液质联用同时测定大鼠尿液中6种苯二氮卓类药物

doi:10.19894/j.issn.1000-0518.210046

摘要/Abstract

摘要：

建立高效液相色谱串联质谱法同时测定大鼠尿液中的6种苯二氮卓类药物（BZDs）。以乙酸乙酯为萃取溶剂，经液-液萃取前处理后检测。以地西泮为内标，流动相为甲醇-0.01%甲酸+5 mmol／L甲酸铵，梯度洗脱，柱温为40℃；流速为0.3 mL／min；进样量为2 μL，质谱采用电喷雾离子源正离子模式（ESI⁺）。8-溴-1-甲基-6-吡啶-2-基-4H-［1，2，4］三唑并［4，3-a］-1，4苯并二氮杂卓（Pyrazolam）、氯硝西泮（5-［2-氯苯基］-l，3-二氢-7-硝基-2H-1，4-苯二氮卓-2-酮，Clonazepam）、8-溴-6-［2-氟苯基］-1-甲基-4H-［1，2，4］三唑［4，3a］［1，4］苯并二氮杂卓（Flubromazolam）、甲氯西泮（（S）-5-［2-氯苯基］-1，3-二氢-3-甲基-7-硝基-2H-1，4-苯并二氮杂卓-2-酮，Meclonazepam）、7-溴-5-［2-氟苯基］-1，3-二氢-2H-1，4-苯并二氮杂-2-酮（Flubromazepam）和2′-氯地西泮（7-氯-5-［2-氯苯基］-1，3-二氢-1-甲基-2H-1，4-苯并二氮杂卓-2-酮，Diclazepam）共6种BZDs在20～2000 ng／mL范围内均有良好的线性，日内和日间精密度均在0.70%～5.60%之间，准确度均在87.00%～103.00%，提取回收率基本均>80.00%，稳定性良好。该方法快速、简便、灵敏和准确，适用于大鼠尿液中6种BZDs的同时测定。

关键词: 苯二氮卓类药物, 大鼠尿液, 高效液相串联质谱

Abstract:

A liquid chromatography tandem mass spectrometry method is established for the simultaneous determination of six benzodiazepines (BZDs) in rat urine. Ethyl acetate is used as the extraction solvent, and it is tested after liquid-liquid extraction pretreatment. Diazepam is used as the internal standard, the mobile phase is methanol- (0.01% formic acid+ 5 mmol/L ammonium formate) with a gradient elution, the column temperature is 40℃; the flow rate is 0.3 mL/min and the injection volume is 2 μL. The electrospray ion source is in positive ion mode (ESI+). The six BZDs (pyrazolam: 8-bromo-1-methyl-6-pyridin-2-yl-4H- [1, 2, 4] triazolo [4, 3-a] [1, 4] benzodiazepine; clonazepam: 5- [2-chlorophenyl] -l, 3 dihydro-7-nitro-2H-benzodiazepin-2-one; flubromazolam: 8-bromo-6- [2-fluorophenyl] -1-methyl-4H- [1, 2, 4] triazolo- [4, 3a] [1, 4] benzodiazepine; meclonazepam: 3 (+) -5- (o-chlorophenyl) -1, 3-dihydro-3-methyl-7-nitro-2H-1, 4-benzodiazpine-2-one; flubromazepam: 7-bromo-5- [2-fluorophenyl] -1, 3-dihydro-2H-1, 4-benzodiazepin-2-one; diclazepam: 7-chloro-5- [2-chlorophenyl] -1-methyl-1, 3-dihydro-2H-1, 4-benzodiazepin-2-one) all have good linearity in the range of 20~2000 ng/mL, and the intraday and interday precisions of both 0.70%~5.60%. In the meantime, the accuracy is between 87.00% and 103.00%, and extraction recoveries of six analytes are greater than 80.00%, and the stability is good. The method is fast, simple, sensitive and accurate, which is suitable for the simultaneous measurement of the concentrations of six BZDs in rat urine.

Key words: Benzodiazepines, Rat urine, High performance liquid chromatography-quadrupole-time-of-flight mass spectrometry

中图分类号:

O652.1

易小翠, 易伟, 雷霖, 曾皓, 蒋一峰, 李维理, 陈春林. 液质联用同时测定大鼠尿液中6种苯二氮卓类药物[J]. 应用化学, 2022, 39(02): 340-348.

Xiao-Cui YI, Wei YI, Lin LEI, Hao ZENG, Yi-Feng JIANG, Wei-Li LI, Chun-Lin CHEN. Simultaneous Determination of Six Benzodiazepines in Rat Urine by Liquid Chromatography Tandem Mass Spectrometry[J]. Chinese Journal of Applied Chemistry, 2022, 39(02): 340-348.

图/表 10

参考文献 21

[1]	赵晓东, 时晶, 杨益昌, 等. 失眠的诊断与中西医治疗[J]. 中华中医药杂志, 2011, 26(11):2641-2643.
	ZHAO X D, SHI J, YANG Y C, et al. Diagnosis and treatment of insomnia by integrated traditional Chinese and western medicine[J]. Chin J Tradit Chinese Med Pharm, 2011, 26(11):2641-2643.
[2]	叶增杰, 梁木子, 高颖怡, 等. 苯二氮卓类药物依赖的诊治进展[J]. 医学与哲学(B), 2018, 39(2):65-68.
	YE Z J, LIANG M Z, GAO Y Y, et al. Progress of diagnosis and treatment of benzodiazepine dependence[J]. Med Philosophy (B), 2018, 39(2):65-68.
[3]	余果. 6种苯二氮卓类药物的联合分析及氯硝西泮在急性中毒大鼠体内的分布研究[D]. 湖北:华中科技大学, 2013.
	YU G. Simultaneous determination of 6 benzodiazepines and distribution of clonazepam in poisoned rats[D]. Hubei:Huazhong University of Science and Technology, 2013.
[4]	严爱花, 李贤良, 郗存显, 等. 液相色谱-串级质谱法同时检测中成药及保健食品中非法添加的22种苯二氮卓类药物[J]. 分析化学, 2013, 41(4):509-516.
	YAN A H, LI X L, XI C X, et al. Simultaneous determination of 22 benzodiazepines in Chinese patent drugs and health-care foods by liquid chromatography-tandem mass spectrometry[J]. Chinese J Anal Chem, 2013, 41(4):509-516.
[5]	HUPPERTZ L M, BISEL P, WESTPHAL F, et al. Characterization of the four designer benzodiazepines clonazolam, deschloroe-tizolam, flubromazolam, and meclonazepam, and identification of their in vitro metabolites[J]. Forensic Toxicol, 2015, 33(2):388-395.
[6]	向平, 沈保华, 严慧, 等. 药物辅助性犯罪案件中新型苯二氮幕卓类策划药2-氯地西泮的鉴定[J]. 法医学杂志, 2018, 34(3):248-252.
	XIANG P, SHEN B H, YAN H, et al. Identification of new designerbenzodiazepine diclazepam in drug facilitated sexual[J]. Assault J Forensic Med, 2018, 34(3):248-252.
[7]	徐仿敏, 姜兆林, 刘凌云, 等. 2′-氯地西泮鉴定方法研究[J]. 中国刑警学院学报, 2018, 144(4):114-118.
	XU F M, JIANG Z L, LIU L Y, et al. Study on the identification method of 2′-diclazepam[J]. J Criminal Police College China, 2018, 144(4):114-118.
[8]	MOOSMANN B, HUTTER M, HUPPERTZ L M, et al. Characterization of the designer benzodiazepine pyrazolam and its detectability in human serum and urine[J]. Forensic Toxicol, 2013, 31(2):263-271.
[9]	陈建旭. Q-Orbitrap高分辨质谱检测生物样品中镇静催眠药含量[D]. 河南:郑州大学, 2017.
	CHEN JX. DeterminationofsedativehypnoticsinbiologicalsamplesbyQ-Orbitraphigh-resolutionmass spectrometry[D]. Henan:Zhengzhou University, 2017.
[10]	MOOSMANN B, BISEl P, AUW?RTER V. Characterization of the designer benzodiazepine diclazepam and preliminary data on its metabolism and pharmacokinetics[J]. Drug Test Anal, 2014, 6(7/8):757-763.
[11]	MOOSMANN B, HUPPERTZ L M, HUTTER M, et al. Detection and identification of the designerbenzodiazepine flubromazepam and preliminary data on its metabolism and pharmacokinetics[J]. J Mass Spectrom, 2013, 48(11):1150-1159.
[12]	VIKINGSSON S, WOHLFARTH A, ANDERSSON M L, et al. Identifying metabolites of meclonazepam by high-resolution mass spectrometry using human liver microsomes, hepatocytes, a mouse model, and authentic urine samples. [J]. AAPSJ, 2017, 19(3):736-742.
[13]	MADELEINE P B, MARKUS R M, OLOF B, et al. Human urinary metabolic patterns of the designer benzodiazepines flubromazolam and pyrazolam studied by liquid chromatography-high resolution mass spectrometry[J]. Drug Test Anal, 2018, 10(3):496-506.
[14]	赵彦彪, 郑晓雨, 郑珲, 等. 新精神活性物5F-AMB和策划药Diclazepam的GC-MS检验方法研究[J]. 刑事技术, 2019, 44(4):289-292.
	ZHAO Y B, ZHENG X Y, ZHEN H, et al. Identifying synthetic cannabis 5F-AMB andbenzodiazepine categorized diclazepam with GC-MS[J]. Forensic Sci Technol, 2019, 44(4):289-292.
[15]	宋爱英. 检验diclazepam及其代谢产物二例[J]. 中国法医学杂志, 2018, 33(5):537-538.
	SONG A Y. Two cases ofdiclazepam and its metabolites[J]. Chinese J Forensic Med, 2018, 33(5):537-538.
[16]	赵丹, 冯峰, 粟有志, 等. 超高效液相色谱-串联质谱法测定螺旋藻多糖的单糖组成[J]. 色谱, 2017, 35(4):413-420.
	ZHAO D, FENG F, SU Y Z, et al. Determination of monosaccharide in spirulina polysaccharide by ultra performance liquid chromatography-tandem mass spectrometry[J]. Chinese J Chromatogr, 2017, 35(4):413-420.
[17]	丁文宇, 唐克慧, 王宇驰, 等. 高效液相色谱法测定地拉罗司有关物质的分析方法研究[J]. 中国新药杂志, 2016, 25(20):2376-2381.
	DING W Y, TANG K H, WANG Y C et al. An HPLC analytical method for determining related substances indeferasirox[J]. Chinese J New Drugs, 2016, 25(20):2376-2381.
[18]	张中印, 赵柳微, 曹葳蕤, 等. QuEChERS前处理方法结合高效液相色谱-串联质谱测定蜂蜜和蜂王浆中14种喹诺酮类药物残留[J]. 食品科学, 2016, 37(16):242-248.
	ZHANG Z Y, ZHAO L W, CAO W R, et al. Simultaneous determination of fluoroquinolone residues in honey and royal jelly by quechers and high performance liquid chromatography-tandem mass spectrometry[J]. Food Sci, 2016, 37(16):242-248.
[19]	冯健男, 杜守颖, 白洁, 等. 生物样品前处理的研究进展[J]. 中国中药杂志, 2014, 39(21):4143-4148.
	FENG J N, DU S Y, BAI J, et al. Research progress of pretreatment of biological samples[J]. China J Chinese Mater Med, 2014, 39(21):4143-4148.
[20]	商婷, 赵灵娟, 李佩, 等. 固相支撑液液萃取-液相色谱-串联质谱法测定尿液中10种单羟基多环芳烃[J]. 分析化学, 2019, 47(6):91-97.
	SHANG T, ZHAO L J, LI P, et al. Determination of 10 kinds of monohydroxylated polycyclic aromatic hydrocarbons in human urine by supported liquid extraction followed by liquid chromatography-tandem mass spectrometry[J]. Chinese J Anal Chem, 2019, 47(6):91-97.
[21]	徐多麒. 生物样品中扎来普隆及其代谢产物检测方法研究[D]. 中国人民公安大学, 2017.
	XU D L. Determination ofzaleplon and Its Metabolites in bio-samples[D]. People′s Public Security University of China, 2017.

化合物	母离子	子离子	碰撞能
Compound	Precursor ion（m／z）	Product ion（m／z）	Collision energy／V
Pyrazolam	354.03	167.07	40
Pyrazolam	354.03	206.03	40
Clonazepam	316.05	270.05	25
Clonazepam	316.05	214.04	35
Flubromazolam	371.03	292.11	20
Flubromazolam	371.03	223.07	40
Meclonazepam	330.06	284.07	20
Meclonazepam	330.06	214.04	40
Flubromazepam	333.00	226.09	40
Flubromazepam	333.00	206.08	40
Diclazepam	319.04	227.05	35
Diclazepam	319.04	154.04	35
Diazepam	285.08	193.09	35
Diazepam	285.08	154.04	35

化合物	母离子	子离子	碰撞能
Compound	Precursor ion（m／z）	Product ion（m／z）	Collision energy／V
Pyrazolam	354.03	167.07	40
Pyrazolam	354.03	206.03	40
Clonazepam	316.05	270.05	25
Clonazepam	316.05	214.04	35
Flubromazolam	371.03	292.11	20
Flubromazolam	371.03	223.07	40
Meclonazepam	330.06	284.07	20
Meclonazepam	330.06	214.04	40
Flubromazepam	333.00	226.09	40
Flubromazepam	333.00	206.08	40
Diclazepam	319.04	227.05	35
Diclazepam	319.04	154.04	35
Diazepam	285.08	193.09	35
Diazepam	285.08	154.04	35

化合物	质控浓度	日内精密度	日内准确度	日间精密度	日间准确度
Compound	QC／（ng·mL^－1）	RSD／%	RE／%	RSD／%	RE／%
Pyrazolam	160	2.42	87.17	2.44	87.47
?	800	3.00	94.17	2.46	94.38
?	1 600	2.78	90.07	2.89	90.73
Clonazepam	160	2.05	96.45	5.18	97.84
?	800	2.99	100.87	2.86	102.46
?	1 600	2.04	93.46	1.92	94.71
Flubromazolam	160	1.45	93.79	1.63	94.18
?	800	0.99	98.20	1.01	98.89
?	1600	1.09	91.16	0.70	91.11
Meclonazepam	160	2.24	98.70	5.51	100.17
?	800	3.10	101.53	3.15	102.88
?	1 600	1.60	93.44	1.08	94.57
Flubromazepam	160	2.23	94.47	3.01	95.44
?	800	2.66	97.18	2.48	98.47
?	1 600	1.36	89.25	1.18	90.08
Diclazepam	160	2.55	93.27	1.09	92.35
?	800	0.72	96.25	1.08	96.52
?	1 600	1.02	87.80	0.72	87.62

化合物	质控浓度	日内精密度	日内准确度	日间精密度	日间准确度
Compound	QC／（ng·mL^－1）	RSD／%	RE／%	RSD／%	RE／%
Pyrazolam	160	2.42	87.17	2.44	87.47
?	800	3.00	94.17	2.46	94.38
?	1 600	2.78	90.07	2.89	90.73
Clonazepam	160	2.05	96.45	5.18	97.84
?	800	2.99	100.87	2.86	102.46
?	1 600	2.04	93.46	1.92	94.71
Flubromazolam	160	1.45	93.79	1.63	94.18
?	800	0.99	98.20	1.01	98.89
?	1600	1.09	91.16	0.70	91.11
Meclonazepam	160	2.24	98.70	5.51	100.17
?	800	3.10	101.53	3.15	102.88
?	1 600	1.60	93.44	1.08	94.57
Flubromazepam	160	2.23	94.47	3.01	95.44
?	800	2.66	97.18	2.48	98.47
?	1 600	1.36	89.25	1.18	90.08
Diclazepam	160	2.55	93.27	1.09	92.35
?	800	0.72	96.25	1.08	96.52
?	1 600	1.02	87.80	0.72	87.62

化合物	质控浓度	回收率	基质效应
Compound	QC／（ng·mL^－1）	Recovery／%	Matrix effect／%
Pyrazolam	160	86.86	68.12
?	800	84.34	69.99
?	1 600	85.18	66.10
Clonazepam	160	86.86	68.12
?	800	89.22	86.56
?	1 600	83.26	88.46
Flubromazolam	160	85.57	96.75
?	800	88.28	96.02
?	1 600	80.98	97.73
Meclonazepam	160	87.41	85.98
?	800	89.37	86.60
?	1 600	82.38	88.00
Flubromazepam	160	86.70	88.81
?	800	89.05	88.54
?	1 600	80.50	92.67
Diclazepam	160	79.33	92.08
?	800	83.67	92.62
?	1 600	79.94	93.46
Diazepam	160	76.73	96.47
?	800	81.63	96.56
?	1 600	81.54	98.12