QuEChERS-超高效液相色谱-串联质谱法同时测定婴幼儿辅食果泥中5种链格孢霉毒素

doi:10.3969/j.issn.1004-1524.2022.12.16

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (12): 2728-2739.DOI: 10.3969/j.issn.1004-1524.2022.12.16

QuEChERS-超高效液相色谱-串联质谱法同时测定婴幼儿辅食果泥中5种链格孢霉毒素

邓涛¹(), 杨华², 肖英平², 汪雯², 吕文涛², 王小骊², 吴振¹^,^*(), 吉小凤²^,^*()

1.宁波大学食品与药学学院,浙江宁波 315000
2.浙江省农业科学院农产品质量安全与营养研究所,浙江杭州 310021

收稿日期:2022-01-24 出版日期:2022-12-25 发布日期:2022-12-26
通讯作者: 吴振,吉小凤
作者简介:吉小凤,E-mail:jixiaofeng@zaas.ac.cn
*吴振,E-mail: wuzhen@nbu.edu.cn;
邓涛(1997—),男,安徽滁州人,硕士研究生,研究方向为食品检测。E-mail:dengtao8822@163.com
基金资助:
国家重点研发计划政府间合作项目(2019YFE0103900)

Simultaneous determination of five Alternaria toxins by QuEChERS-ultra-performance liquid chromatography-tandem mass spectrometry in fruit puree for infants

DENG Tao¹(), YANG Hua², XIAO Yingping², WANG Wen², LYU Wentao², WANG Xiaoli², WU Zhen¹^,^*(), JI Xiaofeng²^,^*()

1. College of Food and Pharmaceutical Sciences, Ningbo University, Ningbo 315000, Zhejiang, China
2. Institute of Agro-Product Safety and Nutrition, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China

Received:2022-01-24 Online:2022-12-25 Published:2022-12-26
Contact: WU Zhen,JI Xiaofeng

摘要/Abstract

摘要：

建立婴幼儿辅食果泥中细交链孢菌酮酸、腾毒素、交链孢酚单甲醚、交链孢酚和交链孢烯5种链格孢霉毒素的QuEChERS-超高效液相色谱-串联质谱分析方法。待测样品经乙腈-水-甲酸混合液(79∶20∶1,V/V/V)提取,4 g 无水硫酸镁、0.5 g 无水乙酸钠和0.5 g 氯化钠脱水盐析,100 mg C18净化,经Waters ACQUITY UPLC^® HSS T3 (2.1 mm×100 mm, 1.8 μm)液相色谱柱分离,以5 mmol·L^-1 的乙酸铵水溶液和甲醇作为流动相梯度洗脱,在电喷雾离子源电离,多反应监测模式条件下进行测定,基质外标法定量。该方法在0.1~200 μg·L^-1 范围内线性关系良好,决定系数(R²)均大于0.998,检测限和定量限分别为0.1 μg·kg^-1 和0.3 μg·kg^-1。不同加标水平的平均回收率为73.8%~118.0%,相对标准偏差为2.3%~8.5%。结果表明,该方法高效、准确、灵敏,适用于婴幼儿辅食果泥中5种链格孢霉毒素确证检测。

关键词: 果泥, 链格孢霉毒素, 食品安全

Abstract:

A QuEChERS-ultra-performance liquid chromatography-tandem mass spectrometry method for the determination of five Alternaria toxins including tenuazonic acid, tentoxin, aletrnariol monomethyl ether, alternariol and altenuene in fruit puree for infants was established. A fruit puree sample was extracted with a mixture solution of acetonitrile∶water∶formic acid (79∶20∶1, V/V/V), followed by a salting-out extraction using 0.5 g anhydrous CH₃COONa and 0.5 g NaCl coupled with a dehydration step with the addition of 4 g anhydrous MgSO₄, and a purification step with 100 mg C18. An ACQUITY UPLC^® HSS T3 column (2.1 mm×100 mm, 1.8 μm) was used for chromatographic separation. The mobile phase consisted of 5 mmol·L^-1 ammonium acetate aqueous solution and methanol and a gradient elution program was applied. The measurement was carried out under the condition of multiple response monitoring mode and electrospray ion source. The matrix-matched external standard was used for quantitative analysis. The linearity of the method was in the range of 0.1-200 μg·L^-1, with the determination coefficients (R²) greater than 0.998. The limits of detection and quantification were 0.1 μg·kg^-1 and 0.3 μg·kg^-1, respectively. The average recoveries were in the range of 73.8% to 118.0% with the relative standard deviations of 2.3% to 8.5%. The results demonstrated that the developed method was efficient, accurate, sensitive, and suitable for the quantitative determination of five Alternaria toxins in fruit puree for infants.

Key words: fruit puree, Alternaria toxin, food safety

中图分类号:

TS201.6

邓涛, 杨华, 肖英平, 汪雯, 吕文涛, 王小骊, 吴振, 吉小凤. QuEChERS-超高效液相色谱-串联质谱法同时测定婴幼儿辅食果泥中5种链格孢霉毒素[J]. 浙江农业学报, 2022, 34(12): 2728-2739.

DENG Tao, YANG Hua, XIAO Yingping, WANG Wen, LYU Wentao, WANG Xiaoli, WU Zhen, JI Xiaofeng. Simultaneous determination of five Alternaria toxins by QuEChERS-ultra-performance liquid chromatography-tandem mass spectrometry in fruit puree for infants[J]. Acta Agriculturae Zhejiangensis, 2022, 34(12): 2728-2739.

图/表 13

图1 标准溶液中5种链格孢霉毒素MRM离子色谱图

Fig.1 Mass spectra of tenuazonic acid (TeA), tentoxin (TEN), aletrnariol monomethyl ether (AME), alternariol (AOH), and altenuene (ALT)

表1 五种链格孢霉毒素质谱参数

Table 1 MS-MS parameters for five Alternaria toxins

真菌毒素 Mycotoxin	母离子 Precursor ion/ (m/z)	子离子 Product ions/ (m/z)	去簇电压 Declustering potential/eV	碰撞能量 Collision energy/eV	ESI扫描方式 ESI Scanning way	保留时间 Retention time/min
TeA	196.0	139.0^*	-50	-28	-	4.01
		112.0	-50	-40
TEN	413.3	271.1	-123	-23	-	6.37
		140.8^*	-126	-27
AME	271.2	256.2^*	-100	-28	-	6.92
		228.2	-100	-42
AOH	257.0	213.2^*	-120	-32	-	6.28
		147.2	-120	-42
ALT	293.0	257.0^*	57	19	+	5.81
		239.0	70	29

图2 提取液对5种链格孢霉毒素回收率的影响(加标浓度20 μg·kg-1)

Fig.2 Effect of extracting solutions on the recoveries of five Alternaria toxins (at spiked level of 20 μg·kg-1)

图3 提取液体积对5种链格孢霉毒素回收率的影响(加标浓度20 μg·kg-1)

Fig.3 Effect of the volume of extracting solutions on the recoveries of five Alternaria toxins (at spiked level of 20 μg·kg-1)

图4 果胶酶用量对5种链格孢霉毒素回收率的影响(加标浓度20 μg·kg-1)

Fig.4 Effect of dosages of pectinase on the recoveries of five Alternaria toxins (at spiked level 20 μg·kg-1)

图5 超声时间对5种链格孢霉毒素回收率的影响(加标浓度20 μg·kg-1)

Fig.5 Effect of ultrasonic time on the recoveries of five Alternaria toxins (at spiked level 20 μg·kg-1)

图6 盐析剂对婴幼儿辅食果泥中5种链格孢霉毒素回收率的影响(加标浓度20 μg·kg-1)

Fig.6 Effects of salting out agents on the recoveries of five Alternaria toxins (at spiked level of 20 μg·kg-1)

图7 净化剂C18 和PSA对5种链格孢霉毒素回收率的影响(加标浓度20 μg·kg-1) *表示差异显著(P<0.05)。

Fig.7 Effect of purifying agent C18 and PSA on the recoveries of five Alternaria toxins (at spiked level of 20 μg·kg-1) * indicated the significant difference (P<0.05).

图8 C18用量对5种链格孢霉毒素回收率的影响(加标浓度20 μg·kg-1)

Fig.8 Effect of dosages of C18 on the recoveries of five Alternaria toxins (at spiked level of 20 μg·kg-1)

表2 五种链格孢霉毒素的线性关系、检出限、定量限

Table 2 Linear relationship, limit of detection, limit of quantitation of five Alternaria toxins

真菌毒素 Mycotoxin	线性范围 Linear range/ (μg·kg^-1)	线性方程 Linearity	决定系数 Determination coefficient (R²)	检测限 Limit of detection/ (μg·kg^-1)	定量限 Limit of quantification/ (μg·kg^-1)
TeA	0.1~200	y=6 518.6x-4 576.6	0.998 8	0.1	0.3
TEN	0.1~200	y=68 726x+242 291	0.998 6	0.1	0.3
AME	0.1~200	y=366 699x+610 963	0.999 6	0.1	0.3
AOH	0.1~200	y=104 261x+95 424	0.999 8	0.1	0.3
ALT	0.1~200	y=87 531x-43 124	0.999 6	0.1	0.3

表3 五种链格孢霉毒素的回收率和精密度

Table 3 Recoveries and intra- and inter-day accuracy and precision for the determination of five Alternaria toxins

真菌毒素 Mycotoxin	添加浓度 Spiked concentration/ (μg·kg^-1)	精密度 Accuracy/%		相对标准偏差RSD/%
真菌毒素 Mycotoxin	添加浓度 Spiked concentration/ (μg·kg^-1)	日内/(n=6) Intra-day/(n=6)	日间/(n=18) Inter-day/(n=18)	日内/(n=6) Intra-day/(n=6)	日间/(n=18) Inter-day/(n=18)
TeA	5	73.8±2.4	78.5±9.0	3.2	8.5
	20	82.6±5.5	83.6±6.3	6.7	7.5
	50	77.3±3.2	81±±5.6	4.1	7.0
TEN	5	115.3±4.8	117.9±5.3	4.1	4.5
	20	116.9±3.9	112.6±4.9	3.3	4.3
	50	117.3±2.7	118.0±8.2	2.3	7.0
AME	5	97.4±4.0	93.1±4.4	4.1	4.8
	20	91.0±5.0	92.8±5.1	5.5	5.8
	50	96.3±5.6	95.5±4.9	5.8	6.1
AOH	5	107.7±2.6	106.3±4.8	2.4	4.5
	20	105.9±2.5	105.5±2.5	2.3	2.4
	50	101.9±8.3	104.2±8.5	8.2	8.2
ALT	5	108.2±6.8	109.6±6.3	6.3	7.7
	20	107.7±3.3	106.0±3.5	3.1	3.3
	50	112.3±3.4	111.9±3.3	3.0	4.3

图9 婴幼儿辅食果泥中5种链格孢霉毒素的基质效应

Fig.9 Matrix effect evaluation for five Alternaria toxins determination in fruit puree

图10 婴幼儿辅食果泥中AME(10.9 μg·kg-1),AOH (17.2 μg·kg-1),TEN (13.1 μg·kg-1),TeA(31.0 μg·kg-1) 检出质谱图

Fig.10 UPLC-MS/MS chromatograms of AME(10.9 μg·kg-1), AOH(17.2 μg·kg-1), TEN(13.1 μg·kg-1), TeA (31.0 μg·kg-1) in positive fruit puree for infants

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QuEChERS-超高效液相色谱-串联质谱法同时测定婴幼儿辅食果泥中5种链格孢霉毒素

Simultaneous determination of five Alternaria toxins by QuEChERS-ultra-performance liquid chromatography-tandem mass spectrometry in fruit puree for infants

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