presence of prolintane Morphine, codeine and methadone and the metabolite OH-phenyl-prolintane (Fig. 1). major metabolite EDDP (2-ethylidene-1,5-dimethyl-3,3- Human biotransformation of prolintane was diphenylpyrrolidine) were founded in three case of death studied from 1992 by Ruker et al. (11) on human related to drug abuse, two of them with polydrug use. volunteers. A number of 26 metabolites were identified, Diazepam and the main metabolite nordiazepam were some found in all volunteers. Metabolism pathways were identified in two cases of death by drug overdose. proposed [11,12]; most important biotransformation Most of the compounds were identified in positive reaction can be illustrated by the following scheme (Fig. 2): ESI mode. Under negative ESI mode no appreciable As products of aromatic hydroxylation, OH- signals were produced for the analytes with the exception phenyl-prolintane is one of the major metabolite together of THCCOOH. to oxo-prolintane. The GC-MS fragmentation pattern of An adequate mass tolerance of 5 ppm prevents OH-phenyl-prolintane includes ions m/z 190, 126, 105, mass distortion and false negative/positive results. 84 [11]. According to literatures, the main characteristic Average mass accuracy error (∆ppm) ranged between ions of prolintane in the GC–MS full-scan mass spectra: 0.15 and 1,5 ppm, as presented in Table 2. m/z 126, 174, 91, 84 [13]. The GC-MS fragmentation Confirmation by HRMS-MS analysis pattern of α-PVP is: m/z 126, 77, 84, 105 and 188 [13,14]. A targeted HRMS/MS analyse was performed. As ions m/z 126, 105 and 84 are common for The exact masses of all "suspect" analytes were added to prolintane, α-PVP and hydroxyphenyl-prolintane in the inclusion list and time windows were set on the basis the GC-MS spectra, processing software indicated the of expected retention time for each analyte. The t-MS2 presence of prolintane metabolite. mode was set at an RP of 17,500 FWHM at m/z 200 to On the other hands, the LC-HRMS full scan measure product ions of target compounds. Different analysis of the blood/urine samples in the same cases fragmentation voltages during LC/HRMS were applied to indicated the presence of α-PVP m/z 232.1701 [M+H]+ study the accurate masses of the obtained characteristic and three metabolites: OH-α-PVP m/z 234.1857 fragments. The transitions of at least four product ions [M+H]+, 2-oxo -α-PVP m/z 246.14940 [M+H]+ and the were monitored (Table 2). compound C15H21NO2 m/z 248.16503 [M+H]+ (Fig. 3). Each chromatogram was carefully evaluated. None of samples was positive for prolintane. The α-PVP Comparisons with pure standards available for: metabolite 1-phenyl-2-(pyrrolidin-1-yl)pentan-1-ol OH- morphine, codeine, methadone, diazepam and THC- PVP (m/z 233,1779), formed by reduction of the ketone COOH, allowed the identification through the mass structure to a corresponding alcohol, was found to be spectra and retention times (RT). the most abundant species in the analysed human urine A pre-confirmatory step was performed for the samples and in vitro experiments for the prediction of case of no standard available using the HighChem Mass metabolic pathways [14 – 16] (Fig. 4) . Frontier 7.0 software to generate possible fragmentation As can be seen in both Figures 2 and 4, some of path-ways of the detected compounds. prolintane and α-PVP metabolites share the same formula False positive results were observed in full but have different structures: OH-phenyl-prolintane and scan HRMS screening for the following compounds: OH -α-PVP with the common formula C15H23NO have ethylphenidate, methylone, ethylone, synthetic same exact mass of [M+H]+ 234.18579; oxo-prolintane cannabinoid J200. The MS-MS analysis and the use of and α-PVP with the common formula C15H21NO have predicted fragmentation together with the low abundance same exact mass of [M+H]+ 232.17014. The hydroxylation observed, suggest that the unknown compound might products of both α-PVP and prolintane have the same be impurities or secondary products resulting from the formula C15H21NO2 with mass of [M+H]+ 248.16503. synthesis process. However, the metabolites produced by degradation of pyrolidine ring are different: α-PVP metabolite is

DISCUSSIONS the compound C11H15NO m/z 178.12319 [M+H]+

and prolintane metabolite is the compound C11H17N Since inconsistences were observed between the m/z 164.14399 [M+H]+. The ion m/z 178.121319 was results of the available GS-MS and HRMS methods result identified in the urine samples for all cases with a mass especially for NPSs identification, different strategies error lower than 0.5, while the ion m/z 164.14399 was were used to get the correct conclusions: metabolites not identified in any sample (Fig. 4). According to the identification, followed by HRMS-MS analysis assisted metabolite identification analysis in the debated cases, by software tools. α-PVP exposure is more likely than prolintane exposure. In four cases (samples no. 7 and 13, 16, 17)

177 Chiţescu C.L. et al. New psychoactive substances (NPSs) abuse in Romania

Table 1. Results of the toxicological analysis Analysed Toxicological analysis Nr. Case history sample GC-MS HRMS full scan / HRMS-MS α - PVP Blood Negative Male, 35 years old, known as drug abuser. α - PVP metabolites 1 Deceased α - PVP Urine Prolintane α - PVP metabolites Blood Negative Not carried out Male, 35 years old, homeless; known as drug 2 α - PVP abuser. Deceased Urine Prolintane α - PVP metabolites α - PVP Blood Negative Male, 32 years old, homeless; known as drug α - PVP metabolites 3 abuser. Deceased α - PVP Urine Prolintane α - PVP metabolites Female, 15 years old, brought in the emergency Blood Negative Nor-ephedrine 4 room after unknown drug ingestion. Gastric lavage Negative Ephedrine Male, 25 years old, possible drug overdose; α - PVP 5 Urine Pyrovalerone hospitalised for 9 hours. Deceased α - PVP metabolites Blood Nordiazepam, Diazepam Male, 36 years old, known as drugs abuser, Metamizol Nordiazepam 6 drug overdose. Deceased in prison. Diazepam Urine Metamizol Nordazepam Male, 36 years old, suspicion of NPSs Blood Negative Negative 7 ingestion. Suicide. Deceased Urine Negative MDPV and metabolites Morphine Morphine Blood Codeine Diazepam Male 19 years old, known as drugs abuser, Diazepam Nordiazepam 8 drug overdose. Morphine Morphine Diazepam Deceased Urine Codeine Nordiazepam α - PVP α - PVP metabolites Female 28 years old, known as heroine abuser. 9 Blood Methadone Methadone Deceased. α - PVP Blood Prolintane Male 34 years old, known as drugs abuser, drug α - PVP metabolites 10 overdose. Deceased. α - PVP Urine Prolintane α - PVP metabolites Blood Negative Negative Male, 16 years old, brought in the emergency 11 room after NPSs use. 11-nor-9- Urine Negative carboxy-THC Morphine Morphine Codeine Blood Methadone Methadone Codeine Pyrovalerone Male 44 years old, known as heroine and NPSs 12 Morphine abuser. Deceased. Morphine Codeine Urine Methadone Methadone Codeine EDDP Pyrovalerone Male, 31 years old, known as drugs abuser, Blood Not carried out MDPV 13 suicide. Deceased. Urine Not carried out MDPV and four metabolites Male, 22 years old, driving under the influence 14 Urine Not carried out 11-nor-9-carboxy-THC of drugs Male 29 years old, driving under the influence MDMA 15 Urine Not carried out HMMA (active metabolite) of drugs MDA (active metabolite) Blood Not carried out Negative Female 17 years old, victim of claimed sexual 16 MDPV assault. Suicide attempt. Urine Not carried out MDPBP Blood Not carried out Negative Male 26 years old, aggressor of claimed sexual 17 MDPV assault Urine Not carried out MDPBP

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Table 2. Detected compounds, retention time, mass error and the main product ions after CID Detected Elemental Average mass Precursor (m/z) RT Product ions (m/z) compounds composition error 257.08; 228.05; 193.08; Diazepam C H ClN O 7.64 0,36 285.0794 [M + H]+ 16 13 2 182.03; 154.04 243.06; 208.10; 226.04; Nordiazepam C H ClN O 7.43 0,25 271.0638 [M + H]+ 15 11 2 165.02; 140.02 268.13; 255.10; 237.09; Morphine C H NO 3.10 0,43 286.1443 [M + H]+ 17 19 3 211.07; 173.06 282.14; 243.10; 225.09; Codeine C H NO 3,48 0,31 300.1599 [M + H]+ 18 21 3 187.08; 137.06

+ Methadone C21H27NO 6.13 0,40 310.2171 [M + H] 187.11; 265.15; 247.14; 159.11 + EDDP C20H23N 4.99 0,63 278.1910 [M + H] 249.15; 234.12; 186.12 214.16; 172.11; 161.09; α-PVP C H NO 4.23 0,31 232.1696 [M + H]+ 15 21 126.19; 105.03; 91.05 C H NO 216.17; 186.13; 145.10; OH-α-PVP 15 23 4.29 0,30 234.1856 [M + H]+ 117.07; 91.05 258.15; 154.12; 138.09; MDPV C H NO 6,02 0,15 276.1594 [M + H]+ 16 21 3 123.04; 112.078; 84.08 + MDPBP C15H19NO3 3.17 -0.27 262.1438[M +H] 244.13; 202.11; 145.08; 84.08 228.17; 175.11; 157.10; Pyrovalerone C H ON 4.70 0.38 246.1858 [M+H]+ 16 23 133.06; 119.04; 91.05

MDMA C11H15NO2 4.30 0,25 194.1181 [M+H]+ 135.04; 163.08; 137.06; 117.07

HMMA C11H17NO2 5.40 0,30 196.1337 [M+H]+ 165.09; 137.06;123.04; 58.07 162.12; 132.09; 117.07; Ephedrine C H NO 3.71 0,16 166.1231 [M+H]+ 10 15 107.05; 91.05

Norephedrine C9H13NO 3.58 0,15 152.1075 [M+H]+ 134.10; 117.07; 109.06; 91.05; 11-nor-9-carboxy 325.18; 299.20; 245.15; C H O 9.80 1,35 343.1909 [M-H]- THC 21 28 4 191.10; 179.10; 161.09

Figure 2. Proposed scheme for the major metabolic pathways Figure 1. Mass spectrum and predicted fragmentation in GC- of prolintane according to Ruker et al., 1992 and Espartero et MS for hydroxyphenil-prolintane. al., 1997. HRMS analysis in full scan mode disclosed the presence identified in the urine samples of both involved persons, of MDPV. GC-MS analysis carried out for samples no. 7 in a case or rape tentative (Fig. 6). This was in agreement was negative for MDPV. with previous published data [18, 19]. Both persons According to literature [7, 11, 17], ten MDPV involved admitted that having snorting a powdered drug metabolites were identified in consumers‘ urine. Phase I that was distributed under the name "Pure by Magic". and phase II metabolites were found by HRMS analyses in urine samples positives for MDPV (Fig. 5). B. HRMS-MS analysis MDPBP, a recent identified synthetic substance Although high mass resolution and mass accuracy of the pyrrolidinophenone type, together with the main alone are very useful, the information provided is not metabolites: demethylenil MDPBP, C14H19NO3 (m/z sufficient for reliable identification of the compounds [17]. 249.1365), and demethylenil-methyl-deamino-oxo- Without fragmentation, no structural information can be dihydro MDPBP, C11H14O4 (m/z 211.09703) were obtained, thus, the MS-MS analysis were performed for

179 Chiţescu C.L. et al. New psychoactive substances (NPSs) abuse in Romania

confirmation. Without the reference standards, the tentative identification of the detected compound by the identification of fragments that can be acquired with software tools is commonly feasible [20-22]. Identification of the pharmaceutical residues substances based on a combination of accurate-mass determination on a TOF-MS system and fragmentation patterns generated by in-source CID have been described [23]. In the present work HRMS-MS analysis and HighChem Mass Frontier 7.0 software were used for presumptive confirmation. The identification was based on the MS/MS spectral match of the suspect compound to the profile of predicted MS/MS spectra. Multiple fragmentation mechanisms were monitored as follow (Fig. 7). Figure 3. LC-HRMS full scan chromatogram of α-PVP and its metabolites In all positive samples the fragmentation found in urine sample no 2; from the top to the bottom: α-PVP, 232. 232.1701 pattern of α-PVP (Fig. 7 A) and was in agreement m/z, RT = 4.48; OH- α-PVP, 234.1857 m/z, RT =4.57; 2-oxo- α-PVP with Mass Frontier Software prediction (Fig. 7 246.1494 m/z, RT 5.52; N,N bis-dialkyl α-PVP 178.1231 m/z, RT 4.34. B) and with α-PVP reference standard MS-MS analysis (Fig. 7 C). Based on data analysis, we conclude that α-PVP was the drug of abuse in the presented cases (no. 1-5, 10). The same procedure was applied for de identification and presumptive confirmation of OH- α-PVP, MDPV, MPBPB, MDMA and metabolites, pyrovalerone, ephedrine. Reference standards, available for α-PVP and prolintane 2 month after the samples analysis performing, allowed the confirmation of the used approach (Fig. 7 C). For opiates and diazepam reference Figure 4. Proposed scheme for the major metabolic pathways of α-PVP standards were used to compare the responses according to Grapp et al., 2016 and Negreira et al., 2015. of positive samples. Those compounds were identified in biological samples by both GC- MS and HRMS screening methodology and confirmed by GC-MS in SIM mode and respectively by HRMS-MS analysis. HRMS technique allows the identification of ephedrine and MDPV for the case 4 respective 7, not founded by GC-MS analysis.

CONCLUSIONS

We report an analytical approach for the identification of psychoactive substances in biological sampled by collaborative toxicology laboratories using GC-MS and HRMS. GC-MS analyses were useful in identifying characteristic fragments mainly in the case of opiates, THC, benzodiazepines. New Figure 5. LC-HRMS full scan chromatogram of MDPV and the methods and an upgrade of the libraries for metabolites M2, M5, M6, M9 (according to Ibáñez et al., 2016) found in NPSs are necessary. urine sample no. 6; from the top to the bottom: MDPV, 276.1594 m/z, Further analyses of the samples via LC- RT = 6.01; M2, 278.1747 m/z, RT = 6.22 M5, 306.1331 m/z, RT = 3.98; HRMS resulted in the identification of synthetic M6, 292.1554 m/z, RT = 6.60; M9, 294.1704 m/z, RT = 4.91.

180 Romanian Journal of Legal Medicine Vol. XXVI, No 2(2018) cathinones as α-PVP, MDPV, MDPBP, pyrovalerone. was performed for confirmatory purpose. Metabolites were identified, leading to much more The overall information given by reliable way to confirm the drug’s use. MS-MS analysis HRMS/HRMS-MS analysis (full-spectrum acquisition, accurate mass of protonated molecule and relevant fragment ions) together with a predictive approaches allowed the presumptive identification of the detected compounds without a reference standards a priori. Searching a large number of target drugs on the basis of their exact mass appears to be one of the most efficient ways in drug identification. High-resolution mass spectrometry (HRMS), using an Orbitrap analyzer, proved to be a valuable screening tool, as it provides sensitive, full-spectrum MS data with high mass resolution and mass accuracy. The possibility of performing screening is very useful, as laboratories do not need to purchase all reference standards before analysis, with the subsequent problem of high costs. The study results proved that Romanian Figure 6. LC-HRMS full scan chromatogram of MDPV, MDBPB youth follow the current trend of the new and metabolites; from the top to the bottom: MDPV, 276.1594 generation of drugs of abuse, which impose state m/z, RT = 6.01; M2, 278.1747 m/z; MDPBP 262.1438 m/z, RT = of art laboratory equipment purchase and new 3.18; M1 250.14532 m/z, RT = 3.26; M2 211.09703 m/z, RT = 5.97.

B C

Figure 7. LC/HRMS-MS spectra and postulated fragments α-PVP. A. extracted ion MS-MS chromatogram at 5 Δppm for blood sample (case no. 3). B. The fragments and possible structures assigned by HighChem Mass Frontier 7. C. extracted ion MS-MS chromatogram at 5 Δppm for α-PVP reference standard solution.

181 Chiţescu C.L. et al. New psychoactive substances (NPSs) abuse in Romania analytical method development and validation. Acknowledgements. The present research was supported in by the project POSCCE ID 1815, in terms of Conflict of interest. The authors declare that providing facilities. The authors thanks to the “Dunarea there is no conflict of interest. de Jos” University of Galati for technical support.

References 1. ESPAD Report 2015: Results from the European School Survey Project on Alcohol and Other Drugs. Luxembourg: Publications Office of the European Union, 2016. 2. 2016 National Report. Ministry of Administration and Interior National Anti-drug Agency Romanian Monitoring Centre for Drugs and Drugs Addiction, Bucharest, Romania 2017. 3. 2015 National Report. Ministry of Administration and Interior National Anti-drug Agency Romanian Monitoring Centre for Drugs and Drugs Addiction, Bucharest, Romania 2016. 4. Meyer MR, Peters FT, Maurer HH. Automated mass spectral deconvolution and identification system for GC-MS screening for drugs, poisons, and metabolites in urine. Clinical chemistry 2010: 56: 575–584. 5. Wu AH, Gerona R, Armenian P, French D, Petrie M, Lynch KL. Role of liquid chromatography-high-resolution mass spectrometry (LC- HR/MS) in clinical toxicology. Clin. Toxicol. 2012; 50(8):733-742. 6. Ojanperä I, Kolmonen M, Pelander A. Current use of high-resolution mass spectrometry in drug screening relevant to clinical and forensic toxicology and doping control. Anal Bioanal Chem. 2012; 403:1203–1220. 7. Francesco B, Boscolo MR, Guido B, Fabio M, Favretto VD. A mixed MDPV and benzodiazepine intoxication in a chronic drug abuser: determination of MDPV metabolites by LC-HRMS and discussion of the case. Forensic Sci. Int. 2014; 243:149-55. 8. Rossi SS, Odoardi S, Gregori A, Peluso G, Ripani L, Ortar G, Serpelloni G, Romolo FS. An analytical approach to the forensic identification of different classes of new psychoactive substances (NPSs) in seized materials. Rapid Commun. Mass Spectrom. 2014; 28:1904–1916. 9. Tyrkkö E, Pelander A, Ojanperä I. Differentiation of structural isomers in a target drug database by LC/Q-TOFMS using fragmentation prediction. Drug Test. Anal. 2010: 2:259-20. 10. Tsadik TA, Barnes AJ, Lowe RH, Kolbrich Spargo EA, Milman G, Pirnay SO. Urinary MDMA, MDA, HMMA, and HMA Excretion Following Controlled MDMA Administration to Humans. J Anal Toxicol. 2009; 33(8):439–446. 11. Ruckers G, Neugebauer M, Zhong D. Study on the metabolism of racemic prolintane and its optically pure enantiomers. Xenobiotica 1992; 22:143-152. 12. Espartero AG, Pérez JA, Zapardiel A, Bermejo E, Hernández L. Direct determination of prolintane and its metabolite oxoprolintane in human urine by capillary zone electrophoresis and beta-cyclodextrin-modified micellar electrokinetic chromatography. J Chromatogr A. 1997; 778(1-2):355-361. 13. Grapp M, Sauer C, Vidal C, Muller D. GC-MS analysis of the designer drug pyrrolidinovalerophenone and its metabolites in urine and blood in an acute poisoning case. Forensic Sci Int. 2016; 259:14-19. 14. Meyer MR, Du P, Schuster F, Maurer HH. Studies on the metabolism of the α-pyrrolidinophenone designer drug methylenedioxypyrovalerone (MDPV) in rat and human urine and human liver microsomes using GC-MS and LC-high-resolution MS and its detectability in urine by GC-MS. J. Mass Spectrom. 2010; 45:1426-1442. 15. Hasegawa K, Suzuki O, Wurita A, Minakata K, Yamagishi I, Nozawa H, Gonmori K, Watanabe K. Postmortem distribution of a-pyrrolidinovalerophenone and its metabolite in body fluids and solid tissues in a fatal poisoning case measured by LC–MS–MS with the standard addition method. Forensic Toxicol. 2014; 32:225-234. 16. Negreira N, Erratico C, Alexander TK, van Nuijs LN, Heath E, Neels H, Covaci A. In vitro Phase I and Phase II metabolism of α-pyrrolidinovalerophenone (α-PVP), methylenedioxypyrovalerone (MDPV) and methedrone by human liver microsomes and human liver cytosol. Anal Bioanal. Chem. 2015; 407:5803–5816. 17. Ibáñez M, Pozo Ó J, Sancho J V, Orengo T, Haro G, Hernández F. Analytical strategy to investigate 3,4-methylenedioxypyrovalerone (MDPV) metabolites in consumers’ urine by high-resolution mass spectrometry. Anal Bioanal Chem. 2016; 408(1):151-64. 18. Balikova M, Zidkova M, Oktabec Z, Maresova V, Linhart I, Himl M, Novotny M. The Abuse of 3,4-Methylenedioxypyrrolidinobutyrophenone (MDPBP): A Case Report. J Forensic Toxicol Pharmacol doi:10.4172/2325-9841.1000108, 2013. 19. Meyer MR, Mauer S, Meyer GM, Dinger J, Klein B, Westphal F, Maurer HH. The in vivo and in vitro metabolism and the detectability in urine of 3',4'-methylenedioxy-alpha-pyrrolidinobutyrophenone (MDPBP), a new pyrrolidinophenone-type designer drug, studied by GC- MS and LC-MS. Drug Test Anal.2014; 6(7-8):746-56. 20. Ojanperä S, Pelander A, Pelzing M, Krebs I, Vuori E, Ojanperä I. Isotopic pattern and accurate mass determination in urine drug screening by liquid chromatography/time-of-flight mass spectrometry. Rapid Commun Mass Spectrom. 2006; 20(7):1161-1167. 21. Pelander A, Ristima J, rasanen I, Vuori E, Ojanperä I. Screening for basic drugs in hair of drug addicts by liquid chromatography/time-of- flight mass spectrometry. Ther Drug Monit. 2008; 30(6):717-724. 22. Brandt SD, Sumnall HR, Measham F, Cole J. The confusing case of NRG-1, second generation mephedrone, Br. Med. J. 2010; 341:c3564. 23. Zedda M, Zwiener C. Is nontarget screening of emerging contaminants by LC-HRMS successful? A plea for compound libraries and computer tools. Anal. Bioanal. Chem. 2012; 403:2493–2502.

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