Introduction
============

The new psychoactive substances (NPS) 3-methoxyphen-cyclidine (3-MeO-PCP, Fig. **[1a](#F1){ref-type="fig"}**) and 3-methoxyrolicyclidine (3-MeO-PCPy, Fig. **[1b](#F1){ref-type="fig"}**) are derivatives of phencyclidine (PCP, Fig. **[1c](#F1){ref-type="fig"}**), which is known for over 60 years. As reviewed by Morris and Wallach \[[@r1]\], PCP was synthesized first in 1956 and approved in the following years as a non-narcotic anesthetic in veterinary medicine under the trade names Sernyl or Sernylan. The use for human treatment was short-lived and limited due to unfavorable side effects such as agitation, hallucination, and delirium-like conditions. In 1967, phencyclidine appeared as a street drug in the USA under names such as "Peace Pill", "Angle Dust", "Blue Dust", or "Killer Weed", and in Germany, recreational use appeared in 1977. To overcome national narcotic laws, the structure of phencyclidine was continuously modified and included modifications of the amine moiety, the phenyl, or the piperidine ring \[[@r2], [@r3]\]. Since the derivative phenylcyclohexylpyrrolidine (also known as rolicyclidine or PCPy, Fig. **[1d](#F1){ref-type="fig"}**) has been scheduled in the USA in the 1970s, other modifications were encountered that included alkoxy derivatives \[[@r1], [@r4]\]. According to reports from the European Monitoring Center for Drugs and Drug Addiction (EMCDDA), 3-MeO-PCP \[[@r5]\] and 4-MeO-PCP have been identified in 2012 and 2011 as NPS, respectively \[[@r6], [@r7]\]. 3-MeO-PCP and 3-MeO-PCPy are usually snorted, smoked as a PCP-laced marijuana cigarette, ingested as a tablet, or injected intravenously or subcutaneously \[[@r1]\]. Effects such as euphoria and analgesia might share some similarities with PCP \[[@r8], [@r9]\] although some differences might also exist regarding their psychopharmacological profile \[[@r1]\]. Mechanisms of action include uncompetitive antagonism of the excitatory *N*-methyl-D-aspartate (NMDA) receptor and reuptake inhibition of noradrenaline, serotonin, and dopamine \[[@r10], [@r11]\].

However, a current challenge is that such NPS cannot be detected reliably in toxicological urine screening procedures as excretion products are usually unknown. Therefore, the aims of our study were the elucidation of the phase I metabolism of 3-MeO-PCP and 3-MeO-PCPy in rat and human liver microsomes using high resolution LC-MS^n^ (LC-HR-MS^n^), the identification of the involved CYP isoenzymes, and the investigation of the detectability of common users' doses in standard urine screening approaches (SUSA) using gas chromatography-mass spectrometry (GC-MS), liquid chromatography-multi-stage mass spectrometry (LC-MS^n^), and liquid chromatography-high-resolution tandem mass spectrometry (LC-HR-MS/MS).

Experimantal procedures
=======================

Chemicals and Reagents
----------------------

3-MeO-PCP and 3-MeO-PCPy were synthesized and characterized as reported previously \[[@r12]\]. NADP^+^ and isocitrate dehydrogenase were obtained from Biomol (Hamburg, Germany), Isolute Confirm HCX cartridges (130 mg, 3 mL) from Biotage (Uppsala, Sweden), ammonium sulfate from Fluka (Buches, Switzerland), acetonitrile (LC-MS grade), ammonium formate (analytical grade), dichloromethane, diethyl ether, formic acid (LC-MS grade), isocitrate, magnesium chloride, pyridine, superoxide dismutase, sodium hydroxide, and Tris buffer from Sigma-Aldrich (Taufkirchen, Germany), acetic anhydride, acetic acid, ethyl acetate, glucuronidase/arylsulfatase, hydrochloric acid (37%), isopropanol, methanol (LC-MS grade), aqueous ammonia (32%), and sodium dihydro-gen phosphate and all other chemicals and biochemicals from VWR (Darmstadt, Germany). The baculovirus-infected insect cell microsomes (Supersomes), containing 1 nmol/mL of human cDNA-expressed CYP 1A2, CYP 2A6, CYP 2B6, CYP 2C8, CYP 2C9, CYP 2C19, CYP 2D6, or 2 nmol/mL CYP 2E1, CYP 3A4, CYP 3A5, and pooled human liver microsomes (pHLM, 20 mg microsomal protein/mL, 400 pmol total CYP/mg protein), were obtained from BD Biosciences (Heidelberg, Germany). After delivery, the microsomes were thawed at 37°C, aliquoted, snap-frozen in liquid nitrogen, and stored at -80°C until use.

Urine Samples
-------------

Studies were performed using rat urine samples from male Wistar rats (Charles River, Sulzfeld, Germany) for toxicological diagnostic reasons according to the corresponding German law. The compounds were administered once in an aqueous suspension by gastric intubation in a dose of 10 mg/kg body mass (BM) for the identification of the metabolites and once in a 1 mg/kg BM representing common users' doses, calculated and downscaled using an allometric dose-by-factor approach described by Sharma and McNeill \[[@r13]\].

The rats were housed in metabolism cages for 24 h, having water ad libitum. Urine was collected separately from feces over a 24-h period. Blank urine samples were collected before drug administration to confirm the absence of interfering compounds. The samples were directly analyzed and then stored at -20°C.

Sample Preparation for Identification of Phase I and II Metabolites by LC-HR-MS^n^
----------------------------------------------------------------------------------

The first of two preparations of high dosed rat urine samples was performed in analogy to a published procedure \[[@r14]\]. A 2-mL aliquot of urine sample was adjusted to pH 5.2 with 1 M acetic acid, a 50-µL aliquot of a mixture (100,000 Fishman units per mL) of glucuronidase (EC No. 3.2.1.31) and arylsulfatase (EC No. 3.1.6.1) from Helix pomatia L was added, and subsequently incubated at 50°C for 2 h. Afterwards, the sample was centrifuged at 5,000 rpm for 5 min, the supernatant loaded onto a cation exchange Confirm HCX solid phase extraction (SPE) cartridge, previously conditioned with 1 mL methanol and 1 mL water. After passing the SPE cartridge, washing steps were performed with 1 mL water, 1 mL 0.01 M hydrochloric acid, 1 mL water, and 1 mL methanol. The retained basic compounds were eluted using 1 mL of a freshly prepared mixture of methanol/32% aqueous ammonia (98:2, *v/v*). The eluate was subsequently evaporated to dryness under a stream of nitrogen at 70°C and reconstituted with 50 µL of methanol. A 10-µL aliquot was injected for LC-HR-MS^n^ analysis. Conditions were as described below.

The second sample preparation was performed according to Wissenbach *et al*. \[[@r15]\]. A 100-µL aliquot of the high dosed rat urine sample was mixed with 500 µL of acetonitrile and vortexed for 2 min. Afterwards, the sample was centrifuged for 2 min at 10,000 *g*, the supernatant evaporated to dryness under a gentle stream of nitrogen at 70°C, and the residue reconstituted with a mixture of mobile phase A and B (1:1, *v/v*) described below for LC-MS^n^. A 5-µL aliquot was injected onto the LC-HR-MS^n^ system. Conditions were as described below.

Microsomal Incubations for pHLM and Initial CYP Activity Screening Studies
--------------------------------------------------------------------------

The incubations were performed according to a published procedure \[[@r14]\] using CYP 1A2, CYP 2A6, CYP 2B6, CYP 2C8, CYP 2C9, CYP 2C19, CYP 2D6, CYP 2E1, CYP 3A4, CYP 3A5, or pHLM for 30 min at 37°C. The final incubation mixture consisted of 90 mM buffer (Tris buffer for CYP 2A6 and CYP 2C9, phosphate buffer for all other incubations), 25 µM drug as substrate, 200 U/mL superoxide dismutase, 75 pmol/mL CYP isoenzyme, and regenerating system consisted of 5 mM magnesium chloride, 5 mM isocitrate, 1.5 mM NADP^+^, and 0.5 U/mL isocitrate dehydro-genase in a final volume of 50 µL. Reaction was initiated by addition of regenerating system and stopped with 50 µL ice-cold acetonitrile. Afterwards, the sample was centrifuged for 5 min at 10,000 *g*, the supernatant transferred to a glass vial, and a 10-µL aliquot was injected onto the LC-MS^n^ system. Conditions were as described below.

Identification of Phase I and II Metabolites in Urine by LC-HR-MS^n^
--------------------------------------------------------------------

Phase I and II metabolites in urine were identified by a system consisted of an Orbitrap Velos Pro (Thermo Fischer Scientific, Dreieich, Germany) equipped with a heated electrospray ionization (HESI)-II source coupled to a Dionex Ultimate 3000 LC system. The used conditions and gradient elution were according to a published procedure \[[@r14]\], gradient program was composed according to Wissenbach *et al*. \[[@r15]\]. A parent mass list was used containing proposed metabolites whereby the list was divided into two separate experiments to reduce the amount of preferred ions for monitoring. The system was running Thermo Scientific Xcalibur 2.2.

Analysis of the Metabolites in Microsomal Incubations by LC-MS^n^
-----------------------------------------------------------------

LC-MS^n^ analysis of the metabolites in microsomal incubations was performed according to a published procedure \[[@r14]\]. The system consisted of a LXQ linear ion trap MS equipped with an HESI-II source coupled to an Accela LC system consisting of a degasser, a quaternary pump, and an autosampler (all from ThermoFisher Scientific, Dreieich, Germany). The gradient program was composed according to Wissenbach *et al*. \[[@r15]\] and the same as described for LC-HR-MS^n^. The system was running Thermo Scientific Xcalibur 2.0.7.

GC-MS SUSA
==========

According to a published procedure \[[@r16]\], a 5-mL aliquot of the low dosed rat urine sample was divided into two aliquots, the first was submitted to acid hydrolysis for 15 min and basified to pH 9, followed by combining with the second and subsequent extraction with a dichloromethane-isopropanol-ethyl acetate mixture (1:1:3, *v/v/v*). After evaporation, the residue was acetylated with an acetic anhydride-pyridine mixture (3:2, *v/v*) under microwave irradiation, again evaporated and reconstituted in 100 µL of methanol. A 1-µL aliquot was injected onto the GC-MS system, consisted of a Hewlett-Packard (HP; Agilent, Waldbronn, Germany) 5890 Series II gas chromatograph combined with an Agilent 5972 MSD mass spectrometer running HP MS ChemStation (DOS series) B.02.05. GC and MS conditions were according to a published procedure \[[@r16]\].

The full scan data files were evaluated by use of the automated mass spectral deconvolution and identification system (AMDIS, <http://chemdata.nist.gov/mass-spc/amdis/>) in simple mode. The target library was a modified version of the Maurer/Pfleger/Weber MPW_2016 library \[[@r17]\]. The deconvolution settings were according to Meyer *et al*. \[[@r18]\].

LC-MSN SUSA
===========

In accordance to Wissenbach *et al*. \[[@r19]\], a 100-µL aliquot of the low dosed urine sample was worked up as described for the identification of phase I and II metabolites in urine. A 10-µL aliquot was injected onto the LC-MS^n^ system. Conditions were as described for analysis of the metabolites in microsomal incubations.

For data acquisition, ThermoFisher ToxID 2.1.1 for automatic target screening in the MS^2^ screening mode was used. The settings were according to Wissenbach *et al*. \[[@r19]\]. ToxID was run automatically after file acquisition by using an Xcalibur processing method starting the software tool. The target library was a modified version of the Maurer/Wissenbach/Weber MWW_2014 library \[[@r20]\].

LC-HR-MS/MS SUSA
================

In accordance to Helfer *et al*. \[[@r21]\], a 100-µL aliquot of the low dosed rat urine sample was mixed with 500 µL of acetonitrile and vortexed for 2 min. After centrifugation for 2 min at 10,000 *g*, the supernatant was evaporated to dryness under a gentle stream of nitrogen at 70°C and the residue reconstituted with a mixture of mobile phase A and B (1:1, *v/v*) described below. A 10-µL aliquot was injected onto the LC-HR-MS/MS system, consisted of a Q-Exactive system equipped with an HESI-II source (ThermoFisher Scientific, Dreieich, Germany) coupled to an Accela LC system, consisting of a degasser, a quaternary pump, and an HTC PAL auto-sampler (CTC Analytics AG, Zwingen, Switzerland). Gradient elution and MS conditions were according to a published procedure \[[@r21]\]. The system was running Thermo Scientific Xcalibur 3.0.63. For data acquisition, ThermoFisher TraceFinder Clinical Research 3.2 software was used as described by Helfer *et al*. \[[@r21]\].

Determination of the Detection Limits of the Parent Drugs in Urine for SUSA
---------------------------------------------------------------------------

For assessing the general performance of the SUSA, the determination of the limits of detection (LOD) for the parent drugs were determined \[[@r22]\]. For this purpose, 3-MeO-PCP and 3-MeO-PCPy were spiked in rat urine in increasing concentrations, respectively, and analyzed *via* GC-MS, LC-MS^n^, and LC-HR-MS/MS SUSA. The concentration level with a signal-to-noise of three was defined as LOD.

Results and discussion
======================

Identification of Phase I and II Metabolites by LC-HR-MS^n^
-----------------------------------------------------------

With the SPE-based sample preparation, only basic compounds were retained leading to cleaner extracts. For the non-basic compounds and the phase II metabolites, the second sample preparation was performed after simple precipitation.

The proposed structural formulas were deduced by comparing the spectra of the metabolites with those of the parent compounds. Precursor masses (PM) are from MS^1^ spectra, fragment ions from MS^2^ spectra. All masses are given with the calculated exact masses. The phase I metabolites found for 3-MeO-PCP are shown in Table **[1](#T1){ref-type="table"}**, those for 3-MeO-PCPy in Table **[2](#T2){ref-type="table"}**. The phase II metabolites found for 3-MeO-PCP are shown in Table **[3](#T3){ref-type="table"}**, those for 3-MeO-PCPy in Table **[4](#T4){ref-type="table"}**. Overall, for 3-MeO-PCP, 30 phase I and seven phase II metabolites, and for 3-MeO-PCPy, 26 phase I and eight phase II metabolites were detected.

Proposed Fragmentation Patterns for Identification of the Phase I and II Metabolites by LC-HR-MS^n^
---------------------------------------------------------------------------------------------------

### 3-MeO-PCP

In general, the spectrum of the parent compound (no. 1 in Table **[1](#T1){ref-type="table"}**) revealed a characteristic fragmentation pattern, whereby a benzylic cleavage led to a 3-methoxy-phenyl-cyclohexyl fragment represented by the fragment ion of *m/z* 189.1279 and a piperidine fragment represented by the fragment ion of *m/z* 86.0967. In cases where the 3-methoxy-phenylcyclohexyl fragment was unchanged, fragment ions could be observed at *m/z* 189.1279 (nos. 6, 13, 20, and 30), if monohydroxylated at *m/z* 205.1229 (nos. 7, 14, 15, and 24-29), if *O*-demethylated at *m/z* 175.1123 (nos. 2, 3, 8-10, and 16), and if hydroxylated and *O*-demethylated at *m/z* 191.1072 (nos. 4, 5, 11, 12, 17-19, and 21-23). In cases where the piperidine fragment was unchanged, fragment ions could be observed at *m/z* 86.0970 (nos. 2, 4, and 7), if monohydro-xylated at *m/z* 102.0919 (nos. 3, 6, 11, 12, 14, and 15), and if dihydroxylated at *m/z* 118.0868 (nos. 8-10, 13, 17-19, and 24). Aliphatic hydroxylations were proposed by the elimination of water resulting in unsaturated fragments at *m/z* 187.1123 (205.1229 -- 18.0100 u; nos. 7, 14, 15, and 24-29), 173.0966 (191.1072 -- 18.0100 u; nos. 4, 5, 11, 12, 17, 19, and 21-23), and 84.0813 (102.0919 -- 18.0100 u; nos. 3, 6, 11, 12, and 14).

A metabolite with PM of *m/z* 260.2014 revealed a spectrum of an *O-*demethyl metabolite (no. 2) with a fragment ion of *m/z* 175.1123. Two hydroxy metabolites were revealed by PM of *m/z* 290.2120. One isomer revealed a piperidine-hydroxy metabolite (no. 6) and one a cyclohexyl-hydroxy metabolite (no. 7). Four dihydroxy metabolites were revealed by PM of *m/z* 306.2069, one piperidine-dihydroxy (no. 13) and two isomeric cyclohexyl-hydroxy piperidine-hydroxy metabolites (nos. 14 and 15). One trihydroxy metabolite was revealed by PM of *m/z* 322.2018 representing a cyclohexyl-hydroxy piperidine-dihydroxy metabolite (no. 24). In addition, a hydroxylation at the α-position to the amine at the piperidine ring would led to a rather instable hemiaminal species followed by ring opening and oxidation to an aliphatic carboxy metabolite. This mechanism had already been described for PCP \[[@r23]\] and for diverse PCP derivatives by Sauer *et al*. \[[@r24]-[@r29]\]. The observed corresponding metabolites were a carboxy metabolite (no. 20) with PM of *m/z* 320.1862, five isomeric methyl artifacts of carboxy cyclohexyl-hydroxy metabolites (nos. 25-29) with PM of *m/z* 336.2175, and one carboxy alkyl-hydroxy metabolite (no. 30) with PM of *m/z* 336.2175. For metabolite no. 7, only one isomer was detected most probably due to the lower formation rate in contrast to the metabolites nos. 25-29. Moreover, in combination with *O-*demethylations ring opened methyl artifacts of carboxy metabolites could be observed with PM of *m/z* 306.2069 (no. 16) and after additional cyclohexyl-hydroxylation (methyl artifact) with PM of *m/z* 322.2018 (nos. 21-23). The existence of some carboxy metabolites was proposed after finding artificially formed methyl ester structures. One explanation for these formations could be a methylation of the carboxy group and nucleophilic attack during the work-up procedure, where the samples were evaporated in methanol at 70°C. Further confirmation was obtained when methanol was replaced with ethanol, leading to ethyl ester structures giving spectra with methylene shifts of the corresponding fragment ions.

Combinations of *O-*demethylation and hydroxylation could also be proposed. Two *O*-demethyl hydroxy isomers occurred with PM of *m/z* 276.1964, in particular one piperidine- (no. 3) and one cyclohexyl-hydroxy metabolite (no. 4), and five *O*-demethyl dihydroxy metabolites with PM of *m/z* 292.1913. Three isomers were observed with both hydroxy groups at the piperidine ring (nos. 8-10) and two isomers with one hydroxy group at the piperidine and the cyclohexyl ring, respectively (nos. 11 and 12). Spectra with PM of *m/z* 308.1862 were revealed after threefold hydroxylation, represented by three *O*-demethyl cyclohexyl-hydroxy piperidine-dihydroxy metabolites (nos. 17-19). One product of the metabolites nos. 8-10 after oxidation and elimination of water could be observed with PM of *m/z* 288.1600 (no. 5).

Concerning phase II metabolism, seven glucuronides were found (nos. 57-63 in Table **[3](#T3){ref-type="table"}**). These conjugates were identified by the PM shift of +176.0322 u. The fragment ions were identical to those of the underlying phase I metabolites. One of these glucuronides was postulated as *O*-demethyl aryl-hydroxy glucuronide (no. 58). The corresponding phase I metabolite could not be detected, either due to low concentrations or insufficient conjugate cleavage during work-up procedure for this particular metabolite. The position in the aromatic ring system could be confirmed with performed LC-HR-MS/MS analysis, revealing a spectrum with an additional fragment ion of *m/z* 123.0440 (C~7~H~7~O~2~), representing a dihydroxy tropylium ion.

### 3-MeO-PCPy

The parent compound spectrum (no. 31 in Table **[2](#T2){ref-type="table"}**) showed a characteristic fragmentation pattern analogous to 3-MeO-PCP. The 3-methoxy-phenyl-cyclohexyl fragment was represented by the fragment ion of *m/z* 189.1279, but the corresponding pyrrolidine fragment at *m/z* 72.0813 was not detected. In analogy to 3-MeO-PCP, an unchanged 3-methoxy-phenyl-cyclohexyl fragment was represented at *m/z* 189.1279 (nos. 37, 39, 44, and 51), after monohydroxylation at *m/z* 205.1229 (nos. 45, 46, and 52-55), after *O*-demethylation at *m/z* 175.1123 (nos. 32, 33, 38, and 40), and after hydro-xylation and *O*-demethylation at *m/z* 191.1072 (nos. 34-36, 41-43, and 47-49). Although the pyrrolidine fragment was not detectable in the spectrum of the unchanged molecule, the corresponding fragments were observed in the spectra of the metabolites. When the pyrrolidine fragment was unchanged, fragment ions could be observed at *m/z* 72.0813 (no. 32), if monohydroxylated at *m/z* 88.0762 (nos. 33 and 37), and if dihydroxylated at *m/z* 104.0712 (nos. 38, 39, 41-43, 45, and 46). Again, an aliphatic hydroxylation was proposed if water elimination was observed. These fragments were represented at *m/z* 187.1123 (205.1229 -- 18.0100 u; nos. 45, 46, and 52-55), 173.0966 (191.1072 -- 18.0100 u; nos. 34-36, 41-43, and 47-50), and 70.0657 (88.0762 -- 18.0100 u; nos. 33 and 37).

A metabolite with PM of *m/z* 246.1858 revealed an *O-*demethyl metabolite (no. 32). One pyrrolidine-hydroxy metabolite was revealed by PM of *m/z* 276.1958 (no. 37), one pyrrolidine-dihydroxy metabolite by PM of *m/z* 292.1913 (no. 39), and two trihydroxy metabolite by PM of *m/z* 308.1862, both representing cyclohexyl-hydroxy pyrrolidine-dihydroxy isomers (nos. 45 and 46). In contrast to 3-MeO-PCP, monohydroxylation at the cyclohexyl ring could not be detected being an intermediate to further steps. In accordance to 3-MeO-PCP metabolism, aliphatic carboxy metabolites could be observed as carboxy metabolite (no. 44) with PM of *m/z* 306.2069, as four isomeric methyl artifacts of carboxy cyclohexyl-hydroxy metabolites (nos. 52-55) with PM of *m/z* 322.2018, as one methyl artifact of a carboxy cyclohexyl-dihydroxy metabolite (no. 56) with PM of *m/z* 338.1967, and as one carboxy alkyl-hydroxy metabolite (no. 51) with PM of *m/z* 322.2018. *O-*Demethylation and hydroxylation reactions could be observed for four *O*-demethyl hydroxy isomers with PM of *m/z* 262.1807, whereby one isomer is postulated as the *O*-demethyl pyrrolidine-hydroxy metabolite (no. 33) and three isomers as *O*-demethyl cyclohexyl-hydroxy metabolites (nos. 34-36). Moreover, after dihydroxylation, one *O*-demethyl pyrrolidine-dihydroxy metabolite with PM of *m/z* 278.1756 (no. 38) and after trihydroxylation three *O*-demethyl cyclohexyl-hydroxy pyrrolidine-dihydroxy meta-bolites with PM of *m/z* 294.1705 (nos. 41-43) could be observed. Furthermore, one methyl artifact of an *O*-demethylated carboxy metabolite could be observed with PM of *m/z* 292.1949 (no. 40) and four isomers after additional cyclohexyl-hydroxylation with PM of *m/z* 308.1862 (nos. 47-50). In analogy to 3-MeO-PCP metabolism studies, the methyl artifacts, postulated as methyl ester structures, were confirmed as described above.

Concerning phase II metabolism, eight glucuronides were found, whereby corresponding phase I metabolites of five of them could not be detected (nos. 66-70 in Table **[4](#T4){ref-type="table"}**). The reason for the lack of corresponding phase I metabolites for the two aryl hydroxylated metabolites (nos. 66 and 67) were the same already described above. Again, the position of the hydroxy groups were confirmed by additional fragment ions

Proposed Metabolic Pathways
---------------------------

The metabolic pathways, which could be proposed according to the identified metabolites, are given in Fig. (**[2](#F2){ref-type="fig"}**) for 3-MeO-PCP and in Fig. (**[3](#F3){ref-type="fig"}**) for 3-MeO-PCPy, respectively. Numbers correspond to those given in Tables **[1](#T1){ref-type="table"}**-**[4](#T4){ref-type="table"}**.

### 3-MeO-PCP

Hydroxylation steps could be detected at the cyclohexyl ring (no. 7 in Fig. **[2](#F2){ref-type="fig"}**), at the piperidine ring (no. 6), or at both rings forming two isomeric metabolites (nos. 14 and 15). The metabolite with monohydroxylated piperidine ring (no. 6) as well as both isomers of cyclohexyl-hydroxy piperidine-hydroxy (nos. 14 and 15) could further get hydroxylated at the piperidine ring to a piperidine-dihydroxy metabolite (no. 13) or to isomers of cyclohexyl-hydroxy piperidine-dihydroxy metabolite, whereby only one isomer was detected (no. 24). If hydroxylation of metabolite no. 6 took place at α-position to the amine, the resulting hemiaminal could perform a ring opening, and the formed aldehyde could be further oxidized to the corresponding carboxylic acid (no. 20). Further hydroxylation of this metabolite at the alkyl side chain (no. 30) or at the cyclohexyl ring to five isomeric structures (nos. 25-29) could be observed. *O*-Demethylation (no. 2) could be detected as another pathway, followed by hydroxylation at the cyclohexyl ring (no. 4), at the piperidine ring (no. 3), or at both rings, again into two isomeric metabolites (nos. 11 and 12). In analogy to the pathway without *O*-demethylation, the metabolite with the monohydroxylated piperidine ring (no. 3) as well as both cyclohexyl-hydroxy piperidine-hydroxy isomers (nos. 11 and 12) could further get hydroxylated at the piperidine ring to three isomeric piperidine-dihydroxy metabolites (nos. 8-10) or to three isomeric cyclohexyl-hydroxy piperidine-dihydroxy metabolites (nos. 17-19). Meta-bolite no. 5 could be explained as a product of one of the metabolites nos. 17-19 after oxidation of one hydroxy group and elimination of water. If hydroxylation of metabolite no. 3 led to a hemiaminal, one ring opened carboxy metabolite (no. 16) and after further hydroxylation at the cyclohexyl ring three isomers (nos. 21-23) could be detected.

Glucuronidation could be observed for metabolites nos. 6 (to 61), 2 (to 57), 3 (to 59 or 60), 11 or 12 (to 63), and 8-10 (to 62). For the glucuronide no. 58, the corresponding precursor should be an aryl-hydroxy of metabolite no. 2, which could not be detected possibly for reasons already described above.

### 3-MeO-PCPy

The metabolic pathways were similar to those of 3-MeO-PCP. Hydroxylation could be detected at the pyrrolidine ring after monohydroxylation (no. 37 in Fig. (**[3](#F3){ref-type="fig"}**)) and dihydro-xylation (no. 39). At the cyclohexyl ring, monohydroxylation was not observed but two isomers in combination with a dihydroxy pyrrolidine ring (nos. 45 and 46). Again, after ring opening, one carboxylic acid metabolite (no. 44), one after further hydroxylation at the alkyl side chain (no. 51), four cyclohexyl-monohydroxy isomers (nos. 52-55), and one cyclohexyl-dihydroxy isomer (no. 56) were detected. *O*-Demethylation (no. 32) followed by hydroxylation at the piperidine ring led to one monohydroxy (no. 33) and one dihydroxy isomer (no. 38). At the cyclohexyl ring, three monohydroxy (nos. 34-36), but no dihydroxy isomers were detected. Combinations only occurred with dihydroxy pyrrolidine ring, again resulting in three isomeric structures (nos. 41-43) fitting with the three precursor isomers nos. 34-36. *O*-Demethylation in combination with ring opening led to metabolite no. 40 and to the three isomers nos. 47-50 after further cyclohexyl-monohydroxylation.

Glucuronidation could be observed for metabolites nos. 45 or 46 (to 71), 32 (to 64), and 33 (to 65). The precursor of no. 68 was a pyrrolidine-monohydroxy metabolite in α-position (no. 37) followed by oxidation to the lactams, those of nos. 69 and 70 after further cyclohexyl-hydroxylation. Metabolite no. 66 resulted by aryl-hydroxylation of the *O*-demethyl metabolite no. 32 followed by glucuronidation. Metabolite no. 67 resulted from metabolite no. 66 after additional lactam formation.

Microsomal Incubations and Initial CYP Activity Screening
---------------------------------------------------------

Incubations with pHLM were carried out for comparison of the formed rat phase I metabolites with those of humans. For 3-MeO-PCP, the metabolites nos. 2, 3, 4, 7, and 13 from Table **[1](#T1){ref-type="table"}**, for 3-MeO-PCPy, the metabolites nos. 32 and 39 from Table **[2](#T2){ref-type="table"}** were detected after pHLM incubation. Essentially, *O*-demethylation and hydroxylation at the pyrrolidine and/or the cyclohexyl ring were observed for both species. However, ring opening steps could not be detected in pHLM, what could either be explained by low formation rates or by interindividual isomeric variability that could lead to the absence of hydroxylation products in the α-position in humans, and thus, absence of detectable carboxy metabolites.

To test the involvement of single CYP enzymes in the initial metabolic steps, the proposed metabolites were detected after incubations with the ten most important human hepatic CYP enzymes. The involvement of the corresponding CYPs is shown in Table **[5](#T5){ref-type="table"}** for 3-MeO-PCP and for 3-MeO-PCPy. The relative involvement of individual CYPs was defined in relation to the highest peak abundances during precursor ion monitoring of the formed metabolites. CYPs forming the relative highest peak abundances are given with "++" and all others with "+".

For 3-MeO-PCP, CYP 2B6 was involved in the formation of hydroxylation at the cyclohexyl ring as well as at the piperidine ring. *O-*Demethylation was performed by CYP 2C19, 2B6, and CYP 2D6. Regarding 3-MeO-PCPy, hydro-xylation at the pyrrolidine ring was again catalyzed by CYP 2B6. However, cyclohexyl-hydroxylation was not detected. Concerning *O-*demethylation, CYP 2C9, 2B6, and CYP 2D6 were involved. As most involved enzymes were polymor-phically expressed, pharmacogenomic variations might occur, but clinical data are needed for further studies.

Toxicological Detection by GC-MS, LC-MS^n^, and LC-HR-MS/MS
-----------------------------------------------------------

To test for toxicological detectability after common users' doses of approximately 10 mg (<https://www.erowid.org>, <http://bluelight.org>), rat urines were screened after administration of compound doses scaled by the dose-by-factor approach of Sharma and McNeill \[[@r13]\] *via* GC-MS, LC-MS^n^, and LC-HR-MS/MS SUSA. The detected targets for both compounds are given in Tables **[6](#T6){ref-type="table"}**-**[8](#T8){ref-type="table"}**.

For 3-MeO-PCP, detection was possible *via* the parent compound (no. 1) and metabolites nos. 2 and 3 by GC-MS, *via* metabolites nos. 8, 13, 57, 58, and 61 by LC-MS^n^, and nos. 2, 3, 8, 13, 24, 57, 58, 59, and 61 by LC-HR-MS/MS. Administration of 3-MeO-PCPy could be monitored *via* the metabolites no. 32 by GC-MS, nos. 32, 38, 39, 64, and 67 by LC-MS^n^, and nos. 32, 38, 39, 45, 64, 66, and 67 by LC-HR-MS/MS. The reason why not all types of metabolites were found for both drugs could be explained by different formation rates, influence of ion suppression, chromato-graphic and/or ionization properties. The risk of overlooking a drug consumption caused by ion suppression could be minimized by screening for several targets *e.g.* metabolites.

For general performance of the SUSAs, increasing concentrations of the parent drugs were analyzed although they were not the main targets chosen for urine analysis. The LODs were determined at a signal-to-noise ratio of 3. In GC-MS, LC-MS^n^, and LC-HR-MS/MS, the LODs were 5, 10, and 0.1 ng/mL for 3-MeO-PCP and 10, 10, and 0.1 ng/mL for 3-MeO-PCPy, respectively.

Conclusion
==========

The PCP analogues 3-MeO-PCP and 3-MeO-PCPy were extensively metabolized in rats *via* aliphatic and aromatic hydroxylation, carboxylation after ring opening, *O*-demethylation, and glucuronidation. The initial steps could be confirmed by detection of the corresponding metabolites in pHLM incubations. The CYP enzymes involved in the metabolism of both compounds were CYP 2B6 and CYP 2D6. In addition, CYP 2C19 was involved in 3-MeO-PCP *O*-demethylation and piperidine-hydroxylation whereas CYP 2C9 in 3-MeO-PCPy *O*-demethylation. As only polymor-phically expressed enzymes were involved, pharmaco-genomic variations might occur, but clinical data are needed to confirm the relevance. Detectability studies showed that all tested SUSAs were able to monitor consumptions of both drugs considering that metabolites were the main targets.

The authors like to thank Markus R. Meyer, Andreas G. Helfer, Lilian H. J. Richter, Lea Wagmann, Carsten Schröder, Gabriele Ulrich, and Armin A. Weber for their support.

CONFLICT OF INTEREST
====================

The authors confirm that this article content has no conflict of interest.

![Structures of 3-MeO-PCP (**a**), 3-MeO-PCPy (**b**), PCP (**c**), and PCPy (**d**).](CN-15-692_F1){#F1}

![Metabolic pathways of 3-MeO-PCP. Undefined positions of hydroxylation are indicated by unspecific bonds. Parent compound is marked by a box. Two arrows indicate a pathway that contains multiple metabolism steps.](CN-15-692_F2){#F2}

![Metabolic pathways of 3-MeO-PCPy. Undefined positions of hydroxylation are indicated by unspecific bonds. Parent compound is marked by a box. Two arrows indicate a pathway that contains multiple metabolism steps. Structures in brackets are postulated intermediate metabolites.](CN-15-692_F3){#F3}

###### 

3-MeO-PCP and its phase I metabolites detected in rat urine by LC-HR-MS^n^ with protonated precursor mass (PM), characteristic fragment ions (FI), calculated exact masses, proposed elemental composition, mass error, relative intensity, and retention times (RT).

  ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
  **No.**   **Metabolite**                                                               **Measured Accurate**\   **Calculated Exact**\   **Error (ppm)**   **Elemental Composition**   **Relative Intensity (%)**   **RT (min)**
                                                                                         **Mass (*m/z*)**         **Mass (*m/z*)**                                                                                   
  --------- ---------------------------------------------------------------------------- ------------------------ ----------------------- ----------------- --------------------------- ---------------------------- --------------
  1         3-MeO-PCP                                                                    10.6                                                                                                                        

                                                                                         274.2166                 274.2171                -1.82             C~18~H~28~NO                100                          

                                                                                         86.0963                  86.0970                 -8.13             C~5~H~12~N                  100                          

                                                                                         189.1277                 189.1279                -1.06             C~13~H~17~O                 40                           

  2         3-MeO-PCP-M (*O*-demethyl-)                                                  7.3                                                                                                                         

                                                                                         260.2006                 260.2014                -3.07             C~17~H~26~NO                100                          

                                                                                         86.0964                  86.0970                 -6.97             C~5~H~12~N                  100                          

                                                                                         175.1122                 175.1123                -0.57             C~12~H~15~O                 26                           

  3         3-MeO-PCP-M (*O*-demethyl-piperidine-HO-)                                    6.1                                                                                                                         

                                                                                         276.1955                 276.1964                -3.26             C~17~H~26~NO~2~             100                          

                                                                                         84.0808                  84.0813                 -5.95             C~5~H~10~N                  8                            

                                                                                         102.0914                 102.0919                -4.90             C~5~H~12~NO                 100                          

                                                                                         175.1122                 175.1123                -0.57             C~12~H~15~O                 30                           

  4         3-MeO-PCP-M (*O*-demethyl-cyclohexyl-HO-)                                    5.1                                                                                                                         

                                                                                         276.1959                 276.1964                -1.81             C~17~H~26~NO~2~             100                          

                                                                                         86.0964                  86.0970                 -6.97             C~5~H~12~N                  100                          

                                                                                         173.0965                 173.0966                -0.58             C~12~H~13~O                 30                           

                                                                                         191.1071                 191.1072                -0.52             C~12~H~15~O~2~              6                            

  5         3-MeO-PCP-M (*O*-demethyl-cyclohexyl-HO-dehydro-oxo-piperidine-)             5.7                                                                                                                         

                                                                                         288.1598                 288.1600                -0.69             C~17~H~22~NO~3~             100                          

                                                                                         80.0497                  80.0500                 -3.75             C~5~H~6~N                   5                            

                                                                                         98.0601                  98.0606                 -5.10             C~5~H~8~NO                  100                          

                                                                                         173.0965                 173.0966                -0.58             C~12~H~13~O                 6                            

                                                                                         191.1073                 191.1072                0.52              C~12~H~15~O~2~              1                            

  6         3-MeO-PCP-M (piperidine-HO-)                                                 8.9                                                                                                                         

                                                                                         290.2115                 290.2120                -1.72             C~18~H~28~NO~2~             28                           

                                                                                         84.0808                  84.0813                 -5.95             C~5~H~10~N                  1                            

                                                                                         102.0914                 102.0919                -4.90             C~5~H~12~NO                 18                           

                                                                                         189.1278                 189.1279                -0.53             C~13~H~17~O                 8                            

  7         3-MeO-PCP-M (cyclohexyl-HO-)                                                 7.6                                                                                                                         

                                                                                         290.2177                 290.2170                2.41              C~18~H~28~NO~2~             60                           

                                                                                         86.0964                  86.0970                 -6.97             C~5~H~12~N                  100                          

                                                                                         187.1122                 187.1123                -0.53             C~13~H~15~O                 52                           

                                                                                         205.1227                 205.1229                -0.98             C~13~H~17~O~2~              14                           

  8         3-MeO-PCP-M (*O*-demethyl-piperidine-di-HO-) isomer 1                        7.3                                                                                                                         

                                                                                         292.1906                 292.1913                -2.40             C~17~H~26~NO~3~             30                           

                                                                                         101.0598                 101.0603                -4.95             C~5~H~9~O~2~                18                           

                                                                                         118.0864                 118.0868                -3.39             C~5~H~12~NO~2~              100                          

                                                                                         175.1121                 175.1123                -1.14             C~12~H~15~O                 20                           

  9         3-MeO-PCP-M (*O*-demethyl-piperidine-di-HO-) isomer 2                        8.1                                                                                                                         

                                                                                         292.1911                 292.1913                -0.68             C~17~H~26~NO~3~             100                          

                                                                                         101.0598                 101.0603                -4.95             C~5~H~9~O~2~                38                           

                                                                                         118.0865                 118.0868                -2.54             C~5~H~12~NO~2~              100                          

                                                                                         175.1121                 175.1123                -1.14             C~12~H~15~O                 28                           

  10        3-MeO-PCP-M (*O*-demethyl-piperidine-di-HO-) isomer 3                        9.2                                                                                                                         

                                                                                         292.1912                 292.1913                -0.34             C~17~H~26~NO~3~             26                           

                                                                                         101.0600                 101.0603                -2.97             C~5~H~9~O~2~                1                            

                                                                                         118.0865                 118.0868                -2.54             C~5~H~12~NO~2~              1                            

                                                                                         175.1119                 175.1123                -2.28             C~12~H~15~O                 1                            

  11        3-MeO-PCP-M (*O*-demethyl-cyclohexyl-HO-piperidine-HO-) isomer 1             2.4                                                                                                                         

                                                                                         292.1909                 292.1913                -1.37             C~17~H~26~NO~3~             100                          

                                                                                         84.0807                  84.0813                 -7.14             C~5~H~10~N                  1                            

                                                                                         102.0914                 102.0919                -4.90             C~5~H~12~NO                 100                          

                                                                                         173.0965                 173.0966                -0.58             C~12~H~13~O                 14                           

                                                                                         191.1071                 191.1072                -0.52             C~12~H~15~O~2~              2                            

  12        3-MeO-PCP-M (*O*-demethyl-cyclohexyl-HO-piperidine-HO-) isomer 2             4.1                                                                                                                         

                                                                                         292.1909                 292.1913                -1.37             C~17~H~26~NO~3~             78                           

                                                                                         84.0808                  84.0813                 -5.95             C~5~H~10~N                  1                            

                                                                                         102.0913                 102.0919                -5.88             C~5~H~12~NO                 100                          

                                                                                         173.0963                 173.0966                -1.73             C~12~H~13~O                 20                           

                                                                                         191.1070                 191.1072                -1.05             C~12~H~15~O~2~              3                            

  13        3-MeO-PCP-M (piperidine-di-HO-)                                              10.2                                                                                                                        

                                                                                         306.2064                 306.2069                -1.63             C~18~H~28~NO~3~             50                           

                                                                                         101.0597                 101.0603                -5.94             C~5~H~9~O~2~                16                           

                                                                                         118.0864                 118.0868                -3.39             C~5~H~12~NO~2~              100                          

                                                                                         189.1276                 189.1279                -1.59             C~13~H~17~O                 56                           

  14        3-MeO-PCP-M (cyclohexyl-HO-piperidine-HO-) isomer 1                          4.2                                                                                                                         

                                                                                         306.2065                 306.2069                -1.31             C~18~H~28~NO~3~             100                          

                                                                                         84.0806                  84.0813                 -8.33             C~5~H~10~N                  1                            

                                                                                         102.0913                 102.0919                -5.88             C~5~H~12~NO                 100                          

                                                                                         187.1120                 187.1123                -1.60             C~13~H~15~O                 24                           

                                                                                         205.1226                 205.1229                -1.46             C~13~H~17~O~2~              8                            

  15        3-MeO-PCP-M (cyclohexyl-HO-piperidine-HO-) isomer 2                          5.8                                                                                                                         

                                                                                         306.2065                 306.2069                -1.31             C~18~H~28~NO~3~             38                           

                                                                                         102.0914                 102.0919                -4.90             C~5~H~12~NO                 100                          

                                                                                         187.1122                 187.1123                -0.53             C~13~H~15~O                 30                           

                                                                                         205.1228                 205.1229                -0.49             C~13~H~17~O~2~              10                           

  16        3-MeO-PCP-M (*O*-demethyl-carboxy-) methyl artifact                          8.7                                                                                                                         

                                                                                         306.2062                 306.2069                -2.29             C~18~H~28~NO~3~             100                          

                                                                                         115.0756                 115.0759                -2.61             C~6~H~11~O~2~               30                           

                                                                                         132.1022                 132.1025                -2.27             C~6~H~14~NO~2~              100                          

                                                                                         175.1122                 175.1123                -0.57             C~12~H~15~O                 18                           

  17        3-MeO-PCP-M (*O*-demethyl-cyclohexyl-HO-piperidine-di-HO-) isomer 1          4.9                                                                                                                         

                                                                                         308.1856                 308.1862                -1.95             C~17~H~26~NO~4~             100                          

                                                                                         101.0598                 101.0603                -4.95             C~5~H~9~O~2~                1                            

                                                                                         118.0864                 118.0868                -3.39             C~5~H~12~NO~2~              100                          

                                                                                         173.0965                 173.0966                -0.58             C~12~H~13~O                 14                           

                                                                                         191.1070                 191.1072                -1.05             C~12~H~15~O~2~              2                            

  18        3-MeO-PCP-M (*O*-demethyl-cyclohexyl-HO-piperidine-di-HO-) isomer 2          5.1                                                                                                                         

                                                                                         308.1858                 308.1862                -1.30             C~17~H~26~NO~4~             20                           

                                                                                         101.0598                 101.0603                -4.95             C~5~H~9~O~2~                1                            

                                                                                         118.0864                 118.0868                -3.39             C~5~H~12~NO~2~              4                            

                                                                                         191.1068                 191.1072                -2.09             C~12~H~15~O~2~              1                            

  19        3-MeO-PCP-M (*O*-demethyl-cyclohexyl-HO-piperidine-di-HO-) isomer 3          5.5                                                                                                                         

                                                                                         308.1857                 308.1862                -1.62             C~17~H~26~NO~4~             78                           

                                                                                         101.0597                 101.0603                -5.94             C~5~H~9~O~2~                1                            

                                                                                         118.0864                 118.0868                -3.39             C~5~H~12~NO~2~              100                          

                                                                                         173.0964                 173.0966                -1.16             C~12~H~13~O                 20                           

                                                                                         191.1070                 191.1072                -1.05             C~12~H~15~O~2~              3                            

  20        3-MeO-PCP-M (carboxy-) methyl artifact                                       11.7                                                                                                                        

                                                                                         320.2216                 320.2226                -3.12             C~19~H~39~NO~3~             100                          

                                                                                         115.0756                 115.0759                -2.61             C~6~H~11~O~2~               30                           

                                                                                         132.1022                 132.1025                -2.27             C~6~H~14~NO~2~              100                          

                                                                                         189.1278                 189.1279                -0.53             C~13~H~17~O                 40                           

  21        3-MeO-PCP-M (*O*-demethyl-carboxy-cyclohexyl-HO-) methyl artifact isomer 1   5.2                                                                                                                         

                                                                                         322.2016                 322.2018                -0.62             C~18~H~28~NO~4~             100                          

                                                                                         115.0755                 115.0759                -3.48             C~6~H~11~O~2~               28                           

                                                                                         132.1021                 132.1025                -3.03             C~6~H~14~NO~2~              100                          

                                                                                         173.0963                 173.0966                -1.73             C~12~H~13~O                 6                            

                                                                                         191.1069                 191.1072                -1.57             C~12~H~15~O~2~              1                            

  22        3-MeO-PCP-M (*O*-demethyl-carboxy-cyclohexyl-HO-) methyl artifact isomer 2   5.8                                                                                                                         

                                                                                         322.2012                 322.2018                -1.86             C~18~H~28~NO~4~             100                          

                                                                                         115.0755                 115.0759                -3.48             C~6~H~11~O~2~               26                           

                                                                                         132.1021                 132.1025                -3.03             C~6~H~14~NO~2~              100                          

                                                                                         173.0964                 173.0966                -1.16             C~12~H~13~O                 14                           

                                                                                         191.1070                 191.1072                -1.05             C~12~H~15~O~2~              2                            

  23        3-MeO-PCP-M (*O*-demethyl-carboxy-cyclohexyl-HO-) methyl artifact isomer 3   6.2                                                                                                                         

                                                                                         322.2011                 322.2018                -2.17             C~18~H~28~NO~4~             58                           

                                                                                         115.0755                 115.0759                -3.48             C~6~H~11~O~2~               30                           

                                                                                         132.1022                 132.1025                -2.27             C~6~H~14~NO~2~              100                          

                                                                                         173.0965                 173.0966                -0.58             C~12~H~13~O                 20                           

                                                                                         191.1071                 191.1072                -0.52             C~12~H~15~O~2~              2                            

  24        3-MeO-PCP-M (cyclohexyl-HO-piperidine-di-HO-)                                3.8                                                                                                                         

                                                                                         322.2016                 322.2018                -0.62             C~18~H~28~NO~4~             100                          

                                                                                         100.0758                 100.0762                -4.00             C~5~H~10~NO                 1                            

                                                                                         118.0864                 118.0868                -3.39             C~5~H~12~NO~2~              100                          

                                                                                         187.1120                 187.1123                -1.60             C~13~H~15~O                 24                           

                                                                                         205.1226                 205.1229                -1.46             C~13~H~17~O~2~              8                            

  25        3-MeO-PCP-M (carboxy-cyclohexyl-HO-) methyl artifact isomer 1                7.2                                                                                                                         

                                                                                         336.2175                 336.2175                0.00              C~19~H~30~NO~4~             36                           

                                                                                         115.0756                 115.0759                -2.61             C~6~H~11~O~2~               34                           

                                                                                         132.1023                 132.1025                -1.51             C~6~H~14~NO~2~              100                          

                                                                                         187.1124                 187.1123                0.53              C~13~H~15~O                 20                           

                                                                                         205.1229                 205.1229                0.00              C~13~H~17~O~2~              4                            

  26        3-MeO-PCP-M (carboxy-cyclohexyl-HO-) methyl artifact isomer 2                8.0                                                                                                                         

                                                                                         336.2173                 336.2175                -0.59             C~19~H~30~NO~4~             52                           

                                                                                         115.0755                 115.0759                -3.48             C~6~H~11~O~2~               30                           

                                                                                         132.1021                 132.1025                -3.03             C~6~H~14~NO~2~              100                          

                                                                                         187.1121                 187.1123                -1.07             C~13~H~17~O                 24                           

                                                                                         205.1226                 205.1229                -1.46             C~13~H~17~O~2~              8                            

  27        3-MeO-PCP-M (carboxy-cyclohexyl-HO-) methyl artifact isomer 3                8.5                                                                                                                         

                                                                                         336.2172                 336.2175                -0.89             C~19~H~30~NO~4~             80                           

                                                                                         115.0756                 115.0759                -2.61             C~6~H~11~O~2~               10                           

                                                                                         132.1022                 132.1025                -2.27             C~6~H~14~NO~2~              34                           

                                                                                         187.1122                 187.1123                -0.53             C~13~H~15~O                 20                           

                                                                                         205.1228                 205.1229                -0.49             C~13~H~17~O~2~              4                            

  **No.**   **Metabolite**                                                               **Measured Accurate**\   **Calculated Exact**\   **Error (ppm)**   **Elemental Composition**   **Relative Intensity (%)**   **RT (min)**
                                                                                         **Mass (*m/z*)**         **Mass (*m/z*)**                                                                                   

  28        3-MeO-PCP-M (carboxy-cyclohexyl-HO-) methyl artifact isomer 4                8.9                                                                                                                         

                                                                                         336.2172                 336.2175                -0.89             C~19~H~30~NO~4~             24                           

                                                                                         115.0755                 115.0759                -3.48             C~6~H~11~O~2~               32                           

                                                                                         132.1022                 132.1025                -2.27             C~6~H~14~NO~2~              100                          

                                                                                         187.1122                 187.1123                -0.53             C~13~H~15~O                 18                           

                                                                                         205.1227                 205.1229                -0.98             C~13~H~17~O~2~              2                            

  29        3-MeO-PCP-M (carboxy-cyclohexyl-HO-) methyl artifact isomer 5                9.1                                                                                                                         

                                                                                         336.2171                 336.2175                -1.19             C~19~H~30~NO~4~             45                           

                                                                                         115.0755                 115.0759                -3.48             C~6~H~11~O~2~               20                           

                                                                                         132.1021                 132.1025                -3.03             C~6~H~14~NO~2~              60                           

                                                                                         187.1122                 187.1123                -0.53             C~13~H~15~O                 8                            

                                                                                         205.1226                 205.1229                -1.46             C~13~H~17~O~2~              100                          

  30        3-MeO-PCP-M (carboxy-alkyl-HO-)                                              10.5                                                                                                                        

                                                                                         336.2172                 336.2175                -0.89             C~19~H~30~NO~4~             50                           

                                                                                         99.0439                  99.0446                 -7.07             C~5~H~7~O~2~                1                            

                                                                                         131.0704                 131.0708                -3.05             C~6~H~11~O~3~               6                            

                                                                                         148.0970                 148.0974                -2.70             C~6~H~14~NO~3~              100                          

                                                                                         189.1277                 189.1279                -1.06             C~13~H~17~O                 28                           
  ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

###### 

3-MeO-PCPy and its phase I metabolites detected in rat urine by LC-HR-MS^n^ with protonated precursor mass (PM), characteristic fragment ions (FI), calculated exact masses, proposed elemental composition, mass error, relative intensity, and retention times (RT).

  ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
  **No.**   **Metabolite**                                                                **Measured Accurate**\   **Calculated Exact**\   **Error (ppm)**   **Elemental Composition**   **Relative Intensity (%)**   **RT (min)**
                                                                                          **Mass (*m/z*)**         **Mass (*m/z*)**                                                                                   
  --------- ----------------------------------------------------------------------------- ------------------------ ----------------------- ----------------- --------------------------- ---------------------------- --------------
  31        3-MeO-PCPy                                                                    10.0                                                                                                                        

                                                                                          260.2010                 260.2014                -1.54             C~17~H~26~NO                42                           

                                                                                          189.1276                 189.1279                -1.59             C~13~H~17~O                 100                          

  32        3-MeO-PCPy-M (*O*-demethyl-)                                                  6.6                                                                                                                         

                                                                                          246.1850                 246.1858                -3.25             C~16~H~24~NO                100                          

                                                                                          72.0807                  72.0813                 -8.32             C~4~H~10~N                  68                           

                                                                                          175.1121                 175.1123                -1.14             C~12~H~15~O                 100                          

  33        3-MeO-PCPy-M (*O*-demethyl-pyrrolidine-HO-)                                   6.1                                                                                                                         

                                                                                          262.1802                 262.1807                -1.91             C~16~H~24~NO~2~             100                          

                                                                                          70.0651                  70.0657                 -8.56             C~4~H~8~N                   1                            

                                                                                          88.0756                  88.0762                 -6.81             C~4~H~10~NO                 100                          

                                                                                          175.1120                 175.1123                -1.71             C~12~H~15~O                 24                           

  34        3-MeO-PCPy-M (*O*-demethyl-cyclohexyl-HO-) isomer 1                           4.4                                                                                                                         

                                                                                          262.1801                 262.1807                -2.29             C~16~H~24~NO~2~             70                           

                                                                                          173.0963                 173.0966                -1.73             C~12~H~13~O                 100                          

                                                                                          191.1069                 191.1072                -1.57             C~12~H~15~O~2~              18                           

  35        3-MeO-PCPy-M (*O*-demethyl-cyclohexyl-HO-) isomer 2                           4.6                                                                                                                         

                                                                                          262.1801                 262.1807                -2.29             C~16~H~24~NO~2~             100                          

                                                                                          173.0963                 173.0966                -1.73             C~12~H~13~O                 100                          

                                                                                          191.1062                 191.1072                -5.23             C~12~H~15~O~2~              14                           

  36        3-MeO-PCPy-M (*O*-demethyl-cyclohexyl-HO-) isomer 3                           5.6                                                                                                                         

                                                                                          262.1806                 262.1807                -0.38             C~16~H~24~NO~2~             64                           

                                                                                          173.0966                 173.0966                0.00              C~12~H~13~O                 4                            

                                                                                          191.1070                 191.1072                -1.05             C~12~H~15~O~2~              20                           

  37        3-MeO-PCPy-M (pyrrolidine-HO-)                                                8.8                                                                                                                         

                                                                                          276.1958                 276.1964                -2.17             C~17~H~26~NO~2~             60                           

                                                                                          70.0659                  70.0657                 2.85              C~4~H~8~N                   2                            

                                                                                          88.0756                  88.0762                 -6.81             C~4~H~10~NO                 100                          

                                                                                          189.1277                 189.1279                -1.06             C~13~H~17~O                 70                           

  38        3-MeO-PCPy-M (*O*-demethyl-pyrrolidine-di-HO-)                                6.5                                                                                                                         

                                                                                          278.1754                 278.1756                -0.72             C~16~H~24~NO~3~             84                           

                                                                                          87.0440                  87.0446                 -6.89             C~4~H~7~O~2~                14                           

                                                                                          104.0706                 104.0712                -5.77             C~4~H~10~NO~2~              100                          

                                                                                          175.1120                 175.1123                -1.71             C~12~H~15~O                 50                           

  39        3-MeO-PCPy-M (pyrrolidine-di-HO-)                                             9.5                                                                                                                         

                                                                                          292.1905                 292.1913                -2.74             C~17~H~26~NO~3~             18                           

                                                                                          87.0440                  87.0446                 -6.89             C~4~H~7~O~2~                10                           

                                                                                          104.0707                 104.0712                -4.80             C~4~H~10~NO~2~              94                           

                                                                                          189.1277                 189.1279                -1.06             C~13~H~17~O                 100                          

  40        3-MeO-PCPy-M (*O*-demethyl-carboxy-) methyl artifact                          8.0                                                                                                                         

                                                                                          292.1907                 292.1913                -2.05             C~17~H~26~NO~3~             100                          

                                                                                          101.0598                 101.0603                -4.95             C~5~H~9~O~2~                38                           

                                                                                          118.0865                 118,0868                -2.54             C~5~H~12~NO~2~              100                          

                                                                                          175.1122                 175.1123                -0.57             C~12~H~15~O                 30                           

  41        3-MeO-PCPy-M (*O*-demethyl-cyclohexyl-HO-pyrrolidine-di-HO-) isomer 1         3.0                                                                                                                         

                                                                                          294.1710                 294.1705                1.70              C~16~H~24~NO~4~             22                           

                                                                                          87.0440                  87.0446                 -6.89             C~4~H~7~O~2~                10                           

                                                                                          104.0706                 104.0712                -5.77             C~4~H~10~NO~2~              100                          

                                                                                          173.0963                 173.0966                -1.73             C~12~H~13~O                 20                           

                                                                                          191.1071                 191.1072                -0.52             C~12~H~15~O~2~              4                            

  42        3-MeO-PCPy-M (*O*-demethyl-cyclohexyl-HO-pyrrolidine-di-HO-) isomer 2         4.0                                                                                                                         

                                                                                          294.1703                 294.1705                -0.68             C~16~H~24~NO~4~             94                           

                                                                                          87.0440                  87.0446                 -6.89             C~4~H~7~O~2~                10                           

                                                                                          104.0706                 104.0712                -5.77             C~4~H~10~NO~2~              100                          

                                                                                          173.0964                 173.0966                -1.16             C~12~H~13~O                 44                           

                                                                                          191.1069                 191.1072                -1.57             C~12~H~15~O~2~              12                           

  43        3-MeO-PCPy-M (*O*-demethyl-cyclohexyl-HO-pyrrolidine-di-HO-) isomer 3         4.4                                                                                                                         

                                                                                          294.1701                 294.1705                -1.36             C~16~H~24~NO~4~             92                           

                                                                                          87.0441                  87.0446                 -5.74             C~4~H~7~O~2~                12                           

                                                                                          104.0707                 104.0712                -4.80             C~4~H~10~NO~2~              100                          

                                                                                          173.0965                 173.0966                -0.58             C~12~H~13~O                 30                           

                                                                                          191.1072                 191.1072                0.00              C~12~H~15~O~2~              6                            

  44        3-MeO-PCPy-M (carboxy-)                                                       11.0                                                                                                                        

                                                                                          306.2058                 306.2069                -3.59             C~18~H~28~NO~3~             100                          

                                                                                          101.0597                 101.0603                -5.94             C~5~H~9~O~2~                40                           

                                                                                          118.0864                 118.0868                -3.39             C~5~H~12~NO~2~              100                          

                                                                                          189.1277                 189.1279                -1.06             C~13~H~17~O                 70                           

  45        3-MeO-PCPy-M (cyclohexyl-HO-pyrrolidine-di-HO-) isomer 1                      4.7                                                                                                                         

                                                                                          308.1856                 308.1862                -1,95             C~17~H~26~NO~4~             20                           

                                                                                          86.0440                  87.0446                 -6,89             C~4~H~7~O~2~                6                            

  45        3-MeO-PCPy-M (cyclohexyl-HO-pyrrolidine-di-HO-) isomer 1                      4.7                                                                                                                         

                                                                                          104.0707                 104.0712                -4,80             C~4~H~10~NO~2~              100                          

                                                                                          187.1121                 187.1123                -1,07             C~13~H~15~O                 42                           

                                                                                          205.1227                 205.1229                -0,98             C~13~H~17~O~2~              14                           

  46        3-MeO-PCPy-M (cyclohexyl-HO-pyrrolidine-di-HO-) isomer 2                      6.3                                                                                                                         

                                                                                          308.1858                 308.1862                -1.30             C~17~H~26~NO~4~             82                           

                                                                                          87.0441                  87.0446                 -5.74             C~4~H~7~O~2~                2                            

                                                                                          104.0707                 104.0712                -4.80             C~4~H~10~NO~2~              44                           

                                                                                          187.1121                 187.1123                -1.07             C~13~H~15~O                 20                           

                                                                                          205.1227                 205.1229                -0.98             C~13~H~17~O~2~              6                            

  47        3-MeO-PCPy-M (*O*-demethyl-carboxy-cyclohexyl-HO-) methyl artifact isomer 1   5.0                                                                                                                         

                                                                                          308.1855                 308.1862                -2.27             C~17~H~26~NO~4~             100                          

                                                                                          101.0598                 101.0603                -4.95             C~5~H~9~O~2~                30                           

                                                                                          118.0865                 118.0868                -2.54             C~5~H~12~NO~2~              100                          

                                                                                          173.0966                 173.0966                0.00              C~12~H~13~O                 20                           

                                                                                          191.1072                 191.1072                0.00              C~12~H~15~O~2~              4                            

  48        3-MeO-PCPy-M (*O*-demethyl-carboxy-cyclohexyl-HO-) methyl artifact isomer 2   5.4                                                                                                                         

                                                                                          308.1852                 308.1862                -3.24             C~17~H~26~NO~4~             100                          

                                                                                          101.0597                 101.0603                -5.94             C~5~H~9~O~2~                40                           

                                                                                          118.0864                 118.0868                -3.39             C~5~H~12~NO~2~              100                          

                                                                                          173.0964                 173.0966                -1.16             C~12~H~13~O                 18                           

                                                                                          191.1070                 191.1072                -1.05             C~12~H~15~O~2~              2                            

  49        3-MeO-PCPy-M (*O*-demethyl-carboxy-cyclohexyl-HO-) methyl artifact isomer 3   6.3                                                                                                                         

                                                                                          308.1857                 308.1862                -1.62             C~17~H~26~NO~4~             88                           

                                                                                          87.0440                  87.0446                 -6.89             C~4~H~7~O~2~                1                            

                                                                                          101.0598                 101.0603                -4.95             C~5~H~9~O~2~                38                           

                                                                                          118.0864                 118.0868                -3.39             C~5~H~12~NO~2~              100                          

                                                                                          173.0963                 173.0966                -1.73             C~12~H~13~O                 6                            

                                                                                          191.1070                 191.1072                -1.05             C~12~H~15~O~2~              16                           

  50        3-MeO-PCPy-M (*O*-demethyl-carboxy-cyclohexyl-HO-) methyl artifact isomer 4   10.2                                                                                                                        

                                                                                          308.1859                 308.1862                -0.97             C~17~H~26~NO~4~             34                           

                                                                                          101.0599                 101.0603                -3.96             C~5~H~9~O~2~                8                            

                                                                                          118.0864                 118.0868                -3.39             C~5~H~12~NO~2~              24                           

                                                                                          173.0965                 173.0966                -0.58             C~12~H~13~O                 4                            

  51        3-MeO-PCPy-M (carboxy-alkyl-HO-) methyl artifact                              9.7                                                                                                                         

                                                                                          322.2015                 322.2018                -0.93             C~18~H~28~NO~4~             80                           

                                                                                          102.0553                 102.0555                -1.96             C~4~H~8~NO~2~               1                            

                                                                                          117.0548                 117.0551                -2.56             C~5~H~9~O~3~                26                           

                                                                                          134.0814                 134.0817                -2.24             C~5~H~12~NO~3~              100                          

                                                                                          189.1278                 189.1279                -0.53             C~13~H~17~O                 30                           

  52        3-MeO-PCPy-M (carboxy-cyclohexyl-HO-) methyl artifact isomer 1                6.1                                                                                                                         

                                                                                          322.2012                 322.2018                -1.86             C~18~H~28~NO~4~             34                           

                                                                                          101.0597                 101.0603                -5.94             C~5~H~9~O~2~                30                           

                                                                                          118.0863                 118.0868                -4.23             C~5~H~12~NO~2~              100                          

                                                                                          187.1119                 187.1123                -2.14             C~13~H~15~O                 20                           

                                                                                          205.1225                 205.1229                -1.95             C~13~H~17~O~2~              6                            

  53        3-MeO-PCPy-M carboxy-cyclohexyl-HO-) methyl artifact isomer 2                 7.1                                                                                                                         

                                                                                          322.2014                 322.2018                -1.24             C~17~H~28~NO~4~             32                           

                                                                                          101.0597                 101.0603                -5.94             C~5~H~9~O~2~                30                           

                                                                                          118.0863                 118.0868                -4.23             C~5~H~12~NO~2~              100                          

                                                                                          187.1119                 187.1123                -2.14             C~13~H~15~O                 48                           

                                                                                          205.1225                 205.1229                -1.95             C~13~H~17~O~2~              12                           

  54        3-MeO-PCPy-M (carboxy-cyclohexyl-HO-) methyl artifact isomer 3                7.8                                                                                                                         

                                                                                          322.2016                 322.2018                -0.62             C~18~H~28~NO~4~             100                          

                                                                                          101.0598                 101.0603                -4.95             C~5~H~9~O~2~                32                           

                                                                                          118.0865                 118.0868                -2.54             C~5~H~12~NO~2~              100                          

                                                                                          187.1122                 187.1123                -0.53             C~13~H~15~O                 30                           

                                                                                          205.1228                 205.1229                -0.49             C~13~H~17~O~2~              6                            

  55        3-MeO-PCPy-M (carboxy-cyclohexyl-HO-) methyl artifact isomer 4                8.3                                                                                                                         

                                                                                          322.2016                 322.2018                -0.62             C~18~H~28~NO~4~             100                          

                                                                                          101.0598                 101.0603                -4.95             C~5~H~9~O~2~                10                           

                                                                                          118.0865                 118.0868                -2.54             C~5~H~12~NO~2~              30                           

                                                                                          187.1120                 187.1123                -1.60             C~13~H~15~O                 8                            

                                                                                          205.1227                 205.1229                -0.98             C~13~H~17~O~2~              100                          

  56        3-MeO-PCPy-M (carboxy-cyclohexyl-di-HO-) methyl artifact                      5.4                                                                                                                         

                                                                                          338.1958                 338.1967                -2.66             C~18~H~28~NO~5~             4                            

                                                                                          101.0597                 101.0603                -5.94             C~5~H~9~O~2~                24                           

                                                                                          118.0863                 118.0868                -4.23             C~5~H~12~NO~2~              100                          

                                                                                          203.1069                 203.1072                -1.48             C~13~H~15~O~2~              40                           

                                                                                          221.1174                 221.1178                -1.81             C~13~H~17~O~3~              70                           
  ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

###### 

3-MeO-PCP and its phase II metabolites detected in rat urine by LC-HR-MS^n^ with protonated precursor mass (PM), characteristic fragment ions (FI), calculated exact masses, proposed elemental composition, mass error, relative intensity, and retention times (RT).

  --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
  **No.**   **Metabolite**                                                        **Measured Accurate**\   **Calculated Exact**\   **Error (ppm)**   **Elemental Composition**   **Relative Intensity (%)**   **RT (min)**
                                                                                  **Mass (*m/z*)**         **Mass (*m/z*)**                                                                                   
  --------- --------------------------------------------------------------------- ------------------------ ----------------------- ----------------- --------------------------- ---------------------------- --------------
  57        3-MeO-PCP-M (*O*-demethyl-) glucuronide                               5.3                                                                                                                         

                                                                                  436.2320                 436.2335                -3.44             C~23~H~34~NO~7~             100                          

                                                                                  175.1117                 175.1123                -3.43             C~12~H~15~O                 100                          

                                                                                  351.1436                 351.1444                -2.28             C~18~H~23~O~7~              3                            

  58        3-MeO-PCP-M (*O*-demethyl-aryl-HO-) glucuronide                       4.6                                                                                                                         

                                                                                  452.2272                 452.2284                -2.65             C~23~H~34~NO~8~             100                          

                                                                                  175.1117                 175.1123                -3.43             C~12~H~15~O                 100                          

                                                                                  191.1066                 191.1072                -3.14             C~12~H~15~O~2~              30                           

                                                                                  367.1386                 367.1393                -1.91             C~18~H~23~O~8~              60                           

  59        3-MeO-PCP-M (*O*-demethyl-piperidine-HO-) glucuronide isomer 1        5.7                                                                                                                         

                                                                                  452.2272                 452.2284                -2.65             C~23~H~24~NO~8~             100                          

                                                                                  175.1119                 175.1123                -2.28             C~12~H~15~O                 3                            

                                                                                  278.1235                 278.1240                -1.80             C~11~H~20~NO~7~             100                          

  60        3-MeO-PCP-M (*O*-demethyl-piperidine-HO-) glucuronide isomer 2        5.9                                                                                                                         

                                                                                  452.2275                 452.2284                -1.99             C~23~H~34~NO~8~             100                          

                                                                                  175.1118                 175.1123                -2.86             C~12~H~15~O                 3                            

                                                                                  278.1234                 278.1240                -2.16             C~11~H~20~NO~7~             100                          

  61        3-MeO-PCP-M (piperidine-HO-) glucuronide                              8.1                                                                                                                         

                                                                                  466.2432                 466.2441                -1.93             C~24~H~36~NO~8~             100                          

                                                                                  189.1275                 189.1279                -2.11             C~13~H~17~O                 6                            

                                                                                  278.1235                 278.1240                -1.80             C~11~H~20~NO~7~             100                          

  62        3-MeO-PCP-M (*O*-demethyl-piperidine-di-HO-) glucuronide              5.5                                                                                                                         

                                                                                  468.2226                 468.2234                -1.71             C~23~H~34~NO~9~             10                           

                                                                                  175.1117                 175.1123                -3.43             C~12~H~15~O                 2                            

                                                                                  351.1440                 351.1444                -1.14             C~18~H~23~O~7~              100                          

  63        3-MeO-PCP-M (*O*-demethyl-cyclohexyl-HO-piperidine-HO-) glucuronide   4.3                                                                                                                         

                                                                                  468.2263                 468.2234                6.19              C~23~H~34~NO~9~             14                           

                                                                                  191.1067                 191.1072                -2.62             C~12~H~15~O~2~              90                           

                                                                                  367.1389                 367.1393                -1.09             C~18~H~23~O~8~              100                          
  --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

of *m/z* 123.0440 (C~7~H~7~O~2~) in LC-HR-MS/MS. Furthermore, the other three glucuronides (nos. 68-70) showed keto groups at the pyrrolidine ring. The fact, that the corresponding phase I metabolites could not be detected after conjugate cleavage with subsequent SPE, non-basic structures could be proposed, which led to the suggestion of lactam ring formation following hydroxylation at the α-position and further oxidation.

###### 

3-MeO-PCPy and its phase II metabolites detected in rat urine by LC-HR-MS^n^ with protonated precursor mass (PM), characteristic fragment ions (FI), calculated exact masses, proposed elemental composition, mass error, relative intensity, and retention times (RT).

  ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
  **No.**   **Metabolite**                                                            **Measured Accurate**\   **Calculated Exact**\   **Error (ppm)**   **Elemental Composition**   **Relative Intensity (%)**   **RT (min)**
                                                                                      **Mass (*m/z*)**         **Mass (*m/z*)**                                                                                   
  --------- ------------------------------------------------------------------------- ------------------------ ----------------------- ----------------- --------------------------- ---------------------------- --------------
  64        3-MeO-PCPy-M (*O*-demethyl-) glucuronide                                  5.1                                                                                                                         

                                                                                      422.2169                 422.2179                -2.37             C~22~H~32~NO~7~             100                          

                                                                                      175.1118                 175.1123                -2.86             C~12~H~15~O                 100                          

                                                                                      351.1411                 351.1444                -9.40             C~18~H~23~O~7~              1                            

  65        3-MeO-PCPy-M (*O*-demethyl-pyrrolidine-HO-) glucuronide                   5.8                                                                                                                         

                                                                                      438.2121                 438.2128                -1.60             C~22~H~32~NO~8~             60                           

                                                                                      175.1118                 175.1123                -2.86             C~12~H~15~O                 1                            

                                                                                      264.1078                 264.1083                -1.89             C~10~H~18~NO~7~             100                          

  66        3-MeO-PCPy-M (*O*-demethyl-aryl-HO-) glucuronide                          4.5                                                                                                                         

                                                                                      438.2119                 438.2128                -2.05             C~22~H~32~NO~8~             100                          

                                                                                      191.1067                 191.1072                -2.62             C~12~H~15~O~2~              50                           

                                                                                      367.1388                 367.1393                -1.36             C~18~H~23~O~8~              100                          

  67        3-MeO-PCPy-M (*O*-demethyl-aryl-HO-pyrrolidine-2-oxo-) glucuronide        10.1                                                                                                                        

                                                                                      452.1915                 452.1920                -1.11             C~22~H~30~NO~9~             30                           

                                                                                      191.1067                 191.1072                -2.62             C~12~H~15~O~2~              100                          

                                                                                      367.1387                 367.1393                -1.63             C~18~H~23~O~8~              72                           

  68        3-MeO-PCPy-M (cyclohexyl-HO-pyrrolidine-2-oxo-) glucuronide               10.8                                                                                                                        

                                                                                      466.2072                 466.2077                -1.07             C~23~H~32~NO~9~             50                           

                                                                                      205.1222                 205.1229                -3.41             C~13~H~17~O~2~              100                          

                                                                                      381.1543                 381.1549                -1.57             C~19~H~25~O~8~              12                           

  69        3-MeO-PCPy-M (cyclohexyl-di-HO-pyrrolidine-2-oxo-) glucuronide isomer 1   4.6                                                                                                                         

                                                                                      482.2020                 482.2026                -1.24             C~2~3H~32~NO~10~            10                           

                                                                                      203.1068                 203.1072                -1.97             C~13~H~15~O~2~              40                           

                                                                                      221.1173                 221.1178                -2.26             C~13~H~17~O~3~              100                          

                                                                                      397.1492                 397.1499                -1.76             C~19~H~25~O~9~              8                            

  70        3-MeO-PCPy-M (cyclohexyl-di-HO-pyrrolidine-2-oxo-) glucuronide isomer 2   5.2                                                                                                                         

                                                                                      482.2020                 482.2026                -1.24             C~23~H~32~NO~10~            14                           

                                                                                      203.1065                 203.1072                -3.45             C~13~H~15~O~2~              44                           

                                                                                      221.1170                 221.1178                -3.62             C~13~H~17~O~3~              100                          

                                                                                      397.1482                 397.1499                -4.28             C~19~H~25~O~9~              4                            

  71        3-MeO-PCPy-M (cyclohexyl-HO-pyrrolidine-di-HO-) glucuronide               8.0                                                                                                                         

                                                                                      484.2178                 484.2183                -1.03             C~23~H~34~NO~10~            60                           

                                                                                      205.1224                 205.1229                -2.44             C~13~H~17~O~2~              100                          

                                                                                      381.1545                 381.1549                -1.05             C~19~H~25~O~8~              20                           
  ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

###### 

General involvement of human CYP isoenzymes in initial metabolic steps of 3-MeO-PCP and 3-MeO-PCPy.

  -----------------------------------------------------------------------------------------------------------------------------------------
                              **CYP**\   **CYP**\   **CYP**\   **CYP**\   **CYP**\   **CYP**\   **CYP**\   **CYP**\   **CYP**\   **CYP**\
                              **1A2**    **2A6**    **2B6**    **2C8**    **2C9**    **2C19**   **2D6**    **2E1**    **3A4**    **3A5**
  --------------------------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
  **3-MeO-PCP**                                                                                                                  

  *O-*Demethylation                                 \+                               ++         \+                               

  Piperidine-hydroxylation                          ++                               \+                                          

  Cyclohexyl-hydroxylation                          ++                                                                           

  **3-MeO-PCPy**                                                                                                                 

  *O*-Demethylation                                 \+                    \+                    ++                               

  Pyrrolidine-hydroxylation                         ++                                                                           
  -----------------------------------------------------------------------------------------------------------------------------------------

###### 

Proposed targets for GC-MS SUSA monitoring 3-MeO-PCP or 3-MeO-PCPy, with molecular masses, most abundant fragment ions, their relative intensities, and retention indices (RI) according to Kovats \[[@r16]\]. The numbers correspond to those in [Figs. (2](#F2){ref-type="fig"} and [3](#F3){ref-type="fig"}).

  ----------------------------------------------------------------------------------------------------------------------------------------
  **No.**   **Target for SUSA**                              **Molecular**\   **GC-MS Fragment Ions (*m/z*),**\                   **RI**
                                                             **Mass (u)**     **and Relative Intensity (%)**                      
  --------- ------------------------------------------------ ---------------- --------------------------------------------------- --------
  2         3-MeO-PCP                                        273              273 (40), 230 (100), 161 (52), 121 (41)             2120

  2         3-MeO-PCP-M (*O*-demethyl-) AC                   301              84 (18), 166 (21), 244 (16), 258 (100), 301 (28)    2210

  3         3-MeO-PCP-M (*O*-demethyl-piperidine-HO-) 2 AC   359              164 (16), 258 (21), 300 (100), 316 (12), 359 (15)   2510

  32        3-MeO-PCPy-M (*O*-demethyl-) AC                  287              70 (38), 107 (60), 152 (38), 244 (100), 287 (22)    2160
  ----------------------------------------------------------------------------------------------------------------------------------------

###### 

Proposed targets for LC-MS^n^ SUSA monitoring 3-MeO-PCP or 3-MeO-PCPy, with protonated precursor ions, characteristic MS^2^ and MS^3^ fragment ions, and retention times (RT). The numbers correspond to those in [Figs. (2](#F2){ref-type="fig"} and [3](#F3){ref-type="fig"}).

  -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
  **No.**   **Target for SUSA**                                     **Precursor**\     **MS^2^ Fragment Ions (*m/z*), and Relative Intensity (%)**   **MS^3^ Fragment Ions (*m/z*), and Relative Intensity (%)**   **RT (min)**
                                                                    **Ions (*m/z*)**                                                                                                                               
  --------- ------------------------------------------------------- ------------------ ------------------------------------------------------------- ------------------------------------------------------------- --------------
  8         3-MeO-PCP-M (*O*-demethyl-piperidine-di-HO-)            292                101 (20), 118, (100), 175 (25)                                175: 81 (10), 107 (100)                                       8.6

  13        3-MeO-PCP-M (piperidine-di-HO-)                         306                101 (20), 118 (100), 189 (56)                                 189: 81 (20), 121 (100)                                       11.6

  57        3-MeO-PCP-M (*O*-demethyl-) glucuronide                 436                175 (100), 315 (70), 391 (10)                                 175: 81 (20), 107 (100)                                       5.5

  58        3-MeO-PCP-M (*O*-demethyl-aryl-HO-) glucuronide         452                175 (20), 191 (90), 367 (100)                                 191: 81 (6), 123 (100)                                        4.6

  61        3-MeO-PCP-M (piperidine-HO-) glucuronide                466                189 (6), 278 (100)                                            278: 84 (53), 102 (90), 242 (100)                             9.0

  32        3-MeO-PCPy-M (*O*-demethyl-)                            246                72 (100), 175 (60)                                            175: 81 (15), 107 (100)                                       8.2

  38        3-MeO-PCPy-M (*O*-demethyl-pyrrolidine-di-HO-)          278                87 (14), 104 (100),\                                          175: 81 (20), 107 (100)                                       7.3
                                                                                       175 (50), 232 (30)                                                                                                          

  39        3-MeO-PCPy-M (pyrrolidine-di-HO-)                       292                87 (10), 104 (94), 189 (100)                                  189: 81 (20), 121 (100)                                       10.9

  64        3-MeO-PCPy-M (*O*-demethyl-) glucuronide                422                175 (100), 315 (1)                                            175: 81 (30), 107 (100)                                       4.9

  67        3-MeO-PCPy-M\                                           452                191 (70), 367 (100)                                           191: 81 (6), 123 (100)                                        10.6
            (*O*-demethyl-aryl-HO-pyrrolidine-2-oxo-) glucuronide                                                                                                                                                  
  -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

###### 

Proposed targets for LC-HR-MS/MS SUSA monitoring 3-MeO-PCP or 3-MeO-PCPy, with protonated precursor ions, characteristic MS^2^ fragment ions, and retention times (RT). The numbers correspond to those in [Figs. (2](#F2){ref-type="fig"} and [3](#F3){ref-type="fig"}).

  ---------------------------------------------------------------------------------------------------------------------------------------------------------------
  **No.**   **Target for SUSA**                                                  **Precursor**\     **MS^2^ Fragment Ions (*m/z*)**\                  **RT**\
                                                                                 **Ions (*m/z*)**   **and Relative Intensity (%)**                    **(min)**
  --------- -------------------------------------------------------------------- ------------------ ------------------------------------------------- -----------
  2         3-MeO-PCP-M (*O*-demethyl-)                                          260.2014           81.0701 (15), 86.0966 (100), 107.0490 (73),\      4.4
                                                                                                    175.1111 (37), 260.2001 (4)                       

  3         3-MeO-PCP-M (*O*-demethyl-piperidine-HO-)                            276.1964           81.0701 (20), 84.0810 (18), 102.0913 (99),\       4.0
                                                                                                    107.0490 (100), 175.1111 (54)                     

  8         3-MeO-PCP-M (*O*-demethyl-piperidine-di-HO-)                         292.1913           79.0545 (16), 84.0810 (18), 102.0914 (100),\      3.1
                                                                                                    107.0491 (41), 173.0954 (58)                      

  13        3-MeO-PCP-M (piperidine-di-HO-)                                      306.2069           81.0701 (23), 101.0597 (14), 118.0860 (25),\      5.1
                                                                                                    121.0645 (100), 189.1266 (48)                     

  24        3-MeO-PCP-M (cyclohexyl-HO-piperidine-di-HO-)                        322.2018           79.0545 (41.88), 101.0597 (34), 118.0861 (66),\   4.3
                                                                                                    121.0645 (68), 187.1111 (100)                     

  57        3-MeO-PCP-M (*O*-demethyl-) glucuronide                              436.2335           81.0702 (10), 86.0966 (100), 107.0491 (23),\      4.0
                                                                                                    141.0176 (9), 175.1112 (22)                       

  58        3-MeO-PCP-M (*O*-demethyl-aryl-HO-) glucuronide                      452.2284           81.0701 (8), 86.0966 (55), 123.0438 (100),\       3.8
                                                                                                    191.1059 (76), 367.1369 (3)                       

  59        3-MeO-PCP-M (*O*-demethyl-piperidine-HO-) glucuronide                452.2284           84.0810 (56), 102.0914 (52), 107.0491 (90),\      4.0
                                                                                                    175.1111 (37), 278.1225 (100)                     

  61        3-MeO-PCP-M (piperidine-HO-) glucuronide                             466.2441           84.0810 (45), 102.0914 (38), 121.0646 (100),\     4.7
                                                                                                    189.1268 (41), 278.1225 (79)                      

  32        3-MeO-PCPy-M (*O*-demethyl-)                                         246.1858           72.0812 (100), 81.0702 (16), 107.0491 (86),\      4.3
                                                                                                    175.1112 (45), 246.1844 (4)                       

  38        3-MeO-PCPy-M (*O*-demethyl-pyrrolidine-di-HO-)                       278.1756           70.0656 (14), 79.0546 (15), 88.0759 (100),\       3.1
                                                                                                    107.0491 (38), 173.0956 (64)                      

  39        3-MeO-PCPy-M (pyrrolidine-di-HO-)                                    292.1913           81.0702 (24), 87.0443 (9), 104.0706 (18),\        5.0
                                                                                                    121.0646 (100), 189.1268 (46)                     

  45        3-MeO-PCPy-M (cyclohexyl-HO-pyrrolidine-di-HO-)                      308.1862           79.0545 (42), 87.0443 (29), 104.0706 (57),\       4.1
                                                                                                    121.0646 (66), 187.1111 (100)                     

  64        3-MeO-PCPy-M (*O*-demethyl-) glucuronide                             422.2179           72.0812 (100), 81.0701 (11), 107.0491 (27),\      3.7
                                                                                                    175.1112 (26), 422.2160 (13)                      

  66        3-MeO-PCPy-M (*O*-demethyl-aryl-HO-) glucuronide                     438.2128           72.0812 (43), 81.0701 (8), 123.0438 (100),\       3.5
                                                                                                    191.1059 (78), 367.1375 (3)                       

  67        3-MeO-PCPy-M (*O*-demethyl-aryl-HO-pyrrolidine-2-oxo-) glucuronide   452.1920           81.0702 (8), 86.0603 (22), 123.0439 (100),\       6.2
                                                                                                    149,0592 (9), 191.1060 (78)                       
  ---------------------------------------------------------------------------------------------------------------------------------------------------------------
