同位素稀释-液相色谱-串联质谱法在小麦粉细交链孢菌酮酸和腾毒素标准物质制备和定值中的应用

doi:10.3724/SP.J.1123.2019.12005

摘要/Abstract

摘要：

建立了小麦粉中细交链孢菌酮酸（TeA）和腾毒素（TEN）标准物质的研制和定值方法，为开展粮食中交链孢霉毒素基体标准物质的研制提供重要方法学借鉴。该标准物质样品为天然污染交链孢霉毒素的小麦籽粒，定值目标物为TeA和TEN，采用同位素稀释-液相色谱-串联质谱法（ID-LC-MS/MS）进行定值测量，多个实验室合作定值。所研制的标准物质具有常温避光保存、定值不确定度小等特点。该标准物质是目前国际上唯一一种天然污染TeA和TEN的小麦粉标准物质，可用于食品安全风险监测、产品质量检测等领域相关分析方法的评价和测量质量控制等。

关键词: 液相色谱-串联质谱, 标准物质, 细交链孢菌酮酸, 腾毒素, 交链孢霉毒素, 小麦粉

Abstract:

A method for the preparation and certification of the reference material of tenuazonic acid (TeA) and tentoxin (TEN) in wheat flour was developed. This method provided methodological references to develop of standard material for analyzing alternaria toxins in grains. The wheat flour reference materials were based on wheat grains which were naturally contaminated with alternaria toxins. The certified values for TeA and TEN were determined by isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) and cooperated certification of multiple laboratories. The wheat flour reference materials were stored at room temperature, protected from light and characterized by low uncertainty. The wheat flour reference materials are currently the only wheat flour reference materials that naturally contaminate TeA and TEN, and it can be used in the evaluating related analytical methods involved in food safety risk monitoring, product quality testing and quality control measurements.

Key words: liquid chromatography-tandem mass spectrometry (LC-MS/MS), reference material, tenuazonic acid (TeA), tentoxin (TEN), alternaria toxins, wheat flour

谢继安, 刘柏林, 赵紫微, 张磊, 杨大进, 赵云峰. 同位素稀释-液相色谱-串联质谱法在小麦粉细交链孢菌酮酸和腾毒素标准物质制备和定值中的应用[J]. 色谱, 2020, 38(7): 833-840.

XIE Ji'an, LIU Bolin, ZHAO Ziwei, ZHANG Lei, YANG Dajin, ZHAO Yunfeng. Application of isotope dilution-liquid chromatography-tandem mass spectrometry in the preparation and certification of reference material of tenuazonic acid and tentoxin in wheat flour[J]. Chinese Journal of Chromatography, 2020, 38(7): 833-840.

图/表 11

图1 TeA和TEN的结构式

Fig. 1 Structural formulas of tenuazonic acid (TeA) and tentoxin (TEN)

表1 TeA、13C2-TeA、TEN和D3-TEN的质谱参数

Table 1 MS parameters of the TeA, 13C2-TeA, TEN and D3-TEN

Compound	Parent ion (m/z)	Product ions (m/z)	Cone voltage/V	Collision energies/eV
* Quantitative ion.
TeA	196.0	139.1^*, 112.1	52	20, 24
¹³C₂-TeA	198.1	141.1	52	20
TEN	413.2	141.1^*, 271.1	50	22, 18
D₃-TEN	416.4	141.1	50	22

图2 小麦粉中TeA和TEN标准物质的生产制备工艺流程图

Fig. 2 Flow chart for preparation and certification of TeA and TEN in wheat flour

图3 提取时间对小麦粉中TeA和TEN含量的影响

Fig. 3 Effect of extraction time on the contents of TeA and TEN in wheat flour

图4 HLB柱规格对小麦粉中TeA(250 μg/kg)和TEN(50 μg/kg)回收率的影响

Fig. 4 Effect of HLB column specifications on the recoveries of TeA (250 μg/kg)and TEN (50 μg/kg) in wheat flour

图5 样品溶液中有机溶剂体积分数对HLB固相萃取柱净化能力的影响

Fig. 5 Effect of volume fractions of organic solvent in sample solution on the purification capacity of HLB SPE column

图6 MRM模式下TeA、13C2-TeA、TEN和D3-TEN的色谱图

Fig. 6 Chromatograms of TeA, 13C2-TeA, TEN and D3-TEN in MRM mode

图7 TeA、13C2-TeA、TEN和D3-TEN的质谱图

Fig. 7 Mass spectra of TeA, 13C2-TeA, TEN and D3-TEN

表2 采用基质标准曲线法和同位素内标稀释法时的检测结果

Table 2 Detected results when using matrix standard curve and isotope internal standard dilution methods

Compound	Blank/ (μg/kg)	Added/ (μg/kg)	Detected/(μg/kg)
Compound	Blank/ (μg/kg)	Added/ (μg/kg)	Matrix standard curve method	Isotope internal standard dilution method
TeA	＜0.5	10	9.68	10.04
	＜0.5	50	48.8	50.2
	＜0.5	200	197.5	200.6
TEN	＜0.1	2.0	1.93	2.01
	＜0.1	10	9.73	10.07
	＜0.1	40	38.9	40.2

表3 阴性小麦粉样品中TeA和TEN的加标回收率和相对标准偏差(n=6)

Table 3 Spiked recoveries and RSDs of the TeA and TEN in negative wheat flour samples (n=6)

Compound	Added/ (μg/kg)	Content/ (μg/kg)	Recovery/ %	RSD/ %
TeA	10	9.89	98.9	3.8
	50	51.72	103.4	2.4
	200	209.12	104.6	1.5
TEN	2.0	1.99	99.6	3.1
	10	10.08	100.8	2.2
	40	41.47	103.7	1.4

表4 小麦粉中TeA和TEN标准物质含量定值及不确定度

Table 4 Certified values and uncertainties of the TeA and TEN in wheat flour

Level	Compound	Relative uncertainties/%					Uncertainty (k=2)/(μg/kg)	Standard value/ (μg/kg)
Level	Compound	u_hb	u_s	u_char	u_cal	u	Uncertainty (k=2)/(μg/kg)	Standard value/ (μg/kg)
u_hb: homogeneity uncertainty; u_s: stability uncertainty; u_char: multi-laboratory characterizations uncertainty; u_cal: standard curve measurement uncertainty; u: total uncertainty; k: coverage factor.
Low	TeA	3.26	1.14	2.00	1.59	4.30	9	98
	TEN	2.73	1.03	2.21	0.79	3.75	2	19
High	TeA	3.40	1.14	1.90	0.78	4.13	17	198
	TEN	2.30	0.58	2.09	0.40	3.20	3	39

参考文献

1	EFSA Panel on Contaminants in the Food Chain . EFSA J, 2011, 9 (10): 2407
2	Tiemann U , Tomek W , Schneider F , et al. Toxicol Lett, 2009, 186 (2): 139
3	Davide A , Mari E , Jose A G R . EFSA J, 2016, 14 (12): 4654
4	GB 2761-2017
5	Zhao Y , Li N , Xie Y Y , et al. China Standardization, 2019 (19): 185
5	赵艳, 李娜, 谢艳艳, et al. 中国标准化, 2019 (19): 185
6	GB 1355-86
7	JJG 1006-94
8	JJF 1343-2012
9	Zhao H , Hao S J , Liu Y D , et al. Chemical Analysis and Meterage, 2018, 27 (S1): 101
9	赵辉, 郝淑杰, 刘原栋, et al. 化学分析计量, 2018, 27 (S1): 101
10	Zhao M T . Journal of Chinese Mass Spectrometry Society, 2004, 25 (Suppl): 167
10	赵墨田. 质谱学报, 2004, 25 (Suppl): 167
11	Xu R F , Fang X , Xu B , et al. Journal of Chinese Mass Spectrometry Society, 2000, 21 (3/4): 167
11	徐锐锋, 方向, 徐蓓, et al. 质谱学报, 2000, 21 (3/4): 167
12	Xu J J , Huang B F , Cai Z X , et al. Chinese Journal of Food Hygiene, 2016, 28 (3): 291
12	许娇娇, 黄百芬, 蔡增轩, et al. 中国食品卫生杂志, 2016, 28 (3): 291
13	Wang X Y , Zhang L , Li J G , et al. Chinese Journal of Food Hygiene, 2014, 26 (3): 205
13	王向勇, 张磊, 李敬光, et al. 中国食品卫生杂志, 2014, 26 (3): 205
14	Lu X B , Chen J P , Wang S Q , et al. Chinese Journal of Chromatography, 2012, 30 (9): 915
14	卢宪波, 陈吉平, 王淑秋, et al. 色谱, 2012, 30 (9): 915
15	Raimund M , Waterbeemd H . J Comput Aided Mol Des, 2001, 15 (4): 337
16	赵凯.[博士学位论文].北京: 中国疾病预防控制中心, 2015
17	Sun D L , Qiu N N , Zhou S , et al. Toxins (Basel), 2019, 11 (3): 166
18	Guo W B , Fan K , Nie D X , et al. Toxins (Basel), 2019, 11 (2): 87
19	Xing L J , Zou L J , Luo R F , et al. Food Chem, 2019, 311: 125975
20	Zhang Y T , Li H , Zhang J , et al. Environ Sci Pollut Res Int, 2019, 26 (22): 22485

[1]	赵芮, 黄晴雯, 余智颖, 韩铮, 范楷, 赵志辉, 聂冬霞. QuEChERS-超高效液相色谱-串联质谱法同时测定水果中36种真菌毒素[J]. 色谱, 2023, 41(9): 760-770.
[2]	岳超, 赵超群, 毛思浩, 王展华, 施贝, 徐欣丰, 梁晶晶. 通过式固相萃取净化-超高效液相色谱-串联质谱法测定液体乳中10种氨基甲酸酯类农药残留[J]. 色谱, 2023, 41(9): 807-813.
[3]	孟二琼, 念琪循, 李峰, 张秋萍, 许茜, 王春民. 磺酸化磁性氮化碳固相萃取-超高液相色谱-串联质谱筛检淡水鱼中孔雀石绿和隐色孔雀石绿[J]. 色谱, 2023, 41(8): 673-682.
[4]	王秋旭, 冯启言, 朱雪强. 加速溶剂萃取-固相萃取净化结合超高效液相色谱-串联质谱法测定沉积物中双酚类化合物[J]. 色谱, 2023, 41(7): 582-590.
[5]	夏宝林, 汪仕韬, 殷晶晶, 张维益, 杨娜, 刘强, 吴海晶. 自动上样固相萃取-超高效液相色谱-串联质谱法同时测定水中9类43种抗菌药物残留[J]. 色谱, 2023, 41(7): 591-601.
[6]	陈东洋, 张昊, 张磊, 王一红, 王小丹, 冯家力, 梁静, 钟旋. 固相萃取-超高效液相色谱-串联质谱法测定发酵食品中的曲酸[J]. 色谱, 2023, 41(7): 632-639.
[7]	童学智, 陈东洋, 冯家力, 范翔, 张昊, 杨胜园. QuEChERS-超高效液相色谱-串联质谱法快速测定水产品中5种卤代苯醌[J]. 色谱, 2023, 41(6): 490-496.
[8]	曾永福, 陈美芳, 邵雨, 闫永欢, 张海超, 王敬, 艾连峰, 康维钧. 植物中27种典型药品及个人护理品多残留检测方法的建立及其在芽苗菜中迁移规律的分析[J]. 色谱, 2023, 41(5): 386-396.
[9]	吴少明, 欧阳立群, 孟鹏, 何孟杭, 林钦, 陈言凯, 刘文菁, 苏晓明, 戴明. 固相萃取净化-超高效液相色谱-串联质谱法测定畜肉中18种卡因类麻醉剂[J]. 色谱, 2023, 41(5): 434-442.
[10]	孙佳林, 牛宇敏, 高群, 张晶, 邵兵. 超高效液相色谱-串联质谱法测定灰尘中26种双酚类化合物[J]. 色谱, 2023, 41(5): 417-425.
[11]	邵曼, 余晓琴, 黄丽娟, 姚欢, 李澍才. 增强型脂质去除净化剂结合超高效液相色谱-串联质谱法测定食品中8种大麻素类化合物[J]. 色谱, 2023, 41(5): 426-433.
[12]	李振环, 胡小键, 陆一夫, 谢琳娜, 朱英. 基于高通量全自动固相萃取的超高效液相色谱-串联质谱法测定人尿中16种抗生素和4种β-受体激动剂[J]. 色谱, 2023, 41(5): 397-408.
[13]	宋新力, 王宁, 何飞燕, 程灿玲, 王飞, 王京龙, 张立华. 碳纳米管复合材料结合分散固相萃取-高效液相色谱-串联质谱法检测环境水样中痕量全氟化合物[J]. 色谱, 2023, 41(5): 409-416.
[14]	王娇, 吴桐, 王新全, 刘真真, 徐浩, 汪志威, 狄珊珊, 赵慧宇, 齐沛沛. QuEChERS-液相色谱-串联质谱法测定中草药中22种三唑类农药残留[J]. 色谱, 2023, 41(4): 330-338.
[15]	朱卫红, 王超, 张霖琳, 袁懋. 超高效液相色谱-串联质谱法直接测定水中5种黄原酸[J]. 色谱, 2023, 41(4): 339-347.