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Abstract ［Objectives］ This study was conducted to establish a liquid chromatography-tandem mass spectrometry method for the determination of coumoxystrobin residue in milk.
［Methods］A sample was ultrasonically extracted with acetonitrile, purified by a solid phase extraction column, concentrated by nitrogen blowing, and tested on a machine.
［Results］ In the range of 0.01-10.00 mg/kg, the linear relationship of coumoxystrobin was good. The linear equation was y=5 045.96x+935.332, r2=0.999. The detection limit was 0.002 mg/kg, and the quantitation limit was 0.006 mg/kg. The method has good stability, high precision, and the detection limit can meet the limit requirement of coumoxystrobin in GB 2763-2019.
［Conclusions］This study can provide technical support for the supervision and monitoring of coumoxystrobin.
Key words Milk; Coumoxystrobin; Residue; Determination
Received: June 10, 2021 Accepted: August 11, 2021
Supported by Special Fund for Innovation Capacity Building of Scientific Research Institutions (QKHFQ［2019］4001); Science and Technology Program of Guizhou Province (QKHZC［2019］2451-8-3).
Meijuan ZHENG (1986-), female, P. R. China, intermediate engineer, devoted to research about chemical analysis.
*Corresponding author. E-mail: 287476045@qq.com.
Coumoxystrobin is a high-end microbicide researched by Shenyang Research Institute of Chemical Industry, developed by Jilin Bada Agrochemical Co., Ltd. It belongs to methoxy acrylate, is a protective microbicide, and has a certain therapeutic effect simultaneously［1-4］. It has the characteristics of broad-spectrum, low toxicity, high efficiency and safety, and has immunizing, disease-preventing, treating, and production- and income-increasing effects. It has special effects on apple tree rot, and it is the most authoritative agent for the prevention and control of rot in China. It has a wide microbicidal spectrum and is effective against downy mildew, late blight, scab, anthracnose and leaf mold of melons, vegetables, fruit trees, and also against ring spot, cotton wilt, rice blast, and sheath blight, wheat common rot and corn southern leaf blight.
At present, there are few related studies on the analysis and testing of coumoxystrobin［5-8］, and there is no detection method for the content of coumoxystrobin in milk. However, milk is one of the products of farm production lines, and it is necessary to detect the residual amount of various chemical agents in the milk flowing to the market. In this study, a liquid chromatography-tandem mass spectrometry method was established for the determination of coumoxystrobin residue in milk, providing technical support for the detection of coumoxystrobin. Materials and Methods
Reagents and equipment
High performance liquid chromatograph-tandem mass spectrometer: Waters XEVO TQ-S micro (Waters Corporation, USA); ultra-high-speed refrigerated centrifuge (Lixin Instruments (Shanghai) Co., Ltd.); nitrogen blowing apparatus (ANPEL Laboratory Technologies (Shanghai) Inc.); vortex oscillator (Shanghai Maiqi Environmental Protection Technology Co., Ltd.); electronic balance (0.000 1 g) (Beijing Hongda Hengye Technology Co., Ltd.); solid phase extraction instrument (Shanghai Yiyao Instrument Technology Development Co., Ltd.); solid phase extraction column (Dikma Technology Co., Ltd.); 0.22 μm organic filter membrane (Dikma Technology Co., Ltd.).
Acetonitrile (chromatographically pure); methanol (chromatographically pure); formic acid (99%); sodium chloride (analytically pure); coumoxystrobin standard products, all from ANPEL Laboratory Technologies (Shanghai) Inc.
Solution preparation
Standard stock solution: First, 0.01 g of coumoxystrobin standard product (CAS: 850881-70-8, purity≥99%) was accurately weighed into a 10 ml volumetric flask, dissolved with acetonitrile, and then diluted to constant volume. The obtained solution was finally prepared into a standard stock solution with a concentration of 1 000 μg/ml, which was stored at -18 ℃ and was valid within 6 months.
Standard working solution: From the standard stock solution, 0.1 ml was accurately pipetted into a 10 ml volumetric flask, diluted with acetonitrile, and then prepared into a standard intermediate solution with a concentration of 10 μg/ml, which was stored at -18 ℃ and was valid within 1 month.
Sample treatment
A 2 g of sample was accurately weighed into a centrifuge tube, and added with 10 ml of acetonitrile and 4 g of NaCl. The mixture was vortex-mixed for 1 min, ultrasonically extracted for 10 min, and centrifuged at 6 000 rpm for 5 min. Then, 5 ml of the supernatant was taken for purification. Next, a solid phase extraction column was activated with acetonitrile, and the supernatant was discarded. The to-be-purified liquid was loaded to the small column, and the effluent was collected and added with 10 ml of acetonitrile. The effluents were combined, and blown with nitrogen in a water bath at 50 ℃ until nearly dry. The obtained residue was dissolved with 1 ml of acetonitrile, and the obtained solution was filtered with a 0.22 μm filter membrane, and then determined by a liquid chromatograph-mass spectrometer. Instrument conditions
LC conditions
Mobile phase: A: 0.2% formic acid water, B: acetonitrile, for gradient elution (Table 1).
Chromatographic column: C18 (2.6 μm, 21 mm×100 mm); flow rate: 0.3 ml/min; injection volume: 1 μl; column temperature: 35 ℃.
Mass spectrometry conditions
Ionization mode: ESI; positive ion mode; curtain gas (CUR): 30.0 Psi; collision gas (CAD): 9.0; ionization voltage (IS): positive ion 4 500 V; spray gas (GSI): 55 Psi; desolvation temperature (TEM): 550℃; auxiliary heating gas (GSI): 55 Psi (Table 2).
Results and Analysis
Determination of instrument conditions
The mobile phase combinations of acetonitrile-water, methanol-water, formic acid water-acetonitrile were selected for comparison, mainly to investigate the resolution of target substance and the response value at the same concentration. When acetonitrile: 0.2% formic acid solution was used as the mobile phase, the resolution effect of each target was the best, and it finally was selected as the mobile phase of this method. The chromatogram of coumoxystrobin is shown in the figure below.
Linear range, standard curve, detection limit and quantitation limit
Standard working solutions of 1, 2, 5, 8, and 10 ng/ml were prepared. A standard curve was drawn with the area of quantitative ion peak as the ordinate and the concentration of standard substance as the abscissa. The detection limit was calculated according to the signal-to-noise ratio S/N=3, and the quantitation limit was calculated according to the signal-to-noise ratio S/N=10. In the range of 0.01-10.00 mg/kg, the linear relationship of coumoxystrobin was good. The linear equation was y=5 045.96*x+935.332, r2=0.999. The detection limit was 0.002 mg/kg, and the quantitation limit was 0.006 mg/kg.
Method accuracy test
In the milk matrix, the target substance was added at the levels of 0.015, 0.030, and 0.100 mg/kg, and the recovery was shown in Table 1. From the results in Table 1, it can be seen that the recovery of coumoxystrobin in milk was in the range of 80.6%-90.7%, which met the testing requirement.
Conclusions
In this study, a liquid chromatography-tandem mass spectrometry method was established for the determination of coumoxystrobin residue in milk. The sample was ultrasonically extracted with acetonitrile, purified by a solid phase extraction column, concentrated by nitrogen blowing, and tested on the machine. In the range of 0.01-10.00 mg/kg, the linear relationship of coumoxystrobin was good. The linear equation was y=5 045.96x+935.332, r2=0.999. The detection limit was 0.002 mg/kg, and the quantitation limit was 0.006 mg/kg. The method has good stability, high precision, and the detection limit can meet the limit requirement of coumoxystrobin in GB 2763-2019, which can provide technical support for the supervision and monitoring of coumoxystrobin. References
［1］ ZHANG L. Determination of coumoxystrobin TC by HPLC［J］. Modern Agrochemicals, 2017, 016(004): 30-31. (in Chinese)
［2］ ZHONG CW, LI ZF, ZHANG RM, et al. Leaching and adsorption characteristics of coumoxystrobin in soil ［J］. Agrochemicals, 2018, 057(009): 650-653. (in Chinese)
［3］ GUAN AY, YANG JC, LIU CL. Fungicide coumoxystrobin［J］. World Pesticides, 2014, 000(004): 62-63. (in Chinese)
［4］ WANG LF,, LI CM, XU JB, et al. Simultaneous determination of 11 strobilurin fungicides in different vegetation types of soil by QuEChERS ultra performance liquid chromatography-tandem mass spectrometry［J］. Chinese Journal of Chromatography, 2018, 36(8): 36-43. (in Chinese)
［5］ WU JL, YUAN Y, LIU Z, et al. Analytical method of coumoxystrobin and tebuconazol 40% SC by HPLC［J］. Pesticide Science and Administration, 2019. (in Chinese)
［6］ SI NG, LIU JL, CHEN L. Application technology of new fungicide coumoxystrobin［J］. Modern Agriculture, 2010, 000(010): 46-47. (in Chinese)
［7］ JIAO B, XU J, LIU XG, et al. Simultaneous determination of pyrisoxazole and five other pesticides in typical types of Chinese soil using ultra-performance liquid chromatography and tandem mass spectrometry［J］. Agro-Environment & Development, 2020, 037(003): 424-431. (in Chinese)
［8］ LUO HL, ZHOU JR, ZHOU S, et al. Residue analysis of coumoxystrobin in orchard soil［J］. Agrochemicals, 2020(2): 113-116. (in Chinese)
Editor: Yingzhi GUANG Proofreader: Xinxiu ZHU
［Methods］A sample was ultrasonically extracted with acetonitrile, purified by a solid phase extraction column, concentrated by nitrogen blowing, and tested on a machine.
［Results］ In the range of 0.01-10.00 mg/kg, the linear relationship of coumoxystrobin was good. The linear equation was y=5 045.96x+935.332, r2=0.999. The detection limit was 0.002 mg/kg, and the quantitation limit was 0.006 mg/kg. The method has good stability, high precision, and the detection limit can meet the limit requirement of coumoxystrobin in GB 2763-2019.
［Conclusions］This study can provide technical support for the supervision and monitoring of coumoxystrobin.
Key words Milk; Coumoxystrobin; Residue; Determination
Received: June 10, 2021 Accepted: August 11, 2021
Supported by Special Fund for Innovation Capacity Building of Scientific Research Institutions (QKHFQ［2019］4001); Science and Technology Program of Guizhou Province (QKHZC［2019］2451-8-3).
Meijuan ZHENG (1986-), female, P. R. China, intermediate engineer, devoted to research about chemical analysis.
*Corresponding author. E-mail: 287476045@qq.com.
Coumoxystrobin is a high-end microbicide researched by Shenyang Research Institute of Chemical Industry, developed by Jilin Bada Agrochemical Co., Ltd. It belongs to methoxy acrylate, is a protective microbicide, and has a certain therapeutic effect simultaneously［1-4］. It has the characteristics of broad-spectrum, low toxicity, high efficiency and safety, and has immunizing, disease-preventing, treating, and production- and income-increasing effects. It has special effects on apple tree rot, and it is the most authoritative agent for the prevention and control of rot in China. It has a wide microbicidal spectrum and is effective against downy mildew, late blight, scab, anthracnose and leaf mold of melons, vegetables, fruit trees, and also against ring spot, cotton wilt, rice blast, and sheath blight, wheat common rot and corn southern leaf blight.
At present, there are few related studies on the analysis and testing of coumoxystrobin［5-8］, and there is no detection method for the content of coumoxystrobin in milk. However, milk is one of the products of farm production lines, and it is necessary to detect the residual amount of various chemical agents in the milk flowing to the market. In this study, a liquid chromatography-tandem mass spectrometry method was established for the determination of coumoxystrobin residue in milk, providing technical support for the detection of coumoxystrobin. Materials and Methods
Reagents and equipment
High performance liquid chromatograph-tandem mass spectrometer: Waters XEVO TQ-S micro (Waters Corporation, USA); ultra-high-speed refrigerated centrifuge (Lixin Instruments (Shanghai) Co., Ltd.); nitrogen blowing apparatus (ANPEL Laboratory Technologies (Shanghai) Inc.); vortex oscillator (Shanghai Maiqi Environmental Protection Technology Co., Ltd.); electronic balance (0.000 1 g) (Beijing Hongda Hengye Technology Co., Ltd.); solid phase extraction instrument (Shanghai Yiyao Instrument Technology Development Co., Ltd.); solid phase extraction column (Dikma Technology Co., Ltd.); 0.22 μm organic filter membrane (Dikma Technology Co., Ltd.).
Acetonitrile (chromatographically pure); methanol (chromatographically pure); formic acid (99%); sodium chloride (analytically pure); coumoxystrobin standard products, all from ANPEL Laboratory Technologies (Shanghai) Inc.
Solution preparation
Standard stock solution: First, 0.01 g of coumoxystrobin standard product (CAS: 850881-70-8, purity≥99%) was accurately weighed into a 10 ml volumetric flask, dissolved with acetonitrile, and then diluted to constant volume. The obtained solution was finally prepared into a standard stock solution with a concentration of 1 000 μg/ml, which was stored at -18 ℃ and was valid within 6 months.
Standard working solution: From the standard stock solution, 0.1 ml was accurately pipetted into a 10 ml volumetric flask, diluted with acetonitrile, and then prepared into a standard intermediate solution with a concentration of 10 μg/ml, which was stored at -18 ℃ and was valid within 1 month.
Sample treatment
A 2 g of sample was accurately weighed into a centrifuge tube, and added with 10 ml of acetonitrile and 4 g of NaCl. The mixture was vortex-mixed for 1 min, ultrasonically extracted for 10 min, and centrifuged at 6 000 rpm for 5 min. Then, 5 ml of the supernatant was taken for purification. Next, a solid phase extraction column was activated with acetonitrile, and the supernatant was discarded. The to-be-purified liquid was loaded to the small column, and the effluent was collected and added with 10 ml of acetonitrile. The effluents were combined, and blown with nitrogen in a water bath at 50 ℃ until nearly dry. The obtained residue was dissolved with 1 ml of acetonitrile, and the obtained solution was filtered with a 0.22 μm filter membrane, and then determined by a liquid chromatograph-mass spectrometer. Instrument conditions
LC conditions
Mobile phase: A: 0.2% formic acid water, B: acetonitrile, for gradient elution (Table 1).
Chromatographic column: C18 (2.6 μm, 21 mm×100 mm); flow rate: 0.3 ml/min; injection volume: 1 μl; column temperature: 35 ℃.
Mass spectrometry conditions
Ionization mode: ESI; positive ion mode; curtain gas (CUR): 30.0 Psi; collision gas (CAD): 9.0; ionization voltage (IS): positive ion 4 500 V; spray gas (GSI): 55 Psi; desolvation temperature (TEM): 550℃; auxiliary heating gas (GSI): 55 Psi (Table 2).
Results and Analysis
Determination of instrument conditions
The mobile phase combinations of acetonitrile-water, methanol-water, formic acid water-acetonitrile were selected for comparison, mainly to investigate the resolution of target substance and the response value at the same concentration. When acetonitrile: 0.2% formic acid solution was used as the mobile phase, the resolution effect of each target was the best, and it finally was selected as the mobile phase of this method. The chromatogram of coumoxystrobin is shown in the figure below.
Linear range, standard curve, detection limit and quantitation limit
Standard working solutions of 1, 2, 5, 8, and 10 ng/ml were prepared. A standard curve was drawn with the area of quantitative ion peak as the ordinate and the concentration of standard substance as the abscissa. The detection limit was calculated according to the signal-to-noise ratio S/N=3, and the quantitation limit was calculated according to the signal-to-noise ratio S/N=10. In the range of 0.01-10.00 mg/kg, the linear relationship of coumoxystrobin was good. The linear equation was y=5 045.96*x+935.332, r2=0.999. The detection limit was 0.002 mg/kg, and the quantitation limit was 0.006 mg/kg.
Method accuracy test
In the milk matrix, the target substance was added at the levels of 0.015, 0.030, and 0.100 mg/kg, and the recovery was shown in Table 1. From the results in Table 1, it can be seen that the recovery of coumoxystrobin in milk was in the range of 80.6%-90.7%, which met the testing requirement.
Conclusions
In this study, a liquid chromatography-tandem mass spectrometry method was established for the determination of coumoxystrobin residue in milk. The sample was ultrasonically extracted with acetonitrile, purified by a solid phase extraction column, concentrated by nitrogen blowing, and tested on the machine. In the range of 0.01-10.00 mg/kg, the linear relationship of coumoxystrobin was good. The linear equation was y=5 045.96x+935.332, r2=0.999. The detection limit was 0.002 mg/kg, and the quantitation limit was 0.006 mg/kg. The method has good stability, high precision, and the detection limit can meet the limit requirement of coumoxystrobin in GB 2763-2019, which can provide technical support for the supervision and monitoring of coumoxystrobin. References
［1］ ZHANG L. Determination of coumoxystrobin TC by HPLC［J］. Modern Agrochemicals, 2017, 016(004): 30-31. (in Chinese)
［2］ ZHONG CW, LI ZF, ZHANG RM, et al. Leaching and adsorption characteristics of coumoxystrobin in soil ［J］. Agrochemicals, 2018, 057(009): 650-653. (in Chinese)
［3］ GUAN AY, YANG JC, LIU CL. Fungicide coumoxystrobin［J］. World Pesticides, 2014, 000(004): 62-63. (in Chinese)
［4］ WANG LF,, LI CM, XU JB, et al. Simultaneous determination of 11 strobilurin fungicides in different vegetation types of soil by QuEChERS ultra performance liquid chromatography-tandem mass spectrometry［J］. Chinese Journal of Chromatography, 2018, 36(8): 36-43. (in Chinese)
［5］ WU JL, YUAN Y, LIU Z, et al. Analytical method of coumoxystrobin and tebuconazol 40% SC by HPLC［J］. Pesticide Science and Administration, 2019. (in Chinese)
［6］ SI NG, LIU JL, CHEN L. Application technology of new fungicide coumoxystrobin［J］. Modern Agriculture, 2010, 000(010): 46-47. (in Chinese)
［7］ JIAO B, XU J, LIU XG, et al. Simultaneous determination of pyrisoxazole and five other pesticides in typical types of Chinese soil using ultra-performance liquid chromatography and tandem mass spectrometry［J］. Agro-Environment & Development, 2020, 037(003): 424-431. (in Chinese)
［8］ LUO HL, ZHOU JR, ZHOU S, et al. Residue analysis of coumoxystrobin in orchard soil［J］. Agrochemicals, 2020(2): 113-116. (in Chinese)
Editor: Yingzhi GUANG Proofreader: Xinxiu ZHU