高效液相色谱-串联质谱法测定动物源性食品中矮壮素残留

doi:10.3724/SP.J.1123.2019.04036

摘要/Abstract

摘要：

建立了高效液相色谱-串联质谱测定动物源性食品中矮壮素残留的分析方法。样品经含1%（v/v）乙酸的乙腈溶液提取、正己烷脱脂、阳离子固相萃取柱（PCX）净化，采用Venusil MP C18（2）色谱柱（150 mm×2.1 mm，3 μm）分离，以乙腈和0.1%（v/v）甲酸水溶液为流动相进行梯度洗脱，采用电喷雾电离、正离子模式扫描，多反应监测模式（MRM）检测，基质匹配标准曲线内标法定量。结果表明：矮壮素在0.200~500 μg/L范围内呈良好线性，相关系数（r²）均不低于0.9993，方法的定量限为0.500 μg/kg；以猪肉、牛肉、羊肉、鸡肉、鸡蛋、猪肾、牛肝、羊肾、鸡肝、牛奶为基质，矮壮素的平均加标回收率为93.4%~101%，相对标准偏差为2.3%~8.0%。该方法基质干扰小，灵敏度高，准确可靠，适用于动物源性食品中矮壮素残留的定量检测。

关键词: 高效液相色谱-串联质谱, 固相萃取, 矮壮素, 动物源性食品

Abstract:

A method for the determination of chlormequat chloride (CCC) residues in animal derived foods by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed. The samples were extracted with acetonitrile containing 1% (v/v) acetic acid and defatted with n-hexane, followed by clean-up on a cationic solid phase extraction column. The analytes were separated on a Venusil MP C18(2) column (150 mm×2.1 mm, 3 μm) under a gradient elution program using acetonitrile and 0.1% (v/v) formic acid aqueous solution as the mobile phases. Then, the analytes were detected by tandem mass spectrometry using a positive electrospray ionization (ESI⁺) source in the multiple reaction monitoring (MRM) mode. Matrix-matched internal standard calibration curves were used for quantitative analysis. The calibration curves showed good linearity in the range of 0.200-500 μg/L for CCC, with correlation coefficients (r²) no less than 0.9993. The limit of quantification (LOQ) of the method was 0.500 μg/kg. The average recoveries of CCC in pork, beef, mutton, chicken, egg, pig kidney, beef liver, sheep kidney, chicken liver and milk matrices at spiked levels of 0.500-500 μg/kg were 93.4%-101%, and the relative standard deviations were 2.3%-8.0%. The method has less matrix interference, with high sensitivity, accuracy and reliability, and it is suitable for the quantitative detection of CCC residues in animal derived foods.

Key words: high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), solid phase extraction (SPE), chlormequat chloride (CCC), animal derived foods

肖泳, 吴海智, 袁列江, 唐吉旺, 王淑霞, 王秀, 邓航, 吴林. 高效液相色谱-串联质谱法测定动物源性食品中矮壮素残留[J]. 色谱, 2020, 38(2): 218-223.

XIAO Yong, WU Haizhi, YUAN Liejiang, TANG Jiwang, WANG Shuxia, WANG Xiu, DENG Hang, WU Lin. Determination of chlormequat chloride residues in animal derived foods by high performance liquid chromatography-tandem mass spectrometry[J]. Chinese Journal of Chromatography, 2020, 38(2): 218-223.

图/表 6

图1 矮壮素的分子结构式

Fig. 1 Molecular structure formula of chlormequat chloride (CCC)

表1 CCC和CCC-D4的质谱参数

Table 1 MS parameters of CCC and CCC-D4

Analyte	Monitoring ion pair (m/z)	Collision energy/eV	Tube lens offset/V
* Quantitative ion.
CCC	122.044＞58.077^*	23	51
	122.044＞62.993	21	51
	124.037＞58.045	28	52
CCC-D4	126.123＞58.157^*	26	58
	126.123＞67.111	22	58

图2 采用不同色谱柱时加标鸡蛋样品中矮壮素(0.500 μ g/kg)的色谱图

Fig. 2 Chromatograms of CCC (0.500 μ g/kg) spiked in egg samples using the different chromatographic columns a. column: Venusil MP C18(2) (150 mm×2.1 mm, 3 μ m); mobile phase: (A) 0.1% (v/v) formic acid aqueous solution (containing 10 mmol/L ammonium acetate) and (B) acetonitrile; gradient elution program: 0-1.00 min, 10%B; 1.00-3.50 min, 10%B-95%B; 3.50-4.00 min, 95%B; 4.00-4.01 min, 95%B-10%B; 4.01-5.00 min, 10%B. b. column: Venusil HILIC (150 mm×2.1 mm, 3 μ m); mobile phase: 0.1% (v/v) formic acid aqueous solution (containing 10 mmol/L ammonium acetate)-acetonitrile (40 : 60, v/v); isocratic elution. c. column: Thermo Hypersil Gold (150 mm×2.1 mm, 3 μ m). Mobile phases and gradient elution program were the same as that in Fig. 2a.

图3 不同洗脱溶液对矮壮素回收率的影响

Fig. 3 Effects of different elution solutions on the recoveries of CCC A. methanol (containing 50 mmol/L ammonium acetate); B. methanol (containing 75 mmol/L ammonium acetate); C. methanol (containing 100 mmol/L ammonium acetate); D. 50% (v/v) methanol aqueous solution (containing 100 mmol/L ammonium acetate); E. 80% (v/v) methanol aqueous solution (containing 100 mmol/L ammonium acetate).

表2 不同基质中矮壮素的线性方程、相关系数、定量限、加标回收率和精密度(n=6)

Table 2 Linear equations, correlation coefficients (r2), LOQs, spiked recoveries and precisions of CCC in the different matrices (n=6)

Matrix	Linear equation	r²	LOQ/ (μg/kg)	Recoveries/%			RSDs/%
Matrix	Linear equation	r²	LOQ/ (μg/kg)	Low	Medium	High	Low	Medium	High
Low, medium, high spiked levels: 0.500, 10.0, 200 μ g/kg for pork, beef, mutton; 0.500, 5.00, 40.0 μ g/kg for chicken; 0.500, 10.0, 100 μ g/kg for egg, chicken live; 0.500, 20.0, 500 μ g/kg for pig kidney, beef liver, sheep kidney, milk; y: peak area ratio of CCC to CCC-D4; x: mass concentration, μ g/L.
Pork	y=0.0740+0.0955x	0.9999	0.500	99.6	95.8	95.6	4.2	4.1	3.2
Beef	y=0.0590+0.0961x	0.9999	0.500	97.5	99.4	93.7	6.7	6.7	3.5
Mutton	y=0.0732+0.0903x	0.9998	0.500	95.6	95.4	98.3	5.4	2.7	4.4
Chicken	y=0.0890+0.0926x	0.9993	0.500	95.9	97.8	96.5	5.4	5.8	3.5
Egg	y=0.0741+0.0911x	0.9999	0.500	99.9	101	97.3	3.5	2.8	2.6
Pig kidney	y=0.1024+0.0864x	0.9998	0.500	99.3	99.0	97.4	4.7	5.1	4.3
Beef liver	y=0.0744+0.0834x	0.9993	0.500	97.2	98.5	93.4	8.0	4.5	4.4
Sheep kidney	y=0.1545+0.0870x	0.9998	0.500	99.2	96.6	96.6	3.9	4.5	5.6
Chicken liver	y=0.0855+0.0881x	0.9999	0.500	99.3	100	98.7	6.1	3.6	3.4
Milk	y=0.0783+0.0909x	0.9995	0.500	98.8	98.4	99.3	5.6	4.7	2.3

图4 不同加标样品中矮壮素(0.500 μ g/kg)的色谱图

Fig. 4 Chromatograms of CCC (0.500 μ g/kg) spiked in the different samples

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