A high-throughput method for detecting pesticides and antibiotics in follicular fluid

Abstract

The application discloses a high-throughput detection method for pesticides and antibiotics in follicular fluid, which adopts saturated NaCl solution salting-out combined with acetonitrile containing 0.1% formic acid, utilizes the synergistic effect of salting-out and acidification to break the binding of target compounds and proteins in the high-protein complex matrix of follicular fluid, realizes one-step protein precipitation-extraction, and then performs quantitative analysis through dispersed solid-phase extraction purification and LC-MS / MS. Compared with the prior art, the application does not need enzymolysis overnight, and the pretreatment time is shortened to less than 1 hour; the external standard method is adopted for quantification, and there is no need to configure expensive isotope internal standards; for the high-protein complex matrix of follicular fluid, the binding of target compounds and proteins is broken through salting-out and acidification, and the average recovery rate of polar compounds such as neonicotinoids and carbamates is significantly improved. The method has the advantages of small sample consumption, simple operation, reliable precision and accuracy, and is suitable for trace analysis and detection of various pesticides and antibiotics in follicular fluid.

Description

Technical Field

[0001] This invention belongs to the field of environmental analysis and biological sample detection technology, specifically relating to a high-throughput detection method for pesticides and antibiotics in follicular fluid. Background Technology

[0002] Follicular fluid is a crucial microenvironment for oocyte development and maturation, containing energy substances, hormones, and signaling molecules, and plays a key role in female reproductive health and the developmental quality of the next generation. However, with the widespread use of pesticides and antibiotics in agriculture and medicine, pregnant women may be exposed to these exogenous chemicals through diet, environment, or drug treatment. Some chemicals can accumulate in follicular fluid, potentially affecting oocyte quality, embryo implantation, and offspring health. Therefore, establishing an efficient, sensitive, and high-throughput detection method for comprehensively assessing pesticide and antibiotic residue levels in follicular fluid is of great significance for revealing the reproductive risks of environmental pollutants.

[0003] The SCIEX Triple Quad 7500 system is a next-generation ultra-sensitive liquid chromatography-tandem mass spectrometry (LC-MS / MS) platform designed for multi-residue detection and trace analysis. This system offers higher detection sensitivity, a wider linear range, and a superior signal-to-noise ratio, enabling precise quantification of trace pesticides and antibiotics in complex matrices. Compared to traditional mass spectrometers, the Triple Quad 7500 employs more advanced ion source and detector technologies, effectively reducing matrix interference and improving method reliability and reproducibility, making it particularly suitable for complex biological matrices such as follicular fluid, which is rich in proteins and metabolites. This system allows for the simultaneous and rapid screening and quantification of multiple target compounds, providing strong technical support for high-throughput detection of pesticide and antibiotic residues in follicular fluid.

[0004] In the prior art, several pretreatment and analytical methods have been reported in literature and patents for the detection of multiple contaminants in follicular fluid. For example, patent application number 202510990920, "A Method for Detecting Multiple Contaminants in Follicular Fluid," discloses a detection strategy based on overnight enzymatic hydrolysis combined with organic solvent extraction. This method first mixes the follicular fluid sample with a mixed internal standard solution, then adds β-glucuronidase, and performs enzymatic hydrolysis by shaking at 37 °C for 16 hours to hydrolyze bound metabolites. The target analytes are then extracted multiple times using an acetonitrile solution containing 0.1% formic acid. After drying with nitrogen and reconstitution with acetonitrile / water, the sample is analyzed using a quadrupole liquid chromatography-mass spectrometry (LC-MS) system. While this method can detect multiple types of contaminants in follicular fluid, its long enzymatic hydrolysis time (requiring 16 hours overnight) results in a long pretreatment cycle and low efficiency. Furthermore, the quantification using the internal standard method requires the additional preparation of multiple isotope internal standard solutions, increasing the complexity and cost of the method. In addition, the solvent extraction step of this patent is repeated many times, which is cumbersome and consumes a lot of samples, making it unsuitable for high-throughput screening of small-volume precious samples.

[0005] Therefore, existing technologies still suffer from problems such as long enzymatic digestion time, complex internal standard systems, low pretreatment efficiency, and limited detection throughput. There is an urgent need to develop a new method for rapid screening of multiple pollutants in follicular fluid that does not require enzymatic digestion, is easy to operate, has reliable quantification, and high detection efficiency, in order to meet the needs of clinical and environmental exposure studies for high-throughput, multi-component analysis. Summary of the Invention

[0006] To address the aforementioned technical problems in existing technologies, the purpose of this application is to provide an analytical method for detecting the residues of various pesticides and antibiotics in follicular fluid. This method achieves accurate quantitative analysis of multiple compounds in follicular fluid through standard curve establishment, sample pretreatment, and liquid chromatography-tandem mass spectrometry (LC-MS / MS) detection.

[0007] The method of this invention employs external standard quantification, enabling simultaneous detection of multiple compounds. The pretreatment process utilizes the synergistic effect of saturated NaCl salting-out and formic acid acidification to disrupt the hydrogen bonds and electrostatic interactions between the target compounds and proteins in the complex high-protein matrix of follicular fluid. Combined with dispersed solid-phase extraction purification technology, this significantly improves the recovery rate and purification efficiency of polar compounds. The detection step uses a high-sensitivity mass spectrometry platform to achieve high-throughput detection of trace amounts and multiple types of contaminants in follicular fluid.

[0008] The technical solution adopted in this invention is: A high-throughput detection method for pesticides and antibiotics in follicular fluid includes the following steps: Step 1: After thawing the follicular fluid, add a saturated NaCl aqueous solution and acetonitrile containing 0.05-1% formic acid by volume. Utilize the synergistic effect of salting out by saturated NaCl and acidification by formic acid to break the hydrogen bonds and electrostatic interactions between the target compound and the protein in the complex high-protein matrix of the follicular fluid, achieving one-step protein precipitation-extraction. After thorough mixing, centrifuge to separate the supernatant. Blow the supernatant to near dryness with nitrogen, and then reconstitute it with a solvent. Step 2: The complex solution obtained in Step 1 is subjected to dispersion solid-phase extraction purification treatment, and then solid-liquid separation is performed to obtain the test solution that can be used for LC-MS / MS analysis; Step 3: The test solution obtained in Step 2 is quantitatively detected using LC-MS / MS technology.

[0009] Further, in step 1, the volume ratio of the thawed follicular fluid to the saturated NaCl aqueous solution is 1:0.8-2, preferably 1:1-1.5; the volume of acetonitrile in step 1 is 2-6 times the volume of the thawed follicular fluid, preferably 3-4 times.

[0010] Further, in step 1, the volume fraction of formic acid in the acetonitrile is 0.05-1%, preferably 0.1%; under this acidification condition, the average recovery rate of polar compounds (neonicotinoids, antibiotics) in the follicular fluid is not less than 85%.

[0011] Furthermore, in step 1, saturated NaCl salting-out and formic acid acidification work synergistically to the high-protein matrix of follicular fluid. Compared with the condition of using only acetonitrile extraction (salt-free control), the recovery rate of neonicotinoids and antibiotic polar compounds is increased by 40%-50%, and the average recovery rate of 10 representative compounds is increased from 53.8% to over 89.4% (see Example 2, Table 3).

[0012] Furthermore, the solvent used for resolution in step 1 is a methanol-water mixture, with a methanol-water volume ratio of 1:0.5-2, preferably 1:1.

[0013] Further, in step 2, the dispersion solid-phase extraction purification process involves transferring the complex solution into a dSPE tube pre-filled with C18 packing material, PSA packing material, and anhydrous MgSO4 for purification. After thorough vortex mixing, the supernatant is collected by centrifugation. The supernatant is then filtered through a 0.22 µm organic filter membrane to obtain the test solution. The dSPE tube does not contain graphitized carbon black (GCB) to avoid irreversible adsorption loss of aromatic target compounds (cypermethrin, bifenthrin, etc.).

[0014] Furthermore, in the dispersed solid-phase extraction purification system, the amount of C18 packing is 40-60 mg, the amount of PSA packing is 40-60 mg, the amount of anhydrous MgSO4 is 120-180 mg, and it does not contain graphitized carbon black GCB.

[0015] Further, in step 3, the detection conditions for LC-MS / MS technology are as follows: the chromatographic column is a Pfimek Kinetex F5 pentafluorophenyl reversed-phase column (1.7 µm, 2.1 × 150 mm), which is used to simultaneously separate target compounds with polarity differences spanning five orders of magnitude, achieving simultaneous high-throughput detection of pesticides and antibiotics; mobile phase A is 0.2 wt% formic acid aqueous solution, mobile phase B is methanol, and the flow rate is 0.3 mL / min; the column temperature is 30–40℃; gradient elution conditions are: 0–2.0 min, 90% A; 2.0–7.0 min, 90%→25% A; 7.0–7.2 min, 25%→5% A; 7.2–9.1 min, 5% A; 9.1–11.0 min, 5%→90% A; 11.0–12.0 min, 90% A; mass spectrometry conditions are: electrospray ionization (ESI), positive ion mode, and multiple reaction monitoring (MRM) mode.

[0016] Furthermore, the detection method of the present invention also includes the preparation of a standard curve, the steps of which are as follows: S1: Prepare standard stock solutions of the target pesticide and antibiotic standards using hexane as a solvent; S2: Take each standard stock solution and prepare a mixed pesticide-antibiotic standard stock solution using acetonitrile as a solvent; Select a follicular fluid sample that has been pre-screened as "not detected" as a matrix blank, and complete the blank extraction and reconstitution according to the pretreatment method in step 1 to obtain a matrix extract that is completely consistent with the matrix composition of the actual sample; Use the matrix extract as a solvent to gradually dilute the mixed pesticide-antibiotic standard stock solution to prepare matrix-matched standard solutions of different concentrations; S3: Analyze using LC-MS / MS, and establish a matrix-matched standard curve using the external standard method to quantitatively calculate the residual amount of the corresponding target compound in the sample.

[0017] Furthermore, the mixed pesticide-antibiotic concentration range of the multiple matrix-matched standard solutions is 1-100 ng / mL, covering the trace detection range of pesticides and antibiotics in actual follicular fluid samples (limit of quantitation 0.3-1.5 ng / mL).

[0018] Furthermore, the pesticides and antibiotics detected by this invention include at least one of neonicotinoids, carbamates, organophosphates, and triazoles, and at least one of antibiotics; the pesticides and antibiotics are detected simultaneously under the same pretreatment process and the same chromatographic-mass spectrometry analysis conditions; preferably, they include at least one of thiamethoxam, imidacloprid, carbofuran, aldicarb, chlorpyrifos, dichlorvos, cypermethrin, bifenthrin, ciprofloxacin, and sulfadiazine.

[0019] The detection method of this invention can be well applied in the following fields: follicular fluid quality assessment in assisted reproductive technology; research on the impact of environmental exposure on reproductive health; and monitoring of pesticide and antibiotic residue levels in follicular fluid.

[0020] Compared with the prior art, the beneficial effects achieved by this application are: 1) Pre-processing time is significantly reduced: This invention employs a one-step extraction method combining saturated NaCl salting-out with acidified acetonitrile, eliminating the need for overnight enzymatic hydrolysis. The pretreatment time is reduced from 16+ hours in existing technologies to ≤1 hour, significantly improving detection efficiency and making it suitable for rapid screening of clinical samples.

[0021] 2) The recovery rate of polar compounds is significantly improved: To address the complex, high-protein matrix of follicular fluid, this invention utilizes the synergistic effect of saturated NaCl salting-out and 0.1% formic acid acidification to disrupt the hydrogen bond / electrostatic interactions between the target compound and the protein, promoting the release of protein-bound compounds. Experiments demonstrate that, compared to the salt-free control group (average recovery rate 53.8%), the saturated NaCl solution group showed a 40-50 percentage point increase in the recovery rate of polar compounds such as neonicotinoids and antibiotics, with an average recovery rate of 89.4%, significantly superior to other salting-out agents (MgSO4, Na2SO4).

[0022] 3) Simplified quantitative system, smaller sample volume, and reduced cost: This invention employs an external standard method for quantification, eliminating the need for expensive isotope internal standards, significantly reducing detection costs and methodological complexity. Simultaneously, matrix effects are effectively corrected through matrix-matched standard curves, ensuring quantitative accuracy. Only 100-200 µL of sample is required, making it suitable for batch analysis of valuable clinical samples. The dSPE purification step is rapid and efficient, avoiding the cumbersome operations of traditional solid-phase extraction (SPE).

[0023] 4) Broad coverage and scalability for biological exposure-risk research: The method of this invention enables simultaneous high-throughput detection of pesticides and antibiotics under the same pretreatment process and chromatographic-mass spectrometry conditions, covering multiple pesticide groups such as neonicotinoids, carbamates, organophosphates, and triazoles, as well as antibiotics. Candidates can be rapidly added according to research needs. It is suitable for rapid screening and quantification of multiple exogenous chemicals in follicular fluid, providing methodological support for obtaining evidence of reproductive-related health risks. Detailed Implementation

[0024] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0025] Reagents and materials: acetonitrile (LC-MS grade), methanol (LC-MS grade), formic acid (analytical grade), saturated sodium chloride aqueous solution (25℃), anhydrous magnesium sulfate (MgSO4), 0.22 µm organic filter membrane.

[0026] Example 1: LC-MS / MS Detection Conditions and Methodology Validation 1.1 Sample Pretreatment Follicular fluid samples were obtained from the Department of Obstetrics and Gynecology, Zhejiang Provincial People's Hospital (with ethical approval and participant authorization). After collection, the samples were frozen at -80℃ and thawed in an ice bath protected from light before analysis. Pretreatment was performed as follows: Step 1: Add 200 µL of follicular fluid to a 2 mL screw-top centrifuge tube, along with 200 µL of saturated NaCl solution and 800 µL of acetonitrile (containing 0.1% formic acid by volume). Utilize the synergistic effect of salting out with saturated NaCl and acidification with formic acid to disrupt the hydrogen bonds and electrostatic interactions between the target compound and proteins in the complex high-protein matrix of the follicular fluid. Vortex thoroughly for 2 min, then sonicate for 10 min under ice bath conditions, and centrifuge at 4 ℃ and 12000 r / min for 10 min. Transfer the supernatant to a clean tube and purge to near dryness with nitrogen at 40 ℃; subsequently, reconstitute with 200 µL of methanol / water (1:1, v / v) and vortex for 30 s.

[0027] Step 2: The reconstituted solution obtained in Step 1 is transferred into a dSPE tube pre-filled with C18 packing (50 mg) / PSA packing (50 mg) / anhydrous MgSO4 (150 mg). After vortexing for 1-2 min, it is centrifuged at 12000 r / min for 5 min. The supernatant is then filtered through a 0.22 µm organic filter membrane to obtain the test solution that can be used for LC-MS / MS analysis. LC-MS / MS analysis is then performed.

[0028] 1.2. LC-MS / MS conditions Chromatographic analysis was performed using a Kinetex F5 column (1.7 µm, 2.1 × 150 mm) at a column temperature of 35 °C and a mobile phase flow rate of 0.30 mL / min. Mobile phase A was 0.2% formic acid aqueous solution, and mobile phase B was methanol. The gradient elution program was as follows: 0–2.0 min, maintaining 90% A; 2.0–7.0 min, linearly decreasing from 90% A to 25% A; 7.0–7.2 min, rapidly decreasing from 25% A to 5% A; 7.2–9.1 min, maintaining 5% A; 9.1–11.0 min, linearly increasing from 5% A to 90% A; ending at 11.0 min and performing column equilibration. Mass spectrometry detection was performed using an electrospray ionization (ESI) source in positive ion mode, with multiple reaction monitoring (MRM) as the acquisition method. The spray voltage was set to 4.5 kV, and the ion source temperature was controlled at 300–350 °C.

[0029] 1.3 Preparation of standard solutions and matrix matching standard curve S1: Accurately weigh each of the target pesticide and antibiotic standards shown in Table 1, and prepare a single standard stock solution of 1.0 mg / mL with n-hexane; inject each single standard stock solution into the same volumetric flask, dilute to the mark with acetonitrile, and prepare a mixed standard stock solution (the concentration of each compound is 5.0 mg / L), and store at -20℃.

[0030] S2: Select several follicular fluid samples that were pre-screened as "not detected" and combine them into a matrix blank. Perform blank extraction and reconstitution according to steps 1 and 2 in Section 1.1 to obtain a matrix extract with a matrix composition completely consistent with the actual sample matrix. Using this matrix extract as a solvent, prepare matrix-matched standard solutions of 8 concentrations (compound concentrations: 1, 2, 5, 10, 20, 40, 50, 100 ng / mL) by serially diluting the mixed standard stock solution. Perform LC-MS / MS analysis and detection in ascending order of concentration, and record the MRM peak area of ​​each target analyte. Plot a calibration curve with the concentration (ng / mL) of each compound in the mixed standard working solution as the x-axis and the corresponding peak area as the y-axis, using weighted least squares (1 / x)... 2 The regression yields the linear regression equation and the correlation coefficient.

[0031] 1.4 Methodological Validation 1.4.1 Linear Range and Correlation Coefficient Ten representative compounds showed good linearity in the range of 1-100 ng / mL, with correlation coefficients r ≥ 0.995 (see Table 1).

[0032] 1.4.2 Limit of Detection and Limit of Quantification The limits of detection (LOD) and limits of quantitation (LOQ) were calculated using signal-to-noise ratios (S / N) of 3 and 10, respectively. The LOD for representative compounds was 0.1–0.5 ng / mL, and the LOQ was 0.3–1.5 ng / mL (see Table 1).

[0033] 1.4.3 Spiking Recovery and Precision Recovery experiments were performed in a matrix blank at three spiking levels (1, 10, and 100 ng / mL). Blank extraction and reconstitution were performed according to steps 1 and 2 of Section 1.1 above, and LC-MS / MS conditions were applied. Each level was measured in triplicate. The recoveries of representative compounds ranged from 85.1% to 95.2%, with relative standard deviations (RSDs) of 4.2% to 14.8% (see Table 1).

[0034] Table 1. Linear range, correlation coefficient, accuracy, precision, and limit of quantitation. .

[0035] 1.5 Actual Sample Testing and Application Validation To verify the practical application performance of this method, ten clinical follicular fluid samples (from the Department of Obstetrics and Gynecology, Zhejiang Provincial People's Hospital) were selected and subjected to blank extraction and reconstitution followed by steps 1 and 2 in Section 1.1 above, and then analyzed under LC-MS / MS conditions. The results showed that different types of pesticide and antibiotic residues were detected in multiple samples, with concentrations ranging from 0.18 to 51.13 ng / mL. Specific detection results are shown in Table 2. The results indicate that the method of this invention performs well in the analysis of complex biological matrix samples, effectively detecting trace pesticide and antibiotic residues in follicular fluid, demonstrating the practicality and reliability of the method.

[0036] Table 2. Detection results of representative compounds in follicular fluid samples (ng / mL) ; Note: nd indicates not detected.

[0037] Example 2: Optimization of salting-out conditions for pretreatment method. The purpose of the experiment was to compare the effects of different salting-out agents on the recovery rate of the target compound and to screen the optimal salting-out conditions.

[0038] Several follicular fluid samples that were pre-screened as "not detected" were selected and combined into a blank follicular fluid matrix.

[0039] The experimental method involved selecting 10 representative compounds shown in Table 3. Each representative compound was spiked into a blank follicular fluid matrix (final spike concentration 10 ng / mL). The following four experimental groups were used for pretreatment (all groups included subsequent dSPE purification steps): ① Group A (salt-free control): directly extracted with acetonitrile (0.1% formic acid). The sample pretreatment steps were repeated in Example 1, except that "in step 1 of the sample pretreatment in section 1.1, the addition of saturated NaCl solution was omitted". The final sample solution was used for LC-MS / MS analysis.

[0040] ②Group B (saturated NaCl): The sample pretreatment steps are the same as in Example 1.

[0041] ③ Group C (saturated MgSO4): The sample pretreatment steps are the same as in Example 1, except that "the saturated NaCl solution is replaced with an equal volume of saturated MgSO4 solution".

[0042] ④ Group D (saturated Na2SO4): The sample pretreatment steps are the same as in Example 1, except that "the saturated NaCl solution is replaced with an equal volume of saturated Na2SO4 solution".

[0043] The test solutions obtained from the above four groups of experiments were analyzed and detected by LC-MS / MS according to the method in Example 1, and quantitative calculations were performed according to the standard curve prepared in Example 1.

[0044] The above four groups of experiments were performed in parallel six times each. The average recovery rate and relative standard deviation were calculated, and the results are shown in Table 3.

[0045] Table 3. Effects of different salting-out agents on the recovery rate of the target compound. .

[0046] Analysis of the results in Table 3 shows that the average recovery rate of the salt-free control group was only 53.8%, with the recovery rates of polar compounds (such as neonicotinoids and antibiotics) generally below 50%. After adding saturated NaCl, the average recovery rate increased to 89.4%, significantly better than other salting-out conditions (p<0.01). Furthermore, MgSO4 and Na2SO4 had similar effects, but overall, they were still inferior to NaCl. In summary, NaCl had the most significant "salting-out enhancement" effect on polar compounds, increasing their recovery rate by approximately 40-50 percentage points. The conclusion is that saturated NaCl solution is the optimal salting-out agent.

[0047] Example 3: Optimization of acidification conditions for pretreatment method. The purpose of the experiment was to optimize the formic acid concentration in the extraction solvent and balance the recovery rate and matrix effect.

[0048] Several follicular fluid samples that were pre-screened as "not detected" were selected and combined into a blank follicular fluid matrix.

[0049] The experimental method involved selecting 10 representative compounds shown in Table 4. Each representative compound was spiked into a blank follicular fluid matrix (final spike concentration 10 ng / mL). The following six experimental groups underwent pretreatment, with the sample pretreatment steps repeating those in Example 1. The only difference was that in step 1 of the sample pretreatment in section 1.1, the acid composition of the acetonitrile (containing 0.1% formic acid by volume) was changed; all other conditions remained the same. The pretreatment for the following six experimental groups involved salting out with saturated NaCl followed by extraction with acetonitrile under different acidification conditions: Group E contains pure acetonitrile (acid-free); Group F contains acetonitrile with 0.05% formic acid; Group G contains acetonitrile with 0.1% formic acid; Group H contains acetonitrile with 0.5% formic acid; Group I contains acetonitrile with 1% formic acid; and Group J contains acetonitrile with 0.1% acetic acid.

[0050] The test solutions obtained from the above 6 groups of experiments were analyzed and detected by LC-MS / MS according to the method in Example 1, and quantitative calculations were performed according to the standard curve prepared in Example 1.

[0051] The above 6 groups of experiments were performed in parallel 6 times each. The average recovery rate and relative standard deviation were calculated, and the results are shown in Table 4.

[0052] Table 4. Effect of different acidification conditions on the recovery rate of the target compound ; Note: * indicates that carbamates tend to degrade under strong acid conditions.

[0053] Analysis of the results in Table 4 shows that the average recovery rate under acid-free conditions (Group E) was 77.6%, with relatively low recoveries of polar compounds. The highest average recovery rate (89.4%) was achieved with the addition of 0.1% formic acid (Group G), and the recovery rates for different types of compounds were more balanced. In contrast, strong acid conditions (Groups H and I) reduced the recovery rate of carbamate compounds (such as carbofuran and aldicarb), suggesting a potential risk of acid-catalyzed degradation. The overall performance of the acetic acid system (Group J) was slightly inferior to that of the 0.1% formic acid group. The conclusion is that acetonitrile containing 0.1% formic acid is the optimal extraction solvent.

[0054] Example 4: Comparison of purification materials for pretreatment methods. The purpose of the experiment was to optimize the combination of dSPE purification materials and balance the target analyte recovery rate and matrix removal effect.

[0055] Several follicular fluid samples that were pre-screened as "not detected" were selected and combined into a blank follicular fluid matrix.

[0056] The experimental method involves selecting 10 representative compounds shown in Table 5. Each representative compound is spiked into a blank follicular fluid matrix (final spike concentration 10 ng / mL). The following four sets of experiments are used for pretreatment: Group K C18 alone (100 mg): The sample pretreatment steps are repeated in Example 1, except that "in step 2 of section 1.1, the dSPE tube is pre-filled with C18 packing (100 mg)", and the other conditions remain unchanged, and the final solution is a test solution that can be used for LC-MS / MS analysis.

[0057] Group L PSA alone (100 mg): The sample pretreatment steps are repeated in Example 1, except that "in step 2 of section 1.1, the dSPE tube is pre-filled with PSA packing material (100 mg)", and the other conditions remain unchanged, and the final solution can be used for LC-MS / MS analysis.

[0058] Group M C18 / PSA / MgSO4 combination (50 / 50 / 150 mg): The sample pretreatment steps were repeated in Example 1.

[0059] Group N contains GCB combinations (C18 / PSA / GCB / MgSO4, 50 / 50 / 50 / 150 mg): The sample pretreatment steps are the same as in Example 1, except that "in step 2 of section 1.1, 50 mg of GCB is further added to the dSPE tube", and the other conditions remain unchanged, finally obtaining the test solution that can be used for LC-MS / MS analysis.

[0060] The test solutions obtained from the above four groups of experiments were analyzed and detected by LC-MS / MS according to the method in Example 1, and quantitative calculations were performed according to the standard curve prepared in Example 1.

[0061] The above four sets of experiments were performed in parallel six times each. The average recovery rate and relative standard deviation were calculated, and the results are shown in Table 5. The recovery rate of representative compounds was determined, and the phospholipid removal rate was evaluated (by monitoring characteristic phospholipid ions by LC-MS / MS).

[0062] Table 5. Effects of different purification materials on the recovery rate of the target compound .

[0063] Analysis of the results in Table 5 shows that using C18 packing alone resulted in significant adsorption loss of polar compounds and limited phospholipid removal (62.3%). While PSA alone exhibited good phospholipid removal (88.5%), it led to some loss of alkaline compounds. In contrast, the C18 / PSA / MgSO4 combination achieved the best overall performance, simultaneously maintaining both target analyte recovery (87.6%) and phospholipid removal (91.2%). Although the combination containing GCB slightly improved phospholipid removal (94.8%), it resulted in significant adsorption loss of aromatic compounds (such as cypermethrin and bifenthrin), causing the average recovery rate to drop to 72.3%. Therefore, the C18 / PSA / MgSO4 combination (50 / 50 / 150 mg) was selected as the optimal purification material, ensuring both high target analyte recovery and effective removal of matrix interference.

[0064] Example 5: Comparison with existing technology (enzymatic hydrolysis method) Experimental objective: To compare the differences between the method of this invention and the enzymatic hydrolysis method disclosed in patent application number 202510990920 in terms of pretreatment time, recovery rate and operational complexity.

[0065] The experimental method is as follows: The sample pretreatment of the method of the present invention is carried out under optimal conditions, that is, extraction is performed using saturated NaCl solution and acetonitrile containing 0.1% formic acid, and purification is performed using dSPE pre-loaded with C18 / PSA / MgSO4. For specific treatment steps, please refer to section 1.1 of Example 1.

[0066] The control method, overnight enzymatic hydrolysis, was performed according to patent application number 202510990920. Enzymatic hydrolysis was carried out at 37 °C with constant shaking for 16 h, followed by extraction with a mixed solution of ethyl acetate / methyl tert-butyl ether containing 0.1% formic acid. Spiked samples of follicular fluid containing 10 representative compounds (10 ng / mL) were used for assays. The pretreatment time, recovery rate, and number of steps for both methods were recorded and compared.

[0067] Table 6 Comparison with enzymatic hydrolysis method .

[0068] The results analysis shows that the pretreatment time of the method of the present invention can be controlled within ≤1 h, while the traditional enzymatic digestion method usually requires more than 16 h, improving the overall efficiency by more than 16 times. In terms of methodological performance, the average recovery rate of the present invention for representative compounds is 88.7%, which is better than the 82.5% of the enzymatic digestion method. In addition, the operation process of the present invention is simpler, mainly including only 5 key steps, while the enzymatic digestion method usually requires 8 steps and additional isotope internal standards. The present invention uses the external standard method for quantification, eliminating the need for expensive isotope internal standards, thereby significantly reducing the detection cost.

[0069] The method of this invention is significantly superior to existing enzymatic digestion methods in terms of pretreatment time, recovery rate, ease of operation and cost, and is more suitable for rapid high-throughput screening of clinical samples.

[0070] Example 6: Comparison Experiment of Extraction Solvents Experimental objective: To compare the effects of "acetonitrile (containing 0.1% formic acid)" used in this invention and "ethyl acetate / methyl tert-butyl ether mixed solution containing 0.1% formic acid" used in reference patent 202510990920 as the extraction solvent.

[0071] Experimental design: Several follicular fluid samples that were pre-screened as "not detected" were selected and combined into a blank follicular fluid matrix.

[0072] The experimental method involves selecting 10 representative compounds shown in Table 7. Each representative compound was spiked into a blank follicular fluid matrix (the final spike concentration was uniformly 10 ng / mL). The following two sets of experiments were used for pretreatment: Group P (extraction solvent of the present invention): its sample pretreatment steps are repeated in Example 1; Group Q (control patent extraction solvent): The sample pretreatment steps were repeated in Example 1, except that in step 1 of the sample pretreatment in section 1.1, 800 µL of acetonitrile (containing 0.1% formic acid by volume) was replaced with 800 µL of a mixed solution of ethyl acetate / methyl tert-butyl ether (1:1, v / v) containing 0.1% formic acid. All other conditions remained unchanged (including saturated NaCl salting-out and subsequent dSPE purification steps). The test solutions obtained from the above two groups of experiments were analyzed by LC-MS / MS according to the method in Example 1, and quantitative calculations were performed based on the standard curve prepared in Example 1.

[0073] The above two sets of experiments were performed in parallel for six times each. The average recovery rate and relative standard deviation were calculated, and the results are shown in Table 7.

[0074] Table 7. Effect of different extraction solvents on the recovery rate of the target compound .

[0075] The results in Table 7 show that the average recovery rate of acetonitrile (containing 0.1% formic acid) used as the extraction solvent in this invention is 89.4%, which is significantly higher than the 77.9% recovery rate of the ethyl acetate / methyl tert-butyl ether mixed solution used in the control patent, with an average increase of 11.5 percentage points.

[0076] Especially for polar compounds (neonicotinic acid derivatives, antibiotics), acetonitrile shows a more significant advantage in recovery rate, with an increase of 12-17 percentage points. This is because: 1. Acetonitrile has moderate polarity: As a polar protic solvent, acetonitrile has a strong ability to dissolve polar to moderately polar compounds, while ethyl acetate / MTBE mixed solution has low polarity and limited extraction ability for polar compounds.

[0077] 2. Good water miscibility: Acetonitrile is completely miscible with water, making it more suitable for extraction of high-water-content matrices such as follicular fluid; while ethyl acetate and MTBE have poor water miscibility, which may lead to incomplete phase separation.

[0078] 3. Better acidification effect: 0.1% formic acid in acetonitrile has a better acidification effect than in ethyl acetate / MTBE mixed solution, and can more effectively break the binding of the target substance to the protein.

[0079] 4. Excellent LC-MS compatibility: Acetonitrile is the most commonly used organic phase in reversed-phase chromatography. Direct extraction with acetonitrile avoids solvent conversion and reduces sample loss.

[0080] Conclusion: The present invention selects acetonitrile (containing 0.1% formic acid) as the extraction solvent. Compared with the ethyl acetate / MTBE mixed solution of the reference patent, the average recovery rate of the target compound is increased by 11.5%, especially the recovery rate of polar compounds is significantly improved, which proves the rationality and superiority of the extraction solvent selection of the present invention.

Claims

1. A high-throughput detection method for pesticides and antibiotics in follicular fluid, characterized in that, Includes the following steps: Step 1: After thawing the follicular fluid, add a saturated NaCl aqueous solution and acetonitrile containing 0.05-1% formic acid by volume. Utilize the synergistic effect of salting out by saturated NaCl and acidification by formic acid to break the hydrogen bonds and electrostatic interactions between the target compound and the protein in the complex high-protein matrix of the follicular fluid, achieving one-step protein precipitation-extraction. After thorough mixing, centrifuge to separate the supernatant. Blow the supernatant to near dryness with nitrogen, and then reconstitute it with a solvent. Step 2: The complex solution obtained in Step 1 is subjected to dispersion solid-phase extraction purification treatment, and then solid-liquid separation is performed to obtain the test solution that can be used for LC-MS / MS analysis; Step 3: The test solution obtained in Step 2 is quantitatively detected using LC-MS / MS technology.

2. The high-throughput detection method for pesticides and antibiotics in follicular fluid as described in claim 1, characterized in that, In step 1, the volume ratio of the thawed follicular fluid to the saturated NaCl aqueous solution is 1:0.8-2, preferably 1:1-1.5; the volume of acetonitrile in step 1 is 2-6 times the volume of the thawed follicular fluid, preferably 3-4 times.

3. The detection method as described in claim 1, characterized in that, In step 1, the acetonitrile contains 0.1% formic acid by volume.

4. The high-throughput detection method for pesticides and antibiotics in follicular fluid as described in claim 1, characterized in that, The solvent used for resolution in step 1 is a methanol-water mixture with a methanol-water volume ratio of 1:0.5-2, preferably 1:

1.

5. The detection method as described in claim 1, characterized in that, In step 2, the dispersion solid-phase extraction purification process involves transferring the complex solution into a dSPE tube pre-filled with C18 packing material, PSA packing material, and anhydrous MgSO4 for purification. After thorough vortex mixing, the supernatant is collected by centrifugation and then filtered through a 0.22 µm organic filter membrane to obtain the test solution.

6. The high-throughput detection method for pesticides and antibiotics in follicular fluid as described in claim 5, characterized in that, In the system of dispersion solid phase extraction purification treatment, the amount of C18 packing is 40-60 mg, the amount of PSA packing is 40-60 mg, the amount of anhydrous MgSO4 is 120-180 mg, and it does not contain graphitized carbon black GCB.

7. The detection method as described in claim 1, characterized in that, In step 3, the detection conditions for LC-MS / MS technology are as follows: The chromatographic column was a Pfimek Kinetex F5 pentafluorophenyl reversed-phase column (1.7 µm, 2.1 × 150 mm). The mobile phase A was 0.2 wt% formic acid aqueous solution, and the mobile phase B was methanol. The flow rate was 0.3 mL / min, and the column temperature was 30-40℃. Gradient elution conditions: 0 to 2.0 min, 90% A; 2.0 to 7.0 min, 90% → 25% A; 7.0 to 7.2 min, 25% → 5% A; 7.2 to 9.1 min, 5% A; 9.1 to 11.0 min, 5% → 90% A; 11.0–12.0 min, 90% A; Mass spectrometry conditions: electrospray ionization (ESI), positive ion mode, multiple reaction monitoring (MRM) mode.

8. The high-throughput detection method for pesticides and antibiotics in follicular fluid as described in claim 1, characterized in that, The detection method also includes the preparation of a standard curve, the steps of which are as follows: S1: Prepare standard stock solutions of the target pesticide and antibiotic standards using n-hexane as a solvent; S2: Take each standard stock solution and use acetonitrile as a solvent to prepare a mixed pesticide-antibiotic standard stock solution; select the follicular fluid sample that was pre-screened as "not detected" as the matrix blank, and complete the blank extraction and reconstitution according to the pretreatment methods of steps 1 and 2 of claim 1 to obtain a matrix extract that is completely consistent with the matrix composition of the actual sample; use the matrix extract as a solvent to dilute the mixed pesticide-antibiotic standard stock solution stepwise to prepare matrix-matched standard solutions of different concentrations; S3: LC-MS / MS was used to analyze and obtain chromatograms. The chromatographic peak signal intensity of each target compound was recorded. A matrix-matched standard curve was established using the external standard method to quantitatively calculate the residual amount of the corresponding target compound in the sample.

9. The high-throughput detection method for pesticides and antibiotics in follicular fluid as described in claim 8, characterized in that, The specific procedures for preparing the matrix-matched standard solutions in steps S1 and S2 are as follows: M1: Accurately weigh different pesticide and antibiotic standards, prepare single pesticide or antibiotic standard stock solutions of 0.5-2.0 mg / mL with n-hexane, and store at -15 ℃ to -25 ℃. M2: Individually pipette the pesticide and antibiotic standard stock solutions into the same volumetric flask, dilute to the mark with acetonitrile, and prepare a mixed pesticide-antibiotic standard stock solution of 2.0-8.0 mg / L. Store at -15 ℃ to -25 ℃. Take the follicular fluid sample that was pre-screened as "not detected" as a matrix blank, and perform a blank extraction and reconstitution according to the pretreatment methods of steps 1 and 2 in claim 1 to obtain a matrix extract. Dilute the mixed pesticide-antibiotic standard stock solution stepwise with the matrix extract to prepare multiple matrix-matched standard solutions of corresponding concentrations. The mixed pesticide-antibiotic concentration range of the multiple matrix-matched standard solutions is 1-100 ng / mL.

10. The detection method as described in claim 1, characterized in that, The pesticides and antibiotics to be detected include at least one of neonicotinoids, carbamates, organophosphates, and triazoles, as well as at least one of antibiotics. The pesticides and antibiotics are detected simultaneously under the same pretreatment process and the same chromatographic-mass spectrometry analysis conditions to achieve high-throughput screening of multiple categories of exogenous chemicals with significant polarity differences in follicular fluid. The pesticides and antibiotics to be detected are preferably at least one of thiamethoxam, imidacloprid, carbofuran, aldicarb, chlorpyrifos, dichlorvos, cypermethrin, bifenthrin, ciprofloxacin, and sulfadiazine.