通过式固相萃取-超高效液相色谱-串联质谱法测定水产品中地西泮

doi:10.3724/SP.J.1123.2019.11028

摘要/Abstract

摘要：

建立了通过式固相萃取-超高效液相色谱-串联质谱测定水产品中地西泮残留量的分析方法。样品用乙腈直接提取，经Prime HLB通过式固相萃取柱（60 mg/3 mL）净化，以Acquity UPLC BEH C₁₈（100 mm×2.1 mm，1.7 μm）为色谱柱，甲醇-0.1%（v/v）甲酸水溶液为流动相进行梯度洗脱。在多反应监测（MRM）、正离子电离模式下检测，采用基质匹配标准曲线外标法进行定量分析。结果显示：在0.1~10 ng/mL范围内，地西泮线性关系良好（r²>0.99）。在1.5、3.0和15.0 μg/kg 3个加标水平下，地西泮的加标回收率为88.2%~101.1%，日间和日内精密度（RSD）均在10%以下。该法简便快速，灵敏度高，可用于水产品中地西泮的准确测定。

关键词: 超高效液相色谱-串联质谱, 通过式固相萃取, 地西泮, 水产品

Abstract:

A method was developed for the determination of diazepam in aquatic products by pass-through solid phase extraction-ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The analyte was extracted with acetonitrile directly and purified on a Prime HLB solid phase extraction column (60 mg/3 mL). The separation was performed on an Acquity UPLC BEH C₁₈ column (100 mm×2.1 mm, 1.7 μm)using methanol-0.1% (v/v) formic acid aqueous solution as the mobile phase in gradient elution mode. Qualitative analysis was performed in the multiple reaction monitoring (MRM) mode. The analyte was quantified by matrix-matched external standard curves. The results showed good linear relationship in the range of 0.1-10 ng/mL, and the correlation coefficient (r²) was greater than 0.99. The spiked recoveries of diazepam were 88.2%-101.1% at the spiked levels of 1.5, 3.0 and 15.0 μg/kg, and both the intra-and inter-day precisions were less than 10%. The developed method is simple, rapid and accurate, and it can meet the requirements for diazepam determination in aquatic product samples.

Key words: ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS), pass-through solid phase extraction, diazepam, aquatic products

宿书芳, 孙立臻, 薛霞, 公丕学, 魏莉莉, 李新玲, 祝建华, 刘艳明, 张峰. 通过式固相萃取-超高效液相色谱-串联质谱法测定水产品中地西泮[J]. 色谱, 2020, 38(7): 791-797.

SU Shufang, SUN Lizhen, XUE Xia, GONG Pixue, WEI Lili, LI Xinling, ZHU Jianhua, LIU Yanming, ZHANG Feng. Determination of diazepam in aquatic products by ultra performance liquid chromatography-tandem mass spectrometry with pass-through solid phase extraction[J]. Chinese Journal of Chromatography, 2020, 38(7): 791-797.

图/表 11

图1 地西泮的化学结构式

Fig. 1 Chemical structure of diazepam

表1 地西泮的母离子、子离子及其他质谱参数

Table 1 Precursor ion, daughter ion and other MS parameters of diazepam

Analyte	Precursor ion (m/z)	Daughter ion (m/z)	Declustering potential/V	Collision energy/eV
*Quantitative ion.
Diazepam	285.2	154.0	100	37
		193.0^*	100	38

图2 地西泮标准溶液(1 ng/mL)的提取离子流色谱图

Fig. 2 Extracted ion current chromatograms of diazepam in the standard solution (1 ng/mL)

表2 采用不同提取方式时地西泮的回收率(n=6)

Table 2 Recoveries of diazepam using different extraction methods (n=6)

Pretreatment method	Recoveries/% (RSDs/%)
Pretreatment method	Yellow croaker	Carp	Crucian	Shrimp
Enzymolysis	82.1 (6.9)	79.5 (9.2)	80.6 (7.5)	92.5 (5.7)
Non enzymolysis	103.0 (5.4)	109.0 (4.3)	94.5 (8.3)	101.0 (2.9)

图3 不同提取剂对地西泮回收率的影响

Fig. 3 Effect of different extraction solvents on the recoveries of diazepam Solvent 1: acetonitrile; solvent 2: acetonitrile (containing 1% (v/v) acetic acid); solvent 3: acetonitrile (containing 1% (v/v) ammonium hydroxide); solvent 4: ethyl acetate.

表3 采用不同净化方式时地西泮的基质效应

Table 3 Matrix effects of diazepam using different purification methods

Purification method	Matrix effects/%
Purification method	Yellow croaker	Carp	Crucian	Shrimp 1	Shrimp 2
Prime HLB	6.7	3.9	7.6	10.1	5.5
QuEChERS	11.8	13.5	9.9	16.3	15.0
Non purification	55.2	58.9	37.6	68.0	56.2

图4 不同规格净化柱对地西泮回收率的影响

Fig. 4 Effect of purification columns with different specifications on the recoveries of diazepam

图5 上样溶液中乙腈的体积分数对地西泮回收率的影响

Fig. 5 Effect of volume fractions of acetonitrile on the recoveries of diazepam in sample loading solutions

图6 氮吹对不同样品中目标物回收率的影响(n=3)

Fig. 6 Effect of the nitrogen blowing on the recoveries of the different samples (n=3)

表4 地西泮的线性方程、相关系数、检出限和定量限

Table 4 Linear equations, correlation coefficients (r2), LODs and LOQs of diazepam

Matrix	Linear equation	r²	LOD/(μg/kg)	LOQ/(μg/kg)
Y: peak area; X: mass concentration, ng/mL.
Yellow	Y=1.826×10⁵X+1.463×10⁴	0.9992	0.5	1.5
croaker
Shrimp	Y=2.021×10⁵X+5.873×10³	0.9970	0.5	1.5

表5 水产品中地西泮的回收率及精密度(n=6)

Table 5 Recoveries and precisions of diazepam in aquatic products (n=6)

Matrix	Spiked level/(μg/kg)	Average recovery/%	Intra-day RSD/%	Inter-day RSD/%
Yellow croaker	1.5	95.4	7.0	5.9
	3.0	93.5	4.8	3.3
	15.0	101.1	5.2	5.1
Shrimp	1.5	88.2	2.3	7.7
	3.0	95.6	3.8	5.2
	15.0	97.3	6.4	5.0

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