Table 3.

Analytical techniques for the identification and quantification of AMB-FUBINACA.

Sample	Other Substance(s) Analysed	Sample Preparation/Extraction	Method for Analysis	Method Validation Parameters	Results From the Study	Reference (Year)
Serum, whole blood, and urine samples from 8 patients among the 18 who were transported to local hospitals; and a sample of the herbal “incense” product “AK-47 24 Karat Gold”, which was implicated in the so-called “Zombie” Outbreak in New York, 2016	De-esterified acid metabolite of AMB-FUBINACA	-	LC–QTOF/MS	Internal standard: AMB-FUBINACA	AMB-FUBINACA was identified in AK-47 24 Karat Gold at 16.0 ± 3.9 mg/g. The de-esterified acid metabolite was found in the serum or whole blood of all eight patients, with concentrations ranging from 77 to 636 ng/mL in serum; and in urine of one patient at 165 ng/mL	[3] (2017)
Cayman Chemical drug standards	α-PVP and other NPS	2 µL of 0.1 mM AMB-FUBINACA were mixed with 4 µL of silver nanoparticles and 2 µL of MgCl2	SERS	LOD: 1 nM	Identification of these drugs in a combination pose a challenge for SERS, however this technique is very useful for detecting individual drugs	[57] (2018)
Samples from human liver microsomes in vitro and zebrafish models in vivo	Metabolites of AMB-FUBINACA	Human liver microsomes: AMB-FUBINACA at 5 mM (in methanol) was diluted 200× in microsome suspension and incubated for 1 h at 37 °C. Then, uridine diphosphate glucuronic acid trisodium salt was added and incubated for another half an hour. To terminate the reaction 200 µL of acetonitrile was added, followed by centrifugation (13,000 g; for 10 min). 100 µL of the supernatant was used after membrane filtering for analysis Zebrafish (6–10 months; 0.8–1.2 g): After exposure to 0.1, 0.5, and 1 µg/mL of AMB-FUBINACA (24 °C) for 24 h, zebrafish were removed, cleaned with water and euthanised. The zebrafish were homogenised with a ball mill, and the samples were loaded onto a SPE-Pak@Vac PSA extraction column, which had been conditioned with 1 mL of methanol and 1 mL of water. The column was washed with 1 mL of acetonitrile. The eluent was dried by evaporation at 60 °C under a stream of nitrogen. The residue was reconstituted in 100 µL of flow phase composed of acetonitrile, and 10 µL of the reconstituted solution was injected for analysis	HPLC	n.d.	The precision, simplicity and efficiency of the technique proved advantages for the identification of 17 metabolites, making it a useful tool for the detection of polar metabolites, in clinical and forensic contexts	[39] (2019)
Cayman Chemical standards	CUMYL-PICA, 5F-CUMYL-PICA, MDMB-FUBINACA, NNEI and MN-18	Each SC was dissolved in acetonitrile at 0.5 mg/mL, and 16 µL were added to a quartz capillary tube loaded into the thermolysis autosampler that passed the individual samples to the thermolysis probe equilibrated at 50 °C, which was then rapidly heated (20 °C/second) to the desired temperature. The samples were heated sequentially to 200, 400, 600, and 800 °C	GC-MS; LC-MS/MS	n.d.	SCs heated above 400 °C produce thermolytic, potentially toxic degradants, such as naphthalene, 1-naphthylamine, cyanide and toluene	[40] (2019)
Human blood from real cases	29 SCs; 4 amphetamines; ∆9-THC; ∆9-THC-COOH	To 200 μL of sample, 20 μL of internal standard was added for a final concentration of 5 ng/mL. After the addition of 200 μL of 100 mM sodium acetate buffer (pH 5.0), 300 μL of the conditioned blood was loaded onto a supported-liquid-extraction cartridge. Analytes were eluted with 700 μL methyl terc-butyl ether (× 2). 20 μL of 0.1 M methanolic HCl was then added to elute SCs, and all extracts were dried at 30 °C under nitrogen flow. Residues were reconstituted in 80 μL of 50:50 (v/v) water: methanol and vortexed prior to centrifugation (3000 rpm, 5 min)	LC–MS/MS	Internal standards: JWH-018; N5HP-d5; LOQ: 1 ng/mL; LOD: 0.1 to 6.0 ng/mL; Accuracy: 5.8% at 1 ng/mL; 19.1% at 5 ng/mL; Precision: 10.5% at 1 ng/mL; 9.6% at 5 ng/mL; Linearity: r2: 0.999 with a confirmation ranged between 1 to 6 ng/mL	The validated method allowed for the simultaneous confirmation of 29 SCs and metabolites, 4 amphetamines, and 2 phytocannabinoids in human whole blood. The five most commonly detected SCs in toxicological samples in New Zealand in 2018 were AMB-FUBINACA and/or its acid metabolite, 5F-ADB and/or its acid metabolite, ADB-FUBINACA, 5F-MDMB-PICA acid metabolite, and MDMB-FUBINACA acid metabolite	[65] (2020)

α-PVP: α-Pyrrolidinopentiophenone; ∆⁹-THC: trans-∆⁹-Tetrahydrocannabinol; ∆⁹-THC-COOH: 11-nor-9-Carboxy-∆⁹-tetrahydrocannabinol; GC-MS: Gas chromatography–mass spectrometry; HPLC: High-performance liquid chromatography; LC-MS/MS: Liquid chromatography–tandem mass spectrometer; LOD: Limit of detection; LOQ: Limit of quantification; n.d.: No data (method not validated); SC: Synthetic cannabinoid; SERS: Surface-enhanced Raman spectroscopy.