First metabolic profile of PV8, a novel synthetic cathinone, in human hepatocytes and urine by high-resolution mass spectrometry
Novel psychoactive substances (NPS) are ever changing on the drug market, making it difficult for toxicology laboratory methods to stay current with so many new drugs. Recently, PV8, a synthetic pyrrolidinophenone, was detected in seized products in Japan (2013), The Netherlands (2014), and Germany (2014). There are no controlled PV8 administration studies, and no pharmacodynamic and pharmacokinetic data. The objective was to determine PV8’s metabolic stability in human liver microsome (HLM) incubation and its metabolism following human hepatocyte incubation and high-resolution mass spectrometry (HRMS) with a Thermo Scientific Q-Exactive. Data were acquired with a full-scan data-dependent mass spectrometry method. Scans were thoroughly data mined with different data processing algorithms and analyzed in WebMetaBase. PV8 exhibited a relatively short 28.8 min half-life, with an intrinsic 24.2 μL/min/mg microsomal clearance. This compound is predicted to be an intermediate clearance drug with an estimated human 22.7 mL/min/kg hepatic clearance. Metabolic pathways identified in vitro included: hydroxylation, ketone reduction, carboxylation, N-dealkylation, iminium formation, dehydrogenation, N-oxidation, and carbonylation. The top three in vitro metabolic pathways were di-hydroxylation > ketone reduction > γ-lactam formation. Authentic urine specimen analyses revealed the top three metabolic pathways were aliphatic hydroxylation > ketone reduction + aliphatic hydroxylation > aliphatic carboxylation, although the most prominent peak was parent PV8. These data provide useful urinary metabolite targets (aliphatic hydroxylation, aliphatic hydroxylation + ketone reduction, aliphatic carboxylation, and di-hydroxylation) for forensic and clinical testing, and focus reference standard companies’ synthetic efforts to provide commercially available standards needed for PV8 biological specimen testing.
KeywordsPV8 Novel psychoactive substances Metabolic profiling HRMS Hepatocytes Synthetic cathinones
Novel psychoactive substances (NPS), popularly referred to as “designer drugs” or “legal highs,” are ever changing on the drug market. The European Union’s (EU) Early Warning System (EWS) now monitors over 450 NPS, with 182 compounds identified between 2013 and 2014 alone . NPS encompass drugs in various classes, including synthetic stimulants, synthetic cannabinoids, and synthetic opioids . Acute intoxication with synthetic cathinones, a growing class of synthetic stimulants, can result in hyperthermia, agitation, psychosis, seizures, and even death [2, 3, 4, 5, 6, 7]. In the US, the first three popular synthetic cathinones, mephedrone (4-methylmethcathinone), methylone (3,4-methylenedioxymethcathinone), and MDPV (3,4-methylenedioxypyrovalerone), were added to the list of Schedule I Controlled Substances in 2011 . Since then, 10 other cathinones were temporarily registered as Schedule I compounds, including 4-MEC (4-methyl-N-ethylcathinone), α-PBP (α-pyrrolidinobutiophenone), and α-PVP (α-pyrrolidinovalerophenone).
Pharmacodynamic and pharmacokinetic profiles of most NPS are not available and controlled administration studies cannot be conducted without adequate preclinical studies, limiting data available for predicting metabolites for identification and helping to interpret test results . Synthetic cathinones inhibit norepinephrine (NET), dopamine (DAT), and serotonin (SERT) monoamine transporters [14, 15, 16]. MDPV behaves similar to cocaine as a potent DAT/NET transporter blocker [15, 16]. Recently, Marusich et al. examined the in vitro and in vivo pharmacological effects of three pyrrolidinophenones: α-PVP, α-PBP, and α-PPP (α-pyrrolidinopropiophenone) . These pyrrolidinophenones are DAT/NET transporter blockers that increase in potency with increasing carbon chain length (α-PPP < α-PBP < α-PVP) . We hypothesize that PV8 would behave similarly to MDPV and α-PVP in vitro and in vivo. As PV8 possesses a longer aliphatic chain, it could be more potent than α-PVP with a high risk of addiction. However, predicting the toxicity and activity of psychoactive substances is challenging .
Metabolic profiling of NPS is crucial to understanding pharmacokinetic profiles and pharmacodynamic effects. Metabolism of several α-pyrrolidinophenones, including MDPV, α-PVP, α-PBP, α-PPP, 4-methoxy-α-pyrrolidinopropiophenone (MOPPP), 4-methyl-α- pyrrolidinopropiophenone (MPPP), and 4-methyl-α-pyrrolidinohexiophenone (MPHP), were characterized with in vivo rat urine and in vitro human liver microsome (HLM) studies [18, 19, 20, 21, 22, 23]. Zaitsu et al. recently reviewed the pharmacology of pyrrolidinophenones and concluded the prominent biotransformations included ketone reduction and oxidation on the pyrrolidine ring . PV8 metabolism data are not yet available for in silico, in vitro, or in vivo models.
Understanding NPS metabolic profiles is important for identifying unique markers of their intake and understanding potential adverse effects. Since controlled administration studies are unlikely due to the lack of basic toxicity data, metabolism studies are needed to identify PV8 intake markers to incorporate into analytical methods and to be synthesized as reference standards. Incubations with human hepatocytes generate comprehensive metabolism profiles, and analysis via liquid chromatography high-resolution mass spectrometry (LC-HRMS) with software-assisted data mining are useful for metabolite identification [25, 26, 27, 28]. Human hepatocyte incubations are advantageous over microsomes as hepatocytes are capable of producing relevant phase I and phase II metabolites at concentrations comparable to in vivo findings . Analysis by HRMS is useful for structural elucidation of known and unknown metabolites with accurate-mass abilities and the opportunity for retrospective analysis, depending on acquisition method. The study objective was to determine PV8’s metabolic stability with HLM incubations and to identify human metabolites in hepatocyte incubations and HRMS in order to recommend unique intake markers.
Materials and methods
Chemicals and reagents
PV8·HCl was purchased from Cayman Chemicals (Ann Arbor, MI, USA), and water (LC-MS grade), acetonitrile (LC-MS grade), and formic acid from Fisher Scientific (Fair Lawn, NJ, USA). Diclofenac was obtained from Toronto Research Chemicals (Toronto, ON, Canada). Water for sample preparation was passed through an ELGA Purelab Ultra Analytic purifier (Siemens Water Technologies, Lowell, MA, USA).
In silico metabolite prediction
PV8 molecular structure was uploaded in MetaSite software (v. 5.0.3; Molecular Discovery, Pinner, UK), and predictions were generated with the P450 liver model, reactivity correction enabled, and common biotransformations included. Sites of metabolism were predicted based on CYP3A4, CYP2D6, CYP2C9, and flavin-containing monooxygenase 3 (FMO3), taking into consideration kinetic and thermodynamic factors. Predictions included a molecular structure, biodegradation, logD at pH 4, monoisotopic mass, and probability score (max 100 %) for each predicted metabolite. Only metabolites >50 Da were considered in the final summary.
Metabolic stability assessment with human liver microsomes
To determine PV8 metabolic stability, the drug was incubated in duplicate with HLM (50-donor pooled), obtained from BioReclamation IVT (Baltimore, MD, USA) according to previously published studies [27, 30]. Briefly, samples (100 μL) were collected at eight timepoints between 0 and 60 min and the reaction was stopped with ice-cold acetonitrile [27, 30]. For analysis, samples were diluted 1:100 with aqueous mobile phase (water with 0.1 % formic acid) and injected onto a Thermo Scientific Ultimate 3000 RSLCnano system coupled to a Thermo Scientific QExactive mass spectrometer (Thermo Scientific, Fremont, CA, USA) operating under the same source and mass spectrometric settings as previously published [27, 30]. The experiment was prepared in duplicate and injected twice, allowing us to examine reproducibility. Full-scan data-dependent MSMS (ddMS2) data were acquired with the previously described HLM LC-HRMS method [27, 30] from m/z 100 to 600 at 70,000 and 17,500 resolutions for full-scan and MS2 data, respectively.
Metabolic profiling in human hepatocytes
For metabolic profiling, PV8 was incubated with pooled cryopreserved human hepatocytes obtained from Bioreclamation IVT according to previously published studies [27, 30]. Briefly, samples (500 μL) were collected, based on HLM half-life, after 0, 30 and 120 min, and reactions stopped with ice-cold acetonitrile. Diclofenac also was incubated alongside as a positive control to ensure hepatocytes were viable under the given conditions. For analysis, samples were centrifuged to pellet cell debris and then diluted 5-fold with aqueous mobile phase and injected onto the LC-HRMS using the previously described hepatocyte full-scan ddMS2 LC-HRMS method [27, 30] with the same acquisition parameters as described above for HLM samples. A longer linear gradient and longer chromatographic column were utilized compared with HLM LC conditions to ensure optimal chromatographic separation of metabolites with similar accurate mass, and chemical and physical properties with close retention times. MetaSite predicted the formation of in silico metabolites that would elute after PV8, so an extended gradient afforded us the opportunity to capture more metabolites.
Excel was used to calculate microsomal half-life (T1/2) by plotting percent PV8 remaining (natural logarithmic scale) versus time. Formulas published by Baranczewski et al. and McNaney et al. were used to calculate intrinsic clearance (Clint), microsomal intrinsic clearance (Clint, micr), human hepatic clearance (CLhep), and the extraction ratio (ER) [31, 32].
WebMetaBase (v. 2.0.2; Molecular Discovery, Pinner, UK), an automated software extracting exact masses from parent compound and potential metabolites and examining MS/MS spectra to determine possible biotransformations , was used to analyze raw data files generated by the QExactive. Hepatocyte samples (0, 30, and 120 min) were compared with a blank mobile phase and a neat PV8 standard using the processing settings summarized in the Electronic Supplementary Material (ESM) Table S1 and in Ellefsen et al. . Potential metabolites were automatically filtered by mass error (<5 ppm). Each candidate was considered based on MS (exact mass), MS/MS (considering shifted and non-shifted fragments), and retention time. Prominent metabolites were ranked based on relative abundance. Candidates without MS/MS spectrum were not considered as they could not be structurally elucidated and were of low abundance. The software-predicted structures also were examined when elucidating each metabolite and determining the biotransformations. Additionally, potential metabolites were disqualified if present in the t0 control. In silico predictions and the published literature were consulted for final consideration of potential metabolites. HLM data files also were imported into WebMetaBase as a batch with similar processing settings, except that microsomes were designated as the metabolic system with a retention time range of 2–18 min. Metabolite peak areas <0.2 % of PV8 were not included in metabolic analysis.
Authentic urine specimens
Three urine specimens containing PV8, previously screened by a validated LC-quadrupole time-of-flight (Q-TOF) mass spectrometric screening method, were analyzed. The authentic urine specimens evaluated here were forensic urine specimens, not from human experimental investigations. Specimens were aliquoted, anonymized, and de-identified prior to shipment to the laboratory for analysis as part of an official National Institutes of Health Materials Transfer Agreement between the National Board of Forensic Medicine and the National Institute on Drug Abuse and for exemption from ethical review. For identification of metabolites, urine aliquots were extracted via a solid-phase extraction (SPE) method previously reported by Concheiro et al. with minor modifications . A portion of the t = 2 h hepatocyte incubation was extracted to ensure extraction recovery of metabolites, as previously described in Ellefsen et al. and Swortwood et al. [27, 30]. Briefly, hepatocyte samples were diluted with mobile phase and then treated with phosphate buffer prior to extraction. Authentic urine specimens (100 μL) were fortified with internal standard solution and treated with phosphate buffer prior to extraction. Samples were loaded onto preconditioned SPE cartridges (SOLA CX, 10 mg/1 mL; Thermo Scientific, Fremont, CA, USA), eluted with 95:5 dichloromethane:isopropanol (v/v) containing 2 % ammonium hydroxide, acidified, then dried under nitrogen. Urine and hepatocyte samples were reconstituted in aqueous mobile phase. Samples (10 μL) were injected onto the QExactive and analyzed with the same LC-HRMS conditions as hepatocyte incubations (to enable retention time comparisons). Data files were imported into WebMetaBase for analysis. Identified metabolites were matched to hepatocyte metabolites based on accurate mass, retention time, and MS2.
In silico metabolite prediction
In silico predicted metabolites for PV8. Second-generation metabolites of P1-P3 indicated by lowercase a-c. An * indicates detected in vitro
Metabolic stability assessment with human liver microsomes
PV8 exhibited a relatively short in vitro 28.8 ± 2.6 min T1/2 in HLM, with a 24.2 ± 2.2 μL/min/mg Clint, micr, 22.7 mL/min/kg Clint, 10.7 mL/min/kg CLhep, and a 0.53 predicted ER.
Metabolic profiling in human hepatocytes
PV8 metabolites identified after incubation with human hepatocytes, sorted by retention time (RT). Top three fragments (based on signal intensity) are listed with accurate mass. Ranking was based on area
Mass Error (ppm)
Ketone reduction + aliphatic hydroxylation
Pyrrolidine ring opening + hydroxylation
Ketone reduction + carbonylation
Authentic urine specimens
PV8 was the most prominent peak identified in all three urine specimens; no other synthetic cathinones were identified during QTOF screening. In urine, PV8 demonstrated 91 % extraction efficiency with the SPE technique employed, with minimal (8 %) ion suppression. The top five metabolites identified, in decreasing abundance, were M2, M1, M3, an M1-isomer, and M9. The following metabolites also were detected in all three urine specimens: M4, M5, M6, M8, M12, and M14; however, M7, M10, M11, and M13 were not present in sufficient abundance to trigger MS2. Additional hydroxylated metabolites were identified but the hydroxyl position could not be confirmed. An M9 isomer also was identified with significant abundance, with both hydroxyl groups on the alkyl chain. Three phase II metabolites (M2/M4-, M8-, and M11-glucuronide conjugates) were identified in one urine specimen at low abundance. Metabolite extraction recovery, estimated by comparing extracted and unextracted hepatocyte 2 h incubation samples, was generally >51 % but ranged from 6 to 97 %. M9 recovery was only 6 %.
Metabolic profiling in human hepatocytes
Identification of hydroxylated metabolites
Metabolites generated by ketone reduction
The ketone moiety was reduced in M11, M1, and M13 metabolites. M11 only underwent ketone reduction, while M1 was the keto-reduced product of M2 (or M4), as seen in ESM Fig. S1. M11 was the second most prominent metabolite in 0.5 and 2 h hepatocyte incubations. The base peak m/z 244.2057 resulted from water loss. The presence of m/z 173.1197 indicated a fragment with water loss at the β-keto carbon similar to what Ellefsen et al. reported for 4-MeO-α-PVP . The m/z 117.0699 and 131.0856 fragments, instead of m/z 119.0492 and 133.0646 as observed for PV8, also indicated a transformation occurred on this portion of the molecule. Ketone reduction is the major metabolic pathway reported for other α-pyrrolidinophenones in humans [24, 30, 35, 36]. Ketone reduction was underestimated in silico and thus not shown in Table 1. Further hydroxylation of M11 generates M1. M1 fragments m/z 72.0813, 84.0811, and 91.0545 rule out hydroxylation on the pyrrolidine or aromatic rings. The base peak m/z 260.2006 resulted from precursor 278.21146 via water loss. The m/z 173.1197 fragment was identical to that observed in M1; however, the location of the hydroxyl group on the aliphatic chain cannot be determined by mass spectrometry alone.
Metabolites with γ-lactam formation
Two metabolites, M14 and M13 were formed from hydroxylation on the pyrrolidine ring and subsequent dehydrogenation to the corresponding γ-lactam, as seen in ESM Fig. S2. M14 (2ʹʹ-oxo-PV8) and M13, the keto-reduced product of M14, were late eluting compounds, as the presence of the lactam makes it much more difficult for the nitrogen atom to accept protons and become charged. M14, the third most prominent metabolite in hepatocyte incubations, is characterized by the non-shifted m/z 91.0546, 105.0338, and 189.1273 ions, indicating transformation did not occur on the aromatic ring or aliphatic chain. Presence of the m/z 86.0604 and 69.0340 fragments indicate lactam formation, as described by Ellefsen et al. for 4-MeO-α-PVP . M14 also was predicted in silico (P7). M13 was a combination of biotransformations from M14 (lactam) and M11 (ketone reduction). Presence of the lactam was confirmed by the m/z 86.0604 and 98.0604 fragments, whereas the ketone reduction was confirmed by the m/z 117.0700 and 173.1324 ions. Reduction of the keto-moiety increases M13’s polarity, as indicated by the slightly earlier retention time in comparison to M14. Lactam formation with ketone reduction also was reported for α-PVT .
Other identified metabolites
Six additional metabolites were identified in hepatocyte incubations, which demonstrated other biotransformations, including oxidation (M3), dehydrogenation (M7 and M10), N-dealkylation (M5 and M8), and N-oxidation (M12), as seen in ESM Fig. S3. M3 is a proposed carboxylic acid on the aliphatic chain. Conserved m/z 70.0656, 84.0812, 91.0545, and 105.0338 fragments indicated preserved aromatic and pyrrolidine rings. Presence of m/z 184.1331 and 201.0910 ions indicated carboxylation on the aliphatic chain (ESM Fig. S3a). However, a fragment indicating loss of HCOOH was not detected with this acquisition method. M3 was predicted in silico (P2a) as a second-generation metabolite of P2 (M2). M5 is a primary amine resulting from N-dealkylation of the pyrrolidine ring. Presence of m/z 91.0545 and 105.0338 indicated an unchanged aromatic ring, whereas the lack of m/z 70.0656, 72.0813, and 84.0812 fragments indicated loss of the pyrrolidine ring. The m/z 188.1433 peak suggested a water loss. A primary amine also was reported in humans for α-PVP [36, 37], MDPV , 4-MeO-α-PVP after O-demethylation , and mephedrone . M7 is proposed to result from iminium ion formation, as indicated by m/z 152.1433 and 216.1382 ions. Conserved m/z 91.0546, 105.0338, and 189.1144 ions indicate biotransformation did not occur on the aromatic ring or aliphatic chain. Iminium formation also was reported for 4-MeO-α-PVP  and predicted in silico (P6). M8 was the product of pyrrolidine ring opening and hydroxylation. The product ion spectrum is characterized by fragments indicating the loss of two water molecules (m/z 260.2007 and 242.1900). Absence of m/z 70.0656, 72.0813, and 84.0812 ions indicates the pyrrolidine ring is not intact, which is further confirmed by the m/z 73.0653 fragment representing the hydroxylated chain that is lost from the nitrogen during fragmentation. A biotransformation of this type also was reported for 4-MeO-α-PVP . In addition to the iminium ion formation in M7, another dehydrogenated metabolite, M10, was identified. We propose the dehydrogenation occurred on the pyrrolidine ring, as indicated by the m/z 70.0656 and 110.0956 ions. The m/z 91.0545 and 105.0336 ions indicate a conserved aromatic ring. However, the exact location of the dehydrogenation cannot be determined. Lastly, M12 was identified as the result of N-oxidation, similar to 4-MeO-α-PVP . This metabolite eluted after the parent compound, as expected, with fragments at m/z 154.1588, 86.0604, and 105.0338. The lack of m/z 70.0656, 72.0813, and 84.0812 also supports a biotransformation on the pyrrolidine ring.
Metabolite confirmation and profiling in authentic urine specimens
While three minor phase II metabolites were identified in a single urine specimen, no other phase II metabolites were detected in vitro or in vivo. Conjugation increases metabolite polarity in order to increase elimination. However, PV8 is already a polar compound and elimination in urine is apparent as it was detected with high abundance in all three urine specimens. In the hepatocyte incubations, no glucuronidated PV8 metabolites were identified in sufficient abundance, which could be a limitation of the short (2 h) experiment. Diclofenac acyl glucuronide production in the positive control confirms that the hepatocytes were viable to produce phase II metabolites. No phase II metabolites were observed with 4-MeO-α-PVP or α-PVT [27, 30].
Metabolic stability assessment with human liver microsomes
PV8 is predicted to be an intermediate-clearance drug based on T1/2, Clint, and ER. Compounds with 15–40 mL/min/kg Clint and 20–60 min T1/2 are considered as intermediate clearance by McNaney et al. . Additionally, Lavé et al. classifies intermediate-clearance compounds by a 0.3–0.7 ER . Based on these data, we predict PV8 metabolites could be detected in urine for a few days after intake. However, hepatic clearance can vary among individuals, due to enzymatic polymorphisms, or differences in plasma protein binding, drug metabolism/elimination, and hepatic blood flow.
Comparison with other synthetic cathinones
PV8 exhibited metabolic patterns identified in other structurally similar synthetic cathinones. In 2010, MDPV metabolism was characterized in vitro with HLM [21, 37, 40] and in vivo with rat and human urine . Both groups reported demethylenation and subsequent methylation followed by glucuronidation, while Meyer et al. also reported aromatic and aliphatic hydroxylation, oxidation of the pyrrolidine ring to corresponding lactam, and ring opening to corresponding carboxylic acid . α-PVP metabolism was characterized in silico with Meteor software , in vitro with HLM [22, 36, 37], in vivo with rat urine , and in vivo with human urine [35, 36, 41]. The major biotransformations for α-PVP included aliphatic hydroxylation, hydroxylation followed by dehydrogenation to the corresponding lactam, and N-dealkylation to a primary amine [22, 36]. Shima et al. reported ketone reduction with subsequent glucuronidation . Namera et al. reported a time-course of α-PVP and its 2ʹʹ-oxo metabolite after intravenous injection of an unknown amount in a male’s suicide attempt . Urinary metabolites of PV9 (1-phenyl-2-(pyrrolidin-1-yl)octan-1-one), differing from PV8 by one carbon on the alkyl chain included ketone reduction, lactam-formation, aliphatic carboxylation, and hydroxylation + ketone reduction . Metabolic pathways can differ significantly between pyrrolidinophenones because of alkyl chain length . Metabolite identification and structural elucidation are required for each NPS as reference standard manufacturers need direction prior to synthesis of standards for incorporation into analytical methods. Additionally, metabolite structure is useful for future pharmacodynamic and pharmacokinetic studies.
For the first time, PV8 metabolites were identified in HLM, hepatocytes, and urine, and structurally elucidated by LC-HRMS. PV8, a predicted intermediate-clearance drug, exhibited a 28.8 min T1/2 in HLM. Fourteen metabolites were structurally elucidated in vitro and in vivo. Observed metabolic patterns were comparable to other pyrrolidinophenones, including hydroxylation, ketone reduction, lactam formation, and combinations thereof. In addition to parent drug, analytical methods should monitor M2 (aliphatic hydroxylation), M1 (ketone reduction + aliphatic hydroxylation), M3 (aliphatic carboxylation), and M9 (di-hydroxylation) to serve as markers for PV8 intake in biological specimens to assist forensic and clinical investigators. Mapping NPS metabolic pathways is imperative for understanding pharmacological and toxicological significance, as it may lead to a better understanding of adverse effects and improved public health and safety data to educate the public on the dangers of PV8 intake.
The authors thank Tim Moeller of Bioreclamation IVT for his assistance with the hepatocyte incubation, as well as Ismael Zamora and his team at Molecular Discovery for the MetaSite and WebMetabase software. This research was funded by the Intramural Research Program of the National Institute on Drug Abuse, National Institutes of Health.
Conflict of interest
The authors have no conflicts of interest to declare.
- 1.European Monitoring Center for Drugs and Drug Addiction. New psychoactive substances in Europe—an update from the EU Early Warning System. 2015.Google Scholar
- 2.United Nations Office on Drugs and Crime. World Drug Report 2013. Vienna: United Nations; 2013.Google Scholar
- 5.Capriola M. Synthetic cathinone abuse. Clin Pharmacol Adv Appl. 2013;5:109–15.Google Scholar
- 8.Harrigan T. Schedules of controlled substances: temporary placement of 10 synthetic cathinones into Schedule I. Fed Regist. 2014;79(45):12938–43.Google Scholar
- 9.Uchiyama N, Matsuda S, Kawamura M, Shimokawa Y, Kikura-Hanajiri R, Aritake K, et al. Characterization of four new designer drugs, 5-chloro-NNEI, NNEI indazole analog, alpha-PHPP and alpha-POP, with 11 newly distributed designer drugs in illegal products. Forensic Sci Int. 2014;243C:1–13.CrossRefGoogle Scholar
- 10.Roesner P. Designer Drugs Online News: PV8 (1-Phenyl-2-(1-pyrrolidinyl)-1-heptanone). The compound PV8 (1-Phenyl-2-(1-pyrrolidinyl)-1-heptanone) has been found by the Dutch Customs Laboratory. Altenholz: DigiLab Software GmbH; 2014.Google Scholar
- 11.Roesner P. Designer Drugs Online News: PV-8. The compound PV-8 (2-(Pyrrolidin-1-yl)-1phenylheptan-1-one) has been found by the State Office of Criminal. Altenholz: DigiLab Software GmbH.Google Scholar
- 12.Martin T. Report about the number of exhibits containing Emerging Drug compounds from January 2013 to April 2014 analyzed by the Drug Chemistry Branch, USACIL. Personal communication.Google Scholar
- 16.Marusich JA, Antonazzo KR, Wiley JL, Blough BE, Partilla JS, Baumann MH (2014) Pharmacology of novel synthetic stimulants structurally related to the “bath salts” constituent 3,4-methylenedioxypyrovalerone (MDPV). Neuropharmacology. 2014;87:206–13.Google Scholar
- 20.Springer D, Fritschi G, Maurer HH. Metabolism of the new designer drug alpha-pyrrolidinopropiophenone (PPP) and the toxicological detection of PPP and 4'-methyl-alpha-pyrrolidinopropiophenone (MPPP) studied in rat urine using gas chromatography-mass spectrometry. J Chromatogr B. 2003;796(2):253–66.CrossRefGoogle Scholar
- 21.Meyer MR, Du P, Schuster F, Maurer H. Studies on the metabolism of the α-pyrrolidinophenone designer drug methylenedioxy-pyrovalerone (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(12):1426–42.CrossRefGoogle Scholar
- 25.Wohlfarth A, Pang S, Zhu M, Gandhi AS, Scheidweiler KB, Liu HF, et al. First metabolic profile of XLR-11, a novel synthetic cannabinoid, obtained by using human hepatocytes and high-resolution mass spectrometry. Clin Chem. 2013;53(3):423–34.Google Scholar
- 26.Gandhi AS, Zhu M, Pang S, Wohlfarth A, Scheidweiler KB, Liu HF (2013) First characterization of AKB-48 metabolism, a novel synthetic cannabinoid, using human hepatocytes and high-resolution mass spectrometry. AAPS J. 2013;15(4):1091–8.Google Scholar
- 31.Baranczewski P, Stanczak A, Sundberg K, Svensson R, Wallin A, Jansson J, et al. Introduction to in vitro estimation of metabolic stability and drug interactions of new chemical entities in drug discovery and development. Pharmacol Rep. 2006;58(4):453–72.Google Scholar
- 32.McNaney CA, Drexler DM, Hnatyshyn SY, Zvyaga TA, Knipe JO, Belcastro JV, et al. An automated liquid chromatography-mass spectrometry process to determine metabolic stability half-life and intrinsic clearance of drug candidates by substrate depletion. Assay Drug Dev Technol. 2008;6(1):121–9.CrossRefGoogle Scholar
- 33.Backfisch G, Reder-Hilz B, Hoeckels-Messemer J, Angstenberger J, Sydor J, Laplanche L, et al. High-throughput quantitative and qualitative analysis of microsomal incubations by cocktail analysis with an ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometer system. Bioanalysis. 2015;7(6):671–83.CrossRefGoogle Scholar
- 37.Negreira N, Erratico C, Kosjek T, van Nuijs AL, Heath E, Neels H, et al. In vitro phase I and phase II metabolism of alpha-pyrrolidinovalerophenone (alpha-PVP), methylenedioxypyrovalerone (MDPV) and methedrone by human liver microsomes and human liver cytosol. Anal Bioanal Chem. 2015;407(19):5803–16.CrossRefGoogle Scholar
- 38.Meyer MR, Wilhelm J, Peters FT, Maurer HH. Beta-keto amphetamines: studies on the metabolism of the designer drug mephedrone and toxicological detection of mephedrone, butylone, and methylone in urine using gas chromatography-mass spectrometry. Anal Bioanal Chem. 2010;397(3):1225–33.CrossRefGoogle Scholar
- 40.Strano-Rossi S, Cadwallader AB, de la Torre X, Botre F. Toxicological determination and in vitro metabolism of the designer drug methylenedioxypyrovalerone (MDPV) by gas chromatography/mass spectrometry and liquid chromatography/quadrupole time-of-flight mass spectrometry. Rapid Commun Mass Spectrom. 2010;24(18):2706–14.CrossRefGoogle Scholar