A method for simultaneously determining dodecyldimethylbenzyl ammonium chloride and its hydroxylated metabolites in plants

Abstract

The present invention discloses a method for simultaneously determining dodecyldimethylbenzylammonium chloride and its hydroxylated metabolites in plants, comprising the following steps: freeze-drying and low-temperature crushing and grinding of plant samples, adding recovery rate indicators and extractants, ultrasonic extraction, and low-temperature centrifugation to obtain an extract; the extract is purified and then centrifuged, an internal standard substance is added, and an extraction purification liquid is obtained after filtration; the extraction purification liquid is detected by ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry. Compared with the method of only determining the content of dodecyldimethylbenzylammonium chloride in the prior art, this method is simple and fast, and can simultaneously determine the content of dodecyldimethylbenzylammonium chloride and its hydroxylated metabolites in different parts of the plant, thereby more accurately evaluating the plant absorption, accumulation and metabolic behavior of dodecyldimethylbenzylammonium chloride.

Description

Technical Field

[0001] The invention relates to a detection method, in particular to a method for simultaneously determining dodecyldimethylbenzyl ammonium chloride and its hydroxylated metabolites in plants. Background Art

[0002] Quaternary ammonium compounds (QACs) are cationic organic compounds formed by replacing the hydrogen atoms in the ammonium group with alkane or aromatic groups. This type of compound is widely used in industry, agriculture and daily life, and has become a common pollutant in sewage treatment systems. In addition to being used as food preservatives and mineral flotation agents, QACs are also widely used in medical and health disinfection and cosmetics, and have potential human exposure risks. Dodecyldimethylbenzylammonium chloride (DBAC; CAS number: 139-07-1) is one of the QACs with the highest detection rate in the environment. DBAC has a high solubility in water and can have toxic effects on aquatic organisms at high concentrations, affecting the balance of aquatic ecosystems. In addition, long-term exposure or high-concentration contact may cause a series of health problems in humans, such as decreased fertility, lung cell damage and embryonic neural defects. Therefore, it is crucial to deeply study the distribution, behavior and potential ecological effects of DBAC in the environment.

[0003] At present, there is a lack of research on the absorption, accumulation and fate of DBAC in plants. After being absorbed by plants, DBAC can undergo hydroxylation under the action of oxidase to form corresponding hydroxylated products. The two monohydroxy-substituted metabolites are 7-hydroxydodecyldimethylbenzylammonium chloride (7-OH-DBAC) and 11-hydroxydodecyldimethylbenzylammonium chloride (11-OH-DBAC). DBAC and its hydroxylated metabolites in plants may threaten human health through the food chain. Therefore, it is urgent to develop a method that can simultaneously determine DBAC and its hydroxylated metabolites in order to accurately quantify their safety risks in agricultural products.

[0004] At present, there is no report on the detection method of DBAC hydroxylation metabolites in plants at home and abroad. The main problems of the determination method of DBAC in plants are complicated steps, large amount of solvent, high cost, etc., and it is impossible to take into account the determination of hydroxylation products at the same time. Therefore, in order to solve the problem of simultaneous determination of DBAC and its hydroxylation metabolites in plant samples, it is particularly important to develop a rapid, efficient and low-cost detection method. Summary of the invention

[0005] In view of the above technical problems, the present invention provides a method for rapid detection of DBAC and its hydroxylated metabolites in plants. The method comprises the following steps:

[0006] S1: freeze-drying and low-temperature crushing and grinding samples of different plant parts, adding recovery rate indicators and extractants, ultrasonic extraction, and low-temperature centrifugation to obtain extracts; the different plant parts are plant stems, leaves and roots;

[0007] S2: The extract is purified in a centrifuge tube containing a dispersed solid phase extractant, vortexed and centrifuged at low temperature, an internal standard substance is added, and the organic phase is filtered through a filter membrane to obtain an extract purification solution; the purification includes the following steps:

[0008] The extract is subjected to low-temperature high-speed centrifugation;

[0009] Add the centrifuged extract to the centrifuge tube containing the dispersed solid phase extractant and mix thoroughly for purification;

[0010] After vortex mixing, centrifuge at low temperature and high speed;

[0011] After adding the internal standard substance to the purified liquid after centrifugation, the purified liquid is filtered using an organic phase filter membrane to obtain an extracted purified liquid;

[0012] S3: The extracted purified liquid is detected by liquid chromatography-tandem triple quadrupole mass spectrometry.

[0013] Preferably, the hydroxylated metabolites of dodecyldimethylbenzylammonium chloride are 7-hydroxydodecyldimethylbenzylammonium chloride (7-OH-DBAC) and 11-hydroxydodecyldimethylbenzylammonium chloride (11-OH-DBAC).

[0014] Preferably, the extractant is an acetonitrile solution containing acetic acid, and the recovery indicator is deuterated tetradecyldimethylbenzylammonium-d7chloride (TBAC-d7; CAS No.: 1219178-72-9).

[0015] Preferably, the volume concentration of acetic acid in the acetonitrile solution containing acetic acid is 2%.

[0016] Preferably, the dispersed solid phase extractant includes anhydrous magnesium sulfate (CAS No.: 7487-88-9), graphitized carbon black (GCB; CAS No.: 1333-86-4) and N-propylethylenediamine (Primarysecondary amine, PSA; CAS No.: 111-39-7).

[0017] Preferably, the dispersed solid phase extractant comprises the following components in parts by weight: 7.5 parts of graphitized carbon black, 25 parts of N-propylethylenediamine, and 150 parts of anhydrous magnesium sulfate.

[0018] Preferably, the internal standard is tetrabutylammonium nitrate (TBAN; CAS No.: 1941-27-1).

[0019] Preferably, the conditions of the ultra-high performance liquid chromatography are as follows: two C18 liquid chromatography columns are connected in series, respectively used for environmental concentration enrichment and sample concentration analysis and detection, and the dimensions of the chromatographic columns are 1.8 μm×2.1 mm×50 mm and 1.8 μm×2.1 mm×100 mm, respectively; the column temperature is 40°C, the mobile phase A1 is a 0.1% formic acid aqueous solution, and the mobile phase B1 is acetonitrile.

[0020] Preferably, the mass spectrometry conditions are: ESI scanning mode is positive ion mode, spray voltage is +3000V, nebulizer gas temperature is 250°C, nebulizer gas flow rate is 6Lmin -1 , the drying gas temperature is 350℃, the drying gas flow rate is 10Lmin -1 ; Nebulizer pressure was 45 psi, electron multiplier voltage was 400 V; Multiple reaction monitoring mode: MRM mode.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. The present invention provides a pretreatment and mass spectrometry detection method for simultaneously determining a typical quaternary ammonium compound DBAC and its two hydroxylated metabolites in a plant matrix. The method of the present invention can accurately quantify the parent compound and the metabolites with a high recovery rate.

[0023] 2. The detection method of the present invention is applicable to plant stem, leaf and root samples. According to the pigment content in different parts of the plant, by optimizing the dosage of GCB, the pigment purification efficiency is excellent, the interference of matrix impurities such as pigments on instrument detection is reduced, and the accuracy of quantitative detection is improved.

[0024] 3. The detection method of the present invention greatly reduces the use of organic solvents in the entire pre-treatment process, generates less waste liquid, and is more in line with the concept of green environmental protection. In the pigment purification step, the matrix dispersed solid phase extraction filler is used to replace the traditional neutral alumina solid phase extraction column, thereby greatly reducing the cost of sample pre-treatment, and the pigment purification step is simpler, time-saving and easy to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0027] In the attached figure:

[0028] Figure 1 Figure 2 shows the MS spectra of DBAC and its two hydroxylated metabolites in (a) blank solvent (HPLC grade methanol), (b) plant stem and leaf matrix, and (c) plant root matrix. The spiked concentration of DBAC and its two hydroxylated metabolites was 10 μg L -1 . ;

[0029] Figure 2 Comparison of the color and state of the extracts of pumpkin stem samples before and after centrifugation using methanol and acetonitrile as the extractants in the extraction agent optimization experiment: (a) The state of the extraction system before centrifugation using methanol as the extractant; (b) The state of the extraction system before centrifugation using acetonitrile as the extractant; (c) The state of the extraction system after centrifugation using methanol as the extractant; (d) The state of the extraction system after centrifugation using acetonitrile as the extractant.

[0030] Figure 3Color comparison of pumpkin stem samples treated with different component dispersed solid phase extractors: (a) without pigment purification; (b) with pigment purification using PSA and anhydrous magnesium sulfate; (c) with pigment purification using GCB, PSA and anhydrous magnesium sulfate; DETAILED DESCRIPTION

[0031] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention all belong to the scope of protection of the present invention. Unless otherwise specified, the experimental materials, reagents, instruments, etc. used in the present examples are all commercially available; unless otherwise specified, all technical means in the examples of the present invention are conventional means well known to those skilled in the art.

[0032] Example 1

[0033] The present invention provides a method for simultaneously determining DBAC and its hydroxylated metabolites in plants, which specifically comprises the following steps:

[0034] Sample pretreatment process: After the plant samples are processed, they are separated into parts, freeze-dried, and ground into powder;

[0035] Sample extraction process: Accurately weigh 0.5 g of plant stem and leaf sample into a 50 mL centrifuge tube, add 4.5 mL of ultrapure water (or directly take 5 g of fresh plant sample), add 10 μL of recovery indicator TBAC-d7 (concentration 1.0 μg mL -1 ) and 20 mL of extract (acetonitrile extract containing 2% acetic acid, v / v) and 2.0 g of anhydrous magnesium sulfate, 0.5 g of sodium acetate, shake for several times, ultrasonically extract for 3 min, and centrifuge at low temperature (4500 rpm, 5 min, 4-6 ° C) to obtain an extract;

[0036] Purification process: Weigh the dispersed solid phase extraction reagent into a centrifuge tube, transfer the extract of the plant stem and leaf sample, vortex mix for 1 min, centrifuge at low temperature (4500 rpm, 5 min, 4-6 ° C), add 20 μL of internal standard TBAN (concentration 0.1 μg mL -1 ), vortexed again and passed through a 0.22 μm organic phase filter to obtain the purified extract, which was placed in a -20°C refrigerator for testing;

[0037] Ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS, Agilent 1290-6470B) was used for detection, and the internal standard method was used for quantitative analysis. The two liquid chromatography columns used were used to complete the enrichment and analysis processes respectively. The enrichment column model was Agilent's ZORBAX RRHD Eclipse Plus C18 column (1.8μm×2.1mm×100mm), and the enrichment column was externally connected to the pump box. The analytical column model was Agilent's ZORBAX RRHD Eclipse Plus C18 column (1.8μm×2.1mm×50mm), and the analytical column was connected to the column oven; the column temperature was 40°C, the mobile phase A1 was 0.1% formic acid water, and the mobile phase B1 was acetonitrile. The mobile phase gradient is shown in Table 1.

[0038] Table 1 LC-MS / MS elution gradient

[0039]

[0040] The mass spectrometry parameters were as follows: ESI scanning mode was positive ion mode, spray voltage was +3000 V, nebulizer gas temperature (GasTemp) was 350 °C, and nebulizer gas flow rate (Gas Flow) was 6 L min -1 , the sheath gas temperature (Sheath Gas Temp) is 300℃, and the sheath gas flow rate (Sheath Gas Flow) is 10L min -1 ; Nebulizer pressure was 45 psi, electron multiplier voltage (Delta Electron Multiplier Voltage, Delta EMV) was 400 V; Multiple reaction monitoring mode: MRM mode collision energy parameters are shown in Table 2. Data were processed by Agilent Quant One Way software.

[0041] Table 2 Mass spectrometry parameters of quaternary ammonium compound DBAC and its hydroxylated metabolites

[0042]

[0043] Example 2: Applicability Verification

[0044] This example uses pumpkin stem samples as a representative of plant stem and leaf samples, and conducts spike recovery experiments with different concentrations of DBAC and its hydroxylated metabolites to examine the applicability of this method. Specifically, the following steps are included:

[0045] Dilute the pollutant standard stock solution (μg mL -1), and a standard curve of DBAC and its hydroxylated metabolites was prepared (0.2, 0.4, 0.6, 0.8, 1, 2, 4, 6, 8, 10 μg L -1 ), with reference to Example 1, the LC-MS / MS was used for quantitative analysis by the internal standard method, and the standard curve of DBAC and its hydroxylated metabolites was calculated by linear regression. The instrument detection limits (IDLs) of DBAC and its hydroxylated metabolites were calculated as 3 times the signal-to-noise ratio of low-concentration samples, while the method detection limits (MDLs) were calculated as 5 times the relative deviation of the samples after 6 consecutive injections of low-concentration samples.

[0046] Select uncontaminated pumpkin stem samples (i.e. blank matrix samples), freeze-dry them and grind them for later use;

[0047] A blank matrix sample was pre-treated to obtain a blank matrix extraction and purification solution of plant stems and leaves. The specific steps were the same as in Example 1. The matrix extraction and purification solution was then used to dilute the DBAC and its hydroxylated metabolite standard stock solution (10 μg mL -1 ), and DBAC and its hydroxylated metabolites were spiked (10 μg L -1 ), LC-MS / MS was used for determination, and the specific steps of the determination were the same as those in Example 1. The selectivity of the method of the present invention was determined based on the separation of the three target substances in the obtained chromatogram and the degree of interference by matrix impurities.

[0048] Take the blank matrix sample of the plant and add DBAC, 7-OH-DBAC and 11-OH-DBAC standard stock solutions (10 μg mL -1 ), and different blank matrix spiked samples (50, 250 μg L -1 ), after pretreatment, LC-MS / MS was used for determination. The specific steps were the same as those in Example 1. Quantitative analysis was performed by the internal standard method. The recovery was expressed as the ratio of the concentration of the spiked sample to its spiked concentration. The precision was expressed as the relative standard deviation of the concentration of the spiked sample at the same concentration. Five parallel samples were set.

[0049] The results are shown in Tables 3 and 4. As shown in Table 3, DBAC and its hydroxylated metabolites have a high -1 Good linearity within the range (R 2 >0.998). The IDLs of DBAC and its hydroxylated metabolites were 20 ng L -1 (DBAC), 10ng L -1 (7-OH-DBAC) and 10 ng L -1The MDLs of DBAC and its hydroxylated metabolites in plant stem and leaf matrix samples were 18.60 ng g -1 Dry weight (DBAC), 9.89 ng g -1 Dry weight (7-OH-DBAC) and 8.60 ng g -1 Dry weight (11-OH-DDAC). It can be seen that the method of the present invention can determine the content of 11-OH-DDAC in plant stem and leaf matrix. -1 The detection of DBAC and its hydroxylated metabolites at high levels has high sensitivity.

[0050] Table 3 Linear range and detection limit of DBAC and its hydroxylated metabolites

[0051]

[0052]

[0053] Depend on Figure 1 As shown in the figure, DBAC and its hydroxylated metabolites can be effectively separated by retention time and characteristic ions (m / z) in blank solvent spiked samples and plant stem, leaf and root matrix spiked samples, and no chromatographic peaks interfering with the three target pollutants were detected in different matrix solution samples. It can be seen that the method of the present invention has good separation and selectivity.

[0054] As shown in Table 4, DBAC and its hydroxylated metabolites in plant stem and leaf matrix at different concentrations (50, 250 μg L -1 ) The recovery rate in the spiked samples was between 84.36% and 102.90%, and the relative standard deviation (RSD) ranged from 0.39% to 3.16%, which met the requirements of the international standard (DG345SANCO / 12459 / 2011) (recovery rate was between 70% and 120%, RSD ≤ 20%). It can be seen that the method of the present invention has good accuracy and precision.

[0055] Table 4 Recovery (n=3, %) and relative standard deviation (RSD, %) of DBAC and its hydroxylated metabolites spiked samples in plant matrices

[0056]

[0057] Example 3: Extractant Optimization

[0058] During the pretreatment of plant samples, a suitable extraction solvent is selected to efficiently extract the components to be tested through solid-liquid partition enrichment. In this embodiment, pumpkin stem samples are used as representatives of plant stem and leaf matrix samples. DBAC and its hydroxylated metabolites are spiked and recovered to evaluate the effects of different extraction agents and different extraction methods on sample recovery. The specific steps are as follows:

[0059] Select uncontaminated pumpkin stem samples, freeze-dry them, and grind them for later use;

[0060] The blank matrix was homogenized by adding ultrapure water, and the recovery indicator, DBAC, 7-OH-DBAC, and 11-OH-DBAC standard stock solutions were added;

[0061] Four treatment groups were set up, and equal volumes of extractants with different formulations were added respectively, T1: methanol, T2: 0.5% acidified methanol (acetic acid), T3: acetonitrile, T4: 0.5% acidified acetonitrile (acetic acid) for pretreatment to obtain the matrix extract of the pumpkin stem sample, and the specific steps were the same as in Example 1;

[0062] Complete the sample purification process of each treatment group, the specific steps are the same as in Example 1;

[0063] The concentration of the substance to be tested was determined by LC-MS / MS, and the specific steps were the same as in Example 1.

[0064] As shown in Table 5, the recovery rates of DBAC, 7-OH-DBAC, and 11-OH-DBAC in the treatment group with methanol as the extractant were 73.20% to 78.23%, 77.71% to 83.54%, and 69.39% to 73.55%, respectively. The recovery rates of the treatment group with acetonitrile as the extractant were 86.21% to 100.96%, 90.20% to 99.64%, and 86.04% to 98.81%, respectively. In addition, the addition of acetic acid had a positive effect on improving the recovery rate of DBAC and its two hydroxylated metabolites. Therefore, the use of acidified acetonitrile as an extractant has a better extraction effect on organic compounds with strong polarity (DBAC and its hydroxylated metabolites), and the impurity content in the extract is low.

[0065] Table 5: Effect of different extraction agents on the recovery of DBAC and its metabolites (n=3)

[0066]

[0067] During the extraction process, it was found that Figure 2As shown, using acetonitrile as the extractant (a) and adding anhydrous magnesium sulfate and sodium acetate, the organic solvent can be separated from the plant homogenate sample well, that is, the upper layer is a dark green extraction solution and the lower layer is a granular sediment. When methanol or acidified methanol reported in the literature is used as the extractant (b), after adding anhydrous magnesium sulfate and sodium acetate, the extraction solvent system as a whole is milky, and it is difficult to visually see the stratification phenomenon. After low-temperature high-speed centrifugation, although both acetonitrile (c) and methanol treatment group (d) can achieve solid-liquid separation, the volume of the solid phase layer of the methanol extractant treatment group is significantly larger than that of the acetonitrile extractant treatment group. The inorganic salts and plant homogenate in the methanol extractant treatment group form relatively fine particles, which may have a certain adsorption effect on the target compound, thereby resulting in a decrease in the recovery rate. In addition, by comparing the recovery rates of DBAC and its two hydroxylated metabolites in different extractant treatment groups, it can be determined that the best extractant is acidified acetonitrile.

[0068] Example 4: Optimization of purifier components and dosage

[0069] The pigments released during the extraction process of plant stem and leaf samples will interfere with liquid phase mass spectrometry detection. In this example, pumpkin stems were used as the test plant matrix. By conducting a spike recovery experiment of DBAC and its hydroxylated metabolites, the effects of different component purifier ratios and dosages on reducing the interference of exogenous pigments were evaluated. This example aims to determine the optimal purifier component and dosage to improve the accurate detection of DBAC and its hydroxylated metabolites. Specifically, the following steps are included:

[0070] Select uncontaminated pumpkin stem samples, freeze-dry them, and grind them for later use;

[0071] The blank matrix was homogenized by adding ultrapure water, and the recovery indicator, DBAC, 7-OH-DBAC and 11-OH-DBAC standard stock solutions were added, and then pre-treated to obtain the matrix extract of the plant stem sample, and the specific steps were the same as those in Example 1;

[0072] The spiked extract was added to the following 8 treatment groups: (1) T1: 25 mg PSA, 150 mg anhydrous magnesium sulfate; (2) T2: 50 mg PSA, 150 mg anhydrous magnesium sulfate; (3) T3: 100 mg PSA, 150 mg anhydrous magnesium sulfate; (4) T4: 150 mg PSA, 150 mg anhydrous magnesium sulfate; (5) T5: 25 mg PSA, 150 mg anhydrous magnesium sulfate, 7.5 mg GCB; (6) T6: 25 mg PSA, 150 mg anhydrous magnesium sulfate, 10 mg GCB; (7) T7: 25 mg PSA, 150 mg anhydrous magnesium sulfate, 15 mg GCB; (8) T8: 25 mg PSA, 150 mg anhydrous magnesium sulfate, 20 mg GCB; 3 replicates were set for each treatment.

[0073] The concentration of the substance to be tested was determined by LC-MS / MS, and the specific steps were the same as in Example 1.

[0074] As shown in Table 6, in the treatment groups (T1-T4) where the purifiers were PSA and anhydrous magnesium sulfate, the recoveries of DBAC, 7-OH-DBAC and 11-OH-DBAC were 104.55%-118.24%, 94.94%-110.77% and 96.14%-112.53%, respectively. It is not difficult to see that although the amount of PSA had no significant effect on the recovery of DBAC and its hydroxylated metabolites, the pigment purification process could not be completed by adding only anhydrous magnesium sulfate and PSA; in the treatment groups (T5-T8) where the purifiers were GCB, PSA and anhydrous magnesium sulfate, the recoveries of DBAC, 7-OH-DBAC and 11-OH-DBAC were 23.57%-98.52%, 41.41%-107.50% and 24.34%-94.78%, respectively. With the increase in the amount of GCB added, the recoveries of the target pollutants and the recovery indicator decreased significantly.

[0075] GCB is often used to adsorb pigments, polyphenols and other polar impurities. However, excessive GCB will affect the recovery rate of aromatic ring compounds. In this embodiment, by optimizing the proportion and dosage of the purifier, and comprehensively considering the pigment purification effect and the recovery rate results of each target pollutant, the optimal amount of dispersed solid phase extraction agent is determined to be: GCB 7.5mg, PSA 25mg, anhydrous magnesium sulfate 150mg.

[0076] Table 6: Effects of different dosage ratios of solid phase extractants on the recovery of DBAC and its metabolites (n = 3)

[0077]

[0078]

[0079] Pigment purification results are as follows Figure 3 As shown. The sample (a) without pigment purification is very dark and turbid, making it difficult to test on the machine. The sample (b) using only PSA and anhydrous magnesium sulfate for pigment purification is clear but still dark in color, indicating that there is still a lot of pigment left. The sample (c) using GCB, PSA and anhydrous magnesium sulfate is significantly lighter in color, indicating that the content of impurities such as pigments has been significantly reduced.

[0080] Example 5: Determination of actual contaminated plant samples

[0081] Wheat seedlings were exposed to artificial DBAC-contaminated nutrient solution to verify the effectiveness of the detection method of the present invention. The specific results are as follows:

[0082] The contaminated nutrient solution was artificially added and was divided into: (1) B1: concentration of 10 mg L -1 DBAC contamination, exposure for 24 h, blank culture for 24 h; (2) B2: concentration of 10 mg L -1 DBAC contamination, exposure for 24 h, blank culture for 72 h; (3) B3: concentration of 20 mg L -1 DBAC contamination, exposure for 24 h, blank culture for 24 h; (4) B4: concentration of 20 mg L -1 DBAC contamination was treated for 24 hours and then transferred to non-contaminated nutrient solution for 72 hours. Three replicates were set for each treatment, with three wheat plants in each replicate.

[0083] The wheat variety used in this experiment was Nannong 9918, and the seeds were purchased from Nanjing Agricultural University. Wheat seeds with full and uniform grains were selected and soaked for 2 hours, then disinfected with 5% NaClO solution for 10 minutes and washed thoroughly. The seeds were evenly spread in a sterilized culture dish. After 3 days of exposure to light, they were transferred to a pollutant-free 1 / 4 strength Kimura B nutrient solution for nutrient growth. When the seedlings were 14 days old, they were exposed to pollution. During the nutrient growth and pollution exposure period, they were placed in an incubator under the conditions of: 16 hours of light, 25°C, 8 hours of darkness, 20°C, and 60% humidity. After the exposure treatment, they were placed in a 10mM CaCl2 solution to remove the pollutants adsorbed on the surface, and the wheat was divided into aboveground and root samples for the determination of the content of DBAC and its hydroxylated metabolites.

[0084] Accurately weigh 0.5 g of wheat aerial part sample into a 50 mL centrifuge tube, add 4.5 mL of ultrapure water, add 20 mL of extract (acetonitrile extract containing 2% acetic acid, v / v) and 2.0 g of anhydrous magnesium sulfate, 0.5 g of sodium acetate, shake vigorously for several times, ultrasonically extract for 3 min, and centrifuge at low temperature (4500 rpm, 5 min, 4-6 ° C) to obtain the extract.

[0085] 1 mL of wheat aerial part extract was added into a 2 mL centrifuge tube containing dispersed solid phase extraction reagent (GCB 7.5 mg, PSA 25 mg, anhydrous magnesium sulfate 150 mg), vortexed at 2500 rpm for 1 min, and centrifuged at low temperature (4500 rpm, 5 min, 4-6 °C). 490 μL of the purified supernatant was transferred to 10 μL of internal standard TBAN (concentration 0.1 μg mL -1), vortexed again and then filtered through a 0.22 μm organic phase filter to obtain an extract and purification solution, which was placed in a -20°C refrigerator for testing; LC-MS / MS was used to measure the solution using 0.1% formic acid water and acetonitrile as the mobile phase. The results are shown in Table 6. DBAC, 7-OH-DBAC and 11-OH-DBAC were detected in the roots and aerial parts of DBAC-contaminated wheat, of which the DBAC concentration in the aerial parts of wheat was 0.58-32.16 mg g -1 Dry weight, 7-OH-DBAC concentration ranged from 0.15 to 3.22 mg g -1 Dry weight, 11-OH-DBAC concentration ranged from 0.09 to 2.86 mg g -1 Dry weight; DBAC concentration in wheat roots ranged from 30.94 to 140.89 mg g -1 Dry weight, 7-OH-DBAC concentration ranged from 1.64 to 14.34 mg g -1 Dry weight, 11-OH-DBAC concentration ranged from 1.36 to 5.89 mg g -1 In this experiment, DBAC and its hydroxylated metabolites were detected in both the aerial parts and root samples of plants, which proved that this method was sensitive and reliable.

[0086] Table 6 Concentrations of DBAC and its hydroxylated metabolites in different parts of wheat (μg g -1 Dry weight, data in the table are mean ± standard deviation, n = 3)

[0087]

[0088] It can be understood that the above embodiments only express the preferred implementation modes of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the patent scope of the present invention. It should be pointed out that, for ordinary technicians in this field, the above technical features can be freely combined without departing from the concept of the present invention, and several deformations and improvements can be made, which all belong to the protection scope of the present invention. Therefore, all equivalent changes and modifications made to the scope of the claims of the present invention should belong to the coverage of the claims of the present invention.

Claims

1. A method for simultaneously determining dodecyldimethylbenzylammonium chloride and its hydroxylated metabolites in plants, characterized in that: The hydroxylated metabolites of dodecyl dimethyl benzyl ammonium chloride are 7-hydroxy dodecyl dimethyl benzyl ammonium chloride and 11-hydroxy dodecyl dimethyl benzyl ammonium chloride; the process comprises the following steps: S1: freeze-drying and low-temperature crushing and grinding samples of different plant parts, adding recovery rate indicators and extractants, ultrasonic extraction, and low-temperature centrifugation to obtain extracts; the different plant parts are plant stems, leaves and roots; the extractant is an acetonitrile solution containing acetic acid, and the recovery rate indicator is deuterated tetradecyl dimethyl benzyl ammonium chloride; S2: the extract is purified in a centrifuge tube containing a dispersed solid phase extractant, vortexed and centrifuged at low temperature, an internal standard substance is added, and the organic phase is filtered through a filter membrane to obtain an extracted purified liquid; the purification comprises the following steps: the dispersed solid phase extractant comprises anhydrous magnesium sulfate, graphitized carbon black and N-propylethylenediamine; The extract is subjected to low-temperature high-speed centrifugation; Add the centrifuged extract, add a centrifuge tube containing a dispersed solid phase extractant, mix thoroughly and purify; the dispersed solid phase extractant includes anhydrous magnesium sulfate, graphitized carbon black and N-propylethylenediamine; After vortex mixing, centrifuge at low temperature and high speed; After adding the internal standard substance to the purified liquid after centrifugation, the purified liquid is filtered using an organic phase filter membrane to obtain an extracted purified liquid; S3: The extraction and purification liquid is detected by liquid chromatography-tandem triple quadrupole mass spectrometry; the conditions of the liquid chromatography are: a C18 enrichment column and a C18 analysis column; the mobile phase A1 is a 0.1% formic acid aqueous solution, and the mobile phase B1 is acetonitrile; the elution procedure of the mobile phase is: the B phase is 20% at the beginning, increased to 40% at 3 minutes, increased to 70% at 4 minutes, increased to 100% at 5 minutes and maintained for 1 minute, and decreased back to 20% at 6.5 minutes and maintained for 3 minutes; the flow rate is 300μLmin -1 The mass spectrometry conditions are as follows: the ESI scanning mode is positive ion mode, the electron multiplier voltage is 400 V; the multiple reaction monitoring mode is MRM mode.

2. The method according to claim 1, characterized in that: The volume concentration of acetic acid in the acetonitrile solution containing acetic acid is 2%.

3. The method according to claim 1, characterized in that: The dispersed solid phase extractant comprises the following components in parts by weight: 7.5 parts of graphitized carbon black, 25 parts of N-propylethylenediamine, and 150 parts of anhydrous magnesium sulfate.

4. The method according to claim 1, characterized in that: The internal standard is tetrabutylammonium nitrate.

5. The method according to claim 1, characterized in that The conditions of the liquid chromatography are as follows: the dimensions of the chromatographic columns are 1.8 μm×2.1 mm×50 mm and 1.8 μm×2.1 mm×100 mm respectively; and the column temperature is 40°C.

6. The method according to claim 1, characterized in that The mass spectrometry conditions were: spray voltage +3000 V, atomizing gas temperature 250 °C, atomizing gas flow rate 6 L min -1 , the drying gas temperature is 350℃, the drying gas flow rate is 10Lmin -1 ; Sprayer pressure is 45 psi.

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