Method for simultaneously detecting various pesticides and metabolites thereof in biological sample

Abstract

The invention discloses a method for simultaneously detecting various pesticides and metabolites thereof in a biological sample, which comprises the following steps: adding the biological sample into an acetonitrile aqueous solution containing a mixed isotope internal standard, uniformly mixing by vortex, carrying out centrifugal assisted cold induced phase separation, and taking an upper acetonitrile phase to obtain an on-machine detection sample; and analyzing and determining a plurality of pesticides and metabolites thereof in a sample to be detected on a machine by using LC-HRMS (liquid chromatography-high resolution mass spectrometry). According to the method, centrifugation-assisted cold induced phase separation (CA-CIPS) is combined with liquid chromatography and high-resolution mass spectrometry (LC-HRMS), a multi-target single ion monitoring (MSX-tSIM) mode is adopted, precise targeted detection analysis on 38 trace pesticides and metabolites thereof in various biological samples is achieved, and the method has good accuracy and precision and low limit of quantitation.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological sample detection, and particularly relates to a method for simultaneously detecting multiple pesticides and metabolites thereof in a biological sample. BACKGROUND

[0002] With the expansion of global agricultural production scale and the continuous increase of pesticide varieties, the risk of human exposure to pesticides and their metabolites through the environment and dietary approaches continues to rise. Long-term exposure to pesticides and their metabolites can cause a series of potential health risks, including neurotoxicity, endocrine disruption, and immune system damage. Therefore, accurate screening and monitoring of pesticide residues in the human body is particularly important. In recent years, HRMS has gradually become the mainstream analysis technology for efficient screening of pesticide residues due to its high sensitivity, high accuracy and powerful data acquisition capability. However, there are still many challenges in the accurate detection of trace pesticides in complex human biological matrices (such as blood, breast milk, etc.).

[0003] Current analysis methods mainly focus on detecting specific categories of pesticides. However, these methods are often specific and cannot fully address the scarcity and complexity of biological samples. The collection and processing of blood, urine and breast milk samples are not only expensive, but also severely limited in quantity. Notably, current detection methods often ignore metabolite analysis and lack isotope internal standards for quantitative correction, which may affect the accuracy and reliability of the results. With the continuous development of high-resolution mass spectrometry (HRMS), full scan mode shows excellent selectivity and quantitative ability. However, in ultra-trace detection, factors such as matrix effect and ion space effect may affect the accuracy of full scan mode detection.

[0004] Therefore, it is imperative and urgent to develop a universal sample preparation technology that maximizes the use of samples while comprehensively covering target analytes, achieving high sensitivity and high precision detection of more target substances. SUMMARY

[0005] To solve the above problems, the purpose of the present application is to provide a method for simultaneously detecting multiple pesticides and metabolites thereof in a biological sample, which can accurately and targetly detect 38 trace pesticides and metabolites thereof in multiple biological samples, and has good accuracy, precision and low limit of quantification.

[0006] The purpose of the present application is achieved by the following technical solutions:

[0007] A method for simultaneously detecting multiple pesticides and metabolites thereof in a biological sample, comprising the following steps:

[0008] (1) Biological sample pretreatment

[0009] The biological sample is added into an aqueous acetonitrile solution containing a mixed internal standard, vortexed and mixed, centrifugally assisted cold-induced phase separation is performed, the upper acetonitrile phase is taken, and a sample for machine detection is obtained;

[0010] (2) LC-HRMS is used to analyze and determine a plurality of pesticides and metabolites in the sample for machine detection;

[0011] The chromatographic conditions are as follows: an HSS T3 chromatographic column; organic phase A: methanol and aqueous phase B: 0.1% formic acid; elution program: 0-2 min, 2%-30% A; 2-4 min, 30% A; 4-9 min, 30%-50% A; 9-14 min, 50%-80% A; 14-16 min, 80% A; 16-19 min, 80%-100% A; 19-20.5 min, 100% A; 20.5-21 min, 100%-2% A; 21-24 min, 2% A;

[0012] The mass spectrometric conditions are as follows: a heated electrospray ionization source is used, and a multiple target single ion monitoring MSX-tSIM acquisition mode is used.

[0013] The biological sample is urine, serum or breast milk.

[0014] The pesticides and their metabolites according to the present application are acetamiprid (ACE), N-desmethylacetamiprid (ACE-DE), nitenpyram (NIT), dinotefuran (DIN), dinotefuran urea (DIN-urea), desulfinyl-dinotefuran (DIN-DN), imidacloprid (IMI), imidacloprid olefin (IMI-olefin), imidacloprid urea (IMI-urea), desulfinyl-imidacloprid (IMI-DN), 5-hydroxy-imidacloprid (OH-IMI), thiamethoxam (THI), N-desmethylthiamethoxam (THI-DE), thiamethoxam urea (THI urea), clothianidin (CLO), N-desmethylclothianidin (CLO-DE), clothianidin urea (CLO-urea), thiacloprid (THIA), thiacloprid amide (THIA-amide), cycloxaprid (CYC), imidaclothiz (IMIZ), 6-chloronicotinic acid (6-ClCN), fluopyram (FLU) and sulfoxaflor (SUL), fipronil (FIP), fipronil sulfone (FIP sulfone), fipronil sulfide (FIP sulfide), fipronil desulfinyl (FIP desulfinyl), fipronil amide (FIP amide), fipronil detrifluoromethyl sulfinyl (FIP detrifluoromethyl sulfinyl), ethiprole (ETH), ethiprole sulfone (ETH sulfone), flupyradifurone (FLUP), chlorantraniliprole (CHL), flubendiamide (FLUA), cyantraniliprole (CYA) and cyenopyrafen (CYCP), 4-hydroxy-chlorothalonil (4-OH CHT).

[0015] Preferably, in step (1), the freezing temperature in the centrifugal auxiliary cold-induced phase separation is -20 to -12℃, the centrifugal rotation speed is 10000-13000 rpm, and the centrifugal time is 15-30 min.

[0016] Preferably, in step (1), the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 2:3-4:1, preferably 1:1 v 3:2.

[0017] Preferably, in the chromatographic conditions of step (2), the flow rate is 0.25-0.3 mL / min, and the column temperature is 40-50℃.

[0018] Preferably, in step (2), the parameters of the heated electrospray ionization source are as follows: the capillary temperature is 320℃, the heating temperature is 400℃; the capillary voltage is set to 3800V and 3000V in positive and negative ion ESI modes, respectively; the sheath gas and auxiliary gas are set to 40arb and 10arb in ESI+ mode, and 50arb and 10arb in ESI- mode, respectively.

[0019] Preferably, in step (2), the MSX-tSIM acquisition parameters are set as: mass resolution of 70000 FWHM, AGC of 1x10 5 , isolation window of 5 Da, MSX of 10;

[0020] ddMS2 mode acquisition parameters are: mass resolution of 17500 FWHM, AGC of 2x104, TopN of 3, dynamic exclusion of 6s, stepwise NCE of 15, 35 and 55%.

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

[0022] The present application establishes a high-efficiency and high-sensitivity analysis method for simultaneously determining neonicotinoids, phenylpyrazoles, diacylhydrazines insecticides and chlorothalonil fungicides and their metabolites in biological samples (urine, serum or breast milk). The method combines centrifugal assisted cold-induced phase separation (CA-CIPS) with liquid chromatography and high-resolution mass spectrometry (LC-HRMS), adopts multi-target single ion monitoring (MSX-tSIM) mode, and ensures high sensitivity and accuracy. By optimizing the sample preparation parameters, the recovery rate of the analyte is improved and the matrix effect is minimized. The method has good accuracy, precision and low limit of quantification (LLOQ), and the recovery rate in various biological matrices is 72.6% to 109.6% in the range of 0.01 to 0.2 μg / L. The method has good linearity (R2>0.993) and is suitable for the detection of trace pesticide residues in complex biological samples. The method provides a cost-effective and environmentally friendly alternative to traditional biological monitoring methods, and is suitable for large-scale human biological monitoring research and pesticide exposure risk assessment. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Extraction recovery rates of representative analytes for four different extraction solvents;

[0024] Figure 2 TIC comparison analysis of CA-CIPS method (left) and traditional lipidomics (right) sample preparation method: (A) ESI positive ion analysis; (B) ESI negative ion analysis;

[0025] Figure 3 Distribution diagram of enrichment factor (A) and absolute recovery rate (B) of 38 analytes under different acetonitrile concentrations. DETAILED DESCRIPTION

[0026] The present application will be further described in detail by specific embodiments.

[0027] Example 1:

[0028] 1. Experimental materials and reagents

[0029] Milli-Q water purification system (Millipore, Bedford, USA). Methanol and formic acid (LCMS grade) were supplied by Fisher Scientific (California, USA). Acetonitrile, methanol, acetone, ethyl acetate and ammonium acetate (all HPLC grade) were purchased from Sigma-Aldrich (Shanghai, China).

[0030] 24 neonicotinoid pesticide standards, including acetamiprid (ACE), N- desmethylacetamiprid (ACE-DE), nitenpyram (NIT), dinotefuran (DIN), dinotefuran urea (DIN-urea), desnitro-dinotefuran (DIN-DN), imidacloprid (IMI), imidacloprid olefin (IMI-olefin), imidacloprid urea (IMI-urea), desnitro-imidacloprid (IMI-DN), 5-hydroxy-imidacloprid (OH-IMI), thiamethoxam (THI), N- desmethylthiamethoxam (THI-DE), thiamethoxam urea (THI urea), clothianidin (CLO), N- desmethylclothianidin (CLO-DE), clothianidin urea (CLO-urea), thiacloprid (THIA), thiacloprid amide (THIA-amide), cyantraniliprol (CYC), imidaclothiz (IMIZ), 6-chloronicotinic acid (6-ClCN), flupyradifurone (FLU) and sulfoxaflor (SUL), were supplied by Dr. Ehrenstorfer (Augsburg, Germany), Kanto Chemical Co., Inc. (Osaka, Japan) and Tianjin Alfa-Tech Co., Ltd. (Tianjin, China).

[0031] 9 phenylpyrazole insecticide standards, including fipronil (FIP), fipronil sulfone (FIP sulfone), fipronil sulfide (FIP sulfide), fipronil desulfinyl (FIP desulfinyl), fipronil amide (FIP amide), fipronil detrifluoromethyl sulfinyl (FIP detrifluoromethyl sulfinyl), ethiprole (ETH), ethiprole sulfone (ETH sulfone) and flupyradifurone (FLUP), and 4 diamide insecticide standards, including chlorantraniliprole (CHL), flubendiamide (FLUA), cyantraniliprole (CYA) and cyenopyrafen (CYCP), were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany) and Cambridge Isotope Laboratories (Massachusetts, USA).

[0032] Metabolite of the fungicide chlorothalonil, 4-hydroxy-chlorothalonil (4-OH CHT) standard solution (100 mg / L) was purchased from Dr. Ehrenstorfer (Augsburg, Germany).

[0033] 17 isotopically labeled standards, including CLO-D3, THI-D3, IMI-D4, ACE-D3, DIN-D3, THIA-D4, NIT-13C3 15 N2, 6-ClCN-13C6, ACE-DE-13C2 15 N, CLO-DE-13C 15 N2, IMI-olefin-13C 15 N2, THIA-amide-13C6, FIP-13C4 15 N2, FIP sulfone-13C4 15 N2, FIP sulfide-13C4 15 N2, FIP desulfinyl-13C4 15 N2and FIP detrifluoromethyl sulfinyl-13C4 15 N2, purchased from CDN Isotopes, Inc. (Pointe-Claire, Quebec, Canada), TRC (North York, Ontario, Canada) and Cambridge Isotope Laboratories (Andover, MA, USA).

[0034] Solid standard and stable isotope internal standard stock solutions: 0.005 g (accurate to 0.00001 g) of each of the solid standard and stable isotope internal standard was accurately weighed and dissolved in acetonitrile to a final volume of 50 mL. The concentration of each of the 38 pesticide and metabolite standards and stable isotope internal standard stock solutions was 100 mg / L.

[0035] Mixed standard and stable isotope internal standard intermediate solutions: 0.10 mL of each of the 38 standard stock solutions (100 mg / L) was accurately pipetted and diluted to a final volume of 10 mL with acetonitrile to make a mixed standard intermediate solution with a mass concentration of 1.0 mg / L. 0.10 mL of each of the 17 stable isotope internal standard stock solutions (100 mg / L) was accurately pipetted and diluted to a final volume of 10 mL with acetonitrile to make a mixed stable isotope internal standard intermediate solution with a mass concentration of 1 mg / L. Mixed standard and stable isotope internal standard working solutions: the mixed standard intermediate solution was accurately pipetted and diluted with acetonitrile to make a mixed standard working solution with a mass concentration of 0.10 mg / L. The mixed stable isotope internal standard intermediate solution was accurately pipetted and diluted with acetonitrile to make a mixed stable isotope internal standard working solution with a mass concentration of 0.10 mg / L.

[0036] An appropriate amount of mixed standard solution was accurately pipetted and diluted with 10% acetonitrile water to prepare standard series working solutions of different concentrations. The mass concentrations of pesticides and metabolites in the standard series working solutions were 0.005 μg / L, 0.01 μg / L, 0.02 μg / L, 0.05 μg / L, 0.10 μg / L, 0.20 μg / L, 0.50 μg / L, 1.00 μg / L, 2.00 μg / L, 5.00 μg / L, 10.0 μg / L, and the stable isotope internal standard concentration was 1.0 μg / L of mixed isotope internal standard. All solutions were stored in a -20 °C refrigerator.

[0037] 2. Sample pretreatment

[0038] 200 μL of serum, urine or breast milk was transferred to a 0.6 mL centrifuge tube. Then, 300 μL of acetonitrile containing mixed isotope internal standard (0.5 μg / L) was added, and vortexed for 30 s. Finally, the CA-CIPS operation was performed at -12 °C at 10000 rpm for 15 min using a centrifuge (Allegra 64R centrifuge, Beckman Coulter, Shanghai, China). After phase separation, the upper acetonitrile phase was transferred to a sample bottle for further instrument analysis.

[0039] 3. Liquid phase and mass spectrometry conditions

[0040] Determination was performed using a Dionex U3000 liquid chromatograph and a Thermo Q-Exactive high-resolution mass spectrometer detector (Thermo Fisher Scientific, USA).

[0041] Chromatographic conditions: The organic phase A: methanol and the aqueous phase B: 0.1% formic acid were used as the mobile phase, and the flow rate was constant at 0.3 mL / min. The HSS T3 chromatographic column (2.1 mm x 100 mm, 1.8 μm) (Waters, USA) was used to separate the pesticides at a column temperature of 40 °C, and the gradient elution program was as follows: 2% to 30% A (0-2 min), 30% A (2-4 min), 30% to 50% A (4-9 min), 50% to 80% A (9-14 min), 80% A (14-16 min), 80% to 100% A (16-19 min), 100% A (19-20.5 min), 100% to 2% A (20.5-21 min), 2% A (21-24 min). A 35 μL solvent mixer (Thermo, Germany, Lubeck) was inserted between the chromatographic column and the autosampler, and 5 μL of the sample solution was diluted with 0.3 mL of the initial mobile phase (98% aqueous solution) to reduce the solvent effect of the target analyte on the T3 chromatographic column.

[0042] Mass spectrometry conditions: The mass spectrometer was equipped with a heated electrospray ionization source, with a capillary temperature of 320 °C and a heated temperature of 400 °C. The capillary voltage was set to 3800 V and 3000 V in positive and negative ion ESI modes, respectively. The sheath gas and auxiliary gas were set to 40 arb and 10 arb in ESI+ mode, and 50 arb and 10 arb in ESI- mode, respectively. Multiplexed Targeted Selected Ion Monitoring (MSX-tSIM) mode was used for quantification of pesticides. The MSX-tSIM acquisition parameters were set as follows: mass resolution (70000 FWHM), AGC (1 x 105), isolation window (5 Da), and MSX (10).

[0043] In addition, the ddMS2 mode parameters for fragment acquisition of secondary mass spectrometry were as follows: mass resolution (17500 FWHM), AGC (2 x 104), TopN (3), dynamic exclusion (6 s), and stepped NCE (15, 35, and 55%).

[0044] For each compound, the parent ion was used for quantification, while the retention time, halogen isotope peaks, and fragment ions were used for confirmation. The retention time and ions monitored in MSX-tSIM / ddMS2 mode are shown in Table 1.

[0045] Table 1 Chemical properties and LC-HRMS information of target analytes

[0046]

[0047]

[0048]

[0049] 4. Method validation

[0050] The method was validated according to the SANTE (Standardisation, Analysis, Testing and Evaluation) / 11312 / 2021 (V2) guidelines to assess its method specificity, carryover, calibration curve, lower limits of quantification (LLOQ), accuracy (recovery), precision (repeatability and intermediate precision), and matrix effect.

[0051] Table 2 Method validation results for 38 pesticides

[0052]

[0053]

[0054] Table 2 shows the method validation results for all 38 analytes. The calibration curves for all analytes showed excellent linear relationships (R2> 0.993) in the solvent standard curve internal standard method. The method LLOQ for the 38 analytes in the biological samples ranged from 0.01 to 0.2 μg / L. 2 The average relative recoveries ranged from 72.6% to 102.4% at the LLOQ level with acceptable precision (Tables 3-5). The recoveries ranged from 72.6% to 109.6% at all spiked concentration levels in the three matrices. The intra-day precision ranged from 1.2% to 10.7% and the inter-day precision ranged from 1.4% to 13.5%. The overall matrix effect, recovery, and precision results for the method in this study are summarized in Table 2; detailed results data for each matrix are shown in Tables 3-5.

[0055] 5. Matrix effect, recovery, and precision investigation

[0056] The LLOQ for the 38 analytes in the biological samples ranged from 0.01 to 0.2 μg / L. The average relative recoveries ranged from 72.6% to 102.4% at the LLOQ level with acceptable precision (Tables 3-5). The recoveries ranged from 72.6% to 109.6% at all spiked concentration levels in the three matrices. The intra-day precision ranged from 1.2% to 10.7% and the inter-day precision ranged from 1.4% to 13.5%. The overall matrix effect, recovery, and precision results for the method in this study are summarized in Table 2; detailed results data for each matrix are shown in Tables 3-5.

[0057] Table 3 Matrix effect, recovery, and precision of 38 analytes in urine matrix at different spiked levels

[0058]

[0059]

[0060] Table 4 Matrix effect, recovery, and precision of 38 analytes in serum matrix at different spiked levels

[0061]

[0062]

[0063] Table 5 Matrix effect, recovery, and precision of 38 analytes in breast milk matrix at different spiked levels

[0064]

[0065]

[0066] Example 2: Optimization of the pre-treatment method

[0067] (1) Selection of extraction solvent

[0068] Methanol (log Kow= -0.77), acetonitrile (log Kow= -0.34), acetone (log Kow= -0.24), and ethyl acetate (log Kow= 0.73) were evaluated as extraction solvents. As shown in Table 1, methanol and acetonitrile were not suitable for the extraction of the 38 analytes.Figure 1 As shown, the extraction recoveries of polar pesticides and their metabolites were lower using ethyl acetate, while the recoveries of other solvents were higher, ranging from 92% to 111%, which were more excellent. This phenomenon can be attributed to the suitable interaction between these solvents and the analytes (XLogP3 values between -0.6 and 6.5).

[0069] Although all the tested solvents, such as acetone, methanol and acetonitrile, obtained satisfactory recoveries, the error values of acetone and ethyl acetate were relatively high, and the matrix effect of methanol was relatively large. Therefore, acetonitrile was selected as the extraction solvent in the present application.

[0070] (2) Optimization of acetonitrile aqueous solution concentration

[0071] For CA-CIPS technology, the extraction efficiency of pesticides varies with different proportions of acetonitrile / water system, and its effectiveness is usually evaluated by enrichment factor and absolute recovery. In this experiment, the enrichment factor and absolute recovery of different pesticides treated by CA-CIPS in 40%, 50%, 60%, 70% and 80% acetonitrile / water solution were studied. The distribution of enrichment factor and absolute recovery of 38 pesticides is shown in Figure 3

[0072] The results showed that the enrichment factor of 38 pesticides gradually increased with the continuous decrease of the proportion of acetonitrile / water. On the contrary, when the proportion of acetonitrile / water decreased to 40% and 50%, the absolute recoveries of 8 and 5 neonicotinoid pesticides were lower than 50%, respectively. When the proportion of acetonitrile / water reached 60%, the absolute recoveries of other analytes were all above 50% except DIN-DN (44.4%). This is mainly due to the relatively high polarity of these pesticides (XLogP3 values between -0.6 and 0.7), which makes the upper organic phase (88% acetonitrile) cannot effectively extract these pesticides from the lower aqueous phase (33% acetonitrile). In order to meet the requirements of detecting all different types of pesticides and their metabolites at the same time to the greatest extent, the compromise of 60% acetonitrile / water ratio was selected as the extraction condition in this study. Figure 3 As shown, when the proportion of acetonitrile / water was 60%, the absolute recoveries of 75% of pesticides were more than 70%.

[0073] (3) Optimization of centrifugal assisted CIPS method

[0074] ​Traditional lipidomics sample preparation method: 20 μL serum, urine or breast milk was transferred to a centrifuge tube, 120 μL methanol was added, vortexed for 180 s, 360 μL MTBE and 100 μL water were added, shaken for 10 min, and then centrifuged at 15000 g for 15 min at 4°C. 300 μL of the upper lipid extract was transferred to a 1 mL EP tube, and concentrated and dried under reduced pressure (Thermo Scientific, USA). The lipid extract was dissolved with 150 μL of acetonitrile-isopropanol mixture for instrument analysis.

[0075] CA-CIPS method: 200 μL of serum, urine or breast milk was transferred to a 0.6 mL centrifuge tube. Then, 300 μL of acetonitrile containing mixed internal standard (0.5 μg / L) was added, and vortexed for 30 s. Finally, CA-CIPS operation was performed at -12°C at 10000 rpm for 15 min using a centrifuge (Allegra 64R centrifuge, Beckman Coulter, Shanghai, China). After phase separation, the upper acetonitrile phase was transferred to a sample bottle for further instrument analysis.

[0076] The results, as shown in Figure 2 By lipidomics analysis of two different processing methods, the same position chromatographic peaks of triglycerides, glycerophospholipids and sphingolipids and other lipid components were significantly reduced or disappeared after CA-CIPS method processing, indicating that CA-CIPS method can effectively remove triglycerides, glycerophospholipids and sphingolipids and other lipid components.

[0077] Example 3: Optimization of chromatographic conditions

[0078] (1) Optimization of chromatographic column

[0079] In this study, the separation efficiency of 38 pesticides and their metabolites was studied using HSS T3 (2.1 mm x 100 mm, 1.8 μm) and BEH C18 (2.1 mm x 100 mm, 1.7 μm) two kinds of chromatographic column. The results showed that there was no significant difference in mass spectrometry response of the two kinds of chromatographic column to the target analytes. However, when using HSS T3 chromatographic column, the chromatographic retention of target analytes such as DIN with relatively high polarity was better, and was less affected by solvent effect. Therefore, HSS T3 chromatographic column was selected for subsequent analysis.

[0080] (2) Optimization of mobile phase

[0081] Firstly, the effects of methanol and acetonitrile as organic phase on the mass spectrometric response of target analytes in their respective ESI+and ESI-modes were investigated using ultrapure water as the aqueous phase. The results showed that the response values of most compounds were 1.2-3.7 times higher when methanol was used as the mobile phase than when acetonitrile was used. In addition, methanol as the mobile phase had better chromatographic separation effect on FIPs.

[0082] Subsequently, the chromatographic separation and mass spectrometric response of the mobile phase modifier were evaluated. It is worth noting that the acidic mobile phase system (0.1% formic acid) helps to improve the chromatographic retention behavior of polar pesticides such as 6-C1CN. And it helps to improve the mass spectrometric response of NEOs and FIPs in ESI+mode, such as SUL, FIP amide, ETH and ETH sulfone, etc., which can make their mass spectrometric response increase by 2-3 times. The addition of ammonium acetate in the mobile phase is not conducive to the mass spectrometric response of NEOs in ESI+mode, only the mass spectrometric response of 4-OH CHT is improved to some extent, which makes its response value increase by about 2 times. Therefore, methanol and 0.1% formic acid aqueous solution are determined as the mobile phase system for analyzing various types of pesticides.

[0083] (3) Optimization of gradient elution conditions

[0084] Gradient elution program 1: 0-9 min, 2%→50%; 9-19 min, 50%→100% A; 19-20.5 min, 100% A; 20.5-21 min, 100%→2% A; 21-24 min, 2% A;

[0085] Gradient elution program 2: 0-2 min, 2%→30% A; 2-4 min, 30% A; 4-9 min, 30%→50% A; 9-19 min, 50%→100% A; 19-20.5 min, 100% A; 20.5-21 min, 100%→2% A; 21-24 min, 2% A;

[0086] Gradient elution program 3: 0-2 min, 2%→30% A; 2-4 min, 30% A; 4-9 min, 30%→50% A; 9-14 min, 50%→80% A; 14-16 min, 80% A; 16-19 min, 80%→100% A; 19-20.5 min, 100% A; 20.5-21 min, 100%→2% A; 21-24 min, 2% A.

[0087] The results show that under the condition of gradient elution procedure 1, the peak time of 24 neonicotinoid pesticides is within 10 min, and the peak time of the other 14 pesticides is after 10 min, but the two types of pesticides overlap with each other in their respective retention time segments, which is not conducive to the subsequent MSX-tSIM collection, resulting in the lack of mass spectrum collection points; under the condition of gradient elution procedure 2, the chromatographic behavior of 24 neonicotinoid pesticides is further optimized, and good results are obtained, but 13 phenylpyrazole and bisamide pesticides exist in positive ion or negative ion detection in mass spectrometric detection, under the gradient elution procedure 2, the positive and negative ions overlap, resulting in the extension of the mass spectrum collection period in the mass spectrum collection process, which is not conducive to the subsequent accurate determination. Therefore, the gradient elution procedure is further adjusted, and the gradient elution procedure 3 is selected as the best gradient elution procedure, which can better solve the above problems.

[0088] Example 4: Establishment of multiple synchronous detection method

[0089] In this study, 38 compounds and 17 isotope internal standards need to be detected and analyzed at the same time, in order to ensure the reliability of the quantitative results. Based on the limitations of instrument parameter settings, the MSX-tSIM method designed in this application considers the following standards:

[0090] (1) In the method development process, avoid polarity switching in the same scan time. This is mainly because the polarity switching time of QExactive instrument is about 500 ms, which will cause the total cycle time to be too long, and the data points required for quantification cannot be collected. Therefore, ESI+ is used for all 25 NEOs in this study, although clothianidin and its metabolites have better mass spectrometric response in ESI- mode.

[0091] (2) Considering that many compounds in this study contain halogen elements, in order to obtain M +2 isotope peaks, the separation window of the quadrupole is set to 5 m / z in tSIM collection. Since the precursor ions in this isolation window will be filtered together, in the same scan event, only one precursor ion needs to be set for these compounds. For example, in the same scan event, the molecular weight difference between DIN-UF and DIN-DN is less than the isolation window (5 Da), so one of the two compound molecular ion peaks can be set.

[0092] (3) The cumulative number of compounds set as MSX parameters should be greater than the number of compounds in the same scan event. Considering that the maximum setting parameter of MSX in the Q Exactive instrument is 10, when the number of compounds in the same scan event is greater than 10, further ID grouping of MSX is required to achieve the smooth implementation of MSX-tSIM. For example, in the two scan events of 5.80-6.95 min and 14.90-17.00 min, the number of compounds exceeds 10, so it is necessary to group these MSX using ID. Therefore, in this study, the target substances will be grouped, and the isotopic internal standards will be assigned to the second group to achieve the normal operation of MSX-tSIM.

[0093] (4) There is no time overlap between each time scan event, otherwise the time overlap will cause the number of compounds in the time scan event to change, thereby affecting the final accurate quantification.

[0094] Considering these standards, the present application constructs an MSX-tSIM method with a multiplexing degree of 10, as shown in Table 6, 9 segmented scan windows are established.

[0095] Table 6 Specific setting parameters of MSX-tSIM

[0096]

[0097]

[0098]

Claims

1. A method for simultaneously detecting multiple pesticides and their metabolites in biological samples, characterized in that: The following steps are involved: (1) Biological sample pretreatment The biological sample is added to an acetonitrile aqueous solution containing a mixed internal standard, vortexed to mix, and subjected to centrifugal-assisted cold-induced phase separation. The upper acetonitrile phase is collected to obtain the sample for the instrument test; (2) LC-HRMS was used to analyze and determine multiple pesticides and their metabolites in the samples tested on the machine; The chromatographic conditions are as follows: HSS T3 column; organic phase A: methanol and aqueous phase B: 0.1% formic acid aqueous solution as mobile phases; elution program: 0-2 min, 2%-30% A; 2-4 min, 30% A; 4-9 min, 30%-50% A; 9-14 min, 50%-80% A; 14-16 min, 80% A; 16-19 min, 80%-100% A; 19-20.5 min, 100% A; 20.5-21 min, 100%-2% A; 21-24 min, 2% A; The mass spectrometry conditions were as follows: a heated electrospray ionization source and multiple targeted single ion monitoring MSX-tSIM / ddMS2 acquisition mode.

2. The method for simultaneous detection of multiple pesticides and their metabolites in biological samples according to claim 1, characterized in that: The biological sample is urine, serum or breast milk.

3. The method for simultaneous detection of multiple pesticides and their metabolites in biological samples according to claim 1, characterized in that: The various pesticides and their metabolites are acetamiprid, N-demethylacetamiprid, nitenpyram, dinotefuran, dinotefuran uron, denitrosodinotefuran, imidacloprid, imidacloprid olefin, imidacloprid uron, denitrosoimidacloprid, 5-hydroxyimidacloprid, thiamethoxam, N-demethylthiamethoxam, thiamethoxam uron, clothianidin, N-demethylthiamethoxam, clothianidin uron, thiamethoxam, thiamethoxam, thiamethoxam amide, cyclopyrid, chlorothiazolin, 6-chloronicotinic acid, flupyradone, flubendiamide, flubendiamide, flubendiamide sulfide, flubendiamide desulfenyl, flubendiamide, flubendiamide detrifluoromethylsulfenyl, ethiprole, ethiprole sulfone, fluopyram, chlorantraniliprole, flubendiamide, cyanantraniliprole, cyclomethicone, and 4-hydroxychlorothalonil.

4. The method for simultaneous detection of multiple pesticides and their metabolites in biological samples according to claim 1, characterized in that: In step (1), the freezing temperature in the centrifugal-assisted cold-induced phase separation is -20 to -12°C, the centrifugal speed is 10,000 to 13,000 rpm, and the centrifugal time is 15 to 30 min.

5. The method for simultaneous detection of multiple pesticides and their metabolites in biological samples according to claim 1, characterized in that: In step (1), the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 2:3 to 4:1, preferably 1:1 to 3:

2.

6. The method for simultaneous detection of multiple pesticides and their metabolites in biological samples according to claim 1, characterized in that: In the chromatographic conditions of step (2), the flow rate is 0.25-0.3 mL / min and the column temperature is 40-50°C.

7. The method for simultaneous detection of multiple pesticides and their metabolites in biological samples according to claim 1, characterized in that: In step (2), the parameters of the heated electrospray ionization source are as follows: the capillary temperature is 320°C, the heating temperature is 400°C; the capillary voltage is set to 3800V and 3000V in the positive and negative ion ESI modes, respectively; the sheath gas and auxiliary gas are set to 40arb and 10arb in the ESI+ mode, respectively, and to 50arb and 10arb in the ESI- mode, respectively.

8. The method for simultaneous detection of multiple pesticides and their metabolites in biological samples according to claim 1, characterized in that: In step (2), the MSX-tSIM acquisition parameters were set as follows: mass resolution 70000FWHM, AGC 1×10 5 , the isolation window is 5 Da, and the MSX is 10; The acquisition parameters of ddMS2 mode were: mass resolution 17500FWHM, AGC 2×10 4 , TopN is 3, dynamic exclusion is 6s, and the stepped NCE is 15, 35, and 55%.

Images