气相色谱-四极杆/飞行时间质谱筛查确证辣椒中244种农药残留及其代谢物

doi:10.3724/SP.J.1123.2020.11019

摘要/Abstract

摘要：

建立了辣椒中244种农药残留的QuEChERS前处理结合气相色谱-四极杆/飞行时间质谱(GC-Q-TOF/MS)快速筛查确证方法。鲜辣椒和干辣椒样品分别采用经-20 ℃冷冻的乙腈和1%(v/v)乙酸化乙腈提取,经盐析分层、分散固相萃取净化和浓缩后加入内标并复溶,HP-5MS UI色谱柱(30 m×0.25 mm×0.25 μm)分离,程序升温不分流进样,GC-Q-TOF/MS全扫描模式采集,内标法定量。比较了分析保护剂(AP)和基质匹配校准法对基质效应的补偿效果,最终选择采用基质匹配校准法来补偿基质效应并进行样品中农药残留的校准定量。设置定性筛查中的保留时间最大偏差为±0.25 min,精确质量偏差阈值为±20×10 ^-6。对鲜辣椒中244种农药残留和干辣椒中222种农药残留进行了定量方法验证,实验结果表明,采用建立的数据库和分析方法可以对辣椒进行农药残留的高通量筛查和定量分析。在空白辣椒样品中添加不同水平的目标化合物,以信噪比S/N≥10对应的添加水平作为定量限(LOQ)。鲜辣椒中最大残留限量(MRL)≤0.05 mg/kg的44种农药在鲜辣椒中LOQ≤0.010 mg/kg,线性范围在0.01~1.00 mg/L,在1倍和2.5倍LOQ添加水平下,回收率在60%~120%的农药种类占比分别为88.64%和100%;鲜辣椒中暂无MRL规定或MRL>0.05 mg/kg的200种农药在鲜辣椒中LOQ≤0.025 mg/kg,线性范围在0.05~1.00 mg/L,在1倍、2倍和10倍LOQ添加水平下,回收率在60%~120%的农药种类占比分别为49.50%、87.00%和89.50%; 244种农药的线性相关系数(r ²)均大于0.99。222种农药在干辣椒中LOQ≤0.15 mg/kg,线性范围在0.04~1.00 mg/L, r ²≥0.99的比例为95.46%,在1倍、2倍和10倍LOQ添加水平下,回收率在60%~120%占比分别为72.52%、73.42%和81.53%。应用建立的筛查确证方法对市售的12份鲜辣椒样品和14份干辣椒样品进行农药残留筛查分析,从9份鲜辣椒样品和3份干辣椒样品中筛查出8种农药化合物,经人工鉴定均为阳性,定量结果显示,8种农药化合物均未超过其在GB 2763-2019《食品安全国家标准食品中农药最大残留限量》所规定的MRL。方法快速、简单、高效、可靠,适用于鲜辣椒及干辣椒中多种农药残留的筛查分析。

关键词: QuEChERS, 气相色谱-四极杆/飞行时间质谱, 农药残留, 数据库, 辣椒, 筛查确证

Abstract:

QuEChERS pretreatment combined with gas chromatography-quadrupole time-of-flight mass spectrometry (GC-Q-TOF/MS) has been investigated for application in screening 244 pesticide residues in chilli. Fresh chilli samples were extracted with acetonitrile, and dried chilli samples were extracted using an acetonitrile/acetic acid (99∶1, v/v) mixture. The two extraction solvents were stored at -20 ℃. After salting out and cleaning by dispersive solid phase extraction (dSPE), heptachlor epoxide B was added as an internal standard, and the resulting residues were dissolved in 1.00 mL acetone. The dissolved sample solution was loaded onto an HP-5MS UI column (30 m×0.25 mm, 0.25 μm) and eluted by GC-Q-TOF/MS with a programmable temperature vaporizer and splitless injection in the full-scan mode. The compensation effects of the analytical protectant (AP) and matrix-matched calibration method on the matrix effect were established. AP could be used in the fresh chilli matrix to compensate for matrix effects, but it was not effective in the dried chilli matrix. The matrix-matched calibration method was effective in both matrices, which was selected for the quantification of pesticide residues in the samples. Because of the existence of the isomers of one compound and the same characteristic ions of different compounds, analyte detection was based on a flexible retention time deviation of ±0.25 min and accurate mass deviation of ±20×10^-6. Screening was performed by the software in the automatic matching mode. Compound identification and quantitation were based on a database and calibration curve established with reference materials. Suspicious samples were subjected to manual analysis. Quantitative analysis of 244 pesticide residues in fresh chilli and 222 pesticide residues in dried chilli was performed. The results showed that the developed database and method can provide a reference for the high-throughput screening and quantitation of fresh and dried chilli. Different levels of pesticides were added to the blank chilli samples, and the addition level corresponding to a signal-to-noise ratio (S/N) of 10 was used as the limit of quantification (LOQ). The LOQs of 44 pesticides with a maximum residue limit (MRL) ≤0.05 mg/kg in fresh chilli did not exceed 0.010 mg/kg. The linear ranges of these 44 pesticides were 0.01-1.00 mg/L. At spiked levels of the LOQ and 2.5 times the LOQ, the ratios of the 44 pesticides with recoveries of 60% to 120% were 88.64% and 100%, respectively. The LOQs of 200 pesticides with MRLs ≥0.05 mg/kg or without MRLs in fresh chilli did not exceed 0.025 mg/kg. The linear ranges of these 200 pesticides were 0.05-1.00. At spiked levels of the LOQ, twice the LOQ, and 10 times the LOQ, the ratios of the 200 pesticides with recoveries of 60% to 120% were 49.50%, 87.00%, and 89.50%, respectively. The linear correlation coefficients (r ²) of the 244 pesticides in fresh chilli were greater than 0.99. The LOQs of 222 pesticides in dried chilli were less than 0.15 mg/kg, and the linear ranges were 0.04-1.00 mg/L. The ratios of these 222 pesticides with r² greater than 0.99 was 95.46%. At spiked levels of the LOQ, twice the LOQ and 10 times the LOQ in dried chilli, the ratio of the 222 pesticides with recoveries of 60% to 120% were 72.52%, 73.42%, and 81.53%, respectively. The established screening and confirmation method was used to analyze 12 fresh chilli samples and 14 dried chilli samples. Eight pesticides were found in nine fresh chilli samples and three dried chilli samples, all of which were confirmed to be positive after manual identification. The concentrations of these pesticides were lower than the MRLs required by GB 2763-2019: National Food Safety Standard-Maximum Residue Limits for Pesticides in Food. The results demonstrate that the established method is rapid, easy to execute, efficient, and reliable. It can be used for the high-throughput screening and quantitation of pesticide residues in fresh and dried chilli.

Key words: QuEChERS, gas chromatography-quadrupole time-of-flight mass spectrometry (GC-Q-TOF/MS), pesticide residues, database, chilli, screening and confirmation

中图分类号:

O658

曹琦, 张亚珍, 朱正伟, 吴婉琴, 江丰, 余婷婷. 气相色谱-四极杆/飞行时间质谱筛查确证辣椒中244种农药残留及其代谢物[J]. 色谱, 2021, 39(5): 494-509.

CAO Qi, ZHANG Yazhen, ZHU Zhengwei, WU Wanqin, JIANG Feng, YU Tingting. Screening and confirmation of 244 pesticide residues in chilli by gas chromatography-quadrupole time-of-flight mass spectrometry[J]. Chinese Journal of Chromatography, 2021, 39(5): 494-509.

图/表 7

表 1 244种农药化合物的分子式、精确质量数、特征离子、保留时间(tR)、线性相关系数(r2)和定量限(LOQ)

Table 1 Formulas, exact mass, characteristie ions, retention times (tR), linear correlation coefficients (r2),limits of quantification (LOQs) for the 244 pestcide compounds

图 1 244种农药的保留时间分布

Fig. 1 Distribution of retention times of the 244 pesticides

图 2 222种农药在辣椒中的基质效应分布

Fig. 2 Distribution of matrix effects of the 222 pesticides in chilli

图 3 不同基质中敌敌畏(100 μg/L)的色谱图

Fig. 3 Chromatograms of dichlorvos at 100 μg/L in different matricesa. acetone; b. 1 g/L L(+)-gulonic acid gamma-lactone and 0.5 g/L D-sorbitol in acetone; c. blank matrix of fresh chilli; d. blank matrix of dried chilli. Monitoring ion: m/z 109.0043, retention time 6.188 min.

图 4 丙酮中丙环唑和5种拟除虫菊酯类农药标准溶液 (200 μg/L)的特征离子色谱图

Fig. 4 Characteristic ion chromatograms of standard solutions of propiconazole and five pyrethroid pesticides at 200 μg/L in acetone

表 2 244种农药在鲜辣椒中4个水平和干辣椒中3个水平下的加标回收率(n=5)

Table 2 Recoveries of 244 pesticides at four levels in fresh chilli and three levels in dried chilli (n=5)

Sample	Number of pesticides added	Added/ (mg/kg)	Ratios/%
Sample	Number of pesticides added	Added/ (mg/kg)	Recovery<60%	60%≤recovery≤120%	Recovery>120%
Fresh chilli	44¹⁾	0.010	6.82	88.64	4.55
		0.025	0	100.00	0
	200²⁾	0.025	8.00	49.50	42.50
		0.050	5.50	87.00	7.50
		0.25	1.50	89.50	9.00
Dried chilli	222	0.15	15.32	72.52	12.16
		0.30	14.41	73.42	12.17
		1.5	6.76	81.53	11.71

表 3 辣椒样品中农药残留的筛查及定量结果

Table 3 Screening and quantitative results in chilli samples

Sample	Compound	MRL/(mg/kg)	Content/(mg/kg)	Frequency of detection
Fresh chilli	alachlor (甲草胺)	-	<0.025	1
	chlorfenson (杀螨酯)	-	<0.025	1
	diniconazole (烯唑醇)	-	<0.025	1
	difenoconazole (苯醚甲环唑)	1	0.07, 0.09, 0.11	3
	propiconazole (丙环唑)	-	<0.025	2
	hexachlorobenzene (六氯苯)	-	<0.025	1
	tetraconazole (四氟醚唑)	-	<0.025	1
Dried chilli	mevinphos (速灭磷)	-	<0.15	3
	difenoconazole (苯醚甲环唑)	5	0.35	1

参考文献

[1]	Wang L H, Ma Y Q, Zhang B X. China Vegetables, 2019(8):1
[1]	王立浩, 马艳青, 张宝玺. 中国蔬菜, 2019(8):1
[2]	GB 2763-2019
[3]	Guo D D, Yang F, Li J, et al. Chinese Journal of Analysis Laboratory, 2019(10):9
[3]	郭冬冬, 杨方, 李捷, 等. 分析试验室, 2019(10):9
[4]	Qiao Y S, Wang J H, Qiu Y J, et al. Chinese Journal of Chromatography, 2020,38(12):1402
[4]	乔勇升, 王俊虎, 仇雅静, 等. 色谱, 2020,38(12):1402
[5]	Gao S, Chen H, Hu X Y, et al. Chinese Journal of Chromatography, 2019,37(9):955
[5]	高帅, 陈辉, 胡雪艳, 等. 色谱, 2019,37(9):955
[6]	Lei R Q, Niu Y M, Guo Q Z, et al. Chinese Journal of Chromatography, 2020,38(7):805
[6]	雷汝清, 牛宇敏, 郭巧珍, 等. 色谱, 2020,38(7):805
[7]	Wang J, Ling Y, Deng Y M, et al. Chinese Journal of Chromatography, 2020,38(6):655
[7]	王佳, 凌云, 邓亚美, 等. 色谱, 2020,38(6):655
[8]	Ruan H, Rong W G, Song N H, et al. Chinese Journal of Analysis Chemistry, 2014(8):1110
[8]	阮华, 荣维广, 宋宁慧, 等. 分析化学, 2014(8):1110
[9]	Huang H T, Xie S, Tu X T, et al. Chinese Journal of Analysis Chemistry, 2020,48(3):423
[9]	黄合田, 谢双, 涂祥婷, 等. 分析化学, 2020,48(3):423
[10]	Huang Y S, Shi T, Luo X, et al. Food Chem, 2019,275:255
[11]	Xu X, Xu X, Han M, et al. Food Chem, 2019,276:419
[12]	Silvina N, Verónica C, Julia H, et al. J Agric Food Chem, 2014,62(17):3675
[13]	Wang L D. [MS Dissertation]. Guangzhou: South China University of Technology 2019
[13]	王立丹. [硕士学位论文]. 广州: 华南理工大学 2019
[14]	Rúbies A, Guo L, Centrich F, et al. Anal Bioanal Chem, 2016,408:5769
[15]	Antonio V, Ana A, Carmen F, et al. J Agric Food Chem, 2010,58(5):2818
[16]	Zhang C, Deng Y, Zheng J, et al. TrAC-Trends Anal Chem, 2019,118:517
[17]	Rúbie A, Muñoz E, Gibert D, et al. J Chromatogr A, 2015,1386:62
[18]	Urairat K, Kunapon S, Natchanun L. Food Chem, 2014,153:44
[19]	Xiong X, Liu Q, Zhang G W, et al. Journal of Instrumental Analysis, 2018,37(9):1008
[19]	熊欣, 刘青, 张广文, 等. 分析测试学报, 2018,37(9):1008
[20]	Zheng C M, Zhang Y, Wang S X, et al. Journal of Instrumental Analysis, 2012,31(4):383
[20]	郑翠梅, 张艳, 王松雪, 等. 分析测试学报, 2012,31(4):383
[21]	Rocio F, Maria L, Thierry D. Toxins, 2020,12(3):206
[22]	Xu X L, Zhao H X, Li L, et al. Chinese Journal of Chromatography, 2012,30(3):267
[22]	许秀丽, 赵海香, 李礼, 等. 色谱, 2012,30(3):267
[23]	Wang J G, Wei Y X. Chinese Journal of Health Laboratory Technology, 2016,26(18):2603
[23]	王建国, 魏玉霞. 中国卫生检验杂志, 2016,26(18):2603
[24]	Ye X, Ye B, Xu J, et al. J Sep Sci, 2020,43(17):3546
[25]	Fujiyoshi T, Ikama T, Sato T, et al. J Chromatogr A, 2016,1434:136
[26]	Akutsu K, Kitagawa Y, Yoshimitsu M, et al. Anal Bioanal Chem, 2018,410:3145