Novel synthetic opioids – toxicological aspects and analysis

Figures & data

Figure 1. Chemical structure of fenatanyl and their analogues: (A) fentanyl; (B) tetrahydrofuranylfentanyl (THFF); (C) furanylfentanyl; (D) acetylfentanyl; (E) ocfentanil; (F) butyrfentanyl; (G) cyclopropylfentanyl; (H) methoxyacetylfentanyl; (I) acrylfentanyl; (J) para-fluoroisobutyrfentanyl; (K) para-fluoroisobutyrylfentanyl; (L) carfentanil; (M) α-methylfentanyl. The compounds’ potency has been compared to that of morphine (MOR).

Table 1. Fentanyl analogues reported to the United Nations Office on Drugs and Crime (UNODC) Early Warning Advisory (EWA), not yet placed under control and fentanyl and analogues controlled under the 1961 Single Convention on Narcotic Drugs [47].

Control status	Fentanyl analogues
Reported to the UNODC EWA—not yet placed under control	Acrylfentanyl
	para-Fluoroisobutyrfentanyl
	Butyrfentanyl
	Furanylfentanyl
	Ocfentanil
	Carfentanil
Controlled under the 1961 Single Convention on Narcotic Drugs	Remifentanil
	Acetylfentanyl
	α-Methyl-thiofentanyl
	β-Hydroxyfentanyl
	β-Hydroxy-3-methylfentanyl
	3-Methylthiofentanyl
	para-Fluorofentanyl
	Thiofentanyl
	Acetyl-α-methylfentanyl
	α-Methylfentanyl
	3-Methylfentanyl
	Alfentanil
	Sufentanil
	Fentanyl

Figure 2. 4-ANPP.

Figure 3. Other novel synthetic opioids: (A) AH-7921; (B) U-47700; (C) U-49900; (D) U-50488; (E) MT-45. The compounds’ potency has been compared to that of morphine (MOR).

Table 2. Novel synthetic opioids schedule by Drug Enforcement Administration in 2018 [66].

Substance	Schedule^a
3-Methylfentanyl	I
3-Methylthiofentanyl	I
Acetyl fentanyl	I
Acetyl-α-methylfentanyl	I
Acetyldihydrocodeine	I
AH-7921	I
α-Methylfentanyl	I
α-Methylthiofentany	I
Acrylfentanyl	I
β-Hydroxy-3-methylfentanyl	I
β-Hydroxyfentanyl	I
β-Hydroxythiofentanyl	I
Butyryl Fentanyl	I
Carfentanil	II
Cyclopropylfentnyl	I
Fentanyl	II
Furanyl fentanyl	I
Isobutyryl fentanyl	I
Methoxyacetylfentanyl	I
Ocfentanil	I
para-Fluorofentanyl	I
U-47700	I
Valeryl fentanyl	I

^a Schedule I means the drug has a high potential for abuse, has no currently accepted medical use in treatment in the US or lacks accepted safety for use of the drug or other substance under medical supervision; Schedule II means the drug has a high potential for abuse, has a currently accepted medical use in treatment in the US or a currently accepted medical use with severe restrictions, or abuse of the drug may lead to severe psychological or physical dependence.

Table 3. Analytical information about protein precipitation and sample dilution as sample preparation techniques to determine NSO in biological specimens.

Analyte	Sample	Sample volume	Sample preparation information	Extraction conditions: solvent composition, temperature, agitation	Instrumental	LOD	LOQ	Recovery (%)	Reference
Acetylfentanyl, acetylnorfentayl, acrylfentanyl, butyrfentanyl, p-methoxybutyrfentanyl, p-chloroisobutyrfentanyl, despropionyl p-fluorofentanyl, furanyl fentanyl, norfentayl, ocfentanil, o-fluorofentanyl, valeryl fentanyl and N-methylcarfentanil	Whole blood	0.100 g	PP	Acetonitrile; 35ºC; agitation and centrifugation (10 min at 1 000 g)	UHPLC‐QTOF-MS	0.0005–0.001 mg/kg	0.0005 mg/kg	67–81	[120]
Acrylfentanyl	Blood	0.25 mL	PP	Acetonitrile; –; vortex (1 min) and centrifugation (3 min at 3 500 rpm)	GC-MS; UHPLC-MS/MS	0.05 ng/mL	0.10 ng/mL	–	[104]
Carfentanil, norcarfentanil	Blood (carfentanil, norcarfentanil) and urine (carfentanil)	0.25 mL	PP	Ice-cold acetonitrile (1 000 µL); 40ºC; shaking (5 min at 1 400 rpm) and centrifugation (20 min at 20 000 g)	LC-MS/MS	0.03 ng/mL	0.1 ng/mL	–	[89]
U-47700	Urine and blood	0.1 mL	PP and enzymatic hydrolysis (urine)	Acetonitrile (1:3); –; –	UHPLC-MS/MS	–	–	–	[129]
Acrylfentanyl, 4-chloroisobutyrfentanyl, 4-fluoroisobutyrfentanyl and tetrahydrofuranfentanyl	Urine and serum	–	Dilution	–	LC-MS/MS	<0.5 ng/mL	0.5 ng/mL	–	[103]
Butyrfentanyl, 4-fluorobutyrfentanyl	Serum and urine	–	Dilution	–	LC-MS/MS	–	0.5 ng/mL	–	[107]
Furanoylfentany	Urine	1 mL	Dilution	Acetate buffer (150 µL, pH = 9); –; centrifugation (5 min at 12 000 rpm)	LC-HRMS	–	–	–	[146]
U-47700	Urine	0.05mL	Dilution	Water (250 µL); room temperature; vortex	LC-QTOF	1 ng/mL	1 ng/mL	–	[154]
Acetylfentanyl	Urine	–	Dilution	n.s. (1:5); –; –	LC-MS/MS	≤ 0.1 ng/mL	–	–	[138]

NSO: novel synthetic opioids; LOD: limit of detection; LOQ: limit of quantitation; PP: protein precipitation; UHPL-QTOF-MS: ultra-high-performance liquid quadrupole time-of-flight mass spectrometry; –: undetected; GC-MS: gas chromatography mass spectrometry; UHPLC-MS/MS: ultra-high-performance liquid chromatography-tandem mass spectrometry; LC-MS/MS: liquid chromatography-tandem mass spectrometry; LC-HRMS: liquid chromatography-high-resolution mass spectrometry; LC-QTOF: liquid chromatography-quadrupole time-of-flight; n.s.: not specified.

Table 4. Liquid-Liquid extraction applied to biological specimens to determine NSO.

Analyte	Sample	Sample volume	LLE condtions						Instrumental	LOD	LOQ	Recovery (%)	Reference
			Pretreatment	Extraction solvent	Solvent volume	Extraction	Extraction time	Separation
U-47700	Blood and urine	0.5 mL	1.5 mL distilled water and 250 μL concentrated ammonia	1-Chlorobutane	5 mL	Rotating extractor at 80 rpm	10 min	Centrifuged at 3 000 rpm for 15 min	LC-QTOF	0.8 ng/mL	n.s.	91	[131]
Acetyl fentanyl, β-hydroxythiofentanyl, butyryl fentanyl, carfentanil, furanyl fentanyl, MT-45 and U-47700	Whole blood	0.2 mL	400 μL pH 10 Tris-buffer	Methyl tert-butyl ether	1 mL	Pulse vortexing	∼30 s	Centrifuged for 5 min at ∼13 000 rpm	LC-MS/MS (n.s.)	n.s.	0.2 – 10 ng/mL	n.s.	[155]
Cyclopropylfentanyl and crotonylfentanyl	Blood	n.s.	Carbonate buffer (n.s.)	1-Chlorobutane	n.s.	n.s.	n.s.	n.s.	LC-MS	0.05 ng/mL	0.16 mg/mL	n.s.	[115]
Acetylfentanyl, carfentanil, valeryl fentanyl, acetylfentanyl-4-methylphenethyl, furanyl fentanyl, 3-methylfentanyl, butyryl fentanyl, fluorobutyryl fentanyl, acrylfentanyl, MT-45, W-19, AH-7921, U-47700, AH-8533, AH-8529, para-fluorofentanyl, ocfentanil, 4-methoxy butyryl fentanyl, β-hydroxy-thiofentanyl, IC-26	Urine	0.2 mL	Diluted with 0.8 mL 1.25 mol/L NaCl and 0.375% ammonium hydroxide in 50% isopropanol	90% MTBE/10% hexane	1 mL	Vortex mixing	n.s.	n.s.	LC-MS/MS (n.s.)	n.s.	0.1 – 0.4 ng/mL	82.5 – 95.5	[134]
Furanyl fentanyl and 4-ANPP	Blood and urine	1 mL (blood); 0.5 mL (urine)	(First extraction) 2 mL borate buffer (pH 9.0)	1-Chlorobutane	10 mL	Mixed by rotation	20 min	Centrifuged at 1 725 g for 10 min	GC-MS	n.s.	n.s.	n.s.	[87]
			(Second extraction) upper organic layer	3 mol/L HCl	0.4 mL	Mixed by rotation	15 min	Centrifuged at 1 725 g for 10 min
			(Third extraction) aspirate and discard the upper layer, 100 µL saturated ammonium carbonate and 100 µL concentrated ammonium hydroxide	Chloroform	0.075 mL	Vortex mixing	n.s.	n.s.
Acetyl fentanyl	Whole blood and urine	0.2 g	1 mL saturated NaCl, and 1 mL 1% Na2CO3	Diethyl ether	4 mL	Vortex mixing	2 min	Centrifugation at 4 000 rpm for 5 min	GC-MS/MS; LC-MS/MS-ESI+	n.s.	20 ng/g	88 – 93	[101]
Carfentanil	Whole blood	n.s.	Basic back extraction using sodium carbonate buffer	1-Chlorobutane	n.s.	n.s.	n.s.	n.s.	LC-MS/MS-ESI+	n.s.	0.05 ng/mL	n.s.	[113]
Acrylfentanyl	Whole blood	1 g	500 µL 1 mol/L Tris-buffer pH 11	Tert-butylmethylether	3 mL	Horizontal shaker	10 min	Centrifuged for 10 min at 4 000 g	LC-MS/MS-ESI+	n.s.	0.01 ng/g	84 – 94	[44]
Furanylfentanyl	Whole blood	2 g	501 µL 1 mol/L Tris-buffer pH 11	Tert-butylmethylether	3 mL	Horizontal shaker	10 min	Centrifuged for 10 min at 4 000 g	LC-MS/MS-ESI+	n.s.	0.1 ng/g	83 – 92	[31]
U-47700	Femoral blood, heart blood, urine, kidney, liver, lung and brain	n.s.	n.s.	1-Chlorobutane	n.s.	n.s.	n.s.	n.s.	LC-MS/MS (n.s.)	n.s.	n.s.	n.s.	[124]
U-47700	Whole blood and urine	1 mL	(First extraction) 0.1 mol/L sodium hydroxide (200 μL)	1-Chlorobutane	5 mL	Rotated	5 min	Centrifuged at 2 500 rpm for 2 min	LC-MS/MS-ESI+	n.s.	n.s.	n.s.	[127]
			(Second extraction) organic layer	1.0 mol/L formic acid	0.1 mL	Rotated	3 min	Centrifuged at 2 500 rpm for 2 min
U-47700	Whole blood, liver, vitreous, urine and gastric	1 mL	(First extraction) 1mL deionized water; 1mL concentrated ammonium hydroxide	1-Chlorobutane	6 mL	mixed on a mechanical rocker	10 min	Centrifuged for 5 min at ∼2 400 g	GC-MS	∼5 ng/mL	20 ng/mL	n.s.	[125]
			(Second extraction) top organic layer	1.0 mol/L hydrochloric acid	2 mL	n.s.	10 min	Centrifuged for 5 min at ∼2 400 g
			(Third extraction) remaining aqueous portion; 1 mL concentrated ammonium hydroxide	1-Chlorobutane	3 mL	n.s.	10 min	Centrifuged for 5 min at ∼2 400 g
Carfentanil and furanyl fentanyl	Postmortem blood, vitreous and/or urine	2 mL	(First extraction) saturated borate buffer	THIA (78:20:2 mixture of toluene, hexane and isoamyl alcohol)	n.s.	n.s.	n.s.	n.s.	GC-MS	10 ng/mL	n.s.	n.s.	[108]
			(Second extraction) sulfuric acid and neutralization with NaHCO3/K2CO3	ethyl acetate	n.s.	n.s.	n.s.	n.s.
Furanyl fentanyl and metabolites	Urine	0.1 mL	Incubated with glucuronidase from Red Abalone in acetate buffer, pH 5 at 37 °C for 1 h	90% acetonitrile/10% methanol	0.75 mL	n.s.	n.s.	n.s.	LC-(TOF)-MS and LC-MS/MS-ESI+	n.s.	≤0.5 ng/mL	n.s.	[28]
Ocfentanil	Whole blood, vitreous humor, mucous membrane of the nose, urine, stomach content, liver, kidney, brain tissue and bile	0.5 mL or 0.5 g	Kidney, liver, stomach content (semi-solid), bile and brain tissue were homogenized in water at a ratio (m/m) of 1:2 or 1:5 by means of Ultra Turrax; the swab of the mucous membrane of the nose was extracted in 0.5 mL ultrapure water; 1.0 mL potassium carbonate solution	n-Hexane: ethyl acetate (7:3, v/v)	5 mL	Vortex mixing	2 min	Centrifugation at 3 000 rpm for 5 min	LC-MS/MS-ESI+	2.1 ng/mL	2.1 ng/mL	87.6	[117]
Ocfentanil	Whole blood and urine	500 mg whole blood or 50 mg urine	0.9% sodium chloride solution in water was added resulting in 1 mL diluted sample	1-Chlorobutane	3 mL	Gently mixing the sample	n.s.	Centrifugation (10 min, 3 500 rpm)	LC-MS/MS-APCI	n.s.	2.5 ng/mL	n.s.	[36]
Acetyl fentanyl	Whole blood, liver, brain, vitreous humor, and urine	1.0 mL case blood or 1.0 g a 1:4 tissue homogenate	(First extraction) (n.s.) ammonium hydroxide	1-Chlorobutane	n.s.	n.s.	n.s.	n.s.	GC–MS	62.5 ng/mL	125 ng/mL	n.s.	[95]
			(Second extraction) (n.s.) acid back-extraction	n.s.	n.s.	n.s.	n.s.	n.s.
U-47700	Blood and urine	n.s.	Back extraction using sodium carbonate buffer	1-Chlorobutane	n.s.	n.s.	n.s.	n.s.	LC-QTOF-MS/MS	≤78 ng/mL	≤312.5 ng/mL	n.s.	[64]
U-47700	Urine	0.1 mL	Hydrolisis	Methanol	0.5 mL	Vortex mixed	n.s.	n.s.	LC-MS/MS-ESI+	n.s.	n.s.	n.s.	[129]
U-47700	Urine	1 mL	Alkaline conditions (n.s.)	Methylene chloride:cyclohexane:isopropanol (4.5:4.5:1.0)	n.s.	n.s.	n.s.	n.s.	GC/MS/MS and LC-(TOF)-MS	n.s.	n.s.	n.s.	[130]
U-47700	Whole blood and urine	0.5 mL	β-Glucuronidase hydrolyzed urine; 1.0 mL 1 mol/L potassium carbonate	n-Hexane:ethyl acetate (7:3, v/v)	5 mL	Vortex mixing	2 min	Centrifugation at 3 000 rpm for 5 min	LC-MS/MS-ESI+	1.6 ng/mL	1.6 ng/mL	n.s.	[123]
U-47700	Blood and urine	0.5 mL	(First extraction) 0.2 mol/L Na2CO3 solution (0.5 mL, pH 10)	1-Chlorobutane	5 mL	Shaking	3 min	Centrifuging at 3 300 rpm for 3 min	HPLC-DAD	50 ng/mL	312.5 ng/mL	n.s.	[64]
			(Second extraction) supernatant	0.05 mol/L H2SO4	0.1 mL	Shaking	3 min	Centrifuging at 3 300 rpm for 3 min
4-Fluorobutyrfentanyl	Blood and urine	2 mL	4 mL Tris-buffer (pH= 9), 4 mL acetonitrile, and ultrasonicated for 60 min	Ethyl acetate	10 mL	Shaking	120 min	Centrifuged (n.s.)	LC-MS and GC-MS	7 ng/mL	12 ng/mL	n.s.	[119]
4-Fluorobutyrfentanyl	Liver, kidney, brain, and stomach contents	5 g	10 mL aliquots of Tris-buffer (pH = 9) and 10 mL acetonitrile, and ultrasonicated for 60 min	Ethyl acetate	20 mL	Shaking	120 min	Centrifuged (n.s.)	LC-MS and GC-MS	7 ng/mL	12 ng/mL	n.s.	[119]
Acetyl fentanyl and butyryl fentanyl	Peripheral blood, heart blood, vitreous humor and urine	2 mL	(First extraction) (n.s.) saturated borate buffer	78:20:2 mixture of toluene, hexane and isoamyl alcohol	n.s.	n.s.	n.s.	n.s.	GC–MS	n.s.	n.s.	n.s.	[94]
			(Second extraction) top organic layer	sulfuric acid	n.s.	n.s.	n.s.	n.s.
MT-45	Whole blood	0.5 mL	50 μL ammonium hydroxide	1-Chlorobutane/ acetonitrile (4:1, v/v)	4 mL	Rotated	n.s.	Centrifuged (n.s.) for 10 min	GC–MS	1 ng/mL	1 ng/mL	n.s.	[6]
Butyrfentanyl, carboxybutyrfentanyl, hydroxybutyrfentanyl, desbutyrfentanyl, norbutyrfentanyl	Heart blood, urine, muscle, liver, kidney, lung, spleen, adipose tissue, gastric content, frontal lobe, thalamus, cerebellum, centrum semiovale, praecuneus and hair	1 mL (blood), n.s (remaining specimens)	First extraction (pH 7.4)	Butyl acetate/ethyl acetate (1:1, v/v)	n.s.	n.s.	n.s.	n.s.	LC-MS/MS (n.s.)	n.s.	1 ng/mL	n.s.	[40]
			Second extraction (pH 13.5)	Butyl acetate/ethyl acetate (1:1, v/v)	n.s.	n.s.	n.s.	n.s.
Acetyl fentanyl	Whole blood, liver, vitreous and urine	1 mL	1 mL deionized water; 1 mL concentrated ammonium hydroxide	1-Chlorobutane	6 mL	Mixed on a mechanical rocker	10 min	Centrifuged for 5 min at 2 400 g	GC–MS	50 ng/mL	100 ng/mL	n.s.	[34]
Butyr-fentanyl	Whole blood, liver, vitreous, urine and gastric	1 mL	(First extraction) 2 mL deionized water; 1 mL concentrated ammonium hydroxide	1-Chlorobutane	6 mL	Mixed on a mechanical rocker	10 min	Centrifuged for 5 min at 2 400 g	GC–MS	2 ng/mL	10 ng/mL	n.s.	[34]
			(Second extraction) top organic layer	1.0 mol/L hydrochloric acid	2 mL	Mixed on a mechanical rocker	10 min	Centrifuged for 5 min at 2 400 g
			(Third extraction) remaining aqueous portion made alkaline with 1 mL concentrated ammonium hydroxide	1-Chlorobutane	3 mL	Mixed on a mechanical rocker	10 min	centrifuged for 5 min at 2 400 g
AH-7921	Blood	0.5 mL	Borate buffer pH 11, 0.25 mL	Ethylacetate/heptane	1.2 mL	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	[106]
AH-7921	Whole blood, urine, liver, kidney, spleen, heart, lung, brain, bile, gastric content	2 mL or 1 g	(First extraction) 2 mL deionized water; four drops concentrated potassium hydroxide (45%, w/v)	1-Chlorobutane	5 mL	Rotomixer	20 min	Centrifuged for 10 min at 3 000 rpm	GC–MS	n.s.	n.s.	n.s.	[53]
AH-7921	Blood and plasma	0.5 mL	(First extraction) 0.2 mol/L Na2CO3 solution (0.5 mL, pH 10)	1-Chlorobutane	5 mL	Shaking	3 min	Centrifuging at 3 300 rpm for 3 min	HPLC-DAD; LC-MS/MS-ESI+; LC-(TOF)-MS	n.s.	n.s.	n.s.	[156]
			(Second extraction) supernatant	0.05 mol/L H2SO4	0.1 mL	Shaking	4 min	Centrifuging at 3 300 rpm for 3 min
3-Methylfentanyl	Blood (1); urine (2)	1 mL	(1) 400 μL Na2HPO4 buffer (pH 9); (2) hydrolyzed with 20 μL β-glucuronidase for 16 h 4 ºC , and adjusted to pH 7 with a few drops 1 mol/L HCl	Butyl acetate	0.6 mL	Vortex mixer	2 min	n.s.	LC-MS/MS-ESI+	n.s.	n.s.	n.s.	[49]
3-Methylfentanyl	Blood	1 mL	0.3 mL Tris-buffer (pH 11)	Butyl acetate	0.5 mL	Vortex mixer	2 min	n.s.	LC-MS/MS (n.s.)	0.1 ng/mL	n.s.	n.s.	[84]
Alfentanil, fentanyl, p-fluorofentanyl, cis-3-methylfentanyl, trans-3-methylfentanyl, α-methylfentanyl, norfentanyl, remifentanil and sufentanil	Blood (1); urine (2)	1 mL	(1) 400 μL Na2HPO4 buffer (pH 9); (2) hydrolyzed with 20 μL β-glucuronidase for 16 h 46ºC, and adjusted to pH 7 with a few drops 1 mol/L HCl	Butyl acetate	0.6 mL	Vortex mixer	2 min	Centrifuged (n.s.)	LC-MS/MS-ESI+	(1) ≤1 ng/mL; (2) ≤2 ng/mL	(1) ≤7 ng/mL; (2) ≤7 ng/mL	n.s.	[140]
Sufentanil	Serum	1 mL	(First extraction) 100 µL 1 mol/L NaOH	Toluene-2-propanol (10:1, v/v)	2 mL (0.2 mL)	Roller-shaker	20 min	Centrifugation at 4 000 g for 5 min	LC-MS/MS-ESI+	3 pg/mL	10 pg/mL	75	[157]
			(Second extraction) evaporated under vacuum and residue dissolved in 10 µL cyclo-Hexane	1 mol/L HCL	0.2 mL	Intense shaking	10 s	Centrifugation for 2 min at 4 000 g
Remifentanil	Whole blood	1 mL	(First extraction) (n.s.)	Methanol	1 mL	Vortex mixer	5 s	Centrifuged for 15 min at 1 000 g	Capillary GC with nitrogen-specific detection	n.s.	0.2 ng/mL	79 – 85	[158]
			(Second extraction) (supernatant)	1-Chlorobutane	6 mL	Vortex mixer	5 s	Centrifuged for 10 min at 1 000 g
			(Third extraction) (organic phase)	HCl (10 mmol/L)	0.2 mL	Vortex mixer	5 s	Centrifuged for 10 min at 1 000 g
Remifentanil	Whole blood	0.5 mL	0.5 mL 0.1 mol/L phosphate buffer pH 7.4, the sample pH was readjusted back to pH 7	Dichloromethane	2 mL	Shaking mechanically	10 min	Centrifuged at 13 000 rpm for 10 min	LC-MS/MS-ESI+	n.s.	0.1 ng/mL	76	[159]
Alfentanil	Plasma	1 mL	(First extraction) 200 µL potassium hydroxide (0.5 mol/L)	Heptane:isoamyl alcohol, 98:2 v/v)	4 mL	Horizontally shaken mechanically at 2 Hz	10 min	Centrifuged at 420 g for 15 min	LC-UV/Vis	0.25 ng/mL	2 ng/mL	86.3	[160]
			(Second extraction) 3.8 mL the upper organic	Back extractant (0.5 mol/L KH2PO4, pH 2.8, adjusted with 85% H3PO4)	0.2 mL	Horizontally shaken mechanically at 2 Hz	10 min	Centrifuged at 420 g for 15 min
Sufentanil	Blood, urine, vitreous humour, liver and kidney	2 mL	(First extraction) 1 mL 1.5 mol/L NaOH	hexane:ethanol (19:1 v:v)	5 mL	n.s.	n.s.	n.s.	GC-MS	n.s.	n.s.	n.s.	[161]
			(Second extraction) organic layer	O. 1 mol/L HCI	5 mL	n.s.	n.s.	n.s.
			(Third extraction) aqueous layer and 1 mL 1.5 mol/L NaOH	hexane:ethanol	5 mL	n.s.	n.s.	n.s.
α-Methylfentanyl	Liver	10 g	(First extraction) 40 mL water using a mechanical homogenizer; 3 mL 2 mol/L NaOH	hexane:ethanol (19:1)	60 mL	Mechanical shaker	10 min	n.s.	GC-MS	n.s.	n.s.	n.s.	[18]
			(Second extraction) organic layer	0.1 mol/L HCI	4 mL	Mechanical shaker	10 min	n.s.
			(Third extraction) aqueous layer; 1 mL 2 mol/L NaOH	hexane:ethanol (19:1)	6 mL	Mechanical shaker	11 min	n.s.

NSO: novel synthetic opioids; LLE: liquid-liquid extraction; LOD: limit of detection; LOQ: limit of quantitation; n.s.: not specified; LC-MS/MS-ESI+: liquid chromatography-tandem mass spectrometry with positive electrospray ionization; GC-MS: gas chromatography mass spectrometry; UHPL-QTOF-MS: ultra-high-performance liquid quadrupole time-of-flight mass spectrometry; GC-MS: gas chromatography mass spectrometry; LC-(TOF)-MS: liquid chromatography time-of-flight mass spectrometry; LC-MS/MS-APCI: liquid chromatography-tandem mass spectrometry with atmospheric pressure chemical ionization; LC-QTOF-MS/MS: liquid chromatography-quadrupole time-of-flight-tandem mass spectrometry; HPLC-DAD: high performance liquid chromatography-diode array detection; GC: gas chromatography; LC-UV/Vis: liquid chromatography with ultra-violet/visible.

Table 5. SPE applied to biological specimens to determine NSO.

Analyte	Sample	Sample volume/weight	SPE condtions					Instrumental	LOD	LOQ	Recovery (%)	Reference
			Sorbent	Pretreatment	Conditioning	Wash	Elution
U-47700	Serum and urine	0.1 mL	Chromabond^®	Urine samples were treated with β-glucuronidase/arylsulfatase for conjugate cleavage	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	[132]
β-Hydroxythiofentanyl, butyryl/isobutyrylfentanyl, AH-7921, 2-furanylfentanyl, 4-ANPP, U-47700, MT-45, para-methoxybutyrylfentanyl, 4-methylphenethyl acetyl fentanyl, U-50488, acrylfentanyl, valerylfentanyl, carfentanil, para-fluorofentanyl, ortho-fluorofentanyl, para-fluorobutyrylfentanyl/FIBF and α-methylfentanyl	Blood, serum/plasma and urine	0.5 mL	Plexa PCX 3 mL/60 mg	Acetonitrile (1 mL) added to each sample for a protein precipitation step; 1 mL 0.1 mol/L phosphate buffer	2.0 mL acetonitrile; 2.0 mL deionized water	2.0 mL 0.1 mol/L hydrochloric acid and 2.0 mL acetonitrile	2.0 mL 5% ammonium hydroxide in acetonitrile	LC-MS/MS-ESI+	≤0.25 ng/mL	≤0.5 ng/mL	≥56	[88]
Furanylfentanyl, butyrylfentanyl, 4-ANPP, methoxyacetylfentanyl, THFF, FIBF, acrylfentanyl, para-fluorofentanyl, ortho-fluorofentanyl, carfentanil, β-methylfentanyl, isobutyrylfentanyl, para-methylfentanyl, cyclopentylfentanyl, cyclopropylfentanyl, β-hydroxyfentanyl and αmethylfentanyl	Blood	0.5 mL	130 mg CleanScreen^® DAU	2 mL phosphate buffer (pH 6)	3 mL methanol, 3 mL deionized water and 1 mL phosphate buffer	Deionized water (1.5 mL), 0.1 mol/L acetic acid (0.5 mL) and methanol	2 mL ethyl acetate/acetonitrile/ammonium hydroxide (78:20:2)	LC-MS/MS-ESI+	n.s.	n.s.	n.s.	[86]
U-47700 and furanyl fentanyl	Blood	0.5 mL	130 mg CleanScreen^® DAU	2 mL phosphate buffer (pH 6)	3 mL methanol, 3 mL deionized water and 1 mL phosphate buffer	(1.5 mL)	2 mL ethyl acetate/acetonitrile/ammonium hydroxide (78:20:2)	LC-MS/MS (n.s.)	n.s.	n.s.	n.s.	[162]
Acetyl fentanyl and acetyl norfentanyl	Urine	0.08 mL	Strata X-B 33 μ, 30 mg/3 mL	310 μL 0.1 mol/L sodium acetate buffer (pH 5.0) containing β-glucuronidase and incubating for 60 min at 37 °C	n.s.	1 mL a 0.1% formic acid aqueous solution and 1 mL a 70:30 water/methanol solution	Twice with 0.5 mL a basic 50:50 methanol/acetonitrile solution (2% ammonium hydroxide)	LC-MS/MS-ESI+	0.5 – 1 ng/mL	1.06 – 1.62 ng/mL	n.s.	[151]
Cis- and trans-3-methylfentanyl	Blood	0.5 mL	130 mg CleanScreen^® DAU	2 mL phosphate buffer (pH 6)	3 mL methanol, 3 mL deionized water and 1 mL phosphate buffer	Deionized water (1.5 mL), 0.1 mol/L acetic acid (0.5 mL) and methanol	n.s.	LC-QTOF-MS	n.s.	0.1 ng/mL	n.s.	[163]
Acrylfentanyl	Whole blood	0.25 mL	UCT CleanScreen^® CSDAU020	3 mL 0.1 mol/L phosphate buffer (n.s)	5 mL hexane, 3 mL methanol, 3 mL deionized water and 1mL 1.0 mol/L acetic acid	0.1 mol/L phosphate buffer and 1.0 mol/L acetic acid	(n.s.) 50:50 ethyl acetate:hexane and 78:20:2 methylene chloride:isopropanol:ammonium hydroxide	LC-MS/MS-ESI+	0.05 ng/mL	0.10 ng/mL	n.s.	[104]
Carfentanil, 3-methylfentanyl, 2-furanyl fentanyl and acetyl fentanyl	Whole blood	2 mL	200 mg CleanScreen^® ZSDAU020	0.25 mL acetonitrile were vortexed for 7 s, then 2 mL water and vortexed again for 7 s; 3 mL 0.1 mol/L phosphate buffer (pH 6.0)	3 mL methanol, followed by 2 mL deionized water, and 1.5 mL phosphate buffer (pH 6.0)	2 mL deionized water, 1.5 mL 1 mol/L acetic acid, and 2 mL methanol	3 mL dichloromethane/2-propanol/ammonium hydroxide (78:20:2)	LC-MS/MS(n.s.)	n.s.	0.10 ng/mL	n.s.	[85]
Ortho-fluorofentanyl	Serum, blood and urine	0.2 mL	HybridSPE^®-PLus 96-well plates	Precipitated with the addition of acetonitrile	n.s.	n.s.	n.s.	LC-QTOF-MS	≤0.5 ng/mL	≤1 ng/mL	n.s.	[118]
para-fluoroisobutyryl fentanyl, butyryl fentanyl, furanyl fentanyl and carfentanil and U-47700	Whole blood, liver, brain and urine	1 mL or 1 g	CleanScreen^® ZSDAU020	Buffered with 0.1 mol/L, pH 6 sodium phosphate (4 mL)	Methanol (3 mL), deionized water (3mL), and 0.1 mol/L, pH 6 sodium phosphate buffer (1mL)	Deionized water (3 mL) and 1 mol/L acetic acid (1 mL); hexane (2 mL), hexane:ethyl acetate (50:50, 3 mL), and methanol (3 mL)	Dichloromethane:isopropanol:ammonium hydroxide (78:20:2, 3 mL).	LC-Ion Trap-MSn	0.2 – 0.5 ng/mL	n.s.	n.s.	[150]
U-47700, U-50488, furanyl fentanyl	Whole blood	0.5 mL	130 mg CleanScreen^® DAU	2 mL phosphate buffer (pH 6.0)	3 mL methanol, 3 mL deionized water and 1 mL phosphate buffer	1.5 mL deionized water, 0.5 mL 0.1 mol/L acetic acid and 1.5 mL methanol	2 mL ethyl acetate/acetonitrile/ ammonium hydroxide (78:20:2)	LC-MS/MS-ESI+	0.5 ng/mL	1 ng/mL	n.s.	[30]
Acetyl fentanyl	Cardiac blood from the right ventricle, gastric contents or urine	1.0 mL	Waters OASIS HLB C18	n.s.	1 mL methanol; 1 mL distilled water	(n.s.) Methanol (5%) in water	Methanol	GC-MS and LC-MS/MS-ESI+	n.s.	n.s.	n.s.	[93]
Acetyl fentanyl, acetyl norfentanyl and butyryl fentanyl	Peripheral blood, heart blood, vitreous humor, gastric contents, brain, liver, bile and urine	1.0 mL or 1.0 g	SPEC MP3 SPE columns	Bile, gastric contents and urine were diluted 1:10 with deionized water; brain and liver tissue were diluted with 6.0 g deionized water; 1 mL pH 6.0 phosphate buffer	0.4 mL methanol; 0.4 mL 100 mmol/L phosphate buffer (pH 6)	0.4 mL deionized water; 0.4 mL 100 mmol/L acetic acid	1 mL 78:20:2 dichloromethane/isopropanol/ammonia (v:v:v)	LC-MS/MS-(n.s.)	n.s.	1 ng/mL	n.s.	[94]
U-47700	Urine	0.25 mL	PSCX cartridges	25 μL β-glucuronidase and 425 μL 0.1 mol/L sodium acetate buffer; incubated at 60 °C for 45 min	n.s.	0.5 mL water, 0.2 mL 100 mmol/L HCl, 0.1 mL water:methanol (75:25 v:v)	250 μL ethyl acetate:methanol:ammonium hydroxide (78:20:2 v:v:v)	LC-MS-ESI+	1 ng/mL	1 ng/mL	n.s.	[154]
Alfentanil	Serum and blood	0.2 mL	Bakerbond SPE C18 (3 mL, 500 mg)	6 mL phosphate buffer (0.1 mol/L, pH 6)	2 × 3 mL methanol and 2 mL water	1 mL 0.1 mol/L acetic acid	3 × 1 mL dichloromethane/acetone (50:50 v/v); 3 × 1 mL dichloromethane/2-propanol/ammonium hydroxide (40:10:2 v/v/v)	LC-MS-ESI+	0.3 ng/mL	1 ng/mL	49 – 60	[122]
Norfentanyl, acetyl fentanyl and acetyl norfentanyl	Postmortem heart blood, peripheral blood, bile, brain, liver, urine and vitreous humor	1.0 mL or 1.0 g	SPEC MP3 SPE columns	1 mL pH 6.0 phosphate buffer	0.4 mL methanol; 0.4 mL 100 mmol/L phosphate buffer (pH 6)	0.4 mL deionized water; 0.4 mL 100 mmol/L acetic acid	1 mL 78:20:2 dichloromethane/isopropanol/ammonia (v:v:v)	LC-MS/MS-(n.s.)	n.s.	≤1 ng/mL	94 – 118	[92]
AH-7921	Urine	2 mL	CleanScreen^® ZCDAU020	3 mL 0.1 mol/L phosphate buffer (pH 6.0)	3 mL methanol, 3 mL deionized water and 2 mL 0.1 mol/L phosphate buffer (pH 6.0)	(First wash) 2 mL deionized water, 2 mL 20% acetonitrile in deionized water and 2 mL 0.1 mol/L acetic acid	3 mL dichloromethane/isopropanol/ammonium hydroxide (78:20:2)	GC-MS	n.s.	n.s.	n.s.	[53]
						(Second wash) 2 mL hexane followed by 3 mL methanol
Remifentanil	Plasma	0.25 mL	Waters OASIS HLB C18	Acidified with 5 µL phosphoric acid (85%) and diluted to 500 µL with water	1 mL methanol, 2 mL water	(n.s.) water containing 2% acetic acid; 1 mL water containing 5% methanol and 2% acetic acid	1 mL methanol 60% containing 2% acetic acid	LC-MS-ESI+	0.18 ng/mL	0.5 ng/mL	98.4 – 112.9	[149]
Norfentanyl, carfentanil, sufentanil, norsufentanil, norcarfentanil, lofentanil, 3-methylfentanyl, α-methylfentanyl, ohmefentanyl, remifentanil and remifentaniI-COOH	Urine	0.5 mL	1 mL Waters OASIS HLB C18	0.5 mL 0.2 mol/L acetate buffer (pH 4.0) equilibrated for 30 min	1 mL methanol, 1 mL water	(n.s.) 20% methanol in water	1 mL methanol	LC-MS/MS-ESI+	3 – 27 pg/mL	n.s.	10 – 87	[164]
Sufentanil	Plasma	1 mL	Baker, C18, 200 mg	n.s.	3.0 mL methanol; 1.0 mL KOH (1 mol/L); 1.0 mL water and 0.2 mL ethanol /water (5:95, v/v)	0.7 mL K2HPO4 (10 mmol/L) and 1.4 mL water	1.4 mL methanol	LC-MS/MS-ESI+	n.s.	0.3 ng/mL	89.9	[148]
Sufentanil	Hair	50 mg	Chromabond 18 ec	Incubated in 2 mL Soerensen buffer of pH 7.6 for 2 h at 40 °C	n.s.	n.s.	3 times with acetone/dichlormethane (3:1)	GC-MS/MS	n.s.	n.s.	n.s.	[165]

NSO: novel synthetic opioids; SPE: solid phase extraction; LOD: limit of detection; LOQ: limit of quantitation; LC-MS/MS-ESI+: liquid chromatography-tandem mass spectrometry with positive electrospray ionization; LC-QTOF-MS: liquid chromatography-quadrupole time-of-flight mass spectrometry; GC-MS: gas chromatography mass spectrometry; LC-MS-ESI+: liquid chromatography-mass spectrometry with positive electrospray ionization.

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