Simultaneous Determination of Six Benzodiazepines in Rat Urine by Liquid Chromatography Tandem Mass Spectrometry

doi:10.19894/j.issn.1000-0518.210046

Chinese Journal of Applied Chemistry ›› 2022, Vol. 39 ›› Issue (02): 340-348.DOI: 10.19894/j.issn.1000-0518.210046

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Simultaneous Determination of Six Benzodiazepines in Rat Urine by Liquid Chromatography Tandem Mass Spectrometry

Xiao-Cui YI¹, Wei YI², Lin LEI², Hao ZENG², Yi-Feng JIANG¹, Wei-Li LI², Chun-Lin CHEN¹()

^1.Chemistry and Biological Engineering Academy, Yichun University, Yichun 336000, China
^2.Institute of Criminal Science and Technology of Public Security Bureau of Yichun, Yichun 336000, China

Received:2021-01-26 Accepted:2021-07-07 Published:2022-02-10 Online:2022-02-09
Contact: Chun-Lin CHEN

Abstract

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

CLC Number:

O652.1

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.

Figures/Tables 10

References 21

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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

Simultaneous Determination of Six Benzodiazepines in Rat Urine by Liquid Chromatography Tandem Mass Spectrometry

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References 21

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时间	流动相A	流动相B／%
Time／min	Mobile phase A／%	Mobile phase B／%
0	95	5
2	95	5
3	35	65
9	43	57
11	25	75
15	5	95
20	5	95

化合物 Compound	质控浓度 QC／（ng·mL^－1）	室温下20～24 h Room temperature（20～24 h）		－20℃冷冻 Freeze（－20℃）		反复冻融3次 Freeze-thawing（three times）
化合物 Compound	质控浓度 QC／（ng·mL^－1）	精密度 RSD／%	准确度 RE／%	精密度 RSD／%	准确度 RE／%	精密度 RSD／%	准确度 RE／%
Pyrazolam	160	4.05	90.41	4.94	112.71	2.99	96.75
?	800	1.15	94.55	1.53	104.94	4.08	95.31
?	1 600	1.89	90.66	4.34	108.16	3.56	104.21
Clonazepam	160	4.28	111.72	5.21	112.01	4.14	95.22
?	800	4.07	100.51	2.60	104.24	2.77	91.34
?	1 600	1.95	91.87	3.11	104.41	4.43	96.89〗
Flubromazolam	160	4.08	85.19	1.72	89.17	1.31	80.83
?	800	2.10	81.49	2.45	86.57	1.64	78.42
?	1 600	2.00	80.85	1.77	87.46	4.48	83.78
Meclonazepam	160	6.17	112.01	5.18	113.64	2.88	97.26
?	800	5.24	100.50	2.09	104.53	1.99	92.40
?	1 600	2.41	91.09	3.61	105.88	4.77	97.22
Flubromazepam	160	2.36	90.99	5.17	103.68	3.00	103.68
?	800	3.96	89.62	3.25	93.07	2.69	93.07
?	1 600	2.24	83.25	3.13	94.86	4.34	94.86
Diclazepam	160	2.90	85.41	1.47	86.75	2.14	83.00
?	800	1.08	83.49	1.05	82.75	1.17	82.43
?	1 600	1.01	79.72	0.84	83.88	3.05	81.80

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