A method for detecting hexaconazole by ferrofluid extraction combined with high performance liquid chromatography

Abstract

The application develops a ferrofluid-based dispersive liquid-liquid microextraction method, and determines the residues of chiral fungicide hexaconazole in food by high performance liquid chromatography, and belongs to the field of food safety detection.The application comprises four steps, that is, preparation of eutectic solvent, preparation of ferrofluid, self-dispersed ferrofluid-based dispersive liquid-liquid microextraction, and determination of the optimal selection of extraction conditions, and detection of hexaconazole in food by high performance liquid chromatography.The method has the advantages of rapidness, greenness and high efficiency, and the detection limit, the quantitative limit, the recovery rate and the precision of the method meet the analysis requirements.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of food safety detection, and relates to a dispersed liquid-liquid microextraction method based on ferrofluid combined with high performance liquid chromatography for determining the residues of chiral fungicide hexaconazole in food. BACKGROUND

[0002] Spraying pesticides is one of the effective ways to protect crops from being invaded by pathogens, but the long-lasting effect may cause some pesticides to remain in agricultural products. Chiral hexaconazole is a sterol demethylation inhibitor among triazole fungicides, and is mainly used for preventing and controlling crop fungal diseases caused by basidiomycetes and ascomycetes to improve food quality and yield. However, the half-life of hexaconazole in soil is 225 days, and with biological enrichment, hexaconazole can contaminate crops and enter food through processing technology, such as tea, fruit juice, etc., thereby causing harm to human health, showing reproductive toxicity, neurotoxicity, and even causing cancer. Hexaconazole is composed of (+)-hexaconazole and (-)-hexaconazole. According to the literature, the biological activity, toxicity and residue of hexaconazole enantiomers are different. (-)-hexaconazole is more easily accumulated in zebrafish than (+)-hexaconazole; (+)-hexaconazole has a greater risk to soil microorganisms than (-)-hexaconazole. In order to protect the health and safety of consumers, it is urgent to develop a method that can rapidly detect chiral hexaconazole residues in food at the enantiomer level.

[0003] Before detecting chiral hexaconazole, sample pretreatment is an essential step due to its trace residues and complex matrix. Solid phase extraction and liquid phase extraction are both traditional sample pretreatment methods, but they are time-consuming and require a large amount of solvent. In order to solve these problems, people have developed microextraction methods to reduce extraction time and the volume of organic solvent required, including dispersed liquid-liquid microextraction. Dispersed liquid-liquid microextraction is to mix the extraction solvent and the dispersion solvent and then quickly inject them into the sample solution. Under the action of the dispersion solvent, the organic phase and the aqueous phase are in full contact, which improves the extraction efficiency. Dispersed liquid-liquid microextraction technology has the advantages of simplicity, less solvent consumption, strong enrichment capacity, etc.

[0004] However, the dispersion liquid-liquid microextraction needs 10 to 20 minutes of centrifugation time to collect the extraction solvent, which is the most time-consuming step in the dispersion liquid-liquid microextraction. In order to eliminate the centrifugation step, the development of magnetic solvent is of great concern, which is easier to prepare than magnetic materials and has better repeatability. Ferrofluid is an emerging magnetic solvent, which is a stable and uniform fluid with magnetism. As an extraction solvent, ferrofluid can be quickly and accurately moved under the control of a magnet and magnetically separated, which can save the centrifugation step and shorten the separation time. Ferrofluid is composed of a supporting solvent and magnetic particles. In order to meet the requirements of green chemistry, the supporting solvent of ferrofluid should be low-toxic and environmentally friendly. As a new type of green solvent, eutectic solvent has the advantages of simple preparation, high synthesis yield, adjustable structure and biodegradability, and is expected to replace traditional toxic solvents as the supporting solvent of ferrofluid.

[0005] In the current dispersion liquid-liquid microextraction, the dispersion process is usually completed with the help of extraction equipment (vortex, shock, ultrasonic). Although the dispersion method with the help of extraction equipment does not use toxic dispersion solvents (methanol, acetonitrile and acetone), the use of extraction equipment increases the operation time and reduces the efficiency, which is not suitable for on-site rapid analysis. The introduction of compounds with dispersion function into the synthesis step of eutectic solvent is expected to realize the self-dispersion function of eutectic solvent. Therefore, the development of functional extractant with self-dispersion function and magnetism can realize rapid dispersion liquid-liquid microextraction and meet the requirements of environmental protection and shortening of sample pretreatment time. SUMMARY

[0006] (1) Problems to be solved

[0007] In order to solve the above problems of the prior art, the present application provides a method for detecting chiral hexaconazole, specifically a method for detecting chiral hexaconazole in food based on dispersion liquid-liquid microextraction combined with high performance liquid chromatography.

[0008] (2) Technical solutions

[0009] In order to achieve the above purpose, the main technical solutions adopted by the present application include:

[0010] A method for detecting hexaconazole based on a ferrofluid extraction combined with high performance liquid chromatography, comprising the following steps:

[0011] Step 1: Preparation of eutectic solvent

[0012] The eutectic solvent is composed of a monoterpene alcohol, a medium chain fatty acid and a short chain fatty acid. The monoterpene alcohol is fenchol or citronellol or carvol, the medium chain fatty acid is hexanoic acid or heptanoic acid or octanoic acid, and the short chain fatty acid is formic acid or acetic acid or propionic acid. The monoterpene alcohol, the medium chain fatty acid and the short chain fatty acid are mixed and heated in a molar ratio of 1:(0.25-3):(1-6) to obtain a transparent liquid as the eutectic solvent.

[0013] Second step, preparation of ferrofluid

[0014] The fluid obtained by vortexing the prepared eutectic solvent and ferroferric oxide is a ferrofluid.

[0015] Third step, dispersion liquid-liquid microextraction based on self-dispersed ferrofluid

[0016] The sample solution is taken in a centrifuge tube, the ferrofluid is added to the sample solution to complete the rapid extraction, a magnet is placed beside the centrifuge tube to attract the ferrofluid, and then the supernatant is poured off, and the ferrofluid is eluted with methanol.

[0017] Fourth step, the eluate of the ferrofluid is detected by high performance liquid chromatography, and a standard working curve is drawn with the mass concentration of added chiral hexazolol as the abscissa and the corresponding chromatographic peak area as the ordinate, and the content of chiral hexazolol in the unknown sample solution is calculated.

[0018] In the first step, the monoterpene alcohol is fenchol or citronellol or carvol, the medium chain fatty acid is hexanoic acid or heptanoic acid or octanoic acid, and the short chain fatty acid is formic acid or acetic acid or propionic acid, and the molar ratio is 1:(0.25-3):(1-6). Preferably, the eutectic solvent is prepared by mixing fenchol, hexanoic acid and acetic acid in a molar ratio of 1:0.5:3, and heating at 60°C for 10 min.

[0019] In the second step, the ratio of ferroferric oxide to eutectic solvent is (2.5-150) mg:1 mL, preferably 50 mg:1 mL. In an embodiment of the present application, the mass of ferroferric oxide is 2.5-150 mg, preferably 50 mg, and the volume of eutectic solvent is 1 mL, and vortexing is performed for 1 min.

[0020] In the third step, the ferrofluid is added in an amount of 75-250 μL / 5 mL of sample solution, preferably 175 μL / 5 mL of sample solution, and methanol is added in an amount of 150 μL / 5 mL of sample solution. In an embodiment of the present application, the sample volume is 5 mL, the ferrofluid volume is 75-250 μL, preferably 175 μL, and the methanol volume is 150 μL.

[0021] In the fourth step, the high performance liquid chromatography detection conditions are as follows: a cellulose-tris(3,5-dimethylphenylaminoformate) chiral chromatographic column (4.6 mm*250 mm, 5 μm); an ultraviolet detector; an injection volume of 10 μL; a mobile phase of methanol and water at a volume ratio of 80:20; a flow rate of 0.4 mL / min; and a detection wavelength of 220 nm; and the retention time of the chiral hexaconazole is 17.2 and 18.5 min.

[0022] The concentration of hexaconazole in the sample solution is preferably 0.02-2 μg / mL.

[0023] (III) Beneficial effects

[0024] The beneficial effects of the present application are as follows:

[0025] 1. The present application develops a functional extractant with self-dispersing function and magnetic function, and establishes a ferrofluid dispersed liquid-liquid microextraction method. The method does not need to be vortexed, ultrasonicated or shaken, and the organic phase can be completely dispersed in the sample solution, and the separation of the organic phase and the aqueous phase can be realized without complicated and time-consuming centrifugation steps.

[0026] 2. In the preparation of the ferrofluid, the magnetic particles in the ferrofluid do not need to be prepared by complicated 2-24 h ultrasonication, stirring and washing processes, and can directly use the ferroferric oxide material; and the synthesis time of the traditional ferrofluid is shortened from 30 min-1 h to 1 min, which simplifies the preparation process of the ferrofluid.

[0027] 3. The supporting solvent in the ferrofluid is a low eutectic solvent prepared from monoterpene alcohols, medium-chain fatty acids and short-chain fatty acids, which is a new type of green solvent and has the advantages of simple preparation, high synthesis efficiency, adjustable structure and biodegradability.

[0028] 4. The dispersed liquid-liquid microextraction method based on the ferrofluid developed by the present application has an extraction time of 1 s and a separation time of 1 min.

[0029] 5. The present application performs chiral separation and detection on the hexaconazole enantiomers in the sample.

[0030] Drawings of the specification

[0031] Figure 1 Influence of different monoterpene alcohols on recovery rate.

[0032] Figure 2 Influence of different medium-chain fatty acids on recovery rate.

[0033] Figure 3 Influence of different short-chain fatty acids on recovery rate.

[0034] Figure 4 Effect of different ratios of fenchol and hexanoic acid on recovery rate.

[0035] Figure 5 Effect of different ratios of fenchol, hexanoic acid and acetic acid on recovery rate.

[0036] Figure 6 Effect of different mass fractions of ferroferric oxide on recovery rate.

[0037] Figure 7 Effect of different volumes of ferromagnetic fluid on recovery rate. DETAILED DESCRIPTION

[0038] The present application first prepares ferromagnetic fluid with self-dispersing function, adds the ferromagnetic fluid as an extractant into a sample solution to spontaneously carry out dispersive liquid-liquid microextraction, then separates the ferromagnetic fluid from the aqueous phase by attracting the ferromagnetic fluid with a magnet, elutes the ferromagnetic fluid with methanol, and then detects and analyzes by high performance liquid chromatography. The main factors affecting the recovery rate are studied and optimized from two aspects of preparation of ferromagnetic fluid and conditions of dispersive liquid-liquid microextraction, and a dispersive liquid-liquid microextraction method based on self-dispersing ferromagnetic fluid is established, and the residual chiral fungicide hexaconazole in food is determined by high performance liquid chromatography.

[0039] In order to better explain the present application, so as to be understood, the present application is described in detail in combination with the drawings and through specific embodiments.

[0040] Example 1, parameter optimization of the method for detecting hexaconazole by high performance liquid chromatography based on ferromagnetic fluid dispersive liquid-liquid microextraction

[0041] The method for detecting hexaconazole by high performance liquid chromatography based on ferromagnetic fluid dispersive liquid-liquid microextraction. The specific operation is as follows:

[0042] Mix fenchol, hexanoic acid and acetic acid according to the molar ratio of 1:0.5:3, heat at 60℃ for 10 min to obtain a eutectic solvent. Take 50 mg of ferroferric oxide and 1 mL of eutectic solvent, and vortex for 1 min to obtain ferromagnetic fluid. Add 175 μL of ferromagnetic fluid to a centrifuge tube containing 5 mL of sample solution containing 2 μg / mL of hexaconazole, attract the ferromagnetic fluid droplets with a magnet, and then pour out the sample solution. Finally, dilute the ferromagnetic fluid with 150 μL of methanol, and analyze by high performance liquid chromatography.

[0043] The high performance liquid chromatography detection conditions are as follows: a cellulose-tris (3, 5-dimethylphenylaminoformate) chiral chromatographic column (4.6 mm*250 mm, 5 μm) is used as the chromatographic column, an ultraviolet detector is used as the detector, the injection amount is 10 μL, the mobile phase is methanol and water with a volume ratio of 80:20, the flow rate is 0.4 mL / min, the detection wavelength is 220 nm, and the retention time of chavibetol is 17.2 min and 18.5 min.

[0044] The present application has many influencing factors, and a series of optimization experiments need to be carried out, therefore, the type of monoterpenoid alcohol, the type of medium-chain fatty acid, the type of short-chain fatty acid, the ratio of fenchol and hexanoic acid, the ratio of fenchol, hexanoic acid and acetic acid, the mass fraction of ferroferric oxide and the volume of ferrofluid are optimized, and samples are purchased from local supermarkets to verify the reliability of the detection results of the present application in practical application, and the following experiments are carried out:

[0045] 1. Optimization of the type of monoterpenoid alcohol

[0046] In the experiment, three kinds of monoterpenoid alcohols, namely acyclic monoterpenoid alcohol (citronellol), monocyclic monoterpenoid alcohol (carveol) and bicyclic monoterpenoid alcohol (fenchol), are selected to affect the recovery rate of hexoxazole, and the monoterpenoid alcohol, hexanoic acid and acetic acid are mixed according to a molar ratio of 1:0.5:3 to obtain a eutectic solvent. 50 mg of ferroferric oxide and 1 mL of the eutectic solvent are vortexed for 1 min to obtain a ferrofluid. 175 μL of the ferrofluid is added to a centrifuge tube containing 5 mL of a sample solution with a hexoxazole concentration of 2 μg / mL, the ferrofluid droplets are attracted by a magnet, and then the sample solution is poured out. Finally, 150 μL of methanol is used to dilute the ferrofluid, and high performance liquid chromatography analysis is carried out.

[0047] The results (Table 1) show that when the monoterpenoid alcohol is fenchol, the recovery rate of hexoxazole is the highest, as shown in Figure 1 The reason may be that the ring tension affects the hydroxyl group outside the ring of the monoterpenoid alcohol, and then affects the interaction between the prepared ferrofluid and hexoxazole. Therefore, the optimal monoterpenoid alcohol is fenchol.

[0048] Table 1 Influence of the type of monoterpenoid alcohol on the recovery rate of hexoxazole and relative standard deviation

[0049]

[0050] 2. Optimization of the type of medium-chain fatty acid

[0051] The experiment selected three kinds of medium-chain fatty acids, namely hexanoic acid, heptanoic acid and octanoic acid, to study their effects on the recovery rate of hexaconazole. The mixture of opium alcohol, medium-chain fatty acid and acetic acid was heated to obtain a eutectic solvent according to a molar ratio of 1:0.5:3. The ferroferric oxide with a mass of 50 mg and the eutectic solvent with a volume of 1 mL were vortexed for 1 min to obtain a ferrofluid. 175 μL of the ferrofluid was added to a centrifuge tube containing 5 mL of a sample solution with a concentration of 2 μg / mL of hexaconazole. The ferrofluid droplets were attracted by a magnet, and then the sample solution was poured out. Finally, 150 μL of methanol was used to dilute the ferrofluid, and high performance liquid chromatography analysis was performed.

[0052] The results (Table 2) show that when the medium-chain fatty acid is hexanoic acid, the recovery rate of hexaconazole is the highest, such as Figure 2 The reason may be that the reduction of the carbon chain reduces the viscosity of the medium-chain fatty acid. The viscosities of hexanoic acid, heptanoic acid and octanoic acid are measured to be 6.01, 7.86 and 9.30 mPa.s, respectively. The high viscosity of the medium-chain fatty acid reduces the dispersibility of the prepared ferrofluid in the sample solution. Therefore, the optimal medium-chain fatty acid is hexanoic acid.

[0053] Table 2 Effects of the type of medium-chain fatty acid on the recovery rate of hexaconazole and the relative standard deviation

[0054]

[0055]

[0056] 3. Effects of the type of short-chain fatty acid

[0057] The experiment selected three kinds of short-chain fatty acids, namely formic acid, acetic acid and propionic acid, to study their effects on the recovery rate of hexaconazole. The mixture of opium alcohol, hexanoic acid and short-chain fatty acid was heated to obtain a eutectic solvent according to a molar ratio of 1:0.5:3. The ferroferric oxide with a mass of 50 mg and the eutectic solvent with a volume of 1 mL were vortexed for 1 min to obtain a ferrofluid. 175 μL of the ferrofluid was added to a centrifuge tube containing 5 mL of a sample solution with a concentration of 2 μg / mL of hexaconazole. The ferrofluid droplets were attracted by a magnet, and then the sample solution was poured out. Finally, 150 μL of methanol was used to dilute the ferrofluid, and high performance liquid chromatography analysis was performed.

[0058] The results (Table 3) show that when the short-chain fatty acid is acetic acid, the recovery rate of hexaconazole is the highest, such as Figure 3 Therefore, the optimal short-chain fatty acid is acetic acid.

[0059] Table 3 Effects of the type of short-chain fatty acid on the recovery rate of hexaconazole and the relative standard deviation

[0060]

[0061] 4. Optimization of the molar ratio of opium alcohol and hexanoic acid

[0062] The experiment selected six different molar ratios of horseradish alcohol and hexanoic acid, 1:0.25, 1:0.33, 1:0.5, 1:1, 1:2, and 1:3, to study their effects on the recovery rate of hexaconazole. The eutectic solvent was obtained by mixing the different molar ratios of horseradish alcohol and hexanoic acid with acetic acid at a molar ratio of 3 and heating. A mass of 50 mg of ferroferric oxide was used to obtain a ferrofluid by vortexing for 1 min. 175 μL of the ferrofluid was added to a centrifuge tube containing 5 mL of a sample solution with a concentration of 2 μg / mL of hexaconazole. The ferrofluid droplets were attracted by a magnet, and then the sample solution was poured out. Finally, 150 μL of methanol was used to dilute the ferrofluid, which was analyzed by high-performance liquid chromatography.

[0063] The experimental results (Table 4) show that when the molar ratio of horseradish alcohol to hexanoic acid is in the range of 1:0.25 to 1:0.5, the recovery rate of hexaconazole gradually increases. When the molar ratio exceeds 1:0.5, the recovery rate of hexaconazole decreases, as shown in Figure 4 The reason may be that the molar ratio of horseradish alcohol to hexanoic acid can affect the number of hydrogen bonds, thereby affecting the extraction capacity of the ferrofluid. Therefore, the optimal molar ratio of horseradish alcohol to hexanoic acid is 1:0.5.

[0064] Table 4 Effects of the molar ratio of horseradish alcohol to hexanoic acid on the recovery rate of hexaconazole and the relative standard deviation

[0065]

[0066] 5. Optimization of the molar ratio of horseradish alcohol, hexanoic acid, and acetic acid

[0067] The experiment selected six different molar ratios of horseradish alcohol, hexanoic acid, and acetic acid, 1:0.5:1, 1:0.5:2, 1:0.5:3, 1:0.5:4, 1:0.5:5, and 1:0.5:6, to study their effects on the recovery rate of hexaconazole. The eutectic solvent was obtained by mixing horseradish alcohol, hexanoic acid, and different molar ratios of acetic acid and heating. A mass of 50 mg of ferroferric oxide was used to obtain a ferrofluid by vortexing for 1 min. 175 μL of the ferrofluid was added to a centrifuge tube containing 5 mL of a sample solution with a concentration of 2 μg / mL of hexaconazole. The ferrofluid droplets were attracted by a magnet, and then the sample solution was poured out. Finally, 150 μL of methanol was used to dilute the ferrofluid, which was analyzed by high-performance liquid chromatography.

[0068] The experimental results (Table 5) show that when the molar ratio of horseradish alcohol, hexanoic acid, and acetic acid is in the range of 1:0.5:1 to 1:0.5:3, the recovery rate of hexaconazole gradually increases. When the molar ratio exceeds 1:0.5:3, the recovery rate of hexaconazole decreases, as shown in Figure 5The reason could be that when the amount of acetic acid is sufficient, the ferrofluid can be completely dispersed in the sample solution with the help of acetic acid and extract the chiral hexaconazole. However, excessive acetic acid will reduce the polarity of the sample, causing more extraction solvent containing chiral hexaconazole to dissolve into the aqueous phase. Therefore, the optimal molar ratio of fenchol, hexanoic acid and acetic acid is 1:0.5:3.

[0069] Table 5 Influence of molar ratio of fenchol, hexanoic acid and acetic acid on recovery rate of hexaconazole and relative standard deviation

[0070]

[0071]

[0072] 6. Optimization of the mass fraction of ferroferric oxide

[0073] In this experiment, seven mass fractions of ferroferric oxide were selected, i.e. 2.5, 5, 10, 25, 50, 100 and 150 g / L, to study their influence on the recovery rate of hexaconazole. Fenchol, hexanoic acid and acetic acid were mixed and heated to obtain a eutectic solvent at a molar ratio of 1:0.5:3. Different amounts of ferroferric oxide were taken, 1 mL of eutectic solvent was added and vortexed for 1 min to obtain a ferrofluid. 175 μL of ferrofluid was added to a centrifuge tube containing 5 mL of sample solution with a concentration of 2 μg / mL of hexaconazole, and the ferrofluid droplets were attracted by a magnet. Then the sample solution was poured out. Finally, 150 μL of methanol was used to dilute the ferrofluid, and high performance liquid chromatography analysis was performed.

[0074] The experimental results (Table 6) show that when the mass fraction of ferroferric oxide is in the range of 2.5-50 g / L, the recovery rate of hexaconazole gradually increases, and when the mass fraction exceeds 100 g / L, the recovery rate of hexaconazole tends to be stable, as shown in Figure 6 The reason could be that when the amount of ferroferric oxide is insufficient, the extraction solvent and chiral hexaconazole are lost during the magnetic separation process. Therefore, the optimal mass fraction of ferroferric oxide is 50 g / L.

[0075] Table 6 Influence of mass fraction of ferroferric oxide on recovery rate of hexaconazole and relative standard deviation

[0076]

[0077] 7. Influence of ferrofluid volume

[0078] The experiment selected seven volumes of ferrofluid, 75, 100, 125, 150, 175, 200 and 250 μL, to study the effect on the recovery rate of hexaconazole. The eutectic solvent was prepared by mixing oleyl alcohol, hexanoic acid and acetic acid at a molar ratio of 1:0.5:3 and heating. 50 mg of ferroferric oxide and 1 mL of the prepared eutectic solvent were vortexed for 1 min to obtain the ferrofluid. Different volumes of ferrofluid were added to a centrifuge tube containing 5 mL of sample solution with a concentration of 2 μg / mL of hexaconazole. The ferrofluid droplets were attracted by a magnet, and then the sample solution was poured out. Finally, 150 μL of methanol was used to dilute the ferrofluid, and high performance liquid chromatography was performed.

[0079] The experimental results (Table 7) show that when the volume of ferrofluid changes from 75 to 175 μL, the recovery rate of hexaconazole gradually increases, and when the volume exceeds 175 μL, the recovery rate of hexaconazole decreases, as shown in Figure 7 The reason may be that when the volume is less than 175 μL, the effective interaction between the extraction solvent and the chiral hexaconazole cannot be guaranteed, and the collection of the organic phase is hindered. When the volume is greater than 175 μL, the dispersion degree of the ferrofluid decreases, resulting in a decrease in the recovery rate of hexaconazole. Therefore, the optimal volume of ferrofluid is 175 μL.

[0080] Table 7 Effect of volume of ferrofluid on recovery rate of hexaconazole and relative standard deviation

[0081]

[0082] Example 2, Dispersive liquid-liquid microextraction based on ferrofluid combined with high performance liquid chromatography for detection of hexaconazole and its effect verification

[0083] Detection of chiral hexaconazole in actual samples

[0084] (1) Preparation of eutectic solvent

[0085] Oleyl alcohol, hexanoic acid and acetic acid were mixed at a molar ratio of 1:0.5:3, heated at 60°C for 10 min, and a transparent liquid was obtained as the eutectic solvent.

[0086] (2) Preparation of ferrofluid

[0087] 50 mg of ferroferric oxide and 1 mL of the prepared eutectic solvent were weighed and vortexed for 1 min to obtain the ferrofluid.

[0088] (3) Dispersive liquid-liquid microextraction based on self-dispersing ferrofluid

[0089] 5 mL of the prepared sample solution was taken in a centrifuge tube, and 175 μL of the prepared ferrofluid was added to the sample solution to complete the rapid extraction. A magnet was placed next to the centrifuge tube to attract the ferrofluid, and then the supernatant was poured out. 150 μL of methanol was used to elute the ferrofluid.

[0090] (4) Detection and analysis of hexaconazole in food by high performance liquid chromatography

[0091] The sample solution was analyzed according to the above experimental procedure. The high performance liquid chromatography detection conditions were as follows: column: cellulose-tris(3,5-dimethylphenylaminoformate) chiral column (4.6 mm x 250 mm, 5 μm); detector: ultraviolet detector; injection volume: 10 μL; mobile phase: methanol and water, volume ratio 80:20; flow rate: 0.4 mL / min; detection wavelength: 220 nm; retention time of chiral hexaconazole: 17.2 and 18.5 min. The method was verified by evaluating the linear equation, coefficient of determination (R 2 ), limit of detection and limit of quantification of the self-dispersing ferrofluid dispersion liquid-liquid microextraction combined with high performance liquid chromatography under the optimal conditions, and the results are shown in Table 8. Hexaconazole showed good linearity in the linear range of 0.02-2 μg / mL, and the limit of detection and limit of quantification were calculated according to S / N=3 and 10, and the limit of detection and limit of quantification were 0.006 and 0.02 μg / mL, respectively. In order to investigate the reproducibility of the method, the spiked recovery test was carried out at three addition levels, and each level was repeated 5 times in parallel, and the recovery rate and relative standard deviation (RSD) were calculated, and the results are shown in Table 9. The results show that the recovery rate is in the range of 83.0%-101.7%, and the RSD is between 1.3% and 8.6%, indicating that the established method has good reproducibility and is suitable for the detection of chiral hexaconazole in food.

[0092] Table 8 Method evaluation of hexaconazole in actual samples

[0093]

[0094] Table 9 Spiked recovery rate and relative standard deviation of actual samples

[0095]

[0096]

[0097] The above is only a preferred embodiment of the present application, and is not intended to limit the present application in other forms. Any person skilled in the art can modify or alter the above disclosed technical content to equivalent embodiments. However, any simple modification, equivalent change and alteration of the above embodiments made according to the technical essence of the present application without departing from the technical solution of the present application still falls within the protection scope of the present application.

Claims

1. A method for detecting hexaconazole using ferrofluid extraction combined with high performance liquid chromatography, characterized in that: Includes the following steps: Step 1, Preparation of the eutectic solvent: A transparent liquid obtained by heating a mixture of monoterpene alcohols, medium-chain fatty acids, and short-chain fatty acids is the eutectic solvent; the monoterpene alcohols are ferrugin, citronellol, or carvacrol; the medium-chain fatty acids are hexanoic acid, heptanoic acid, or octanoic acid; and the short-chain fatty acids are formic acid, acetic acid, or propionic acid; the molar ratio of monoterpene alcohols, medium-chain fatty acids, and short-chain fatty acids is 1:(0.25~3):(1~6); the mixing and heating temperature is 60 ℃, and the heating time is 10 min; The second step is the preparation of the ferromagnetic fluid: the fluid obtained by vortexing iron oxide and the prepared eutectic solvent is the ferromagnetic fluid; the ratio of iron oxide to eutectic solvent is (2.5~150) mg: 1 mL; The third step is dispersion-liquid microextraction based on self-dispersing ferromagnetic fluid: The prepared sample solution is placed in a centrifuge tube, and the prepared ferromagnetic fluid is added to the sample solution to complete rapid extraction; a magnet is placed next to the centrifuge tube to attract the ferromagnetic fluid, then the supernatant is discarded, and the ferromagnetic fluid is eluted with methanol; the amount of ferromagnetic fluid added is 75~250 mL / 5 mL of sample solution; the amount of methanol added is 150 mL / 5 mL of sample solution. The fourth step involves analyzing the eluent of the ferromagnetic fluid using a high-performance liquid chromatograph (HPLC). A standard working curve is plotted with the mass concentration of chiral hexaconazole added as the x-axis and the corresponding peak area as the y-axis. The content of chiral hexaconazole in the unknown sample solution is then calculated.

2. The method according to claim 1, characterized in that: In the first step, the molar ratio of the monoterpene alcohol, medium-chain fatty acid, and short-chain fatty acid is 1:0.5:3; the monoterpene alcohol is ferrous alcohol, the medium-chain fatty acid is hexanoic acid, and the short-chain fatty acid is acetic acid.

3. The method according to claim 1, characterized in that: In the second step, the ratio of iron(III) oxide to the eutectic solvent is 50 mg: 1 mL.

4. The method according to claim 1, characterized in that: In the second step, the vortex time is 1 minute.

5. The method according to claim 1, characterized in that: In the third step, the amount of ferromagnetic fluid added is 175 mL / 5 mL of sample solution.

6. The method according to claim 1, characterized in that: In the fourth step, the high-performance liquid chromatography (HPLC) detection conditions are as follows: the chromatographic column is a cellulose-tris(3,5-dimethylphenylcarbamate) chiral column; the detector is a UV detector; the injection volume is 10 mL; the mobile phase is methanol and water in a volume ratio of 80:20; the flow rate is 0.4 mL / min; the detection wavelength is 220 nm; and the retention times of chiral hexaconazole are 17.2 and 18.5 min.

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