Method for detecting various pollutants in follicular fluid

Abstract

The invention discloses a method for detecting various pollutants in follicular fluid. The method comprises the steps of enzymolysis, extraction, nitrogen blowing, redissolution and detection on a machine. Mixing the follicular fluid sample with the mixed internal standard solution, performing enzymolysis overnight, and hydrolyzing the bound metabolite by using beta-glucuronidase; extracting by using an ethyl acetate / methyl tert-butyl ether mixed solution containing 0.1% formic acid, and repeating for three times to extract 13 types of 182 pollutants; and carrying out nitrogen blowing drying and acetonitrile / water redissolution, and analyzing through a series quadrupole rod liquid chromatograph-mass spectrometer. According to the method, the detection limit is as low as nanogram / milliliter, quantification is completed within a short time through single sample injection, more than 100 samples can be detected in a single day, high-throughput and high-sensitivity detection is achieved, energy consumption and cost are reduced through program optimization, high efficiency and environmental friendliness are achieved, and a powerful means is provided for studying the follicular fluid pollutant exposure level and correlating with reproductive health.

Description

Technical Field

[0001] The present invention relates to the technical field of pollutant detection, and in particular to a method for detecting multiple pollutants in follicular fluid. Background Art

[0002] Currently, most methods for detecting follicular fluid contaminants face numerous limitations. First, they are limited to the targeted identification of a small number of specific contaminants, making it difficult to comprehensively and systematically assess the synergistic effects of multiple contaminants in complex human exposure environments. This is primarily due to the difficulty of adapting a single ionization mode to compounds with significantly varying physicochemical properties. Identifying multiple contaminant classes requires multiple runs, which significantly increases time and costs. Furthermore, existing methods typically employ a generalized gradient elution procedure, resulting in lengthy analysis cycles. Reducing single-run times while maintaining separation efficiency is a pressing challenge. Second, the physicochemical properties of contaminants in follicular fluid vary significantly, making existing enrichment techniques incapable of achieving efficient and simultaneous extraction of multiple contaminants. Traditional solid-phase extraction methods typically utilize a single adsorbent, effectively targeting only specific contaminants. Mixed-mode column synthesis is complex and expensive. While employing a multi-column cascade strategy can expand the enrichment range, it significantly increases the risk of sample loss and reduces method reproducibility. Third, the dual challenges of matrix interference and trace detection require urgent resolution. The high abundance of proteins in follicular fluid can interfere with analysis. Existing protein precipitation methods are complex and prone to loss of trace amounts of target compounds. A multifunctional reagent combining protein precipitation and broad-spectrum extraction capabilities is needed to improve pretreatment efficiency. Contaminants in follicular fluid are often present at trace levels. Existing technologies struggle to achieve both high sensitivity and low matrix interference when simultaneously detecting multiple substances, resulting in reduced data reliability. Therefore, there is an urgent need for an efficient, rapid, sensitive, and high-throughput detection technology for the analysis of multiple environmental pollutants in follicular fluid. Summary of the Invention

[0003] The purpose of the present invention is to provide a method for detecting multiple pollutants in follicular fluid to solve the above-mentioned problems existing in the prior art and to achieve high-throughput, rapid, sensitive and accurate detection of multiple pollutants in follicular fluid.

[0004] To achieve the above objectives, the present invention adopts the following technical means: A method for detecting multiple pollutants in follicular fluid comprises the following steps: Enzymatic hydrolysis: The follicular fluid sample was mixed with the mixed internal standard solution and then enzymatically hydrolyzed overnight; Extraction: Add ethyl acetate / methyl tert-butyl ether containing formic acid, vortex to mix, centrifuge and let stand, remove the supernatant, and repeat twice; Nitrogen blowing: collect the supernatant three times and blow it to dryness with nitrogen; Reconstitution: Reconstitute with acetonitrile / water, filter through membrane, and transfer to a sample vial; On-machine detection: Analyze the sample in the sample bottle by tandem quadrupole liquid chromatography-mass spectrometry.

[0005] Preferably, the enzymatic hydrolysis step is specifically as follows: thaw the follicular fluid sample in advance, vortex mix, take 0.4 mL into a glass tube, mix with 100 μL 400 ng / mL mixed internal standard solution, after mixing, add 100 μL β-glucuronidase solution, and use a desktop constant temperature shaker to reciprocate and shake at 37°C and 150 rpm for 16 hours.

[0006] Preferably, the mixed internal standard solution is a mixture of isotope internal standard standards of 13 types of pollutants, prepared with acetonitrile and ultrapure water, and stored in a -20°C refrigerator, with a volume ratio of acetonitrile to ultrapure water of 6:4; The enzyme activity of the β-glucuronidase solution is 8500 units / mL, and the pH is 5.0.

[0007] Preferably, in the extraction step, an ethyl acetate / methyl tert-butyl ether mixed solution containing 0.1% formic acid is added, and the volume ratio of the ethyl acetate / methyl tert-butyl ether mixed solution is 1:5; during extraction, vortex mixing is performed for 5 minutes, followed by centrifugation at 4°C and 1000 rpm for 10 minutes.

[0008] Preferably, in the re-dissolution step, 150 μL of a mixture of acetonitrile and ultrapure water in a volume ratio of 6:4 is used for re-dissolution, the re-dissolution operation is repeated once, and filtered using an organic nylon filter, and finally, the filtrate is transferred to a sampling bottle.

[0009] Preferably, the diameter of the organic nylon filter is 13 mm and the pore size of the filter membrane is 0.22 µm.

[0010] Preferably, in the on-machine detection step, the tandem quadrupole liquid spectrometer used is Waters XEVO TQ-S MICRO.

[0011] Preferably, in the on-machine detection step, the liquid phase conditions are: the chromatographic column adopts a reverse phase C18 chromatographic column ACQUITY UPLC BEH C18, and is equipped with a pre-column; the mobile phase A is 2 mmol / L ammonium acetate, the pH value of ammonium acetate is adjusted to 4.6-5 with acetic acid, and the mobile phase B is acetonitrile; gradient elution is adopted: 10% B 0 min, 10% B 2 min, 40% B 3.5 min, 70% B 4.5 min, 95% B 7.5 min, 95% B 8.5 min, 10% B 11 min, 10% B 13 min; column temperature is 40 ° C; flow rate is 0.3 mL / min; injection volume is 5 μL.

[0012] Preferably, in the on-machine detection step, the mass spectrometry conditions are: electrospray ion source and multiple reaction monitoring mode; capillary voltage: 3.5 kV in positive ion mode, 1.5 kV in negative ion mode; desolvation temperature 550°C; cone gas flow rate 50 L / h; desolvation gas flow rate 1000 L / h.

[0013] The present invention has the following beneficial effects: 1. Efficient Extraction of Multiple Pollutants: Using ethyl acetate / methyl tert-butyl ether (MTBE) containing 0.1% formic acid as the extraction solvent, the system effectively extracts 13 distinct classes of pollutants from follicular fluid, including organophosphates, phthalates, per- and polyfluoroalkyl compounds, bisphenols, parabens, antimicrobials, nonylphenols, benzothiazoles, benzotriazoles, synthetic phenolic antioxidants, pyrethroids, neonicotinoids, and antibiotics. Formic acid precipitates proteins, reduces matrix effects, and minimizes damage to analytical instruments. The optimized mixed extractant ratio significantly improves the simultaneous extraction efficiency of multiple pollutants, ensuring sufficient extraction of target analytes. 2. High-throughput and high-sensitivity detection: This method can simultaneously detect 182 pollutants from 13 categories with a detection limit as low as nanograms per milliliter, enabling large-scale trace detection of follicular fluid. Through an optimized enzymatic hydrolysis step, β-glucuronidase is used to hydrolyze bound metabolites, ensuring a comprehensive assessment of human exposure levels. Combined with the high sensitivity of tandem quadrupole liquid chromatography-mass spectrometry, this method significantly improves detection efficiency and range, overcoming the limitations of traditional detection methods, which often have limited detection range and sensitivity. 3. Rapid Detection and Environmental Protection: An optimized gradient elution program combined with rapid polarity switching technology allows for the separation and quantification of all pollutants in a single injection in a short time, enabling over 100 samples to be tested per day. Compared to traditional detection methods, this significantly shortens analysis time and reduces instrument energy and solvent consumption, improving detection efficiency while also complying with green chemistry principles, reducing testing costs, and delivering significant economic and environmental benefits. DETAILED DESCRIPTION

[0014] The technical solution of the present invention will be described clearly and completely below. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0015] A method for detecting multiple pollutants in follicular fluid comprises the following steps: 1. Enzymatic hydrolysis: Thaw the follicular fluid sample in advance, vortex mix, take 0.4 mL into a glass tube, mix with 100 μL of 400 ng / mL mixed internal standard solution, add 100 μL of β-glucuronidase solution (8500 units / mL, pH = 5.0), and use a desktop constant temperature shaker to reciprocate at 37°C and 150 rpm for 16 hours; Among them, the mixed internal standard solution is a mixture of isotopic internal standard standards of 13 types of pollutants, prepared by acetonitrile: ultrapure water (v / v=6:4), and stored in a -20°C refrigerator; the β-glucuronidase working solution is made by diluting the β-glucuronidase (derived from Roman snail, Type H-2, ≥85,000 units / mL) stock solution to a concentration of 8500 units / mL with 1 mol / L ammonium acetate (acetic acid adjusted to pH=5).

[0016] 2. Extraction: Add a mixture of ethyl acetate and methyl tert-butyl ether (v / v = 1:5) containing 0.1% formic acid to the sample and vortex mix for 5 minutes. Subsequently, centrifuge at 1000 rpm at 4°C for 10 minutes. After standing, aspirate the supernatant to ensure sufficient extraction of the target analyte. Repeat this extraction process twice.

[0017] 3. Nitrogen blowing: Collect the supernatant three times and blow it to dryness with gentle nitrogen. Utilize the inertness and dryness of nitrogen to evaporate the extractant by controlling the temperature and airflow to concentrate the target compound while avoiding loss or contamination of the target compound.

[0018] 4. Redissolution: Slowly add 150 μL of acetonitrile: ultrapure water (v / v = 6:4) along the wall of the tube, gently shake the glass tube to mix, repeat this redissolution operation once, use a disposable syringe to draw up the reconstituted product, and filter through an organic nylon filter (13 mm 0.22 µm) to remove flocs and prevent clogging of the analytical instrument flow system. Finally, transfer the filtrate to a sampling bottle.

[0019] 5. On-machine testing: Analyze using a tandem quadrupole liquid chromatography-mass spectrometer (Waters XEVO TQ-S MICRO); Among them, the liquid phase conditions are as follows: the chromatographic column uses a reversed-phase C18 chromatographic column ACQUITY UPLC BEH C18 (2.1 mm × 100 mm, 1.7 μm; Waters), and is equipped with a pre-column (Acquity UPLC BEH C18 1.7 μm VanGuard Pre-Column 3 / Pk, size 2.1 × 5 mm) to adsorb interfering substances and avoid interference with the main analytical column in the analysis of the target compound. Mobile phase A was 2 mmol / L ammonium acetate (pH adjusted to 4.6-5 with acetic acid), and mobile phase B was acetonitrile. Gradient elution was used: 10% B (0 min), 10% B (2 min), 40% B (3.5 min), 70% B (4.5 min), 95% B (7.5 min), 95% B (8.5 min), 10% B (11 min), and 10% B (13 min). Column temperature was 40°C. Flow rate was 0.3 mL / min. Injection volume was 5 μL. Mass spectrometry conditions: electrospray ionization source (positive and negative ion modes) and multiple reaction monitoring mode; capillary voltage: 3.5 kV in positive ion mode (ESI+), 1.5 kV in negative ion mode (ESI-); desolvation temperature: 550°C; cone gas flow rate: 50 L / h; desolvation gas flow rate: 1000 L / h. Example 1

[0020] A method for detecting multiple pollutants in follicular fluid comprises the following steps: Enzymatic hydrolysis: Freshly collected and frozen follicular fluid samples were thawed at room temperature. After thorough mixing using a vortex mixer, 0.4 mL was accurately transferred to a glass tube. To the tube, 100 μL of a 400 ng / mL mixed internal standard solution was added. This mixed internal standard solution, composed of 13 isotopic internal standard standards for pollutants, was prepared in acetonitrile:ultrapure water (v / v = 6:4) and stored at -20°C. Subsequently, 100 μL of β-glucuronidase solution (8500 units / mL, pH = 5.0) was added. This solution was prepared by diluting β-glucuronidase (from Helix pomatia, Type H-2, ≥85,000 units / mL) in 1 mol / L ammonium acetate (pH adjusted to 5 with acetic acid). Place the glass tube in a desktop constant temperature shaker, set the temperature to 37°C and the speed to 150 rpm, and shake it back and forth for 16 hours to allow β-glucuronidase to hydrolyze the bound pollutants into free forms. Extraction: Add a mixture of ethyl acetate and methyl tert-butyl ether (v / v = 1:5) containing 0.1% formic acid to the digested sample and mix using a vortex mixer for 5 minutes. Centrifuge the sample at 1000 rpm at 4°C for 10 minutes. After centrifugation, allow the sample to stand and carefully aspirate as much of the supernatant as possible, avoiding the precipitate below. Repeat this extraction procedure twice to fully extract the target analytes. Nitrogen blowing: Collect the supernatant obtained from three extractions and use a nitrogen blower to blow the supernatant to dryness with mild nitrogen. The inertness and drying properties of nitrogen can be used to remove the solvent in the enrichment solution while protecting the sample from thermal damage or contamination. Reconstitution: Slowly add 150 μL of acetonitrile:ultrapure water (v / v = 6:4) along the wall of the glass tube. Gently shake the tube to dissolve the enriched target compound in the reconstitution solution. Repeat this reconstitution process once. Use a disposable syringe to draw up the reconstituted product and filter it through an organic nylon filter (13 mm, 0.22 μm) to remove flocculent matter and prevent clogging of the analyzer flow system. Finally, transfer the filtrate to an injection vial. On-machine analysis: Analyses were performed using a tandem quadrupole liquid chromatography-mass spectrometer (Waters XEVO TQ-S MICRO). The liquid chromatography conditions were as follows: a reversed-phase C18 column, ACQUITY UPLC BEH C18 (2.1 mm × 100 mm, 1.7 μm; Waters), equipped with a pre-column (Acquity UPLC BEH C18 1.7 μm VanGuard Pre-Column 3 / Pk, size 2.1 × 5 mm). Mobile phase A consisted of 2 mmol / L ammonium acetate (pH adjusted to 4.6–5 with acetic acid), and mobile phase B consisted of acetonitrile. Gradient elution was: 10% B (0 min), 10% B (2 min), 40% B (3.5 min), 70% B (4.5 min), 95% B (7.5 min), 95% B (8.5 min), 10% B (11 min), and 10% B (3.5 min). The HPLC-MS / MS conditions were: 13 min, column temperature 40°C, flow rate 0.3 mL / min, injection volume 5 μL; mass spectrometry conditions included an electrospray ionization source (positive and negative ion modes) in multiple reaction monitoring mode, capillary voltages: 3.5 kV in positive ion mode (ESI+), 1.5 kV in negative ion mode (ESI-), desolvation temperature 550°C, cone gas flow rate 50 L / h, and desolvation gas flow rate 1000 L / h. Quantitative analysis of the target compounds using an internal standard method demonstrated good linearity for all target compounds over the concentration range of 0.04-400 ng / mL (standard curve R² > 0.99), with sensitivity, accuracy, and precision meeting expectations. Example 2 A method for detecting multiple pollutants in follicular fluid comprises the following steps: Enzymatic hydrolysis: Select another batch of frozen follicular fluid samples, thaw and vortex mix, take 0.4 mL and place it in a glass tube, add 100 μL of 400 ng / mL mixed internal standard solution, then add 100 μL of β-glucuronidase solution (8500 units / mL, pH=5.0), place it in a desktop constant temperature shaker, and shake at 37°C and 150 rpm for 16 hours to complete the enzymatic hydrolysis process. Extraction: Add ethyl acetate / methyl tert-butyl ether (v / v = 1:5) containing 0.1% formic acid to the enzymatically digested sample, vortex mix for 5 minutes, centrifuge at 1000 rpm at 4°C for 10 minutes, remove the supernatant, and repeat the extraction twice.

[0021] Nitrogen blowing: Collect the supernatant three times and blow it to dryness with nitrogen. Reconstitution: Repeat the procedure once with 150 μL of acetonitrile: ultrapure water (v / v = 6:4), filter, and transfer to a sample vial.

[0022] On-device testing: Analyses were performed using a tandem quadrupole liquid chromatography-mass spectrometry (Waters XEVO TQ-S MICRO) using the same liquid chromatography and mass spectrometry conditions as in Example 1, with internal standard analysis used for quantitative analysis of the target compounds. 182 pollutants from 13 categories were successfully detected, and the linearity, sensitivity, accuracy, and precision of each target compound met the required standards, validating the effectiveness of this method across different sample batches. Example 3 A method for detecting multiple pollutants in follicular fluid comprises the following steps: Enzymatic hydrolysis: Take a follicular fluid sample from a special source (such as from a specific living environment or a patient with a specific disease), thaw and vortex, take 0.4 mL into a glass tube, add a mixed internal standard solution and β-glucuronidase solution, and shake on a desktop constant temperature shaker at 37°C and 150 rpm for 16 hours for enzymatic hydrolysis. Extraction: Add ethyl acetate / methyl tert-butyl ether (v / v = 1:5) containing 0.1% formic acid to the sample, vortex mix for 5 minutes, centrifuge at 1000 rpm at 4°C for 10 minutes, remove the supernatant, and repeat the extraction procedure twice. Nitrogen blowing: Collect the supernatant three times and blow dry with nitrogen. Reconstitution: Reconstitute with 150 μL of acetonitrile: ultrapure water (v / v = 6:4), repeat the reconstitution, filter and transfer to a sample vial. On-device testing: Tandem quadrupole liquid chromatography-mass spectrometry (LC-MS / MS) was performed using a Waters XEVO TQ-S MICRO instrument, following established liquid chromatography and mass spectrometry conditions. The internal standard method was employed for quantitative analysis of the target compounds. The results demonstrated that this method accurately detected multiple contaminants in the samples, providing reliable data and demonstrating its applicability and effectiveness for follicular fluid samples with diverse characteristics.

[0023] The present invention is provided as an example, not as a limitation of the embodiments. Those skilled in the art will appreciate that other variations or modifications may be made based on the above description. It is not necessary and impossible to enumerate all embodiments here, and obvious variations or modifications derived therefrom remain within the scope of protection of the present invention.

Claims

1. A method for detecting multiple pollutants in follicular fluid, characterized in that: The following steps are involved: Enzymatic hydrolysis: The follicular fluid sample was mixed with the mixed internal standard solution and then enzymatically hydrolyzed overnight; Extraction: Add ethyl acetate / methyl tert-butyl ether containing formic acid, vortex to mix, centrifuge and let stand, remove the supernatant, and repeat twice; Nitrogen blowing: collect the supernatant three times and blow it to dryness with nitrogen; Reconstitution: Reconstitute with acetonitrile / water, filter through membrane, and transfer to a sample vial; On-machine detection: Analyze the sample in the sample bottle by tandem quadrupole liquid chromatography-mass spectrometry.

2. The method for detecting multiple pollutants in follicular fluid according to claim 1, characterized in that: The enzymatic hydrolysis step is specifically as follows: the follicular fluid sample is thawed in advance, vortex mixed, 0.4 mL is taken into a glass tube, and mixed with 100 μL of 400 ng / mL mixed internal standard solution. After mixing, 100 μL of β-glucuronidase solution is added, and a desktop constant temperature shaker is used to reciprocate and shake at 37°C and 150 rpm for 16 hours.

3. The method for detecting multiple pollutants in follicular fluid according to claim 2, characterized in that: The mixed internal standard solution is prepared by mixing isotope internal standard substances of 13 types of pollutants with acetonitrile and ultrapure water, and stored in a -20°C refrigerator. The volume ratio of acetonitrile to ultrapure water is 6:

4. The enzyme activity of the β-glucuronidase solution is 8500 units / mL, and the pH is 5.

0.

4. The method for detecting multiple pollutants in follicular fluid according to claim 1, characterized in that: In the extraction step, an ethyl acetate / methyl tert-butyl ether mixed solution containing 0.1% formic acid was added, and the volume ratio of the ethyl acetate / methyl tert-butyl ether mixed solution was 1:5; during extraction, vortex mixing was performed for 5 minutes, followed by centrifugation at 4°C and 1000 rpm for 10 minutes.

5. The method for detecting multiple pollutants in follicular fluid according to claim 1, characterized in that: In the re-dissolution step, 150 μL of a mixture of acetonitrile and ultrapure water in a volume ratio of 6:4 was used for re-dissolution, the re-dissolution operation was repeated once, and the solution was filtered using an organic nylon filter. Finally, the filtrate was transferred to a sample injection bottle.

6. The method for detecting multiple pollutants in follicular fluid according to claim 5, characterized in that: The diameter of the organic nylon filter is 13 mm, and the pore size of the filter membrane is 0.22 μm.

7. The method for detecting multiple pollutants in follicular fluid according to claim 1, characterized in that: In the on-machine detection step, the tandem quadrupole liquid spectrometer used is Waters XEVO TQ-S MICRO.

8. The method for detecting multiple pollutants in follicular fluid according to claim 7, characterized in that: In the on-machine detection step, the liquid phase conditions are as follows: a reverse-phase C18 chromatographic column ACQUITY UPLC BEH C18 is used as the chromatographic column, and a pre-column is provided; mobile phase A is 2 mmol / L ammonium acetate, the pH value of ammonium acetate is adjusted to 4.6-5 with acetic acid, and mobile phase B is acetonitrile; gradient elution is used: 10% B 0 min, 10% B 2 min, 40% B 3.5 min, 70% B 4.5 min, 95% B 7.5 min, 95% B 8.5 min, 10% B 11 min, and 10% B 13 min; column temperature is 40°C; flow rate is 0.3 mL / min; and injection volume is 5 μL.

9. The method for detecting multiple pollutants in follicular fluid according to claim 7, characterized in that: In the on-machine detection step, the mass spectrometry conditions are: electrospray ion source and multiple reaction monitoring mode; capillary voltage: positive ion mode 3.5 kV, negative ion mode 1.5 kV; desolvation temperature 550°C; cone gas flow rate 50 L / h; desolvation gas flow rate 1000 L / h.