固相萃取-衍生化-气相色谱-质谱联用法同时测定尿液中4种阿片类物质

doi:10.3724/SP.J.1123.2020.06002

摘要/Abstract

摘要：

公安机关用胶体金尿检法对海洛因滥用者的检测常常受到阿片类镇咳药的干扰，使用传统液-液提取法进行实验室检验，操作效率低，灵敏度不高，无法满足公安机关打击涉毒案件的需要。为此，该研究建立了尿液中吗啡、O ⁶ -单乙酰吗啡、可待因和乙酰可待因4种阿片类物质的固相萃取和衍生化技术结合气相色谱-质谱联用（GC-MS）同时检测方法。尿样用磷酸盐缓冲液调节至pH=6后，经MCX固相萃取柱净化，用N -甲基-N -（三甲基硅烷基）三氟乙酰胺（MSTFA）对吗啡、O ⁶ -单乙酰吗啡、可待因进行衍生化，供GC-MS检测。考察了上样和洗脱流速、淋洗液中甲酸体积分数、洗脱液中氨水体积分数、3%（v/v）甲酸甲醇淋洗液体积和固相萃取柱吹干时间对萃取效果的影响。确定上样和洗脱流速1.0 mL/min，淋洗液中甲酸体积分数3%，洗脱液中氨水体积分数5%，3%（v/v）甲酸甲醇淋洗液体积1 mL，吹干时间1 min为最佳条件。在此条件下，4种阿片类物质在0.02~0.8 μg/mL范围内线性关系良好（r ² ≥0.998），检出限（LOD）为0.0016~0.0039 μg/mL，定量限（LOQ）为0.0054~0.0128 μg/mL，当标准添加水平为0.02、0.1、0.2 μg/mL时，回收率为93.0%~110.3%。该方法结合自动化技术，对固相萃取条件精确控制，操作简便、快速、灵敏、准确，适合尿液中吗啡等4种阿片类物质快速测定，可用于海洛因吸食者的大规模监控，并能准确排除因服用含阿片类镇咳药导致的吗啡胶体金尿检假阳性。

关键词: 固相萃取, 衍生化, 气相色谱-质谱, 吗啡, 阿片类物质, 尿液

Abstract:

Police officers currently use the colloidal gold rapid testing method to detect heroin in the urine of drug abusers, but the results are often rendered erroneous due to the presence of antitussive drugs, which contain opioids. The traditional manual liquid-liquid extraction method for urine testing has low efficiency and poor sensitivity, and hence, it fails to meet the requirements of the public security department to crack down on drug abusers. Therefore, to avoid punishment, most rapid-test-positive people make false claims about intaking cough suppressants. It is imperative to establish a highly efficient automatic method for the simultaneous determination of multiple opioids in urine, to rule out the use of heroin. A method based on solid-phase extraction and derivatization coupled with gas chromatography-mass spectrometry (GC-MS) has been developed for the simultaneous detection of morphine, O ⁶ -acetylmorphine, codeine, and acetyl codeine in urine. Since these four opioids exists as cations in acidic aqueous solution, the urine samples collected from dead bodies or drug addicts were adjusted to pH 6 by using phosphate buffer, enriched, and purified by MCX-SPE columns. Then, morphine, O ⁶ -acetylmorphine, and codeine were derivatized by N -methyl-N -(trimethylsilyl) trifluoroacetamide (MSTFA) for GC-MS testing. The effects of sample loading and elution flow rate, percentage of formic acid in the wash solvent (methanol), percentage of ammonia in the eluent (methanol), volume of the wash solvent, and drying time of the cartridge on the extraction efficiency were investigated in detail. The best results were obtained under the following conditions:sample loading and elution flow rate, 1.0 mL/min; volume fraction of formic acid in the wash solvent, 3%; volume fraction of ammonia in the eluent solvent, 5%; volume of 3% (v/v) formic acid in methanol (eluent), 1 mL; and drying time of the cartridge, 1 min. The GC-MS results showed good linearity in the range of 0.02-0.8 μg/mL with correlation coefficients (r ² ) ≥ 0.998. The limits of detection (LODs) and limits of quantification (LOQs) were 0.0016-0.0039 μg/mL and 0.0054-0.0128 μg/mL, respectively. The recoveries of the target analytes were between 93.0% and 110.3% at spiked levels of 0.02, 0.1, and 0.2 μg/mL. As opposed to similar reported methods, our method showed high sensitivity and recovery; furthermore, the matrix interference was eliminated, and the chromatographic peaks of the analytes were completely separated from the impurity peaks at the level of 0.2 μg/mL. The automatic solid-phase extraction equipment is convenient to operate and allows one to process samples in batches. The conditions for solid-phase extraction can be precisely controlled, and the detection accuracy is greatly improved. In addition, a large number of sample tests can be performed by a few experimenters. Hence, this method facilitates simple and rapid forensic toxicology testing and drug abuse monitoring on a large scale.

Key words: solid-phase extraction (SPE), derivatization, gas chromatograph-mass spectrometry (GC-MS), morphine, opioid, urine

高吭, 刘雅珣, 柯威, 刘凯, 倪浏阳, 陶涛. 固相萃取-衍生化-气相色谱-质谱联用法同时测定尿液中4种阿片类物质[J]. 色谱, 2020, 38(11): 1348-1354.

Hang GAO, Yaxun LIU, Wei KE, Kai LIU, Liuyang NI, Tao TAO. Simultaneous determination of four opioids in urine by solid-phase extraction and derivatization coupled with gas chromatography-mass spectrometry[J]. Chinese Journal of Chromatography, 2020, 38(11): 1348-1354.

图/表 12

表1 尿液样品的固相萃取条件

Table 1 Solid phase extraction conditions for the urine samples

Steps	Solvent	Flow rate/(mL/min)	Volume/mL	Time/min
* Drying the cartridges with 0.1 MPa dry nitrogen.
Pre-equilibration 1	methanol	2	3	1.7
Pre-equilibration 2	water	2	3	1.7
Load sample		1	12	12.8
Washing 1	0.2 mol/L phosphate buffer solution (PBS, pH 6)	2	1	0.6
Syringe pump cleaning	3% (v/v) formic acid in methanol	10	3	0.5
Washing 2	3% (v/v) formic acid in methanol	2	1	0.6
Drying^*				1.0
Syringe pump cleaning	5% (v/v) ammonium hydroxide in methanol	10	5	0.7
Elution	5% (v/v) ammonium hydroxide in methanol	1	2.5	2.6
Air push		10	5	0.8
Total time				25.3

表2 4种分析物的保留时间、定性离子和定量离子

Table 2 Retention times, qualitative ions, and quantitative ion of the four analytes

Compound	Retention time/min	Qualitative ions (m /z )	Quantitative ion (m /z )
TMS: trimethylsilyl.
Codeine, TMS derivative	13.31	178, 234, 371	371
Morphine, 2TMS derivative	13.73	146, 236, 429	236
Acetyl codeine	13.86	229, 282, 341	282
O ⁶ -Acetylmorphine,	14.24	287, 340, 399	399
TMS derivative

图1 (a) O 6 -三甲基硅烷基可待因、(b)O 3 , O 6 -双(三甲基硅烷基)吗啡、(c)O 3 -三甲基硅烷基-O 6 -乙酰基吗啡和(d)乙酰可待因的质谱图

Fig. 1 Mass spectra of (a) codeine, TMS derivative, (b) morphine, 2TMS derivative, (c) O 6 -acetylmorphine, TMS derivative, and (d) acetyl codeine

图2 不同上样流速对尿液中4种阿片类物质回收率的影响(n =5)

Fig. 2 Effect of loading flow rate on the recoveries of the four opioids in urine (n =5)

图3 不同洗脱流速对尿液中4种阿片类物质回收率的影响(n =5)

Fig. 3 Effect of elution flow rate on the recoveries of the four opioids in urine (n =5)

图4 不同体积分数的甲酸甲醇淋洗液对尿液中4种阿片类物质回收率的影响(n =5)

Fig. 4 Effect of volume percentage of formic acid in methanol washing solvent on the recoveries of the four opioids in urine (n =5)

图5 不同体积的淋洗液对尿液中4种阿片类物质回收率的影响(n =5)

Fig. 5 Effect of volume of washing solvent on the recoveries of the four opioids in urine (n =5)

图6 不同体积分数的氨水甲醇洗脱液对尿液中4种阿片类物质回收率的影响(n =5)

Fig. 6 Effect of volume percentage of ammonium hydroxide in methanol eluent solvent on the recoveries of the four opioids in urine (n =5)

图7 不同吹干时间对尿液中4种阿片类物质回收率的影响(n =5)

Fig. 7 Effect of cartridge drying time on the recoveries of the four opioids in urine (n =5)

图8 0.2 μg/mL加标的空白尿液经固相萃取净化后的总离子流色谱图

Fig. 8 Total ion current chromatogram of blank urine spiked at 0.2 μg/mL purified by solid phase extraction

表3 4种分析物的线性范围、线性方程、相关系数、检出限和定量限

Table 3 Linear ranges, linear equations, correlation coefficients (r 2 ), LODs, and LOQs of the four analytes

Compound	Linear range/(μg/mL)	Linear equation	r ²	LOD/(μg/mL)	LOQ/(μg/mL)
y : peak area; x : mass concentration, μg/mL.
Codeine, TMS derivative	0.02-0.8	y =41175x -9401.3	0.9991	0.0016	0.0055
Morphine, 2TMS derivative	0.02-0.8	y =36270x -1806.7	0.9983	0.0028	0.0094
Acetyl codeine	0.02-0.8	y =33755x -15069	0.9994	0.0039	0.0128
O ⁶ -Acetylmorphine, TMS derivative	0.02-0.8	y =43331x -26732	0.9993	0.0016	0.0054

表4 加标尿样中4种阿片类物质的回收率、日内精密度(n =5)和日间精密度(n =3)

Table 4 Recoveries and precisions of intra-day (n =5) and inter-day (n =3) of the four analytes in spiked urine

Compound	Added/(μg/mL)	Average recovery/%	RSDs/%
Compound	Added/(μg/mL)	Average recovery/%	Intra-day	Inter-day
Codeine, TMS	0.02	110.3	4.6	5.7
derivative	0.10	94.9	4.6	7.1
	0.20	97.8	3.1	5.5
Morphine, 2TMS	0.02	104.9	8.7	10.4
derivative	0.10	102.1	5.4	6.0
	0.20	99.3	3.9	4.5
Acetyl codeine	0.02	107.1	3.4	7.7
	0.10	101.5	7.7	7.0
	0.20	100.5	5.6	4.4
O ⁶ -Acetylmorphine,	0.02	93.0	2.6	7.8
TMS derivative	0.10	98.4	7.5	6.1
	0.20	105.0	3.2	4.1

参考文献

1	China National Narcotic Control Commission.. Report on the Drug Situation in China in 2018.(2019-06-17)[2019-08-12]. http://www.nncc626.com/2019-06/17/c_1210161797.htm
1	国家禁毒委员会. 2018年中国毒品形势报告. (2019-06-17)[2019-08-12]. http://www.nncc626.com/2019-06/17/c_1210161797.htm
2	Lü Y F..[MS Dissertation]. Beijing: People's Public Security University of China, 2019
3	Zhang W J..[MS Dissertation]. Kunming: Kunming Medical University, 2019
4	Yan F Q , Li X , Cao J P The Chinese Journal of Clinical Pharmacology, 2015, 31 (8): 652
4	闫赋琴 , 李霞 , 曹军平中国临床药理学杂志, 2015, 31 (8): 652
5	Zhang R M , Zhang R L , Zhao M , et al.. Chinese Journal of Drug Abuse Prevention and Treatment, 2017, 23 (1): 1
5	张锐敏 , 张瑞岭 , 赵敏 , et al.. 中国药物滥用防治杂志, 2017, 23 (1): 1
6	Powers D , Erickson S , Swortwood M J J Forensic Sci, 2018, 63 (4): 1229
7	Liu H C , Yang C A , Liu R H , et al.. J Anal Toxicol, 2017, 41 (5): 421
8	Goggin M M , Gozum S D , Miller A , et al.. J Anal Toxicol, 2018, 42 (6): 384
9	Lopez L F , Maldonado A L , Falcon M , et al.. J Pharm Biomed Anal, 2019, 164 636
10	Rosado T , Barroso M , Vieira D N , et al.. J Anal Toxicol, 2019, 43 (6): 465
11	Dong L , Wei Z W , Yun K M , et al.. Chinese Journal of Health Laboratory Technology, 2010, 20 (4): 697
11	董镧 , 尉志文 , 贠克明 , et al.. 中国卫生检验杂志, 2010, 20 (4): 697
12	He G B , Jiang S Q , Luo Z J Journal of Instrumental Analysis, 2008 S1): 11
12	何国标 , 蒋硕勤 , 罗正坚分析测试学报, 2008 S1): 11
13	Gong L H , Li W P , Guo L Q , et al.. Journal of Light Industry, 2017, 32 (3): 14
13	弓丽华 , 李万鹏 , 郭良起 , et al.. 轻工学报, 2017, 32 (3): 14
14	Duan X F , Chen R H , Ling D H , et al.. Chinese Journal of Health Laboratory Technology, 2013, 23 (8): 1879
14	段夏菲 , 陈若虹 , 凌东辉 , et al.. 中国卫生检验杂志, 2013, 23 (8): 1879
15	Wang L..[PhD Dissertation]. Hefei: University of Science and Technology of China, 2014
16	Zhang X Y , Cai X X , Zhang X Y Chinese Journal of Chromatography, 2014, 32 (6): 586
16	张秀尧 , 蔡欣欣 , 张晓艺色谱, 2014, 32 (6): 586
17	Wang Y , Zhang P , Wu S X , et al.. Journal of Chinese Mass Spectrometry Society, 2017, 38 (6): 620
17	王毅 , 张苹 , 吴生秀 , et al.. 质谱学报, 2017, 38 (6): 620
18	Wang J Z , Liu J , Luo X , et al.. Tobacco Science & Technology, 2017, 50 (6): 53
18	王加忠 , 刘剑 , 罗熹 , et al.. 烟草科技, 2017, 50 (6): 53

[1]	岳超, 赵超群, 毛思浩, 王展华, 施贝, 徐欣丰, 梁晶晶. 通过式固相萃取净化-超高效液相色谱-串联质谱法测定液体乳中10种氨基甲酸酯类农药残留[J]. 色谱, 2023, 41(9): 807-813.
[2]	孟二琼, 念琪循, 李峰, 张秋萍, 许茜, 王春民. 磺酸化磁性氮化碳固相萃取-超高液相色谱-串联质谱筛检淡水鱼中孔雀石绿和隐色孔雀石绿[J]. 色谱, 2023, 41(8): 673-682.
[3]	高祎阳, 丁亚丽, 陈鲁玉, 杜芳, 辛绪波, 冯娟娟, 孙明霞, 冯洋, 孙敏. 共价有机框架材料在固相萃取中的最新应用进展[J]. 色谱, 2023, 41(7): 545-553.
[4]	秦童童, 高莉, 赵文杰. 超交联多孔有机聚合物在柱固相萃取中的应用进展[J]. 色谱, 2023, 41(7): 554-561.
[5]	赵原庆, 胡锴, 杨成, 韩鹏昭, 李立新, 刘晓冰, 张振强, 张书胜. 基于Ti₃C₂T_x/聚酰亚胺复合材料的分散固相萃取-液相色谱法测定尿液中儿茶酚胺类神经递质[J]. 色谱, 2023, 41(7): 572-581.
[6]	陈东洋, 张昊, 张磊, 王一红, 王小丹, 冯家力, 梁静, 钟旋. 固相萃取-超高效液相色谱-串联质谱法测定发酵食品中的曲酸[J]. 色谱, 2023, 41(7): 632-639.
[7]	王秋旭, 冯启言, 朱雪强. 加速溶剂萃取-固相萃取净化结合超高效液相色谱-串联质谱法测定沉积物中双酚类化合物[J]. 色谱, 2023, 41(7): 582-590.
[8]	夏宝林, 汪仕韬, 殷晶晶, 张维益, 杨娜, 刘强, 吴海晶. 自动上样固相萃取-超高效液相色谱-串联质谱法同时测定水中9类43种抗菌药物残留[J]. 色谱, 2023, 41(7): 591-601.
[9]	王园媛, 李璐璐, 吕佳, 陈永艳, 张岚. 固相萃取-超高效液相色谱-三重四极杆质谱法测定饮用水中13种卤代苯醌类消毒副产物[J]. 色谱, 2023, 41(6): 482-489.
[10]	宁晓盼, 姚倩, 许忠祥, 殷耀, 柳菡, 张晓燕, 丁涛, 张勇, 侯玉, 王梦茹, 吴丽娜, 汤奇婷. 固相萃取-高效液相色谱法测定水产调味品中7种对羟基苯甲酸酯类防腐剂[J]. 色谱, 2023, 41(6): 513-519.
[11]	王亚婷, 杨阳, 孙秀兰, 纪剑. 基于气相色谱-质谱法的广泛靶向代谢组学方法开发[J]. 色谱, 2023, 41(6): 520-526.
[12]	王禹衡, 张婧文, 郑洪国, 卢思佳, 于素华, 杨瑞琴, 王勇. 快速溶剂萃取-离子色谱-质谱法测定人体血液、尿液中的氟乙酸[J]. 色谱, 2023, 41(6): 497-503.
[13]	吴少明, 欧阳立群, 孟鹏, 何孟杭, 林钦, 陈言凯, 刘文菁, 苏晓明, 戴明. 固相萃取净化-超高效液相色谱-串联质谱法测定畜肉中18种卡因类麻醉剂[J]. 色谱, 2023, 41(5): 434-442.
[14]	吴瑛瑕, 牟燕, 刘佩珊, 张易天, 曾颖炫, 周枝凤. 气相色谱-质谱法测定大鼠肝脏中的39种脂肪酸[J]. 色谱, 2023, 41(5): 443-449.
[15]	李振环, 胡小键, 陆一夫, 谢琳娜, 朱英. 基于高通量全自动固相萃取的超高效液相色谱-串联质谱法测定人尿中16种抗生素和4种β-受体激动剂[J]. 色谱, 2023, 41(5): 397-408.