Analytical and Bioanalytical Chemistry

, Volume 408, Issue 18, pp 4845–4856 | Cite as

First metabolic profile of PV8, a novel synthetic cathinone, in human hepatocytes and urine by high-resolution mass spectrometry

  • Madeleine J. Swortwood
  • Kayla N. Ellefsen
  • Ariane Wohlfarth
  • Xingxing Diao
  • Marta Concheiro-Guisan
  • Robert Kronstrand
  • Marilyn A. Huestis
Paper in Forefront

Abstract

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.

Graphical Abstract

Top four PV8 metabolites identified in vitro. Biotransformations highlighted in blue. Markush structures presented when exact location of biotransformation is unknown

Keywords

PV8 Novel psychoactive substances Metabolic profiling HRMS Hepatocytes Synthetic cathinones 

Introduction

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 [1]. NPS encompass drugs in various classes, including synthetic stimulants, synthetic cannabinoids, and synthetic opioids [2]. 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 [8]. 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).

In 2013, a new Research Chemical, 1-phenyl-2-(1-pyrrolidinyl)-1-heptanone or PV8, was introduced as a “MDPV replacement,” appearing on several online drug forums. That same year, PV8 was detected in illicit products obtained in Japan [9], and in products analyzed by the Dutch Customs Laboratory [10] and State Office of Criminal Investigations Kiel, Germany in 2014 [11]. In December 2013, the Drug Chemistry Branch, US Army Criminal Investigation Laboratory (USACIL) identified PV8 in four seized samples [12]. PV8, as seen in Fig. 1, is a cathinone-analog closely resembling MDPV and α-PVP. The only available PV8 user reports are described online in popular drug forums, including drugs-forum.com, reddit.com, and bluelight.org, and suggest similar potency to α-PVT (α-pyrrolidinopentiothiophenone) and α-PVP. Users detail their reports with dosages (50–200 mg), routes of administration (insufflation), side effects (desired: euphoria, stimulation; undesired: paranoia, insomnia), and duration of side effects (6 h). Unfortunately, anecdotal self-reports are questionable, as users cannot know what drugs they actually are consuming.
Fig. 1

Structure of PV8 and structurally related Schedule I Controlled Substances, MDPV and α-PVP

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 [13]. 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) [16]. These pyrrolidinophenones are DAT/NET transporter blockers that increase in potency with increasing carbon chain length (α-PPP < α-PBP < α-PVP) [16]. 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 [17].

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 [24]. 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 [29]. 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.

Calculations

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].

Metabolite identification

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 [33], 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. [27]. 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 [34]. 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.

Results

In silico metabolite prediction

MetaSite predicted 17 potential metabolites; eight candidates with scores >20 % are summarized in Table 1, in order of decreasing score and probability. The top three metabolites generated in silico were hydroxylated metabolites (aliphatic and aromatic). Additionally, N-dealkylation, iminium formation, and carboxylation were predicted to be >20 % (Table 1). Metabolites with predicted scores <20 % included hydroxylation, N-dealkylation, dehydrogenation, and ketone reduction (structures not shown). The top three predicted metabolites (hydroxylated products) were reprocessed to predict second generation oxidative and hydroxylated metabolites (Table 1).
Table 1

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

Diclofenac acyl glucuronide and 4′-hydroxydiclofenac were identified in the diclofenac hepatocyte incubations, demonstrating hepatocyte viability. Fourteen PV8 metabolites (Table 2) were identified with mass accuracy <2.3 ppm. Metabolites eluted between 7.3 and 16 min, with PV8 eluting at 10.5 min. Metabolites resulted from hydroxylation (M1, M2, M4, M6, M8, and M9), ketone reduction (M1, M11, and M13), carboxylation (M3), N-dealkylation (M5, M8), iminium formation (M7), dehydrogenation (M10), N-oxidation (M12), and carbonylation (M13, M14). Several glucuronides also resulted but intensities were not sufficient to trigger MS2, so structures could not be confirmed. The main sites of metabolism were the aliphatic chain, pyrrolidine ring, and β-ketone moiety. Hydroxylation preferably occurred on the aliphatic chain and pyrrolidine ring. The top three metabolites identified in hepatocyte incubations resulted from hydroxylation (M9), ketone reduction (M11), and γ-lactam formation (M14).
Table 2

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

ID

Metabolic reaction

RT (min)

Elemental composition

m/z

Mass Error (ppm)

Characteristic fragments

Area

Ranking

0.5 h

2 h

0.5 h

2 h

M1

Ketone reduction + aliphatic hydroxylation

7.35

C17H27NO2

278.21094

1.85

260.20062

173.11974

91.05457

1.5e6

1.6e7

14

9

M2

Aliphatic hydroxylation

7.55

C17H25NO2

276.19562

0.67

131.04924

91.05459

105.03381

5.5e7

9.0e7

4

4

M3

Aliphatic carboxylation

7.61

C17H23NO3

290.17480

0.93

91.05458

184.13319

157.06461

6.3e6

3.8e7

9

7

M4

Aliphatic hydroxylation

7.72

C17H25NO2

276.19565

0.56

131.04926

105.03380

91.05460

3.2e7

6.1e7

7

6

M5

N-dealkylation

9.53

C13H19NO

206.15347

2.28

91.05459

118.06524

188.14334

6.2e6

9.1e6

10

14

M6

Hydroxylation

9.76

C17H25NO2

276.19568

0.45

91.05460

105.03383

170.15382

4.4e7

7.6e7

6

5

M7

Iminium formation

9.83

C17H24NO

258.18518

-1.89

91.05463

70.06571

105.03385

5.0e6

9.9e6

12

13

M8

Pyrrolidine ring opening + hydroxylation

9.90

C17H27NO2

278.21146

-0.01

91.05461

188.14345

118.06529

5.3e7

3.0e7

5

8

M9

Di-hydroxylation

10.10

C17H25NO3

292.19034

1.30

91.05456

188.14334

118.06520

4.1e8

1.0e9

1

1

M10

Dehydrogenation

10.75

C17H23NO

258.18475

1.90

105.03366

70.06562

91.05450

2.9e7

1.1e7

8

11

M11

Ketone reduction

10.78

C17H27NO

262.21643

0.42

244.20578

173.11978

72.08134

1.8e8

6.4e8

2

2

M12

N-oxidation

11.21

C17H25NO2

276.19559

0.78

154.15889

86.06047

105.03382

5.0e6

9.3e6

11

13

M13

Ketone reduction + carbonylation

13.80

C17H25NO2

276.19540

1.47

117.0700

91.05459

98.06044

4.1e6

1.2e7

13

10

M14

Carbonylation

16.04

C17H23NO2

274.18011

0.16

91.05460

86.06048

105.03381

9.1e7

1.2e8

3

3

 

Parent

10.56

C17H25NO

260.20068

0.81

91.05460

105.03380

154.15886

5.9e9

3.8e9

-

-

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 %.

Discussion

The MS2 spectrum of PV8, with structures and proposed fragmentation patterns (PV8, [M+H]+, m/z 260.2006), is shown in Fig. 2. The most intense fragment ion, m/z 91.0546, was consistent with the tropylium ion. Cleavage between C1ʹ (β-keto carbon) and C2ʹ bond generated the next highest intensity benzoylium and pyrroloimmonium ions at m/z 105.0338 and 154.1588, respectively. Cleavage between the nitrogen and C2ʹ resulted in m/z 70.0656 and then 119.0492 after an additional cleavage between C2ʹ and C3ʹ. This is the first PV8 electrospray ionization MS2 spectrum reported in the literature.
Fig. 2

Product ion spectrum of PV8 with assigned fragmentation pattern and labeling of metabolic sites as used throughout the text

Metabolic profiling in human hepatocytes

Identification of hydroxylated metabolites

Monohydroxylations on the aliphatic chain (M2 and M4) and on the pyrrolidine ring (M6) and one di-hydroxylated product (M9) were identified in hepatocyte incubations. Fig. 3 highlights the MS2 spectra for these four hydroxylated metabolites. M2 and M4 hydroxylations were predicted on the aliphatic chain, as m/z 72.0813, 91.0545, and 131.0492 fragments were unchanged, ruling out hydroxylation on the aromatic or pyrrolidine rings. M2 and M4 had m/z 170.1538 ions, indicating a +15.9949 on the pyrroloimmonium ion. Without reference standards or more specific mass spectral data, the presence of the hydroxyl group cannot be determined; hence, Markush structures in the figure. However, we hypothesize that M2 is hydroxylated on C7ʹ and M4 on C6ʹ, based on logD from the in silico software. M2 and M4 were both predicted in silico (P2 and P1). Unlike M2 and M4, M6 conserved m/z 91.0546, 105.0338, 119.0491, and 189.1274 fragments, eliminating hydroxylation on the aromatic ring or aliphatic chain, suggesting pyrrolidine hydroxylation. Presence of m/z 86.0604 also indicates hydroxylation on the pyrrolidine ring, which is further confirmed by m/z 170.1538 and non-shifted m/z 189.1274 fragments. The exact hydroxyl location on the pyrrolidine ring cannot be confirmed; however, a recent report by Shima et al. determined a 2ʹʹ-OH-α-PVP metabolite [35], and we hypothesize a similar PV8 metabolite. M9, the most prominent metabolite identified in 0.5 and 2 h hepatocyte incubations, was di-hydroxylated, most likely on the aliphatic chain and/or pyrrolidine ring as m/z 91.0546 and 105.0338 ions rule out hydroxylation on the aromatic ring. However, exact locations of the hydroxyl groups cannot be determined (Fig. 3).
Fig. 3

Product ion mass spectra and assigned fragmentation patterns for hydroxylated PV8 metabolites. (a) Aliphatic hydroxylation, M2; (b) aliphatic hydroxylation, M4; (c) hydroxylation on pyrrolidine ring, M6; (d) di-hydroxylation, M9. Markush structures presented when exact location of biotransformation is unknown

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 [27]. 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 [27]. 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 [30].

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 [37], 4-MeO-α-PVP after O-demethylation [27], and mephedrone [38]. 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 [27] 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 [27]. 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 [27]. 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

PV8 and 10 of 14 metabolites identified in hepatocyte incubations were identified in all three urine specimens and confirmed by accurate mass, retention time, and MS2. Four metabolites generated in vitro were not confirmed in vivo because of low signal intensity. The top metabolite in hepatocytes (M9) was detected in the top five urine metabolites, in addition to M2, M1, M3, and an M1-isomer. An M9-isomer also was detected with high abundance and was not unexpected as hydroxylation is the major biotransformation observed and PV8 has a long alkyl chain. It is important to note that the parent was the most abundant compound detected in all three urine specimens. Most likely, longer hepatocyte incubation time could have produced metabolite abundance more similar to urine. Time since intake is unknown for the authentic specimens. However, identification of metabolites is still important for extending windows of detection and understanding their contribution to potential toxicity. In addition to PV8, the most prominent metabolites to monitor would be M1, M2, M3, and M9, as seen in Fig. 4.
Fig. 4

Proposed metabolic pathways for PV8 in human hepatocytes. Markush structures presented when exact location of biotransformation is unknown. Bold arrows indicate major pathways

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. [32]. Additionally, Lavé et al. classifies intermediate-clearance compounds by a 0.3–0.7 ER [39]. 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 [21]. 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 [21]. α-PVP metabolism was characterized in silico with Meteor software [36], in vitro with HLM [22, 36, 37], in vivo with rat urine [22], 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 [35]. 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 [41]. 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 [42]. Metabolic pathways can differ significantly between pyrrolidinophenones because of alkyl chain length [42]. 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.

Conclusions

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.

Notes

Acknowledgments

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.

Supplementary material

216_2016_9599_MOESM1_ESM.pdf (823 kb)
ESM 1(PDF 822 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg (outside the USA) 2016

Authors and Affiliations

  • Madeleine J. Swortwood
    • 1
  • Kayla N. Ellefsen
    • 1
    • 2
  • Ariane Wohlfarth
    • 1
    • 3
    • 4
  • Xingxing Diao
    • 1
  • Marta Concheiro-Guisan
    • 1
    • 5
  • Robert Kronstrand
    • 3
    • 4
  • Marilyn A. Huestis
    • 1
    • 6
  1. 1.Chemistry and Drug Metabolism, Intramural Research Program, National Institute on Drug AbuseNational Institutes of HealthBaltimoreUSA
  2. 2.Program in ToxicologyUniversity of Maryland BaltimoreBaltimoreUSA
  3. 3.Department of Forensic Genetics and Forensic ToxicologyNational Board of Forensic MedicineLinköpingSweden
  4. 4.Department of Medical and Health Sciences, Faculty of Health SciencesLinköping UniversityLinköpingSweden
  5. 5.Department of Sciences, John Jay College of Criminal JusticeCity University of New YorkNew YorkUSA
  6. 6.University of Maryland Baltimore School of MedicineSeverna ParkUSA

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