高效液相色谱-串联质谱法同时检测血液中3种季铵盐类肌松剂

doi:10.3724/SP.J.1123.2020.09020

摘要/Abstract

摘要：

维库溴铵、罗库溴铵和泮库溴铵是一类广泛使用的非去极化肌松剂,使用过程中引起过敏反应甚至死亡的情况时有发生,快速检测血液中该类肌松剂的浓度,可为临床早期诊断提供有价值的信息。该类肌松剂为强极性的季铵盐类化合物,在反相色谱柱上难以保留,主要采用离子对色谱法进行分离。采用离子对色谱法时,加入的离子对试剂有离子抑制作用,可降低质谱灵敏度,还会污染质谱系统。该文建立了高效液相色谱-串联质谱(HPLC-MS/MS)同时检测血液中3种季铵盐类肌松剂的检测方法。血液样品经稀释、高速离心后,上清液过Bond Elut AL-N固相萃取柱净化,用0.45 μm的微孔滤膜过滤后检测。采用ZIC-cHILIC色谱柱(50 mm×2.1 mm, 3.0 μm)分离,以乙腈和0.1%甲酸水溶液为流动相,梯度洗脱,在ESI⁺、多反应检测(MRM)模式下检测。3种季铵盐类肌松剂在血液中的基质效应为88.1%~95.4%,在各自范围内线性关系良好,相关系数(R²)均大于0.996,检出限为0.2~0.8 ng/mL,定量限为0.5~2.0 ng/mL,加标回收率为92.8%~110.6%,相对标准偏差(RSD)为3.2%~9.4%。该方法灵敏度高,准确性好,操作简便,可用于血液样品中维库溴铵、罗库溴铵和泮库溴铵的快速检测。

关键词: 高效液相色谱-串联质谱, 季铵盐, 维库溴铵, 罗库溴铵, 泮库溴铵, 肌松剂

Abstract:

Vecuronium, rocuronium, and pancuronium are widely used as non-depolarizing muscle relaxants. There have been occasional cases of allergic reactions and even death when using such muscle relaxants. Rapid determination of the concentration of these muscle relaxants in blood can provide valuable information for early clinical diagnosis. As quaternary ammonium compounds, these muscle relaxants are highly polar. Hence, they cannot be retained effectively on reversed-phase chromatographic columns with conventional mobile phases. These quaternary ammonium muscle relaxants are mainly separated by ion-pair chromatography. Using an ion-pairing reagent can help improve the retention capabilities of quaternary ammonium muscle relaxants. Nevertheless, the sensitivity of MS detection is significantly decreased because of ionic inhibition caused by the ion-pairing reagent in the mobile phase. Furthermore, ion-pairing reagents can pollute the MS system. A method based on high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was established for the simultaneous determination of the three quaternary ammonium muscle relaxants in blood. The blood samples were diluted and subjected to high-speed centrifugation. The supernatant was purified on a Bond Elut AL-N solid phase extraction column and then filtered through a 0.45 μm microporous membrane. The quaternary ammonium muscle relaxants were separated on a ZIC-cHILIC analytical column (50 mm×2.1 mm, 3.0 μm) with gradient elution. Acetonitrile and 0.1% formic acid aqueous solution were used as mobile phases. The separated compounds were analyzed by tandem MS with an electrospray ionization (ESI) source in positive and multiple reaction monitoring (MRM) modes. The matrix effects of vecuronium, rocuronium, and pancuronium in blood were 88.1% to 95.4%. The calibration curves for vecuronium, rocuronium, and pancuronium showed good linear relationships in each range, and all correlation coefficients (R²) were > 0.996. The limits of detection of vecuronium, rocuronium, and pancuronium were 0.2-0.8 ng/mL, with the corresponding limits of quantification being 0.5-2.0 ng/mL. The recoveries of vecuronium, rocuronium, and pancuronium were 92.8% to 110.6%, with relative standard deviations (RSDs) of 3.2%-9.4%. This method is sensitive, accurate, and easy to operate, and it can be used to rapidly determine vecuronium, rocuronium, and pancuronium in blood.

Key words: high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), quaternary ammonium, vecuronium, rocuronium, pancuronium, muscle relaxants

中图分类号:

O658

黄永鹏, 唐慧, 宋云扬, 陈博, 钟辉. 高效液相色谱-串联质谱法同时检测血液中3种季铵盐类肌松剂[J]. 色谱, 2021, 39(7): 695-701.

HUANG Yongpeng, TANG Hui, SONG Yunyang, CHEN Bo, ZHONG Hui. Simultaneous determination of three quaternary ammonium muscle relaxants in blood by high performance liquid chromatography-tandem mass spectrometry[J]. Chinese Journal of Chromatography, 2021, 39(7): 695-701.

图/表 7

表 1 3种肌松剂的质谱参数

Table 1 MS parameters for three muscle relaxants

Compound	Precursor ion	Mass transition (m/z)	Fragmentor/ V	Collision energy/ eV
Vecuronium	[M-Br^-+H]²⁺	279.2/100.1	131	24
bromide		279.2/249.2^*		8
Rocuronium	[M-Br^-+H]²⁺	265.2/235.1	122	12
bromide		265.2/244.2^*		9
Pancuronium	[M-2Br^-]²⁺	286.6/100.1^*	123	21
bromide		286.6/256.2		10

图 1 3种肌松剂的全扫描质谱图

Fig. 1 Full scan mass spectra of the three muscle relaxants

图 2 不同色谱柱上3种肌松剂的提取离子流色谱图

Fig. 2 Extracted ion current chromatograms of the three muscle relaxants on different chromatographic columns

图 3 采用不同流动相时3种肌松剂的提取离子流色谱图

Fig. 3 Extracted ion current chromatograms of the three muscle relaxants using different mobile phases

图 4 (a)3种肌松剂混合标准溶液和(b)空白血液样品的总离子流色谱图

Fig. 4 Total ion current chromatograms of (a) the three muscle relaxants mixed standard solution and (b) blank blood sample

表 2 3种肌松剂的线性范围、线性方程、相关系数、检出限、定量限、回收率及相对标准偏差

Table 2 Linear ranges, linear equations, correlation coefficients (R2), LODs, LOQs, recoveries and RSDs of the three muscle relaxants

Compound	Linear range/ (ng/mL)	Linear equation	R²	LOD/ (ng/mL)	LOQ/ (ng/mL)	Recovery/%	RSD (n=6)/%
Vecuronium bromide	0.5-1000	Y=79.68X+945.47	0.9967	0.2	0.5	92.8-108.3	3.2-5.6
Rocuronium bromide	2.0-1000	Y=107.36X-3040.50	0.9972	0.8	2.0	94.3-106.2	4.2-5.4
Pancuronium bromide	1.7-1000	Y=88.68X-1881.70	0.9963	0.7	1.7	97.9-110.6	5.5-9.4

表 3 4个兔子血液样品的测定结果(n=3)

Table 3 Determination results of four rabbit blood samples (n=3)

No.	Vecuronium bromide/(ng/mL)	Rocuronium bromide/(ng/mL)	Pancuronium bromide/(ng/mL)
1	ND	ND	428
2	364	ND	ND
3	ND	450	ND
4	231	173	287

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