Abstract: Simultaneous separation and detection of methyl parathion, ethyl parathion and formazan in vegetables by high performance liquid chromatography, screening and optimization of the chromatographic conditions for sample extraction, purification and detection. The shim-pack VP-ODS was used as the separation column; the mobile phase was V methanol: V water = 85: 15, and the ultraviolet detection wavelength was 250nm. The three kinds of organic phosphorus measured by this method have good linearity in the range of 0.02-2.0μL, the detection limit is 1.0-5.0μL, and the recovery rate is between 97.44% and 101.22%. This method is suitable for the analysis of pesticide residues in various vegetables.
Keywords: pesticide residues; high performance liquid chromatography; vegetables; organophosphorus organophosphorus pesticides are currently one of the most used pesticides in China, mainly used in food, vegetables and fruits. Although organophosphorus pesticides are non-persistent pesticides, their extensive use will inevitably have a negative impact on the environment and pose a serious threat to people and accumulation. Long-term consumption of contaminated food and vegetables is an important cause of diseases such as cancer, arteriosclerosis, cardiovascular disease, fetal malformations, stillbirths, early days, and premature aging; most people do not show symptoms immediately after eating harmful vegetables. But the poison is enriched in the human body, and it will cause serious consequences after a long time. Most of the pesticide poisoning accidents in China in recent years have focused on highly toxic organophosphorus pesticides, with parathion the most, and some regions accounted for about 80%. Therefore, it is of great significance to establish an effective method for detecting residues of organophosphorus pesticides in agricultural and sideline products. The determination of organophosphorus pesticides often uses gas chromatography. However, most organophosphorus pesticides have strong polarity and thermal instability, which makes gas chromatography analysis difficult. In this study, high performance liquid chromatography was used to simultaneously determine methyl parathion, ethyl parathion and formazan in vegetables.
1 Experimental part
1.1 Instruments and reagent instruments. STI5000 high-performance liquid chromatograph (Shanghai Hegong Scientific Instruments), including: UV5000 ultraviolet-visible detector, P5000 high-pressure infusion pump and N2000 chromatography workstation (SP1 version) (Zhejiang University Intelligent Information Engineering Institute); BF2000 nitrogen blow dryer ( Beijing Bafang Century Technology Co., Ltd.); FS-1 electric homogenizer (Jintan Fuhua Instrument Co., Ltd.). Reagents. Methyl parathion standard product (99.0%, National Pesticide Quality Inspection Center (Shenyang)); ethyl parathion standard product (99.0%, Tianjin Pesticide Quality Inspection Center); methamidophos standard product (98.0%, Tianjin Pesticide) Quality Inspection Center); methanol (chromatographically pure, Beijing Chemical Plant); acetonitrile (chromatographically pure, Tianjin Bodi Chemical Co., Ltd.); experimental water is redistilled water.
1.2 Chromatographic conditions The liquid chromatography column is Shim-Pack: VP-ODS (150L × 4.6); the mobile phase is V methanol: V water = 85: 15; the flow rate is 1.0mL / min. The injection volume was 20 μL, and the area composed of the baseline was called the peak area. A = × σ × h = 2.507σh = 1.064 Wh / 2h> quantification by peak area external standard method.
1.3 Preparation of standard solution Accurately weigh a certain amount of pesticide standard, make a certain concentration of stock solution with methanol, and then dilute the stock solution step by step to obtain mass concentrations of 0.10, 0.50, 1.0, 1.5, 2.0 mg / L standard mixture.
1.4 Sample processing method Take 1000g of cabbage, take the edible part, wipe off the attachment on the surface of the sample with a clean gauze, use the diagonal division method, take the diagonal part, and chop it. Accurately weigh three 25g samples, add 50.0mL of acetonitrile respectively, homogenize in a homogenizer for 2min and filter with filter paper. The filtrate is collected in a 100mL stopper cylinder containing 5g of sodium chloride and covered with a stopper. Vibrate vigorously for 1min, and rest to 10min to separate the acetonitrile and water phase. Filter the organic layer containing acetonitrile through a funnel containing 3g of anhydrous sodium sulfate in a 100mL volumetric flask, dilute to the mark with acetonitrile, draw 10mL of the solution from it, and then blow to near dryness with a nitrogen blow dryer to dissolve with methanol Make up to 5mL, filter through 0.45μm filter and inject.
2 Results and discussion
2.1 Selection of measurement wavelengths The standard stock solutions of the three pesticides were sequentially scanned with a UV-1100 ultraviolet / visible spectrophotometer, and the maximum absorption wavelengths of the three pesticides were measured as methyl parathion 265nm, B The base parathion is 259nm, and formazan is 238nm. Considering the maximum absorption value and sensitivity of the three components comprehensively, the ultraviolet detection wavelength is finally selected as 250nm.
2.2 The effect of mobile phase composition on resolution The experiment investigated the effect of different ratios of methanol-water on the separation of methyl parathion, ethyl parathion, and formazan. The experimental results show that as the methanol content increases, the resolution gradually decreases. When methanol: water = 85: 15, the components can still be well separated, and the analysis time is shorter.
2.3 Working curve and detection limit Take the standard solution in section "1.3" and analyze under optimized chromatographic conditions to obtain a standard chromatogram. The same sample was measured 5 times in parallel, the average value was taken, and a working curve was drawn. The peak area A was used to calculate the concentration c (mg / L). The results showed a good linear relationship, regression equation, correlation coefficient and detection limit.
2.4 Accuracy experiment Take a mixed standard with a mass concentration of 1.5mg / L. Under the above chromatographic conditions, conduct continuous measurement for 7 times. After calculation, the determination of methyl parathion, ethyl parathion, and formazan The precision of RSD is 4.6%, 3.2%, 2.9%.
2.5 Determination of recovery rate Add different concentrations of organophosphorus pesticide standards to cabbage samples of known content, prepare the sample solution for measurement according to the method in Section 1.4, deduct the original pesticide content in the sample, and find the addition The standard recovery rate shows that the methyl parathion recovery rate is 95.44% -98.33%; the ethyl parathion recovery rate is 93.33% -101.67%; and the methamidophos recovery rate is 94.00% -97.78%.
2.6 Sample analysis The three pesticides mentioned above were added to cabbage as simulated vegetable samples, and the samples were processed according to the method in Section 1.4 after the chromatographic conditions were determined. Obtained sample chromatogram as shown. The residual amounts of methyl parathion, ethyl parathion, and formazan were 1.75, 2.13, and 1.83 mg / kg, respectively.
references:
[1] Zhao Xiuli, Wang Ruyuan. Delayed neurotoxicity after organophosphorus pesticide poisoning [J]. Journal of Qingdao Medical College, 1995, 16 (4): 37-38.
[2] Henry K Rotich, Zhang Zhuoyong, Zhao Jinying, etc. SPE-HPLC analysis of methyl parathion, parathion and phoxim pesticide residues in environmental water [J]. Analysis and Test Report, 2002, 21 (5): 50-52.
Keywords: pesticide residues; high performance liquid chromatography; vegetables; organophosphorus organophosphorus pesticides are currently one of the most used pesticides in China, mainly used in food, vegetables and fruits. Although organophosphorus pesticides are non-persistent pesticides, their extensive use will inevitably have a negative impact on the environment and pose a serious threat to people and accumulation. Long-term consumption of contaminated food and vegetables is an important cause of diseases such as cancer, arteriosclerosis, cardiovascular disease, fetal malformations, stillbirths, early days, and premature aging; most people do not show symptoms immediately after eating harmful vegetables. But the poison is enriched in the human body, and it will cause serious consequences after a long time. Most of the pesticide poisoning accidents in China in recent years have focused on highly toxic organophosphorus pesticides, with parathion the most, and some regions accounted for about 80%. Therefore, it is of great significance to establish an effective method for detecting residues of organophosphorus pesticides in agricultural and sideline products. The determination of organophosphorus pesticides often uses gas chromatography. However, most organophosphorus pesticides have strong polarity and thermal instability, which makes gas chromatography analysis difficult. In this study, high performance liquid chromatography was used to simultaneously determine methyl parathion, ethyl parathion and formazan in vegetables.
1 Experimental part
1.1 Instruments and reagent instruments. STI5000 high-performance liquid chromatograph (Shanghai Hegong Scientific Instruments), including: UV5000 ultraviolet-visible detector, P5000 high-pressure infusion pump and N2000 chromatography workstation (SP1 version) (Zhejiang University Intelligent Information Engineering Institute); BF2000 nitrogen blow dryer ( Beijing Bafang Century Technology Co., Ltd.); FS-1 electric homogenizer (Jintan Fuhua Instrument Co., Ltd.). Reagents. Methyl parathion standard product (99.0%, National Pesticide Quality Inspection Center (Shenyang)); ethyl parathion standard product (99.0%, Tianjin Pesticide Quality Inspection Center); methamidophos standard product (98.0%, Tianjin Pesticide) Quality Inspection Center); methanol (chromatographically pure, Beijing Chemical Plant); acetonitrile (chromatographically pure, Tianjin Bodi Chemical Co., Ltd.); experimental water is redistilled water.
1.2 Chromatographic conditions The liquid chromatography column is Shim-Pack: VP-ODS (150L × 4.6); the mobile phase is V methanol: V water = 85: 15; the flow rate is 1.0mL / min. The injection volume was 20 μL, and the area composed of the baseline was called the peak area. A = × σ × h = 2.507σh = 1.064 Wh / 2h> quantification by peak area external standard method.
1.3 Preparation of standard solution Accurately weigh a certain amount of pesticide standard, make a certain concentration of stock solution with methanol, and then dilute the stock solution step by step to obtain mass concentrations of 0.10, 0.50, 1.0, 1.5, 2.0 mg / L standard mixture.
1.4 Sample processing method Take 1000g of cabbage, take the edible part, wipe off the attachment on the surface of the sample with a clean gauze, use the diagonal division method, take the diagonal part, and chop it. Accurately weigh three 25g samples, add 50.0mL of acetonitrile respectively, homogenize in a homogenizer for 2min and filter with filter paper. The filtrate is collected in a 100mL stopper cylinder containing 5g of sodium chloride and covered with a stopper. Vibrate vigorously for 1min, and rest to 10min to separate the acetonitrile and water phase. Filter the organic layer containing acetonitrile through a funnel containing 3g of anhydrous sodium sulfate in a 100mL volumetric flask, dilute to the mark with acetonitrile, draw 10mL of the solution from it, and then blow to near dryness with a nitrogen blow dryer to dissolve with methanol Make up to 5mL, filter through 0.45μm filter and inject.
2 Results and discussion
2.1 Selection of measurement wavelengths The standard stock solutions of the three pesticides were sequentially scanned with a UV-1100 ultraviolet / visible spectrophotometer, and the maximum absorption wavelengths of the three pesticides were measured as methyl parathion 265nm, B The base parathion is 259nm, and formazan is 238nm. Considering the maximum absorption value and sensitivity of the three components comprehensively, the ultraviolet detection wavelength is finally selected as 250nm.
2.2 The effect of mobile phase composition on resolution The experiment investigated the effect of different ratios of methanol-water on the separation of methyl parathion, ethyl parathion, and formazan. The experimental results show that as the methanol content increases, the resolution gradually decreases. When methanol: water = 85: 15, the components can still be well separated, and the analysis time is shorter.
2.3 Working curve and detection limit Take the standard solution in section "1.3" and analyze under optimized chromatographic conditions to obtain a standard chromatogram. The same sample was measured 5 times in parallel, the average value was taken, and a working curve was drawn. The peak area A was used to calculate the concentration c (mg / L). The results showed a good linear relationship, regression equation, correlation coefficient and detection limit.
2.4 Accuracy experiment Take a mixed standard with a mass concentration of 1.5mg / L. Under the above chromatographic conditions, conduct continuous measurement for 7 times. After calculation, the determination of methyl parathion, ethyl parathion, and formazan The precision of RSD is 4.6%, 3.2%, 2.9%.
2.5 Determination of recovery rate Add different concentrations of organophosphorus pesticide standards to cabbage samples of known content, prepare the sample solution for measurement according to the method in Section 1.4, deduct the original pesticide content in the sample, and find the addition The standard recovery rate shows that the methyl parathion recovery rate is 95.44% -98.33%; the ethyl parathion recovery rate is 93.33% -101.67%; and the methamidophos recovery rate is 94.00% -97.78%.
2.6 Sample analysis The three pesticides mentioned above were added to cabbage as simulated vegetable samples, and the samples were processed according to the method in Section 1.4 after the chromatographic conditions were determined. Obtained sample chromatogram as shown. The residual amounts of methyl parathion, ethyl parathion, and formazan were 1.75, 2.13, and 1.83 mg / kg, respectively.
references:
[1] Zhao Xiuli, Wang Ruyuan. Delayed neurotoxicity after organophosphorus pesticide poisoning [J]. Journal of Qingdao Medical College, 1995, 16 (4): 37-38.
[2] Henry K Rotich, Zhang Zhuoyong, Zhao Jinying, etc. SPE-HPLC analysis of methyl parathion, parathion and phoxim pesticide residues in environmental water [J]. Analysis and Test Report, 2002, 21 (5): 50-52.
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