Simultaneous determination of five indole/indazole amide-based synthetic cannabinoids in electronic cigarette oil by ultra performance liquid chromatography

doi:10.3724/SP.J.1123.2022.10014

Abstract

Abstract:

Synthetic cannabinoids (SCs), which are considered some of the most widely abused new psychoactive substances available today, are much more potent than natural cannabis and display greater efficacy. New SCs can be developed by adding substituents such as halogen, alkyl, or alkoxy groups to one of the aromatic ring systems, or by changing the length of the alkyl chain. Following the emergence of the so-called first-generation SCs, further developments have led to eighth-generation indole/indazole amide-based SCs. Given that all SCs were listed as controlled substances on July 1, 2021, the technologies used to detect these substances must be quickly improved. Due to the sheer number of SCs, the chemical diversity and the fast update speed, it is challenging to determine and identify the new SCs. In recent years, several types of indole/indazole amide-based SCs have been seized, but systematic research on these compounds remains limited. Therefore, developing rapid, sensitive, and accurate quantitative methods to determine new SCs are of great importance. Compared with high performance liquid chromatography (HPLC), ultra performance liquid chromatography (UPLC) shows higher resolution, better separation efficiency, and faster analysis speeds; thus, it can meet the demand for the quantitative analysis of indole/indazole amide-based SCs in seized materials.

In this study, a UPLC method was developed for the simultaneous determination of five indole/indazole amide-based SCs, including N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-butyl-1H-indazole-3-carboxamide (ADB-BUTINACA), methyl 2-(1-(4-fluorobutyl)-1H-indole-3-carboxamido)-3,3-dimethylbutanoate (4F-MDMB-BUTICA), N-(1-methoxy-3,3-dimethyl-1-oxobutan-2-yl)-1-(5-fluoropentyl)-1H-indole-3-carboxamide (5F-MDMB-PICA), methyl 3,3-dimethyl-2-(1-(pent-4-en-1-yl)-1H-indazole-3-carboxamido)butanoate (MDMB-4en-PINACA), and N-(adamantan-1-yl)-1-(4-fluorobutyl)-1H-indazole-3-carboxamide (4F-ABUTINACA) in electronic cigarette oil; these SCs have been detected with increasing frequency in seized materials in recent years. The main factors influencing the separation and detection performance of the proposed method, including the mobile phase, elution gradient, column temperature, and detection wavelength, were optimized. The proposed method successfully quantified the five SCs in electronic cigarette oil via the external standard method. The samples were extracted using methanol, and the target analytes were separated on a Waters ACQUITY UPLC CSH C18 column (100 mm×2.1 mm, 1.7 μm) at column temperature of 35 ℃ and flow rate of 0.3 mL/min. The injection volume was 1 μL. The mobile phase consisted of acetonitrile and ultrapure water, and gradient elution was employed. The detection wavelengths were 290 and 302 nm. The five SCs were completely separated within 10 min under optimized conditions and showed good linear relationships between 1-100 mg/L, with correlation coefficients (r²) of up to 0.9999. The limits of detection (LOD) and quantification (LOQ) were 0.2 and 0.6 mg/L, respectively. Precision was determined using standard solutions of the five SCs at mass concentrations of 1, 10, and 100 mg/L. The intra-day precision (n=6) was <1.5%, and the inter-day precision (n=6) was <2.2%. Accuracy was determined by spiking electronic cigarette oil with low (2 mg/L), moderate (10 mg/L), and high (50 mg/L) levels of the five SCs, with six replicates per determination. The recoveries of the five SCs were 95.5%-101.9%, and their relative standard deviations (RSDs, n=6) were 0.2%-1.5%, with accuracies ranging from -4.5% to 1.9%. The proposed method showed good performance when applied to the analysis of real samples. It is accurate, rapid, sensitive, and effective for the determination of five indole/indazole amide-based SCs in electronic cigarette oil. Thus, it satisfies the requirements for practical determination and provides a reference for the determination of SCs with similar structures by UPLC.

Key words: ultra performance liquid chromatography (UPLC), synthetic cannabinoids (SCs), indole/indazole amides, electronic cigarette oil, quantitative analysis

CLC Number:

O658

YANG Zhe, LYU Jianxia, WU Yidi, JIANG Liwei, LI Dongmei. Simultaneous determination of five indole/indazole amide-based synthetic cannabinoids in electronic cigarette oil by ultra performance liquid chromatography[J]. Chinese Journal of Chromatography, 2023, 41(7): 602-609.

Figures/Tables 9

Fig. 1 Structural formulas for the five synthetic cannabinoid molecules MDMB-4en-PINACA: methyl 3,3-dimethyl-2-(1-(pent-4-en-1-yl)-1H-indazole-3-carboxamido) butanoate; ADB-BUTINACA: N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-butyl-1H-indazole-3-carboxamide; 4F-MDMB-BUTICA: methyl 2-(1-(4-fluorobutyl)-1H-indole-3-carboxamido)-3,3-dimethylbutanoate; 4F-ABUTINACA: N-(adamantan-1-yl)-1-(4-fluorobutyl)-1H-indazole-3-carboxamide; 5F-MDMB-PICA: N-(1-methoxy-3,3-dimethyl-1-oxobutan-2-yl)-1-(5-fluoropentyl)-1H-indole-3-carboxamide.

Table 1 Gradient elution procedure

Time/min	Flow rate/(mL/min)	φ(A)/%	φ(B)/%
0	0.3	50	50
1	0.3	50	50
8	0.3	0	100
10^*	0.3	50	50

Fig. 2 Chromatograms of the five synthetic cannabinoids under different mobile phases a. acetonitrile-ultrapure water (65∶35, v/v); b. acetonitrile-triethylamine phosphate buffer (65∶35, v/v); c. methanol-ultrapure water (65∶35, v/v); d. methanol-triethylamine phosphate buffer (65∶35, v/v). Peak identifications: 1. ADB-BUTINACA; 2. 4F-MDMB-BUTICA; 3. 5F-MDMB-PICA; 4. MDMB-4en-PINACA; 5. 4F-ABUTINACA.

Fig. 3 Effects of organic phase ratio on (a) retention times and (b) peak widths of the five synthetic cannabinoids under isocratic elution

Fig. 4 Liquid chromatograms of the five synthetic cannabinoids at 302 nm and 290 nm, respectively Peak Nos. are the same as those in Fig. 2; gradient elution procedure is shown in Table 1.

Table 2 Retention times, detection wavelengths, regression equations, correlation coefficients (r2), limits of detection (LODs) and limits of quantitation (LOQs) of the five synthetic cannabinoids

Compound	Retention time/min	Detection wavelength/nm	Regression equation	r²	LOD/ (mg/L)	LOQ/ (mg/L)
ADB-BUTINACA	3.157	302	y=7.49×10³x+6.82×10²	0.9999	0.2	0.6
4F-MDMB-BUTICA	4.145	290	y=7.22×10³x+6.40×10²	0.9999	0.2	0.6
5F-MDMB-PICA	4.711	290	y=6.66×10³x+6.22×10²	0.9999	0.2	0.6
MDMB-4en-PINACA	6.172	302	y=6.78×10³x+6.98×10²	0.9999	0.2	0.6
4F-ABUTINACA	7.003	302	y=6.61×10³x+8.06×10²	0.9999	0.2	0.6

Table 3 Average spiked recoveries, relative standard deviations (RSDs) and accuracies of the five synthetic cannabinoids (n=6)

Compound	Spiked level/ (mg/L)	Recovery/ %	RSD/ %	Accuracy/ %
ADB-BUTINACA	2	95.6	1.5	-4.4
	10	101.6	0.3	1.6
	50	97.6	0.2	-2.4
4F-MDMB-BUTICA	2	96.1	0.6	-4.0
	10	101.3	0.4	1.3
	50	97.1	0.2	-2.9
5F-MDMB-PICA	2	96.9	1.0	-3.1
	10	101.6	0.3	1.6
	50	96.8	0.2	-3.2
MDMB-4en-PINACA	2	96.7	0.5	-3.3
	10	101.9	0.7	1.9
	50	97.3	0.2	-2.7
4F-ABUTINACA	2	95.5	0.5	-4.5
	10	101.4	0.4	1.4
	50	96.8	0.3	-3.2

Table 4 Intra- and inter-day RSDs of the five synthetic cannabinoids at three spiked levels (n=6)

Compound	1 mg/L				10 mg/L				100 mg/L
Compound	Intra-day RSD/%		Inter-day RSD/%		Intra-day RSD/%		Inter-day RSD/%		Intra-day RSD/%		Inter-day RSD/%
ADB-BUTINACA		0.73		1.20		0.33		0.52		0.25		0.55
4F-MDMB-BUTICA		0.82		1.54		0.42		0.80		0.34		0.58
5F-MDMB-PICA		0.50		1.25		0.22		0.71		0.33		0.51
MDMB-4en-PINACA		0.49		1.15		0.28		0.50		0.34		0.41
4F-ABUTINACA		1.43		2.13		0.64		0.67		0.42		0.47

Fig. 5 Chromatograms of the two electronic cigarette oil samples Peak Nos. are the same as those in Fig. 2.

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