In September 2018, a yellow viscous seized material, weighing less than 100 mg, was asked for us to have it analyzed. The material was seized in a clandestine illicit drug manufacturer, being guessed of a failure semi-product of synthetic cannabinoids.

All solvents used were of HPLC grade. Methanol and acetonitrile are products from Fisher Chemical (Fisher Geel, Belgium). Water was purified from a Milli-Q system of Merck Millipore (Molsheim, France). Formic acid, of mass spectrometry grade, was purchased from Sigma-Aldrich (Shanghai, China). Deuterated chloroform (0.03%, v/v tetramethylsilane) for NMR was obtained in ampoules (750 mL) from Sigma-Aldrich (Saint Aubain, France). The regents and solvents for synthesis and purification were of analytical grade. Obtainment from J& K Scientific (Beijing, China) and Aladdin (Shanghai, China) was indazole-3-carboxylic acid methyl ester, 4-florobenzyl bromide and L-tert-leucin amide hydrochloride.

2.3.1 Analysis of the seized material through gas chromatography-mass spectrometry (GC-MS)

For GC-MS analysis, 10 mg of the seized viscous material was added with 10 mL of methanol, afterwards subjected to ultrasonic extraction and centrifuged at 5 000 r/min for 5 min with a Sigma 3K15 centrifuge (Sigma, Osterode am Harz, Germany). The supernatant was analyzed with an Agilent 7890 B gas chromatograph combined of an Agilent 7693 injector and an Agilent 5977B inert mass selective detector (Agilent, California, USA). The chromatographic separation was conducted on a DB-5MS column (30 m × 0.25 mm, 0.25 μ m film thickness, Agilent, California, USA). Helium was the selected carrier gas at a constant flow of 1.0 mL/min. Oven temperature was programmed from 60 ° C to 300 ° C at a stepping rate of 15 ° C/min and held for 15 min, with the total running time of 31 min. The temperatures of the injection port and the detector were set at 280 ° C and 230 ° C, respectively. The transfer line temperature was set at 280 ° C. The injection volume was 1 μ L under the splitting injection mode at a split ratio of 20:1. The scan range for the mass spectrometer was m/z 40~500amu. The obtained mass spectra of the suspected substance were compared to EI-MS spectra libraries (NIST) and an on-line library (SWGDRUG monographs).

2.3.2 Synthesis of ADB-FUBINACA

The ADB-FUBINACA was synthesized according to the published elsewhere ^{[9, 10, 11]} plus modifications, being schematically shown in Fig.2. The synthetic process was as follows. Indazole-3-carboxylic acid methyl ester 1 was deprotonated with potassium tert-butoxide and alkylated with 4-florobenzyl bromide to afford the methyl 1-(4-fluorobenzyl)-1H-indazole-3-carboxylate 2 (a little yellow solid). In the presence of sodium hydroxide, base-catalyzed hydrolysis of the methyl ester 2 gave the 1-(4-fluorobenzyl)-1H-indazole-3-carboxylic acid 3 (a white solid). Finally, amide bond was formed through free acid 3 subjecting to HOBt/EDC coupling with L-tert-leucinamide to give birth of the ADB-FUBINACA (a white powder) after the purification with flash chromatography. The information of the compounds 2 and 3 has already had descriptions somewhere ^[11]. Additionally, the stability was tested under different conditions, showing that the synthesized ADB-FUBINACA powder was stable within a year under the storage condition of freezer (-10~-25° C). Although the methanolic solution of the synthesized reference standard (1.0 mg/mL) could be stored under the condition of freezer (-10~-25° C) for a short time, it would take more than 6 months to decompose into 1-(4-fluorobenzyl)-1H-indazole-3-carboxylic acid.

Fig.2

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	Fig.2 Synthesis of ADB-FUBINACA

2.3.3 Characterization of the synthesized ADB-FUBINACA through Gas chromatography-mass spectrometry (GC-MS)

The synthesized compound was freshly prepared into its methanolic solution (0.1 mg/mL), afterwards analyzed with an Agilent 7890 B series gas chromatograph in combination of an Agilent 7693 injector and an Agilent 5977B inert mass selective detector (Agilent Technologies, California, USA). The method parameters are identical with those described in 2.3.1.

2.3.4 Liquid chromatograph mass spectrometer-ion trap-time of flight (LCMS-IT-TOF)

Same methanolic solution of the synthesized compound as that for the above GC-MS analysis was diluted 100 times with methanol to a final concentration of 1.0 μ g/mL, having it analyzed into LC-MS system that consists of an LC-20AD high performance liquid chromatograph (Shimadzu, Kyoto, Japan) connected to a hybrid mass spectrometer LCMS-IT-TOF equipping with an electrospray ionization source (Shimadzu, Kyoto, Japan). An Inertsil ODS-SP column (150 mm × 4.6 mm, 5 μ m particle size, Shimadzu, Kyoto, Japan) was used to separate the compound. The mobile phase system consisted of water containing 0.05% formic acid (A) and methanol (B). For chromatographic conditions, elution was carried out with 1.0 mL/min at 35 ° C under the isocratic programming setup: 25% A at time 0 min, and being held for 40 min. The injection volume was 10 μ L. For mass spectrometric conditions, the IT-TOF mass spectrometer was operated through an electrospray ionization (ESI) nanointerface in positive mode, with the parameters being as follows: probe voltage of +4.5 kV, both the curved desolvation line (CDL) and the block heater temperature as 200 ° C, CDL voltage of +25 V, drying gas pressure of 200 kPa, and nebulizing gas flow of 1.5 L/min, Argon of the collision gas. This event was performed with the m/z scan range from 100 to 1 000.

2.3.5 Nuclear magnetic resonance spectroscopy (NMR)

10 mg of the synthesized compound was dissolved into 0.6 mL CDCl₃. All NMR spectra including ¹H-NMR, ¹³C-NMR, DEPT-135 (distortionless enhancement by polarization transfer), ¹H-¹H COSY (correlation spectroscopy), ¹H-¹³C HMBC (heteronuclear multiple bond correlation), and ¹H-¹³C HSQC (heteronuclear single quantum correlation) were recorded at 25 ° C via a Bruker Avance III 500 MHz NMR spectrometer (Bruker, Ettlingen, Germany). Chemical shifts were reported in ppm relative to CHCl₃ (¹H: d = 7.26) and CDCl₃ (¹³C: d = 77.18) as internal standard.

2.3.6 High performance liquid chromatography with photo-diode array detection (HPLC-PDA)

The synthesized compound was prepared into a methanolic solution of 1.0 mg/mL. HPLC-PDA analysis was performed from a Shimadzu LC-20AD system coupling with a photo-diode array detector (Shimadzu, Kyoto, Japan). A Shim-pack HRC-ODS column (150 mm × 4.6 mm, 5 μ m particle size, Shimadzu, Kyoto, Japan) was used to separate the compound with the column chamber set at 40 ° C. The injection volume was 10 μ L, and the flow rate was 1.0 mL/min. Isocratic elution was applied through water containing 0.05% TFA (A) and methanol (B), under the programmed setup as follows: 35% A at time 0 min and being held for 35 min. The UV spectra were recorded from 190 to 400 nm, having acquired the chromatograms at 210 nm.

As shown in Fig.3, many peaks were observed in the total ion chromatogram from CG-MS analyzing the methanolic solution of the seized material. Through the obtained major-peak-shown mass spectra being analyzed one by one carefully, most of the contents of seized material cannot be found from NIST 11.0 library. The unknown peak at 19.818 min, as displayed in Fig. 3, showed major signals at m/z (relative intensity %) 109.0 (100), 253.1 (76), 338.1 (52), 309.1 (7) and 145.0 (7). As the signal of m/z 145.0 is a typical signal observed in the EI-MS spectra of synthetic cannabinoids with an indazole-3-carboxamide structure ^{[12, 13, 14, 15]}, the unknown peak was suspected of indazole derivatives. By means of searching in SWGDRUG monographs library and previously published articles, the mass spectrum of ADB-FUBINACA ^{[16, 17, 18]} and that of the unknown peak were of similar signals, suggesting that the seized material was likely containing ADB-FUBINACA.

Fig.3

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	Fig.3 GC-EI-MS total ion chromatogram of seized material (a) and mass spectrum of the unknown peak at retention time 19.818 min (b)

According to the recommendation of UNODC and SWGDRUG, a reliable and scientifically supported identification of seized drugs or chemicals requires appropriate analytical approach that must entail the determination of at least two uncorrelated parameters, with one of the parameters necessary to provide information on the chemical structure of the analyte. Thus, the unknown peak cannot be identified as ADB-FUBINACA unless there is a related reference standard.

The reference standard of ADB-FUBINACA was not available within the permissible time limit. Hence, the synthesis and characterization of ADB-FUBINACA had to be processed. As described in 2.3.2(2.3.3), the synthesized reference standard was characterized with appropriate analyses including GC-MS, LCMS-IT-TOF, NMR and HPLC-PDA, which were summarized as follows.

3.2.1 GC-MS

As shown in Fig.4, the major peak at 19.809 min was observed in the total ion chromatogram of the synthesized compound, demonstrating in accordance with that of ADB-FUBINACA from the earlier literatures ^{[16, 17, 18]}. The proposed fragmentation of the synthesized compound (ADB-FUBINACA?) was displayed in Fig. 4, too.

Fig.4

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	Fig.4 GC-EI-MS total ion chromatogram of the synthesized compound (a) and mass spectrum of the major compound ADB-FUBINACA (?) at retention time 19.809 min with proposed fragmentation (b)

3.2.2 LCMS-IT-TOF

The LCMS-IT-TOF spectral data of the synthesized compound were shown in Fig.5. The major ion at m/z 405.170 3 was identified as quasi-molecular ion corresponding to the molecular formula for the M+Na ion C₂₁H₂₃FN₄O₂Na⁺ (calcd. 405.169 7, error +1.48 ppm). There are two minor ions at m/z 383.186 4 and m/z 787.348 6 in the spectrum, eligible for being assigned as the M+H and 2M+Na ions, respectively. The m/z value of M+H ion is consistent with earlier published lite-rature ^[19].

Fig.5

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	Fig.5 LCMS-IT-TOF total ion chromatogram of the synthesized compound (a) and mass spectrum of the major compound ADB-FUBINACA (?) at retention time 7.12 min (b)

3.2.3 NMR spectroscopy

To try best to have the structural characterization perfect and complete, NMR test was further conducted. The 1-D NMR spectra for ¹H, ¹³C, DEPT-135 in Fig.6 and 2-D NMR spectra of COSY, HMQC, HMBC in Fig.7 were displayed, respectively.

Fig.6

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	Fig.6 1H, 13C and DEPT-135 NMR spectra of the synthesized ADB-FUBINACA (?) and the proposed proton and carbon peak assignment

Fig.7

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	Fig.7 1H-1H COSY, 1H-13C HMQC and 1H-13C HMBC spectra of the synthesized ADB-FUBINACA (?)

Proton and carbon peak assignment can be correlated to the structure and atom numbering as shown in Fig. 6: ¹H NMR (500 MHz, CDCl₃) δ = 8.22 (d, J = 8.15 Hz, 1H, H-12), 7.66 (d, J = 9.45 Hz, 1H, H-8), 7.27 (m, 1H, H-14), 7.23 (d, J = 8.45 Hz, 1H, H-15), 7.18 (m, 1H, H-13), 7.12 (m, 2H, H-21, 21’ ), 6.92 (m, 2H, H-22, 22’ ), 6.53 (s, 1H, H-7-2), 5.73 (s, 1H, H-7-1), 5.51 (s, 2H, H-19), 4.61 (d, J = 9.50 Hz, 1H, H-5), 1.09 (s, 9H, H-1, 2, 3); ¹³C NMR (125 MHz, CDCl₃) δ = 171.9 (C-6), 161.5 (C-9), 161.4 (d, ¹J_CF=245.29 Hz, C-23), 139.7 (C-16), 136.1 (C-10), 130.6 (d, ⁴J_CF=2.85 Hz, C-20), 128.0 (d, ³J_CF=8.21 Hz, C-21, 21’ ), 126.0 (C-14), 122.2 (C-11), 121.9 (C-13), 121.6 (C-12), 114.8 (d, ²J_CF=21.52 Hz, C-22, 22’ ), 58.6 (C-5), 52.0 (C-19), 33.7 (C-4), 25.7 (C-1, 2, 3).

3.2.4 HPLC-PDA

The methanolic solution of the synthesized compound was analyzed via HPLC to obtain the chromatographic profile. As displayed in Fig.8, the synthesized ADB-FUBINACA (?) was detected at 16.79 min as a major peak. The UV spectrum had two absorption maxima at 206 and 301 nm. Although there are several unidentified minor impurities, the purity was determined of 98.7% from HPLC-PDA with area normalization method, indicating that it is pure enough to be eligible as a reference standard.

Fig.8

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	Fig.8 HPLC-PDA chromatogram recorded at 210 nm of the synthesized compound (a) with same UV-spectrum of ADB-FUBINACA at retention time 16.79 min (b)

Based on all the obtained results from the analytical methods applied above, the synthesized compound was validated as ADB-FUBINACA and can be used as reference standard. The methanolic solution of seized material and that of synthesized ADB-FUBINACA were analyzed with GC-MS under the identical conditions. Compared of the retention time and mass spectrum, the unknown compound (RT = 19.818 min) in the seized material was identified as N-(1-amino-3, 3-dimethyl-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamide or ADB-FUBINACA. In total, the whole identification took less than 25 days with such an endeavor we tried.