Detection of Carbamazepine and Its Metabolites in Blood Samples by LC-MS/MS

doi:10.12116/j.issn.1004-5619.2021.310901

Abstract

Abstract:

Objective To establish a method for the detection of carbamazepine and its metabolites 10,11-dihydro-10,11-epoxycarbamazepine and 10,11-dihydro-10-hydroxycarbamazepine in blood samples by liquid chromatography-tandem mass spectrometry （LC-MS/MS）. Methods The blood samples were treated with 1-butyl-3-methylimidazolium hexafluorophosphate as an extraction solvent. The samples were extracted by ultrasound-assisted extraction and separated by ZORBAX Eclipse Plus C18， 95? column. The mobile phase A aqueous solution containing 0.1% formic acid and 10 mmol/L ammonium acetate, and mobile phase B mixed organic solvent containing acetonitrile/methanol （V_acetonitrile∶V_methanol=2∶3） were used for gradient elution at the flow rate of 1.00 mL/min. An electrospray ion source in positive mode was used for detection in the multiple reaction monitoring. Results The linearities of carbamazepine and its metabolites 10,11-dihydro-10,11-epoxycarbamazepine and 10,11-dihydro-10-hydroxycarbamazepine in blood samples were good within the corresponding range, with correlation coefficients （r） greater than 0.995 6. The limits of detection were 3.00， 0.40 and 1.30 ng/mL，respectively. The limit of quantitation were 8.00，1.00 and 5.00 ng/mL，respectively. The extraction recoveries ranged from 76.00% to 106.44%. The relative standard deviations of the intra-day and inter-day precisions were less than 16%. Carbamazepine and its main metabolite 10,11-dihydro-10,11-epoxycarbamazepine were detected in blood samples of death cases with a mass concentration of 2.71 μg/mL and 252.14 ng/mL，respectively. Conclusion This method has high sensitivity and good selectivity， which is suitable for the detection of carbamazepine and its metabolites in blood samples， and can be used for carbamazepine-related forensic identifications.

Key words: forensic toxicological analysis, LC-MS/MS, carbamazepine, metabolites, 10,11-dihydro-10,11-epoxycarbamazepine, 10,11-dihydro-10-hydroxycarbamazepine

CLC Number:

DF795.1

Hai-yan CUI, Chen-xi LÜ, Yan-hua SHI, Ni YUAN, Jia-hao LIANG, Quan AN, Zhong-yuan GUO, Ke-ming YUN. Detection of Carbamazepine and Its Metabolites in Blood Samples by LC-MS/MS[J]. Journal of Forensic Medicine, 2023, 39(1): 34-39.

Figures/Tables 6

Tab. 1 Liquid-phase parameters of gradient elution

时间/min	流动相A/%	流动相B/%	流速/（mL·min^-1）
0	55	45	1
6	55	45	1
7	0	100	1
9	0	100	1
11	55	45	1
13	55	45	1

Tab. 2 The mass spectrometric parameters of CBZ、CBZ-EP、CBZ-10OH and CBZ-DiH

化合物	保留时间/min	分子式	母离子（m/z）	子离子（m/z）	碎裂电压/V	碰撞能/eV
CBZ	8.182	C₁₅H₁₂N₂O	236.9	194.0^1）	120	17
CBZ	8.182	C₁₅H₁₂N₂O	236.9	219.8	120	18
CBZ-EP	4.492	C₁₅H₁₂N₂O₂	253.1	180.0^1）	87	18
CBZ-EP	4.492	C₁₅H₁₂N₂O₂	253.1	209.9	87	14
CBZ-10OH	3.640	C₁₅H₁₄N₂O₂	254.8	194.0^1）	80	19
CBZ-10OH	3.640	C₁₅H₁₄N₂O₂	254.8	236.9	80	20
CBZ-DiH^2）	8.316	C₁₅H₁₄N₂O	239.0	193.9^1）	120	25
CBZ-DiH^2）	8.316	C₁₅H₁₄N₂O	239.0	222.1	120	19

Fig. 1 TIC and MRM atlas of blood samples

Tab. 3 The linear equation，LOD and LQD of CBZ，CBZ-EP and CBZ-10OH in blood samples

化合物	线性方程	线性范围/（ng·mL^-1）	相关系数（r）	检出限/（ng·mL^-1）	定量限/（ng·mL^-1）
CBZ	y=1.669 5x+0.065 3	8.00~300.00	0.995 6	3.00	8.00
CBZ-EP	y=0.536 1x+0.010 9	1.00~120.00	0.999 2	0.40	1.00
CBZ-10OH	y=0.160 6x-0.009 4	5.00~250.00	0.995 9	1.30	5.00

Tab. 4 The accuracy and precision of CBZ，CBZ-EP and CBZ-10OH in blood samples（%）

化合物	添加质量浓度/ （ng·mL^-1）	相对标准偏差		准确度（n=5）
化合物	添加质量浓度/ （ng·mL^-1）	日内精密度（n=5）	日间精密度（n=15）	准确度（n=5）
CBZ	16	13.49	15.15	11.65
	80	5.53	14.48	12.01
	240	3.32	4.01	2.92
CBZ-EP	2	8.87	7.61	8.18
	10	9.57	8.77	2.40
	96	5.58	4.78	3.66
CBZ-10OH	10	15.15	9.01	11.68
	50	14.20	13.04	7.03
	200	8.81	10.63	7.90

Tab. 5 The recovery， matrix effect and stability of CBZ，CBZ-EP and CBZ-10OH in blood samples

化合物	提取回收率			基质效应			稳定性/%
化合物	添加质量浓度/ （ng·mL^-1）	实际响应值/ （n=5， $x ˉ$ $±$ $s$ ， ng·mL^-1）	提取回收率/%	A_b	A_a	基质效应/%	稳定性/%
CBZ	16	12.87±0.53	80.44	5 295.00±47.80	7 277.80±64.05	72.76	168.52
	240	248.57±6.98	103.57	95 768.60±2 366.86	98 265±538.79	97.46	160.03
CBZ-EP	2	1.52±0.09	76.00	372.60±3.97	412.00±4.12	90.44	84.68
	96	96.86±1.93	100.90	17 928.00±409.06	16 681.80±81.63	107.47	169.78
CBZ-10OH	10	8.30±0.76	82.96	550.60±14.08	830.80±19.42	66.27	88.49
	200	212.88±6.55	106.44	13 980.80±296.62	13 104.00±142.84	106.69	87.90

Tab. 5 The recovery， matrix effect and stability of CBZ，CBZ-EP and CBZ-10OH in blood samples

化合物	提取回收率			基质效应			稳定性/%
化合物	添加质量浓度/ （ng·mL^-1）	实际响应值/ （n=5， $x ˉ$ $±$ $s$ ， ng·mL^-1）	提取回收率/%	A_b	A_a	基质效应/%	稳定性/%
CBZ	16	12.87±0.53	80.44	5 295.00±47.80	7 277.80±64.05	72.76	168.52
	240	248.57±6.98	103.57	95 768.60±2 366.86	98 265±538.79	97.46	160.03
CBZ-EP	2	1.52±0.09	76.00	372.60±3.97	412.00±4.12	90.44	84.68
	96	96.86±1.93	100.90	17 928.00±409.06	16 681.80±81.63	107.47	169.78
CBZ-10OH	10	8.30±0.76	82.96	550.60±14.08	830.80±19.42	66.27	88.49
	200	212.88±6.55	106.44	13 980.80±296.62	13 104.00±142.84	106.69	87.90

References 14

1	BERNUS I. Dose-dependent metabolism of carbamazepine in humans[J]. Epilepsy Res，1996，24（3）：163-172. doi：10.1016/0920-1211（96）00011-3 .
2	DELEU D， AARONS L， AHMED I A. Population pharmacokinetics of free carbamazepine in adult Omani epileptic patients[J]. Eur J Clin Pharmacol，2001，57（3）：243-248. doi：10.1007/s0022801 00300 .
3	焦正，钟明康，施孝金，等. 抗癫（癎）药物监测[J].中国临床药学杂志，2004，13（6）：386-389. doi：10.3969/j.issn.1007-4406.2004.06.025 .
	JIAO Z， ZHONG M K， SHI X J， et al. Antiepileptic drug monitoring[J]. Zhongguo Linchuang Yaoxue Zazhi，2004，13（6）：386-389.
4	KERR B M. Human liver carbamazepine metabolism： Role of CYP3A4 and CYP2C8 in 10，11-epoxide formation[J]. Biochem Pharmacol，1994，47（11）：1969-1979. doi：10.1016/0006-2952（94）90071-X .
5	MYLLYNEN P， PIENIMÄKI P， RAUNIO H， et al. Microsomal metabolism of carbamazepine and oxcarbazepine in liver and placenta[J]. Hum Exp Toxicol，1998，17（12）：668-676. doi：10.1177/09603 2719801701204 .
6	SEMAH F， GIMENEZ F， LONGER E， et al. Carbamazepine and its epoxide： An open study of efficacy and side effects after carbamazepine dose increment in refractory partial epilepsy[J]. Ther Drug Monit，1994，16（6）：537-540.
7	RAMBECK B， SCHNABEL R， MAY T， et al. Postmortem concentrations of phenobarbital， carbamazepine， and its metabolite carbamazepine-10，11-epoxide in different regions of the brain and in the serum： Analysis of autoptic specimens from 51 epileptic patients[J]. Ther Drug Monit，1993，15（2）：91-98. doi：10.1097/00007691-199304000-00004 .
8	MCMILLIN G A， JUENKE J M， TSO G， et al. Estimation of carbamazepine and carbamazepine-10，11-epoxide concentrations in plasma using mathematical equations generated with two carbamazepine immunoassays[J]. Am J Clin Pathol，2010，133（5）：728-736. doi：10.1309/ajcpfahvb26vvvte .
9	陈凯云，骆利平，谢文毅，等. 高效液相色谱-质谱串联法同时测定人血清中拉莫三嗪及卡马西平的含量[J].中国医院用药评价与分析，2012，12（12）：1108-1110. doi：10.14009/j.issn.1672-2124.2012.12.033 .
	CHEN K Y， LUO L P， XIE W Y， et al. Simultaneous determination of lamotrigine and carbamazepine in human serum by LC-MS/MS[J]. Zhongguo Yiyuan Yongyao Pingjia Yu Fenxi，2012，12（12）：1108-1110.
10	石银涛，张松，任飞，等. 血液中卡马西平固相萃取及LC-MS/MS定量方法研究[J].分析测试学报，2012，31（12）：1595-1598. doi：10.3969/j.issn.1004-4957.2012.12.023 .
	SHI Y T， ZHANG S， REN F， et al. Quantitative detection of carbamazepine in the blood by solid phase extraction and LC-MS/MS[J]. Fenxi Ceshi Xuebao，2012，31（12）：1595-1598.
11	MIAO X S， METCALFE C D. Determination of carbamazepine and its metabolites in aqueous samples using liquid chromatography-electrospray tandem mass spectrometry[J]. Anal Chem，2003，75（15）：3731-3738. doi：10.1021/ac030082k .
12	向平，沈敏，卓先义. 生物样品分析方法的有效性验证[J].法医学杂志，2008，24（1）：60-65. doi：10.3969/j.issn.1004-5619.2008.01.019 .
	XIANG P， SHEN M， ZHUO X Y. How to validate a bio-analytical method[J]. Fayixue Zazhi，2008，24（1）：60-65.
13	卓先义，吴侔天. 常见药（毒）物临床治疗血液浓度、中毒例血浓浓度、中毒死亡例血液浓度以及人体内半衰期（续一）[J].法医学杂志，1997，13（2）：123-124.
	ZHUO X Y， WU M T. Blood concentration of clinical treatment， poisoning cases， poisoning death cases and half-life in human body of common drugs （poison）（Continued 1）[J]. Fayixue Zazhi，1997，13（2）：123-124.
14	吴莱文. 治疗药物监测[M].北京：人民卫生出版社，1989：216-222.
	WU L W. Therapeutic drug monitoring[M]. Beijing： People’s Medical Publishing House，1989：216-222.

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