Supercritical chromatography-tandem mass spectrometry detection method of triazole bactericide

Abstract

The invention relates to the technical field of analysis and detection, in particular to a supercritical chromatography-tandem mass spectrometry detection method for triazole bactericides. The supercritical chromatography-tandem mass spectrometry detection method can realize stereoselective separation of different enantiomers of ten triazole bactericides at the same time, significantly improves the detection efficiency of the triazole bactericides, and has the advantages of organic reagent saving, cost reduction and safe and environment-friendly operation as the main mobile phase is CO2 without toxic action. The detection method provided by the invention has the advantages of excellent linear relationship, high reproducibility and high recovery rate, and has great application value in environmental protection, crop pest control, food safety quality control and related qualitative and quantitative research.

Description

Technical Field

[0001] The invention relates to the technical field of analysis and detection, and in particular to a supercritical chromatography tandem mass spectrometry detection method for triazole fungicides. Background Art

[0002] Triazole fungicides are a class of broad-spectrum fungicides containing 1,2,4-triazole groups, which are commonly used to prevent fungal diseases such as powdery mildew and rust in fruits, vegetables, beans and grain crops. In the study, the enantiomer differences in their efficacy were found. R-configuration triadimenol has strong fungicidal activity, while S-configuration has a growth regulatory effect; the enantiomers of triazole fungicides are also different in metabolism and accumulation in organisms and the environment. For example, the S-configuration tebuconazole is metabolized faster than the R-configuration, and the inactive enantiomers of triazole fungicides are difficult to be biodegraded, which leads to the enrichment of enantiomers in the ecological environment and even the production of carcinogenic intermediates. Although triazole fungicides are extremely harmful to the environment and human body, they are still used in agricultural production and food preservation. It is of great significance to separate and study the enantiomers of these fungicides.

[0003] To date, several traditional chromatographic methods have been used for chiral separation and determination of fungicides, including gas chromatography, capillary electrophoresis (CE), liquid chromatography with ultraviolet detection (LC-UV), and liquid chromatography-tandem mass spectrometry (LC-MS / MS). However, these methods always have inevitable disadvantages, including low resolution, severe matrix interference, and long retention time. The physical and chemical properties between enantiomers are similar, and it is difficult to separate them by traditional methods. Often, the same separation method can separate a small number of enantiomers, and it may be necessary to switch between different chromatographic columns, which is troublesome, time-consuming and labor-intensive.

[0004] Therefore, how to provide a detection method that can simultaneously detect multiple triazole fungicides and has convenient, safe and environmentally friendly detection operation has become a technical problem that urgently needs to be solved in this field. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a supercritical fluid chromatography tandem mass spectrometry detection method for triazole fungicides, comprising: using supercritical fluid chromatography tandem mass spectrometry to separate and analyze the sample to be tested, the chromatographic column used in the supercritical fluid chromatography tandem mass spectrometry is CHIRALPAK IA chromatographic column; the mobile phase comprises mobile phase A, mobile phase B and compensating mobile phase, mobile phase A is carbon dioxide, mobile phase B is methanol containing formic acid, and the compensating mobile phase is methanol; the gradient elution program is as follows: at 0 min, the volume percentage of mobile phase A is 94%-96%, and the volume percentage of mobile phase B is 4%-6%; during 0.01-35 min, mobile phase A decreases to 84%-86%, and mobile phase B increases to 14%-16%; during 35-35.1 min, mobile phase A rises back to 94%-96%, and mobile phase B decreases to 4%-6%; during 35.1-40 min, mobile phase A is maintained at 94%-96%, and mobile phase B is maintained at 4%-6%; during each time period, the sum of the volume percentages of mobile phase A and mobile phase B is 100%; the detector used in the supercritical fluid chromatography tandem mass spectrometry is a triple quadrupole mass spectrometer.

[0006] The present invention uses supercritical fluid chromatography (SFC) to detect triazole fungicides, especially for triazole fungicides of different chiralities, to achieve effective stereoselective separation. The present invention optimizes and adjusts the mobile phase and gradient elution program in large quantities, and finds that under the above detection conditions, the chromatographic peak baselines of 10 triazole fungicides can be stable, the retention time does not drift, and the separation degree is above 1.5, achieving baseline separation.

[0007] Preferably, the mobile phase B is methanol containing formic acid, and the volume percentage of formic acid in the mobile phase B is 0.02% to 0.08%, more preferably 0.04% to 0.06%.

[0008] Preferably, the column temperature of the chromatographic column is 40-45°C.

[0009] Preferably, the flow rates of mobile phase A and mobile phase B in the gradient elution program are 1.3-1.5 mL / min; and / or the flow rate of the compensating mobile phase is 0.2-0.3 mL / min.

[0010] Preferably, the back pressure of the chromatography system in the supercritical fluid chromatography tandem mass spectrometry is 1400-1600 psi; and / or the injection volume is 2-10 μl.

[0011] Preferably, the mass spectrometry conditions in the supercritical fluid chromatography tandem mass spectrometry include: ionization mode is ESI+; scanning mode is multiple reaction monitoring; capillary voltage is 4000 V and 3500 V; nebulizer pressure is 15 psi; drying gas temperature is 300°C; drying gas flow rate is 14 L / min; sheath gas temperature is 260°C; and sheath gas flow rate is 11 L / min.

[0012] Preferably, the sample to be tested is a fruit or vegetable sample.

[0013] Preferably, the fruit and vegetable samples are cucumber, rapeseed, grape or apple.

[0014] Preferably, the pretreatment method of the sample to be tested includes: mixing the fruit and vegetable samples with acetonitrile, adding anhydrous magnesium sulfate, sodium chloride and sodium citrate dihydrate after shaking, and taking the supernatant after shaking; mixing the supernatant with anhydrous magnesium sulfate and PSA, taking the supernatant and passing it through a 0.22 μm organic microporous filter membrane for detection on a machine.

[0015] Preferably, the triazole fungicide includes at least one of penconazole, propiconazole, myclobutanil, flutriafol, metconazole, fenbuconazole, tetrafluanidazole, clofoconazole, tebuconazole and triadimefon.

[0016] Furthermore, the present invention provides an application of the detection method in at least one of the following aspects: (1) Environmental protection; (2) Control of crop diseases and pests; (3) Food safety and quality control; (4) Qualitative or quantitative study of chiral triazole fungicides.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The detection method of the supercritical chromatography tandem mass spectrometry of the present invention can simultaneously achieve stereoselective separation for different enantiomers of ten triazole fungicides, significantly improving the detection efficiency of triazole fungicides, and the main mobile phase is non-toxic CO2, which has the advantages of saving organic reagents, reducing costs, and safe and environmentally friendly operation. The detection method of the present invention has an excellent linear relationship, high reproducibility, and high recovery rate, and has great application value in environmental protection, crop pest control, food safety quality control and related qualitative and quantitative research. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the total ion current diagram of the detection method of the present invention.

[0019] Figure 2 This is the chromatogram of propiconazole.

[0020] Figure 3 It is the chromatogram of flutriafol.

[0021] Figure 4 This is the chromatogram of fenbuconazole.

[0022] Figure 5 This is the chromatogram of myclobutanil.

[0023] Figure 6 It is the chromatogram of clofoconazole.

[0024] Figure 7 It is the chromatogram of triadimefon.

[0025] Figure 8 It is the chromatogram of tetrafluthriae.

[0026] Fig. 9 This is the chromatogram of penconazole.

[0027] Fig.10 It is the chromatogram of tebuconazole.

[0028] Fig.11 This is the chromatogram of metconazole.

[0029] Fig.12 It is the average recovery result diagram of Comparative Example 4. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] In the examples provided in this specification, if no specific techniques or conditions are specified, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0032] Example 1 This embodiment provides a method for detecting triazole fungicides by supercritical fluid chromatography tandem mass spectrometry, the steps are as follows: using supercritical fluid chromatography tandem mass spectrometry to separate and analyze the sample to be tested, the chromatographic conditions are as follows: the chromatographic column is a CHIRALPAKIA chromatographic column (5µm, 4.6 mm×250 mm), the chromatographic column filler is amylose-tris (3,5-dimethylphenylcarbamate), the mobile phase includes mobile phase A, mobile phase B and compensation mobile phase; mobile phase A is carbon dioxide; B is 0.05% formic acid methanol by volume; the compensation mobile phase is methanol. Gradient elution is adopted, and the following procedures are followed: the volume percentage of mobile phase A at 0min is 95%, and the volume percentage of mobile phase B is 5%; during 0.01~35min, mobile phase A decreases to 85%, and mobile phase B increases to 15%; during 35~35.1min, mobile phase A rises back to 95%, and mobile phase B decreases to 5%; during 35.1~40min, mobile phase A is maintained at 95%, and mobile phase B is maintained at 5%. The flow rate was 1.5 mL / min, the flow rate of the compensated mobile phase was 0.3 mL / min; the system back pressure was 1500 psi; the column temperature was 40 °C; the injection volume was 5 μL; The mass spectrometry conditions were as follows: the detector was a Waters Xevo TQ-XS triple quadrupole mass spectrometer; ionization mode: ESI+; scanning mode: multiple reaction monitoring (dMRM); capillary voltage: 4000 V and 3500 V; nebulizer pressure: 15 psi; drying gas temperature: 300°C; drying gas flow rate: 14 L / min; sheath gas temperature: 260°C; sheath gas flow rate: 11 L / min. The mass spectrometry parameters of each triazole fungicide are shown in Table 1.

[0033] Table 1 Mass spectrometry parameters of triazole fungicides

[0034] Note: The ions marked with * are quantitative ion pairs.

[0035] Ten triazole fungicides (purchased from Tianjin Alta Technology Co., Ltd.) were prepared into a 10 μg / mL mixed standard with mass spectrometry grade acetonitrile and detected using the above detection method. The total ion current (TIC) was obtained as shown in the following figure: Figure 1 As shown in Figure 2, the chromatograms of the 10 triazole fungicides are as follows: Figures 2~11 shown.

[0036] Further, the mixed standard was diluted to 2 μg / L, 5 μg / L, 10 μg / L, 20 μg / L, 40 μg / L, 80 μg / L, 160 μg / L, 320 μg / L, and 640 μg / L as the standard working solution for detection, and the standard curve was obtained as shown in Table 2.

[0037] Table 2 Standard curve results

[0038] It can be seen that the detection method of the present invention can simultaneously achieve stereoselective separation of different enantiomers of ten triazole fungicides, and has an excellent linear relationship and high reproducibility.

[0039] Example 2 This example uses the supercritical chromatography tandem mass spectrometry detection method in Example 1 to test the recovery rates of ten triazole fungicides in four fruit and vegetable samples, namely, cucumber, rapeseed, grape and apple. The steps are as follows: After the blank samples of cucumber, rapeseed, grape and apple were broken by a wall breaker, 10g of sample (accurate to 0.01g) was weighed in a 50mL centrifuge tube, 10mL of acetonitrile and a ceramic homogenizer were added, and the mixture was shaken vigorously for 1min. 6g of anhydrous magnesium sulfate, 1g of sodium chloride and 1g of sodium citrate dihydrate were added, and the mixture was shaken vigorously for 1min and centrifuged at 6000r / min for 5min. 10mL of supernatant was quantitatively drawn into 15mL plastic containing 1500mg of anhydrous magnesium sulfate and 500mg of PSA, and the supernatant was drawn through a 0.22μm organic microporous filter membrane to obtain solvent A. The 5 μg / mL mixed standard in Example 1 was diluted with solvent A to 2 μg / L, 5 μg / L, 10 μg / L, 20 μg / L, 40 μg / L, 80 μg / L, 160 μg / L, 320 μg / L, and 640 μg / L as matrix standard working solutions.

[0040] The blank samples of cucumber, rapeseed, grape and apple were crushed with a wall breaker, and 10g of sample (accurate to 0.01g) was weighed in a 50mL centrifuge tube, 10mL of acetonitrile and a ceramic homogenizer were added, and the mixture was shaken vigorously for 1min. 6g of anhydrous magnesium sulfate, 1g of sodium chloride and 1g of sodium citrate dihydrate were added, and the mixture was shaken vigorously for 1min and centrifuged at 6000r / min for 5min. 2mL of supernatant was quantitatively drawn into 15mL plastic containing 300mg of anhydrous magnesium sulfate and 100mg of PSA, and the supernatant was drawn through a 0.22μm organic microporous filter membrane to obtain a solution as a basic sample.

[0041] The recovery rate of the supercritical chromatography tandem mass spectrometry detection method in Example 1 was tested using the matrix standard working solution and the basic sample. The results are shown in Table 3.

[0042] Table 3 Recovery test results

[0043] It can be seen that the detection method of the present invention has a high recovery rate and can achieve simultaneous determination of different enantiomers of ten triazole fungicides on a variety of fruits and vegetables.

[0044] Comparative Example 1 This comparative example provides a method for detecting triazole fungicides by supercritical chromatography tandem mass spectrometry, the steps of which are different from those of Example 1 only in that: The flow rate was adjusted to 1.1mL / min, 1.3mL / min, 1.5mL / min, 1.7mL / min, and 2.2mL / min. The separation R of different compound peaks was detected at different flow rates. When the separation R<1, the two peaks were not separated; when R=1, the two peaks were separated by 98%, which was basic separation; when R≥1.5, the two peaks were separated by 99.7%, which was baseline separation.

[0045] The test results are shown in Table 4.

[0046] Table 4 Separation test data at different flow rates

[0047] It can be seen that when the flow rate is 1.5mL / min, the separation between the 1st and 2nd peaks of propiconazole is 1.06, and the separation between the 3rd and 4th peaks is 1.19, which cannot achieve baseline separation. Other triazole fungicides all achieve baseline separation. Since the retention time increases with the decrease of flow rate, 1.5mL / min is selected as the separation method flow rate to reduce the separation time.

[0048] Comparative Example 2 This comparative example provides a method for detecting triazole fungicides by supercritical chromatography tandem mass spectrometry, the steps of which are different from those of Example 1 only in that: Adjust the system back pressure to 1500psi, 1750psi, 2000psi, 2250psi, and 2500psi respectively.

[0049] The resolution R of different compound peaks was detected under different system back pressures.

[0050] The test results are shown in Table 5.

[0051] Table 5 Separation test data of different system back pressures

[0052] As shown in Table 5, the maximum resolution is achieved when the back pressure is 1500 psi.

[0053] Comparative Example 3 This comparative example provides a method for detecting triazole fungicides by supercritical chromatography tandem mass spectrometry, the steps of which are different from those of Example 1 only in that: The column temperature was adjusted to 35°C, 37.5°C, 40°C, 42.5°C, and 45°C, respectively.

[0054] The separation R of different compound peaks was detected at different column temperatures.

[0055] The test results are shown in Table 6.

[0056] Table 6 Separation test data at different column temperatures

[0057] As shown in Table 6, when the column temperature was 40 °C, all 10 triazole fungicides reached baseline separation.

[0058] Comparative Example 4 This comparative example provides a method for detecting triazole fungicides by supercritical chromatography tandem mass spectrometry, the steps of which are different from those of Example 1 only in that: During the pretreatment process, PSA was replaced by an equal amount of C18 or a mixture of PSA and C18.

[0059] The detection method of Example 2 was used to test the recoveries of ten triazole fungicides in four fruit and vegetable samples, namely, cucumber, rapeseed, grape and apple. The average recoveries were calculated as follows: Fig.12 shown.

[0060] The results showed that the recovery rate of triazole fungicides in samples with PSA as the purifier was the best.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting triazole fungicides by supercritical chromatography tandem mass spectrometry, characterized in that: include: Supercritical fluid chromatography tandem mass spectrometry is used to separate and analyze the sample to be tested. The chromatographic column used in the supercritical fluid chromatography tandem mass spectrometry is CHIRALPAK IA chromatographic column; the mobile phase comprises mobile phase A, mobile phase B and compensating mobile phase, mobile phase A is carbon dioxide, mobile phase B is methanol containing formic acid, and the compensating mobile phase is methanol; the gradient elution program is as follows: at 0 min, the volume percentage of mobile phase A is 94%-96%, and the volume percentage of mobile phase B is 4%-6%; during 0.01-35 min, mobile phase A decreases to 84%-86%, and mobile phase B increases to 14%-16%; during 35-35.1 min, mobile phase A rises back to 94%-96%, and mobile phase B decreases to 4%-6%; during 35.1-40 min, mobile phase A is maintained at 94%-96%, and mobile phase B is maintained at 4%-6%; during each time period, the sum of the volume percentages of mobile phase A and mobile phase B is 100%; the detector used in the supercritical fluid chromatography tandem mass spectrometry is a triple quadrupole mass spectrometer.

2. The detection method according to claim 1, characterized in that: The column temperature of the chromatographic column is 40-45°C.

3. The detection method according to claim 1, characterized in that: The flow rates of mobile phase A and mobile phase B in the gradient elution program are 1.3-1.5 mL / min; and / or the flow rate of the compensating mobile phase is 0.2-0.3 mL / min.

4. The detection method according to claim 1, characterized in that: The back pressure of the chromatography system in the supercritical fluid chromatography tandem mass spectrometry is 1400-1600 psi; and / or the injection volume is 2-10 μl.

5. The detection method according to claim 1, characterized in that: The mass spectrometry conditions in the supercritical fluid chromatography tandem mass spectrometry include: ionization mode is ESI+; scanning mode is multiple reaction monitoring; capillary voltage is 4000 V and 3500 V; nebulizer pressure is 15 psi; drying gas temperature is 300°C; drying gas flow rate is 14 L / min; sheath gas temperature is 260°C; and sheath gas flow rate is 11 L / min.

6. The detection method according to claim 1, characterized in that: The samples to be tested are fruit and vegetable samples.

7. The detection method according to claim 6, characterized in that: The fruit and vegetable samples are cucumber, rapeseed, grape or apple.

8. The detection method according to claim 6, characterized in that: The pretreatment method of the sample to be tested includes: mixing the fruit and vegetable sample with acetonitrile, adding anhydrous magnesium sulfate, sodium chloride and sodium citrate dihydrate after shaking, and taking the supernatant after shaking; mixing the supernatant with anhydrous magnesium sulfate and PSA, taking the supernatant and passing it through a 0.22 μm organic microporous filter membrane for detection on a machine.

9. The detection method according to any one of claims 1 to 8, characterized in that The triazole fungicide includes at least one of penconazole, propiconazole, myclobutanil, flutriafol, metconazole, fenbuconazole, tetrafluanidazole, clofoconazole, tebuconazole and triadimefon.

10. Use of the detection method according to any one of claims 1 to 9 in at least one of the following aspects: (1) Environmental protection; (2) Control of crop diseases and pests; (3) Food safety and quality control; (4) Qualitative or quantitative study of chiral triazole fungicides.

Images