Introduction

In recent years, various cathinone-derived designer drugs and synthetic cannabinoids have appeared on the drug market in many countries including Japan, and cases of acute intoxication caused by misuse of these substances have been frequently reported [1–5]. These synthetic cathinones can be obtained through Internet websites and local dealers, and are often sold as “fragrance powder” or “aroma liquid” and are labeled “not for human consumption” to circumvent drug abuse legislation. 3,4-Dimethylmethcathinone [1-(3,4-dimethylphenyl)-2-methylaminopropan-1-one, DMMC] has been more recently abused as one of the alternatives to recently controlled cathinones such as methylone, 4-methylmethcathinone, 4-methoxymethcathinone, and methylenedioxypyrovalerone (MDPV) [6]. These substances became regulated in 2012 as “designated substances (Shitei-Yakubutsu)’’ under the Japanese Pharmaceutical Affairs Law, which controls their production, distribution, and importation.

The synthetic cathinones are most commonly taken by oral ingestion or nasal insufflation, and self-reported doses vary widely from a few milligrams to over 1 g of powder [7–10]. Because users cannot be certain of the actual contents or the purity of the drug, the actual exposure is highly variable. The frequent occurrence of acute intoxication might be attributed in part to this factor. On the other hand, we have no published data for the pharmacokinetics and pharmacodynamics of the currently popular synthetic cathinones in humans owing largely to their recent emergence. A limited understanding of the mechanism of drug action is derived mostly from in vitro studies and animal models. Methylone and pyrovalerone were found to have inhibitory effects on presynaptic norepinephrine and dopamine (DA) reuptake [11–15], and 4-methylmethcathinone, structurally similar to DMMC, also inhibited both synaptosomal DA and 5-hydroxytryptamine (5HT) reuptake, which leads to overall stimulation of the central nervous system [16–18]. The pharmacological effects including increased energy, empathy, openness, and increased libido contribute to strong drug addiction, causing criminally serious problems such as murder, robbery, suicide, and traffic accidents in addition to serious public health concerns. To regulate the recreational use of these materials or to determine the cause of death or poisoning, information about metabolic pathways and metabolite analysis of the cathinones is indispensable in the field of forensic science and toxicology. However, despite several researchers having reported on the metabolism and metabolite analysis of other cathinones in humans and rats [19–30], no such data are available for DMMC.

In the current study, utilizing gas chromatography–mass spectrometry (GC–MS) and validated liquid chromatography–tandem mass spectrometry (LC–MS/MS) procedures, DMMC and several phase I metabolites including diastereomers in a user’s urine specimen were identified and quantified using authentic standards synthesized in our laboratory. In addition, other metabolites including phase II metabolites were deduced from product ion mass spectra and relative retention times obtained by LC–MS/MS. Based on these results, the metabolism of DMMC in humans is discussed including stereoisomeric metabolism. The effectiveness of the hydrolysis of conjugate metabolites is also verified. These analytical issues are of considerable importance both in forensic toxicological evaluation and in drug enforcement procedures.

Materials and methods

Reagents

DMMC, 3,4-dimethylcathinone (DMC), 1-(3,4-dimethylphenyl)-2-methylaminopropan-1-ol (β-OH-DMMC), and 2-amino-1-(3,4-dimethylphenyl)propan-1-ol (β-OH-DMC) were synthesized in our laboratory according to the methods of Archer [31] and Pizarro et al. [32] as described below. All synthesized standards were confirmed by 1H nuclear magnetic resonance (NMR) spectroscopy, and ensured as >98 % pure based on 1H NMR and LC–MS analysis by the direct flow injection method. Dibenzylamine (DBA), used as internal standard (IS), was obtained from Wako Pure Chemical Industries (Osaka, Japan). Standard stock solutions of all compounds were prepared in methanol and diluted with distilled water to appropriate concentrations as needed. β-Glucuronidase/sulfatase (Helix pomatia, Type H-1) was purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals and reagents were obtained from Wako Pure Chemical Industries.

All organic and inorganic reagents used were of analytical grade or quality. Distilled water and HPLC-grade methanol were used throughout the experiments.

Chemical synthesis

3,4-Dimethylmethcathinone (DMMC)

To a solution of 3,4-dimethylbenzaldehyde (Wako) in anhydrous tetrahydrofuran (THF), stirred and cooled in an external ice bath, 1.1 equivalents of ethyl magnesium bromide in THF was added dropwise over the course of 5 min. The mixture was stirred for an additional 1 h at room temperature. The reaction was quenched by the addition of saturated NH4Cl solution, and the mixture was extracted with ethyl acetate. The organic extract was washed with brine, dried over anhydrous Na2SO4, and evaporated to obtain crude 1-(3,4-dimethylphenyl)propan-1-ol, which was used without further purification.

To a solution of 1-(3,4-dimethylphenyl)propan-1-ol in a solvent mixture of n-hexane/diethyl ether (1:1, v/v), activated MnO2 was added portionwise, and the mixture was stirred at room temperature for 36 h. The solids were removed by filtration, the filter cake was washed with ethyl acetate, and the filtrate and washings combined. The organic extract was concentrated under vacuum to give 1-(3,4-dimethylphenyl)propan-1-one.

To a solution of 1-(3,4-dimethylphenyl)propan-1-one in CH2Cl2, one drop of bromine in CH2Cl2 was added. This solution was stirred for 5 min for the reaction to initiate. An equimolar amount of bromine solution was added over a period of a further 10 min. The solvent was removed under vacuum to yield 2-bromo-1-(3,4-dimethylphenyl)propan-1-one.

To a solution of 2-bromo-1-(3,4-dimethylphenyl)propan-1-one in THF was added dropwise 2 M methylamine in THF (Aldrich, St Louis, MO, USA). The mixture was stirred at room temperature overnight. The reaction mixture was treated with 10 % HCl aqueous solution to make it acidic and was then washed with diethyl ether. The aqueous layer was then made basic with 10 % Na2CO3 and extracted with ethyl acetate. The organic extract was washed with brine, dried over anhydrous Na2SO4, and evaporated to give crude DMMC as a pale yellow oil. Finally, 10 % HCl methanol solution was added dropwise to the oil. After removal of the solvent, the crude hydrochloride salt was purified by recrystallization from diethyl ether/2-propanol.

1H NMR (300 MHz, CD3OD): δ 7.82 (d, J = 1.5 Hz, 1H), 7.77 (dd, J = 1.5, 7.5 Hz, 1H), 7.36 (d, J = 7.5 Hz, 1H), 5.01 (q, J = 7.2 Hz, 1H), 2.73 (s, 3H), 2.37 (s, 6H), 1.54 (d, J = 7.2 Hz, 3H).

3,4-Dimethylcathinone (DMC)

To a solution of 2-bromo-1-(3,4-dimethylphenyl)propan-1-one (synthetic intermediate of DMMC) in THF was added dropwise 6 M ammonia in methanol (Aldrich). The resultant product DMC was purified as its hydrochloride by recrystallization from diethyl ether/2-propanol.

1H NMR (300 MHz, CD3OD): δ 7.81 (d, J = 2.1 Hz, 1H), 7.77 (dd, J = 2.1, 7.8 Hz, 1H), 7.34 (d, J = 7.8 Hz, 1H), 5.04 (q, J = 6.9 Hz, 1H), 2.36 (s, 6H), 1.53 (d, J = 6.9 Hz, 3H).

2-Amino-1-(3,4-dimethylphenyl)propan-1-ol (β-OH-DMC)

To a solution of 3,4-dimethylbenzaldehyde in methanol, 5 equivalents of nitroethane was added, and the mixture was cooled in an ice bath overnight. After nitroethane was evaporated under vacuum, the solid residue was dissolved in ethyl acetate. The organic extract was then washed with water and brine, dried over anhydrous Mg2SO4, and evaporated to obtain crude 1-(3,4-dimethylphenyl)-2-nitropropan-1-ol.

To a solution of 1-(3,4-dimethylphenyl)-2-nitropropan-1-ol in anhydrous methanol, a catalytic amount of palladium (10 wt % on carbon powder) was added. Three cycles of evacuation–hydrogen purging were performed by using a three-way stopcock connected to a hydrogen balloon on one outlet and a water aspirator on another. The flask was then left under hydrogen atmosphere and stirred for 4 h. The solution was filtered through celite (Wako), and the cake was washed with previously degassed anhydrous methanol. Finally, the solution was concentrated under vacuum to give diastereomic β-OH-DMC [(1RS,2SR)-2-amino-1-(3,4-dimethylphenyl)propan-1-ol; β-OH-DMC-1, (1RS,2RS)-2-amino-1-(3,4-dimethylphenyl)propan-1-ol; β-OH-DMC-2] as a pale yellow solid. The product ratio of the diastereomers was determined by NMR spectroscopy.

1H NMR (300 MHz, CDCl3; compound exists as a mixture of diastereomers, β-OH-DMC-1 denoted by *, β-OH-DMC-2 denoted by §): δ 7.12–7.00 (m, 3H*, 3H§), 4.42 (d, J = 3.6 Hz, 1H*), 4.16 (d, J = 8.1 Hz, 1H§), 3.12 (dq, J = 3.6, 6.3 Hz, 1H*), 2.99 (dq, J = 8.1, 6.3 Hz, 1H§), 2.25 (s, 3H*, 3H§), 2.24 (s, 3H*, 3H§), 0.99 (d, J = 6.3 Hz, 3H*), 0.98 (d, J = 6.3 Hz, 3H§).

1-(3,4-Dimethylphenyl)-2-methylaminopropan-1-ol (β-OH-DMMC)

To a solution of β-OH-DMC in THF, stirred and cooled in an external ice bath, 2 equivalents of triethylamine and 0.01 equivalents of 4-(dimethylamino)pyridine were added under an argon atmosphere. A solution of 2 equivalents of ethyl chloroformate in THF was then added dropwise, and the mixture was left at room temperature for 6 h. The residue was dissolved in diethyl ether and washed with water, 10 % HCl, and saturated NaCl, and then dried over anhydrous Na2SO4. The solution was then evaporated to obtain N-ethoxycarbonyl-1-(3,4-dimethylphenyl)-2-aminopropan-1-ol.

To a well-stirred suspension of lithium aluminum hydride (LAH) in anhydrous THF, a solution of N-ethoxycarbonyl-1-(3,4-dimethylphenyl)-2-aminopropan-1-ol in anhydrous THF was added dropwise. The mixture was then refluxed and stirred for 18 h to obtain a grey suspension. The excess LAH was destroyed by addition of ethyl acetate and water. The solids were removed by filtration, the filter cake was washed with THF, and the pooled filtrate and washings were evaporated under vacuum. The residue was subjected to column chromatography using an aminosilica gel column and an ethyl acetate/n-hexane mixture (1:2, v/v) as eluent to isolate β-OH-DMMC. Finally, the solution was concentrated under vacuum to give diastereomic β-OH-DMMC [(1RS,2SR)-1-(3,4-dimethylphenyl)-2-methylaminopropan-1-ol, β-OH-DMMC-1; (1RS,2RS)-1-(3,4-dimethylphenyl)-2-methylaminopropan-1-ol, β-OH-DMMC-2] as a pale yellow solid. The product ratio of the diastereomers was determined by NMR spectroscopy.

1H NMR (300 MHz, CDCl3; compound exists as a mixture of diastereomers, β-OH-DMMC-1 denoted by *, β-OH-DMMC-2 denoted by §): δ 7.13–7.02 (m, 3H*, 3H§), 4.74 (d, J = 3.6 Hz, 1H*), 4.17 (d, J = 8.1 Hz, 1H§), 3.12 (dq, J = 3.6, 6.3 Hz, 1H*), 2.67 (dq, J = 8.1, 6.3 Hz, 1H§), 2.48 (s, 3H*), 2.46 (s, 3H§), 2.26 (s, 3H*, 3H§), 2.24 (s, 3H*, 3H§), 0.94 (d, J = 6.3 Hz, 3H§), 0.89 (d, J = 6.3 Hz, 3H*).

Urine specimen

A urine specimen obtained from a DMMC user had been submitted to our laboratory for forensic analysis. The specimen was collected from the user 6 h after an intake of DMMC hydrochloride (ca. 30 mg) based on the user’s confession, and was stored at −20 °C until analysis. Drug-free urine used for validation experiments was obtained from healthy volunteers.

Sample preparation

Sample preparation for GC–MS

A 100-μl aliquot of urine was adjusted to pH 9 with a carbonate buffer (1 M, pH 9.5) and saturated with NaCl. To the solution, 500 μl of chloroform/2-propanol (3:1, v/v) was added and the mixture was vortex-mixed for 1 min. After centrifuging (10 min, 7000 g), the organic layer was transferred into a stoppered glass test tube. After addition of 50 μl of acetic acid, the mixture was evaporated to dryness under a nitrogen stream at 40 °C. The residue was reconstituted in 50 μl of ethyl acetate and trifluoroacetylated at 70 °C for 20 min with 50 μl of trifluoroacetic anhydride (TFAA). After evaporation of the derivatization mixture, the residue was dissolved in 50 μl of ethyl acetate, and a 1-μl aliquot was injected into the GC–MS system.

Sample preparation for LC–MS/MS

A 1-ml aliquot of urine was adjusted to pH 5 with acetic acid (1 M) and incubated at 37 °C for 5 h with β-glucuronidase/sulfatase (15,000 and 750 U/ml urine each). To aliquots of prehydrolysis urine and posthydrolysis urine (100 μl each), 100 μl of DBA aqueous solution (0.1 μg/ml) was added as IS. After mixing briefly, 600 μl of methanol was added to each solution, and the mixtures were vortex-mixed for 1 min. The mixtures were centrifuged for 10 min at 7000 g, and the supernatants were each transferred to a stoppered glass test tube and evaporated to dryness under a nitrogen stream at 50 °C. The residues were each dissolved in 100 μl of distilled water. After filtration through a 0.22-μm membrane filter, 5-μl aliquots were used for LC–MS/MS analysis.

Instrumentation

NMR spectroscopy

All NMR spectra of the synthetic compounds were acquired using a Varian GEM300 NMR spectrometer (Polo Alto, CA, USA) in CDCl3.

GC–MS

A GCMS-QP2010 gas chromatograph–mass spectrometer (Shimadzu, Kyoto, Japan) was operated in the electron ionization (EI) mode utilizing a DB-5MS fused-silica capillary column (30 m × 0.25 mm i.d., film thickness 0.25 μm; Agilent, Santa Clara, CA, USA). Injections were made automatically in the splitless mode at an injection port temperature of 250 °C. The carrier gas was high-purity helium at a flow rate of 3.0 ml/min. The column temperature was initially held at 60 °C for 1 min, and increased to 300 °C at 10 °C/min. The ionization energy and interface temperature were set at 70 eV and 250 °C, respectively.

LC–MS/MS

Analysis was performed on a Prominence Series UFLC system (Shimadzu) linked to an API 3200 QTRAP hybrid triple quadruple linear ion-trap mass spectrometer (AB Sciex, Concord, ON, Canada) equipped with an electrospray ionization (ESI) interface. LC separation was carried out using an L-column2 ODS semi-micro column (150 mm × 1.5 mm i.d., 5 μm particles; Chemicals Evaluation and Research Institute, Tokyo, Japan). The analytes were chromatographed by linear gradient elution with 10 mM ammonium formate buffer (pH 5) and methanol at a flow rate of 0.1 ml/min at a column temperature of 40 °C. Details of the gradient conditions accompany the chromatograms presented as figures in this report. ESI–MS/MS was conducted in the positive ion mode, and the protonated molecules were used as precursor ions. Analyses were performed in the enhanced product ion scan (EPI) mode for identification and in the selected reaction monitoring (SRM) mode for quantitation. Nitrogen was used as nebulizer and collision gas, and the declustering potential (DP) and collision energy (CE) were, respectively, set at 20 V and 20 eV for EPI. The SRM parameters for each compound were set as shown in Table 1.

Table 1 Targeted compounds and their optimized SRM parameters
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Time-of-flight mass spectrometry

Time-of-flight mass spectrometry (TOF MS) was carried out on a microTOF II (Bruker Daltonics, Bremen, Germany) equipped with an ESI interface. ESI–TOF MS was performed in the positive mode under the following operating parameters: capillary voltage, 4.5 kV; capillary exit voltage, 100, 150, or 200; skimmer 1 voltage, 33, 50, or 67. Exact masses of protonated molecules and fragment ions were measured by an infusion method and prompting in-source collision-induced dissociation (CID) as needed. Internal mass calibration was performed on the basis of cluster ions of sodium formate.

Results and discussion

Synthesis of DMMC and putative metabolites

We have no information for metabolic pathways and metabolite analysis of DMMC, but have previously reported on the metabolism of cathinone-derived drugs in humans and rats [19–21]. Several other reports have shown metabolic pathways of methcathinone-derived drugs including 4-methylmethcathinone and 3-bromomethcathinone [23, 28, 30]. These findings and preliminary experimental results by GC–MS and LC–MS/MS suggested that metabolic pathways of DMMC include its N-demethylation, reduction of the ketone group, and combination of these steps. In the present study, four compounds needed for metabolic studies, unchanged DMMC, DMC, β-OH-DMMC, and β-OH-DMC, were chemically synthesized because their reference standards were not commercially available. It was expected that two reductive metabolites that have two chiral carbons, β-OH-DMMC and β-OH-DMC, are, respectively, excreted in urine as diastereomers, and their syntheses therefore were performed according to the methods as described in the materials and methods section to obtain the corresponding diastereomers. Table 2 summarizes the results of the NMR analysis and gives the product ratios of the synthesized diastereomers. The product ratios were calculated from the peak areas of Ha and Hb obtained from the NMR spectra.

Table 2 Results of NMR and product ratios of synthesized diastereomers
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Identification of DMMC and its metabolites in urine by GC–MS

In order to achieve high discrimination capability and high sensitivity, trifluoroacetyl (TFA) derivatization with TFAA was adopted as pretreatment for GC–MS. The free base of DMMC and its metabolites without derivatization provided poor sensitivity and few fragment ions. Figure 1 shows the total ion chromatogram (TIC), extracted mass chromatograms, and EI mass spectra obtained from a DMMC user’s urine and a mixture of authentic standards. DMC-TFA, β-OH-DMMC-diTFA, and β-OH-DMC-diTFA as well as DMMC-TFA were detected in the user’s urine. Two diastereomic metabolites, β-OH-DMMC and β-OH-DMC, respectively, produced two peaks [(c) and (c′), (d) and (d′) in Fig. 1], and no significant difference in mass spectra between the diastereomers was observed (data not shown). This finding indicated that racemic DMMC is metabolized to diastereomic β-OH-DMMC and β-OH-DMC, although Meyer et al. [23] reported that 4-methylmethcathinone is reduced to only one diastereomer because of steric hindrance effects in the enzymatic reaction.

Fig. 1
figure 1

A Total ion chromatogram (TIC) obtained from an authentic standard mixture (1 μg/ml each), B TIC and extracted mass chromatograms obtained from a DMMC user’s urine specimen, and C EI mass spectra of TFA derivatives of DMMC and its metabolites. Peaks: a DMMC-TFA, b DMC-TFA, c and c′ β-OH-DMMC-diTFA, d and d′), β-OH-DMC-diTFA. Numbers in parentheses represent magnification ratios

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On the other hand, some studies [20, 33] suggested that TFA derivatives of β-hydroxylated metabolites of cathinones isomerize at the GC inlet, which intimates that GC–MS cannot provide satisfactory reproducibility and reliability for quantitative determinations. To obtain more reliable quantitative results, simultaneous determinations of DMMC and its metabolites were performed on the LC–MS/MS system.

Determination of DMMC and its metabolites by LC–MS/MS

Identification of DMMC and its metabolites

To simultaneously determine DMMC and its metabolites in urine, a sensitive and reliable LC–MS/MS procedure was established. A C18 semi-micro column with a gentle gradient elution program optimized in the present study provided clear separation of all six analytes, including diastereomers, as shown in Fig. 2A.

Fig. 2
figure 2

A Extracted mass chromatograms obtained from a DMMC user’s urine specimen and B product ion mass spectra of DMMC and its metabolites. Peaks: a DMMC, b DMC, c β-OH-DMMC-2, c′ β-OH-DMMC-1, d β-OH-DMC-2, d′ β-OH-DMC-1, IS internal standard. Each protonated molecule was selected as a precursor ion. The 60-min chromatographic run was carried out with a binary mobile phase of methanol and 10 mM ammonium formate buffer using a linear gradient (15 –55 % methanol)

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Based on our additional experiment, the peaks (c), (c′), (d), and (d′), respectively, correspond to β-OH-DMMC-2, β-OH-DMMC-1, β-OH-DMC-2, and β-OH-DMC-1 because diastereomic ephedrines, structurally similar to β-OH-DMMC and β-OH-DMC, that were eluted at later retention times under the same LC conditions are pseudoephedrine, ephedrine, cathine, and norephedrine (data not shown).

The qualitative analysis by MS/MS was performed in the EPI mode, and CE was set at 20 eV to obtain abundant product ions. Figure 2B shows product ion mass spectra of DMMC and its metabolites extracted from a DMMC user’s urine specimen. No significant difference in product ion mass spectra between each pair of the diastereomers ((c) and (c′), (d) and (d′)) was observed (data not shown).These spectra agreed well with those of the authentic standards.

In addition, the elemental compositions of predominant product ions were verified using TOF MS. The measured accurate mass, molecular formula candidate, and the mass error related to DMMC and its metabolites are shown in Table 3. The measured accurate mass of each ion obtained from the analytes was found to correlate well with the calculated exact mass of the proposed ion, [M+H]+, [M+H–H2O]+, [M+H–H2O–CH3]+, [M+H–H2O–NH2CH3]+, or [M+H–H2O–NH3]+.

Table 3 Measured accurate mass of protonated molecular and predominant fragment ions, and their proposed ion structures
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Validation data

The quantitative analysis by MS/MS was performed in the SRM mode. Table 4 summarizes the validation data obtained by analyzing various concentrations of analytes spiked into drug-free urine. The pretreatment of urine specimens with methanol (protein precipitation method) provided high recoveries of not less than 95 %. Calibration curves for LC–MS/MS (in the SRM mode) were linear over a concentration range of 10–5000 ng/ml. When the values for urinary concentrations of analytes in a DMMC user’s urine are above the calibration range, the concentrations should be determined after dilution with drug-free urine to an appropriate concentration. The obtained values for accuracy deviation and precision were less than 15 % for all compounds at all concentrations examined. β-OH-DMC (especially β-OH-DMC-2) has a lower sensitivity to the ESI–MS compared to the other analytes because of its ready elimination of a water molecule from the protonated molecule by in-source CID (data not shown).

Table 4 Validation data for the LC–MS/MS procedure established in the present study
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Concentrations of DMMC and its metabolites in DMMC user’s urine

Table 5 summarizes the results obtained from an actual user’s urine specimen.

Table 5 Concentrations of DMMC and its metabolites in DMMC user’s urine before and after enzymatic hydrolysis
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Unchanged DMMC was excreted in urine at the highest concentration, and β-OH-DMC-1 was the most abundant metabolite of the identified compounds. In the present study, β-hydroxylic metabolites (i.e., β-OH-DMMC and β-OH-DMC), were stereoisomerically separated and determined for the first time in cathinone-derived designer drugs. Interestingly, this result indicates that the urinary concentration of β-OH-DMMC-2 was higher than that of β-OH-DMMC-1, and β-OH-DMC-2 was, in contrast, less abundant than β-OH-DMC-1. The reversal phenomenon in urinary concentration is thought to be caused by a complex relationship of enantioselective and diastereoselective pharmacokinetic properties of DMMC and its metabolites. In the 1980s, Brenneisen et al. [34] reported on the metabolism of α-aminopropiophenone (cathinone) in khat leaves, and concluded that (S)-cathinone and (R)-cathinone are, respectively, metabolized to 1R,2S-(−)-norephedrine and 1R,2R-(+)-norpseudoephedrine by the stereospecific 1R keto reduction. On the basis of this report, all the β-hydroxylic metabolites detected in the DMMC user’s urine might have the same R-configuration at the corresponding position (β-position). These findings potentially contribute to determination of the enantioselective disposition of DMMC.

Effectiveness of hydrolysis of conjugate metabolites

Our previous and other several studies [20, 21, 23, 26] have indicated that cathinone-derived drugs and/or their metabolites increased in concentration after acidic/enzymatic hydrolysis of conjugated metabolites. As shown in Table 5, DMMC and all of the identified metabolites except for β-OH-DMMC-1 increased after enzymatic hydrolysis. This result confirmed that the hydrolysis of a urine sample is partly effective as pretreatment for GC–MS and LC–MS analysis, and is consistent with our previous reports regarding the increase in the unchanged drug. In terms of diastereoselectivity, each increased level of β-OH-DMMC-2 or β-OH-DMC-2 was higher than that of β-OH-DMMC-1 or β-OH-DMC-1, which suggested that β-OH-DMMC-2 and β-OH-DMC-2 were more abundantly conjugated, and then excreted in urine.

Investigation of other metabolites in DMMC user’s urine

To survey other metabolites including conjugated metabolites, a urine specimen before and after enzymatic hydrolysis was analyzed by LC–MS/MS. The putative metabolites were deduced from the product ions and relative retention times.

Figure 3 shows SRM chromatograms and product ion mass spectra of putative metabolites obtained from a hydrolyzed urine specimen. Putative oxidative metabolites of the xylyl group [peak (a), carboxylic form] and their reductive metabolites of the ketone group [peak (b)] were detected at the retention times of 6.1 and 5.9 min, respectively. These broad peaks probably contain the multiple metabolites: peak (a), 3-carboxyl-4-methylmethcathinone and 4-carboxyl-3-methylmethcathinone (positional isomers); peak (b), 3-carboxyl-4-methylephedrine and 4-carboxyl-3-methylephedrine (positional isomers) and their diastereomers. In addition, putative oxidative metabolites of the xylyl group [peaks (c) and (d) in Fig. 3, alcohol] and their reductive metabolites of the ketone group [peaks (e), (e′), (f), and (f′) in Fig. 3] were detected in urine. No significant difference in product ion mass spectra between each pair of the metabolites, positional isomers (c) and (d), diastereomers (e) and (e′), and diastereomers (f) and (f′), was observed. On the other hand, an obvious difference between positional isomers (e) and (f) was observed: only the metabolite (f) produced a distinctive ion of [M+H–2H2O]+ at m/z 174. This may be because the ability to eliminate water from a protonated molecule changes depending on the position (3 or 4) of a [CH2OH] (hydroxymethyl) group on the benzene ring. These findings indicate that the metabolic pathways of DMMC include oxidation of the xylyl group, and are comparable to the recent results of Meyer et al. [23] that the metabolic pathway of 4-methylmethcathinone includes oxidation of the tolyl group.

Fig. 3
figure 3

A Extracted mass chromatograms obtained from a DMMC user’s urine specimen and B product ion mass spectra of putative metabolites. Peaks: a 3-carboxyl-4-methylmethcathinone and/or 4-carboxyl-3-methylmethcathinone, b 3-carboxyl-4-methylephedrine and/or 4-carboxyl-3-methylephedrine, c and d, 4-hydroxymethyl-3-methylmethcathinone and 3-hydroxymethyl-4-methylmethcathinone (random order), e, e′, f, and f′ 4-hydroxymethyl-3-methylephedrine, 3-hydroxymethyl-4-methylephedrine, and their diastereomers (random order). Each protonated molecule was selected as a precursor ion. The 30-min chromatographic run was carried out with a binary mobile phase of methanol and 10 mM ammonium formate buffer using a linear gradient (5–50 % methanol)

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In another experiment, a urine specimen before hydrolysis was analyzed to directly identify conjugated metabolites related to DMMC. Five putative conjugated metabolites were detected as shown in Fig. 4. No significant difference in product ion mass spectra between each pair of the diastereomic metabolites, peaks (b) and (b′) and peaks (c) and (c′), was observed (data not shown). Both β-OH-DMC-glucuronide (Glu) [peaks (b) and (b′)] and β-OH-DMMC-Glu [peaks (c) and (c′)] produced an [M+H–Glu–H2O]+ ion as a base peak, while DMMC-Glu [peaks (a)] produced [M+H–Glu]+ ion as a base peak. On the basis of retention times of β-OH-DMC-2, β-OH-DMC-1, β-OH-DMMC-2, and β-OH-DMMC-1 in Fig. 2, it is estimated that peaks (b), (b′), (c), and (c′), respectively, correspond to β-OH-DMC-2-Glu, β-OH-DMC-1-Glu, β-OH-DMMC-2-Glu, and β-OH-DMMC-1-Glu. The peak areas corresponding to β-OH-DMC-2-Glu (b) and β-OH-DMMC-2-Glu (c) were larger than those of β-OH-DMC-1-Glu (b′) and β-OH-DMMC-1-Glu (c′), respectively, which were well correlated with higher increased levels of β-OH-DMC-2 and β-OH-DMMC-2 after the hydrolysis compared to those of β-OH-DMC-1 and β-OH-DMMC-1. These peaks corresponding to conjugated metabolites disappeared following enzymatic hydrolysis. No change of β-OH-DMMC-1 in urinary concentration between before and after hydrolysis might be attributed to a slight loss of β-OH-DMMC-1 by decomposition during incubation at 37 °C.

Fig. 4
figure 4

A Extracted mass chromatograms obtained from a DMMC user’s urine specimen and B product ion mass spectra of putative conjugated metabolites. Peaks: a DMMC-glucuronide (Glu), b β-OH-DMC-2-Glu, b′ β-OH-DMC-1-Glu, c β-OH-DMMC-2-Glu, c′ β-OH-DMMC-1-Glu. Each protonated molecule was selected as a precursor ion. The 30-min chromatographic run was carried out with a binary mobile phase of methanol and 10 mM ammonium formate buffer using a linear gradient (5–50 % methanol)

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Proposed metabolic pathways

The detection of urinary metabolites in the present study suggested metabolic pathways of DMMC as shown in Fig. 5. The identified metabolites indicate that the metabolic pathways of DMMC include reduction of the ketone group to β-OH-DMMC, N-demethylation to DMC, and combination of these steps to β-OH-DMC. The putative metabolites reveal the following metabolic steps: oxidation of the xylyl group to two corresponding alcohols (4-hydroxymethyl-3-methylmethcathinone and 3-hydroxymethyl-4-methylmethcathinone), followed by further oxidation of the hydroxymethyl group to the respective carboxylic acids (4-carboxyl-3-methylmethcathinone and 3-carboxyl-4-methylmethcathinone), and then the reduction of the keto group to the respective diastereomic compounds. In addition, DMMC and all of the identified metabolites were estimated to be partially conjugated to mainly glucuronide as shown in Fig. 4.

Fig. 5
figure 5

Proposed metabolic pathways of DMMC in humans

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Conclusions

In the present study, DMMC and its five metabolites including diastereomers were separately determined in a DMMC user’s urine using newly synthesized authentic standards. On the basis of the identified and putative metabolites, the main metabolic pathways of DMMC in humans are proposed as follows: N-demethylation to the primary amine, reduction of the keto group to the alcohol, oxidation of the xylyl group to the corresponding alcohol and carboxylic acids, and combination of these steps. The urinary levels of unchanged DMMC and its metabolites increased slightly after hydrolysis, suggesting that these compounds are partially metabolized to respective conjugates (probably glucuronides). These findings will contribute to the establishment of a reliable analytical procedure for proving the intake of newly encountered designer drugs as well as in prediction of metabolic pathways of them.