超高效液相色谱法同时测定电子烟油中的5种吲哚/吲唑酰胺类合成大麻素

doi:10.3724/SP.J.1123.2022.10014

摘要/Abstract

摘要：

合成大麻素是目前世界上滥用最多的新精神活性物质之一,其结构多变,更新迅速,目前已发展至新型第八代吲哚/吲唑酰胺类。近年来与吲哚/吲唑酰胺类合成大麻素相关的案件逐渐增多,在实际案件中对缴获物中合成大麻素的定量分析需求随之增多,但相应的检验鉴定技术仍处于发展阶段。本研究针对电子烟油中5种常见的吲哚/吲唑酰胺类合成大麻素,建立了超高效液相色谱法对其同时进行定量分析测定。实验对流动相的种类、洗脱梯度、柱温、检测波长等色谱条件进行了优化,再结合外标法定量,实现了对5种合成大麻素的定量分析。样品用甲醇提取,在Waters ACQUITY UPLC CSH C18(100 mm×2.1 mm, 1.7 μm)色谱柱上进行分离,柱温35 ℃,流速0.3 mL/min,进样量1 μL,乙腈和超纯水作为流动相进行梯度洗脱,检测波长为290 nm和302 nm。结果表明,采用该方法,5种合成大麻素可在10 min内完全分离,在1~100 mg/L范围内线性关系良好,相关系数(r²)均可以达到0.9999,检出限为0.2 mg/L,定量限为0.6 mg/L,满足实际样品分析需求。采用1、10、100 mg/L 3个水平的5种合成大麻素混合标准溶液进行精密度试验,日内精密度(n=6)均小于1.5%,日间精密度(n=6)均小于2.2%。以空白电子烟油为基质样品,在2、10、50 mg/L 3个加标水平下进行加标回收试验,各待测物的平均加标回收率为95.5%~101.9%,相对标准偏差(RSD, n=6)为0.2%~1.5%,准确度为-4.5%~1.9%。本方法具有准确、快速、灵敏、分离效果好等优点,适用于电子烟油中5种吲哚/吲唑酰胺类合成大麻素的定量测定,可满足相关鉴定工作的要求,也可为具有相似结构的合成大麻素的液相色谱定量分析提供参考。

关键词: 超高效液相色谱, 合成大麻素, 吲哚/吲唑酰胺, 电子烟油, 定量分析

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

中图分类号:

O658

杨哲, 吕建霞, 吴一荻, 蒋力维, 李冬梅. 超高效液相色谱法同时测定电子烟油中的5种吲哚/吲唑酰胺类合成大麻素[J]. 色谱, 2023, 41(7): 602-609.

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.

图/表 9

图 1 5种合成大麻素分子的结构式

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.

表1 梯度洗脱程序

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

图 2 不同流动相下5种合成大麻素的色谱图

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.

图 3 等度洗脱程序下有机相比例对5种合成大麻素(a)保留时间和(b)峰宽的影响

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

图 4 5种合成大麻素在检测波长分别为302 nm和290 nm下的液相色谱图

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.

表2 5种合成大麻素的保留时间、检测波长、线性方程、相关系数(r2)、检出限及定量限

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

表3 5种合成大麻素的平均加标回收率、相对标准偏差和准确度(n=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

表4 5种合成大麻素在3个加标水平下的日内、日间精密度(n=6)

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

图 5 两个电子烟油样品的色谱图

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

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