超高效液相色谱-串联质谱法同时测定血清中12种抗癫痫药物

doi:10.3724/SP.J.1123.2019.12025

摘要/Abstract

摘要：

建立了超高效液相色谱-串联质谱同时测定血清中加巴喷丁、拉莫三嗪、普瑞巴林、拉考沙胺、左乙拉西坦、托吡酯、奥卡西平、氯硝西泮、丙戊酸钠、卡马西平、苯巴比妥、苯妥英钠12种抗癫痫药物的方法。将非那西丁和氯唑沙宗分别作为正离子和负离子模式下的内标物，血清经乙腈沉淀蛋白质后，取上清液进样分析。采用ACQUITY UPLC BEH C₁₈柱分离，流动相为水和甲醇（均含10 mmol/L甲酸铵），流速为0.4 mL/min。采用电喷雾电离源，在多反应监测和正、负离子模式下分段进行扫描分析。结果表明，12种抗癫痫药物在各自范围内线性关系良好，相关系数均大于0.992，在3个添加水平下方法的加标回收率为90.80%~114.0%，批内精密度RSD≤13.2%，批间精密度RSD≤14.8%。该方法专属性强，灵敏度高，可用于12种抗癫痫药物临床血药浓度监测及药代动力学研究。

关键词: 超高效液相色谱-串联质谱, 血药浓度, 抗癫痫药物, 血清

Abstract:

A sensitive, high-throughput method was established for the simultaneous determination of 12 antiepileptics in serum by ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The antiepileptics were gabapentin, lamotrigine, pregabalin, lacosamide, levetiracetam, topiramate, oxcarbazepine, clonazepam, sodium valproate, carbamazepine, phenobarbital and phenytoin sodium. Phenacetin and chlorzoxazone were used as internal standards. The antiepileptics and internal standards were extracted from serum by protein precipitation using acetonitrile as the precipitant. Chromatographic separation was achieved on an ACQUITY UPLC BEH C₁₈ column with a gradient mobile phase comprising 10 mmol/L ammonium formate aqueous solution and methanol (containing 10 mmol/L ammonium formate) at a flow rate of 0.4 mL/min. Detection was performed in multiple reaction monitoring mode with ion mode switching. The results showed good linear trends in their respective concentration ranges and the correlation coefficients (r²) were greater than 0.992. The spiked recoveries of the 12 antiepileptics in serum were 90.80%-114.0% at the three spiked levels. The intra-assay (n=6) and inter-assay (n=3) precisions were less than 13.2% and 14.8%, respectively. The method has high specificity and sensitivity, and it can be used for clinical blood concentration monitoring and pharmacokinetic studies of the 12 antiepileptics.

Key words: ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), blood drug concentration, antiepileptics, serum

代静, 高乐虹, 彭方达, 于丽佳, 王朝东, 王玉平, 丁春光. 超高效液相色谱-串联质谱法同时测定血清中12种抗癫痫药物[J]. 色谱, 2020, 38(8): 900-905.

DAI Jing, GAO Lehong, PENG Fangda, YU Lijia, WANG Chaodong, WANG Yuping, DING Chunguang. Simultaneous determination of twelve antiepileptic drugs in serum by ultra high performance liquid chromatography-tandem mass spectrometry[J]. Chinese Journal of Chromatography, 2020, 38(8): 900-905.

图/表 5

参考文献

1	Wang D L , Song H D , Xu K , et al. Chinese Medical Sciences Journal, 2019 4): 566
1	王栋梁, 宋海栋, 许可, et al. 中国医学科学院学报, 2019 4): 566
2	Zhu Y F , Gu R J . World Latest Medicine Information, 2019, 19(52): 98
2	朱亚峰, 顾仁骏世界最新医学信息文摘, 2019, 19(52): 98
3	Xu B M , Chen B . China Pharmacy, 2017, 28(35): 5036
3	许倍铭, 陈冰中国药房, 2017, 28(35): 5036
4	Patsalos P N , Spencer E P , Berry D J . Ther Drug Monit, 2018, 40(5): 526
5	Tang F P , Wang W J , Li Y F . China Journal of Hospital Pharmacy, 2010, 30(8): 674
5	汤芳萍, 王伟娇, 李艳飞中国医院药学杂志, 2010, 30(8): 674
6	Liu Y , Zhang Y D , Shi J P . Chinese Journal of Chromatography, 2002, 20(1): 94
6	刘阳, 张颖冬, 石静平色谱, 2002, 20(1): 94
7	Liu Y Y , Wan C C , Zhou C H , et al. Chinese Journal of Clinical Pharmacology, 2017, 33(7): 628
7	刘艳艳, 万昶宸, 周春华, et al. 中国临床药理学, 2017, 33(7): 628
8	Wang W , Liu Y S , Qiao S , et al. Chinese Journal of Clinical Pharmacology, 2014, 30(9): 807
8	王伟, 刘永锁, 乔湜, et al. 中国临床药理学, 2014, 30(9): 807
9	Zhou J , Wang Y M , Zhao N M , et al. Chinese Medical Sciences Journal, 2015, 5(5): 39
9	周晶, 王漪檬, 赵宁民, et al. 中国医药科学, 2015, 5(5): 39
10	Liu J J , Li J F , Dai W T , et al. Journal of Clinical Rational Drug Use, 2018, 11(26): 20
10	刘建军, 李俊峰, 代文婷, et al. 临床合理用药, 2018, 11(26): 20
11	Huang J , Zhang Q Z , Qi X W , et al. Journal of Bingtuan Medicine, 2016, 47(1): 49
11	黄健, 张奇洲, 漆新文, et al. 兵团医学, 2016, 47(1): 49
12	Li N , Chen X L , Zhang L , et al. Chinese Journal of Chromatography, 2018, 36(11): 1105
12	李娜, 陈雪蕾, 张磊, et al. 色谱, 2018, 36(11): 1105
13	Yin X X . Therapeutic Drug Monitoring Beijing People's Military Medical Press 2011 173
13	印晓星治疗药物监测北京人民军医出版社 2011 173
14	Wang X Q , Wen H K , Shao S Q , et al. Journal of Radioimmanology, 2012, 25(2): 230
14	王晓秋, 温怀凯, 邵素倩, et al. 放射免疫学杂志, 2012, 25(2): 230
15	Li M Z , Chen Y K . Laboratory Medicine and Clinic, 2011, 8(24): 2959
15	李美珠, 陈棪焜检验医学与临床, 2011, 8(24): 2959
16	Jabbaribar F , Mortazavi S A , Jouyban A . J Chromatogr Sci, 2009, 47: 877
17	Jiang B , Yang J . Guangzhou Chemical Industry, 2016, 44(6): 15
17	江波, 杨洁广州化工, 2016, 44(6): 15

No.	Compound	Retention time/min	Parent ion (m/z)	Product ion (m/z)	Collision energy/eV	Cone voltage/V	Ionization mode	IS
1	pregabalin	2.51	159.97	97.06	14	26	[M+H]⁺	phenacetin
2	gabapentin	2.55	172.13	154.1	10	24	[M+H]⁺	phenacetin
3	levetiracetam	2.77	171.11	126.1	15	10	[M+H]⁺	phenacetin
4	lacosamide	3.90	251.10	108.1	8	8	[M+H]⁺	phenacetin
5	lamotrigine	4.10	255.98	108.9	50	70	[M+H]⁺	phenacetin
6	phenobarbital	4.33	230.99	188.1	8	20	[M+H]^-	chlorzoxazone
7	topiramate	4.51	338.09	77.90	27	50	[M+H]^-	chlorzoxazone
8	oxcarbazepine	4.77	253.06	180.0	32	52	[M+H]⁺	phenacetin
9	phenytoin sodium	5.09	253.20	182.2	15	45	[M+H]⁺	phenacetin
10	carbamazepine	5.29	237.06	194.1	17	58	[M-H]⁺	phenacetin
11	clonazepam	5.34	316.07	270.1	24	45	[M-H]⁺	phenacetin
12	sodium valproic	5.56	143.10	143.1	3	30	[M-H]^-	chlorzoxazone
13	phenacetin	4.35	179.94	110.0	18	48	[M+H]⁺
14	chlorzoxazone	5.34	168.02	132.0	18	44	[M-H]^-

No.	Compound	Retention time/min	Parent ion (m/z)	Product ion (m/z)	Collision energy/eV	Cone voltage/V	Ionization mode	IS
1	pregabalin	2.51	159.97	97.06	14	26	[M+H]⁺	phenacetin
2	gabapentin	2.55	172.13	154.1	10	24	[M+H]⁺	phenacetin
3	levetiracetam	2.77	171.11	126.1	15	10	[M+H]⁺	phenacetin
4	lacosamide	3.90	251.10	108.1	8	8	[M+H]⁺	phenacetin
5	lamotrigine	4.10	255.98	108.9	50	70	[M+H]⁺	phenacetin
6	phenobarbital	4.33	230.99	188.1	8	20	[M+H]^-	chlorzoxazone
7	topiramate	4.51	338.09	77.90	27	50	[M+H]^-	chlorzoxazone
8	oxcarbazepine	4.77	253.06	180.0	32	52	[M+H]⁺	phenacetin
9	phenytoin sodium	5.09	253.20	182.2	15	45	[M+H]⁺	phenacetin
10	carbamazepine	5.29	237.06	194.1	17	58	[M-H]⁺	phenacetin
11	clonazepam	5.34	316.07	270.1	24	45	[M-H]⁺	phenacetin
12	sodium valproic	5.56	143.10	143.1	3	30	[M-H]^-	chlorzoxazone
13	phenacetin	4.35	179.94	110.0	18	48	[M+H]⁺
14	chlorzoxazone	5.34	168.02	132.0	18	44	[M-H]^-

No.	Compound	Linear range/(μg/L)	Linear equation	r²	LOQ/(μg/L)
Y: peak area ratio of the compound to internal standard; X: mass concentration, μg/L.
1	pregabalin	25.0-2500	Y=0.1508X+0.0008	0.992	16.5
2	gabapentin	12.5-2500	Y=0.3420X+0.0022	0.992	10.0
3	levetiracetam	12.5-2500	Y=1.9180X+0.0078	0.995	0.750
4	lacosamide	12.5-2500	Y=4.3480X+0.0160	0.997	0.210
5	lamotrigine	12.5-2500	Y=0.2405X+0.0008	0.996	0.500
6	phenobarbital	25.0-2500	Y=0.0769X+0.0020	0.997	21.0
7	topiramate	12.5-2500	Y=0.7476X+0.0078	0.998	5.30
8	oxcarbazepine	12.5-2500	Y=1.3070X+0.0045	0.996	0.140
9	phenytoin sodium	12.5-2500	Y=0.3903X+0.0018	0.995	2.30
10	carbamazepine	12.5-2500	Y=8.5390X+0.0460	0.995	0.150
11	clonazepam	1.25-250	Y=1.2230X+0.0006	0.995	0.330
12	sodium valproic	125-12500	Y=0.9634X+0.0810	0.997	103

No.	Compound	Linear range/(μg/L)	Linear equation	r²	LOQ/(μg/L)
Y: peak area ratio of the compound to internal standard; X: mass concentration, μg/L.
1	pregabalin	25.0-2500	Y=0.1508X+0.0008	0.992	16.5
2	gabapentin	12.5-2500	Y=0.3420X+0.0022	0.992	10.0
3	levetiracetam	12.5-2500	Y=1.9180X+0.0078	0.995	0.750
4	lacosamide	12.5-2500	Y=4.3480X+0.0160	0.997	0.210
5	lamotrigine	12.5-2500	Y=0.2405X+0.0008	0.996	0.500
6	phenobarbital	25.0-2500	Y=0.0769X+0.0020	0.997	21.0
7	topiramate	12.5-2500	Y=0.7476X+0.0078	0.998	5.30
8	oxcarbazepine	12.5-2500	Y=1.3070X+0.0045	0.996	0.140
9	phenytoin sodium	12.5-2500	Y=0.3903X+0.0018	0.995	2.30
10	carbamazepine	12.5-2500	Y=8.5390X+0.0460	0.995	0.150
11	clonazepam	1.25-250	Y=1.2230X+0.0006	0.995	0.330
12	sodium valproic	125-12500	Y=0.9634X+0.0810	0.997	103

No.	Compound	Added/(mg/L)	Recovery/%	RSDs/%
No.	Compound	Added/(mg/L)	Recovery/%	Intra-assay (n=6)	Inter-assay (n=3)
1	pregabalin	0.05	95.30	5.99	14.2
		0.25	105.0	5.85	5.99
		1.25	98.10	4.75	9.07
2	gabapentin	0.05	93.30	5.53	14.1
		0.25	104.0	5.30	6.60
		1.25	99.70	6.03	9.43
3	levetiracetam	0.05	97.70	4.01	12.8
		0.25	102.7	7.45	6.52
		1.25	96.50	2.58	9.21
4	lacosamide	0.05	94.00	4.46	14.8
		0.25	95.70	5.08	5.43
		1.25	93.90	3.68	9.69
5	lamotrigine	0.05	111.3	4.63	10.5
		0.25	110.3	4.46	5.12
		1.25	108.6	2.63	9.37
6	phenobarbital	0.05	102.0	4.84	12.5
		0.25	101.9	4.85	5.08
		1.25	103.6	5.14	11.0
7	topiramate	0.05	109.3	4.72	14.5
		0.25	102.7	6.31	7.49
		1.25	106.0	4.93	10.8
8	oxcarbazepine	0.05	112.0	4.78	13.5
		0.25	112.5	4.98	12.9
		1.25	109.0	2.23	10.8
9	phenytoin sodium	0.05	96.70	13.2	14.6
		0.25	105.2	6.93	6.29
		1.25	101.9	3.31	9.03
10	carbamazepine	0.05	91.70	10.7	11.7
		0.25	95.90	1.88	5.48
		1.25	90.80	2.39	9.37
11	clonazepam	0.005	110.0	10.5	10.4
		0.025	114.0	5.56	5.40
		0.125	113.1	2.61	6.89
12	sodium valproic	0.25	107.6	9.34	7.32
		1.25	110.0	2.75	4.12
		6.25	108.7	1.99	11.1