A solid-phase extraction-liquid chromatography method for the detection of organic acid salts in assisted reproductive fluids

Abstract

This invention discloses a solid-phase extraction-liquid chromatography (SPE-LC) method for detecting organic acid salts in assisted reproductive fluids, relating to the field of assisted reproductive fluid detection. The method includes the following steps: (1) dissolving and diluting the organic acid salts separately with a mobile phase to obtain a reference standard stock solution; (2) adding an eluent to the reference standard stock solution and concentrating it under nitrogen blowing, then adjusting the volume with the mobile phase to obtain a reference standard solution; (3) mixing the assisted reproductive fluid and a treatment agent, and incubating to obtain a pretreated test sample; performing solid-phase extraction on the pretreated test sample to obtain a test sample solution; and (4) injecting a blank solution, the test sample solution, and the reference standard solution into a high-performance liquid chromatograph (HPLC) and testing under chromatographic conditions. This invention combines pretreatment and solid-phase extraction techniques, reducing the matrix effect of high-concentration protein and other impurities in the detection of organic acid salts in assisted reproductive fluids, protecting the instrument, improving the selectivity of the detection method, and enhancing the accuracy of qualitative and quantitative detection.

Description

Technical Field

[0001] This invention relates to the field of fluids for in vitro assisted reproduction, and more specifically to a solid-phase extraction-liquid chromatography method for detecting organic acid salts in fluids for in vitro assisted reproduction. Background Technology

[0002] The main methods for determining organic acids include capillary electrophoresis, ion chromatography, gas chromatography, and high-performance liquid chromatography (HPLC). Gas chromatography often requires derivatization of organic acids before separation to increase their volatility due to their high boiling points and difficulty in vaporization, making sample pretreatment cumbersome and time-consuming. Ion chromatography, due to the special properties of the eluent and column packing material, has strict limitations on the protein content in samples and is not suitable for complex sample analyses. Compared with other analytical methods, capillary electrophoresis still has shortcomings in reproducibility. High-performance liquid chromatography remains the most mature method for the determination of organic acids.

[0003] Traditional liquid-liquid extraction methods suffer from drawbacks such as high solvent consumption, low efficiency, and difficulty in automation. Solid-phase extraction (SPE), based on chromatographic separation principles, is a highly efficient and selective sample pretreatment method developed by researchers to address the core challenges of severe matrix interference in complex matrices, low target analyte concentrations, and the need to protect precision analytical instruments. SPE offers advantages such as strong purification capabilities, high enrichment rates, low solvent consumption, and ease of automation, and is widely used in modern analytical laboratories for processing complex matrix samples. Furthermore, with the continuous development of novel adsorption materials and operating modes, SPE technology continues to evolve to meet increasingly complex analytical needs.

[0004] Assisted reproductive fluids are often complex cell culture media with numerous components, many of which exist in trace amounts. For example, vitrification and thawing solutions contain high concentrations of proteins, sugars, salts, amino acids, and buffer systems. Direct dilution or precipitation of these fluids introduces a large number of coexisting impurities into the chromatographic system, potentially leading to peak overlap, decreased column efficiency, high baseline noise, and limited sensitivity. Therefore, there is an urgent need to develop a method for detecting organic acid salts in assisted reproductive fluids that reduces matrix effects, protects the instrument, offers high selectivity, and provides high qualitative and quantitative accuracy. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a solid-phase extraction-liquid chromatography method for detecting organic acid salts in assisted reproductive fluids.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] A solid-phase extraction-liquid chromatography method for the detection of organic acid salts in assisted reproductive fluids, comprising the following steps:

[0008] Step (1): Dissolve and dilute the organic acid salts separately with the mobile phase to obtain the reference stock solution;

[0009] Step (2): After adding the reference standard stock solution to the eluent and concentrating it by nitrogen blowing, the solution is diluted to volume with the mobile phase to obtain the reference standard solution;

[0010] Step (3): Mix the assisted reproductive fluid and the treatment agent, and incubate to obtain a pretreated test sample; perform solid-phase extraction on the pretreated test sample to obtain a test sample solution;

[0011] Step (4): Inject the blank solution, test solution, and reference solution into the high performance liquid chromatograph and test them under chromatographic conditions.

[0012] The solid-phase extraction-liquid chromatography method for detecting organic acid salts in the assisted reproductive fluid includes the following specific steps:

[0013] Step (1): Take organic acid salts, dissolve and dilute them with the mobile phase to prepare solutions, thus obtaining the reference stock solution;

[0014] Furthermore, the organic acid salt is selected from at least one of sodium pyruvate, sodium citrate, and sodium lactate;

[0015] Furthermore, the concentrations of each organic acid salt used in the reference stock solution should be as follows: sodium pyruvate at 2-2.5 mg / mL, sodium citrate at 3-3.5 mg / mL, and sodium lactate at 33-33.5 mg / mL.

[0016] Step (2): Take the reference standard stock solution and place it in a centrifuge tube. Add eluent and concentrate it under nitrogen blowing at 30-32℃. Then, make up to volume with the mobile phase to obtain the reference standard solution.

[0017] Furthermore, the concentrations of each organic acid salt used in the reference solution should be prepared as follows: sodium pyruvate at 10 μg / mL, sodium citrate at 30 μg / mL, and sodium lactate at 495 μg / mL.

[0018] Step (3): Pretreatment: Mix the assisted reproductive fluid and treatment agent, incubate at room temperature for 8-10 min, centrifuge, and take the supernatant to obtain the pretreated test sample; perform solid-phase extraction on the obtained pretreated test sample, specifically including the following steps:

[0019] Activation: The SPE column was activated sequentially with methanol and water;

[0020] Sample loading: When the liquid surface flows over the upper sieve plate, the pretreated sample is drawn up and loaded into the activated column, and the effluent is discarded;

[0021] Rinsing: When the liquid flows over the upper sieve plate, rinse the column with water and then with a 50% methanol aqueous solution in sequence. Discard the outflow. Wait until the liquid is dry, then use a vacuum pump to apply negative pressure for 10-12 minutes.

[0022] Elution: Connect the needle to the lower needle seat, elute the column with eluent, connect the graduated centrifuge tube to the outflow liquid, and after the liquid has drained, use a vacuum pump to extract the remaining liquid in the column under negative pressure to obtain the eluent.

[0023] Concentration: The centrifuge tube containing the eluent is concentrated by nitrogen blowing at 30°C, and the volume is brought up with the mobile phase. After vortexing, the liquid in the centrifuge tube is transferred to a syringe and filtered into a brown bottle to obtain the test solution.

[0024] Further, the ratio of the assisted reproductive fluid to the treatment agent is 100-105 mL: 20-25 mL; the treatment agent is prepared by mixing polyethylene glycol, polyvinylpyrrolidone, sodium chloride, and deionized water in a ratio of 2-2.4 g: 1-1.2 g: 0.6-0.8 g: 10-12 mL, stirring at 45-50°C for 30-40 min, and cooling to room temperature.

[0025] Furthermore, the eluents mentioned in steps (2) and (3) are both 10-30% by mass trifluoroacetic acid methanol solution or 10-30% by mass formic acid methanol solution;

[0026] Step (4): Accurately measure blank solution, test solution, and reference solution, and inject them into the high performance liquid chromatograph. Test under chromatographic conditions and record the chromatograms. Calculate the peak areas using the external standard method and record the results.

[0027] Further, the blank solution is the mobile phase; the chromatographic conditions include: an ion-phase column (Rezex ROA-Organic Acid H+ (8%), 7.8*300mm) using sulfonated polystyrene-divinylbenzene resin as the packing material; mobile phase; detection wavelength of 210 nm; flow rate of 0.4 mL / min; column temperature of 60 °C; and injection volume of 10 μL.

[0028] Furthermore, the elution order of the reference solution in the chromatogram is sodium citrate, sodium pyruvate, and sodium lactate, and the resolution between sodium citrate and sodium pyruvate should be no less than 1.5.

[0029] Furthermore, the mobile phase described in the chromatographic conditions of steps (1), (2), (3), and (4) is a sulfuric acid solution of 1.84-7.36 mmol / L;

[0030] The beneficial effects of this invention are as follows: This invention discloses a solid-phase extraction-liquid chromatography (SPE-LC) method for detecting organic acid salts in assisted reproductive fluids. This invention combines pretreatment and SPE techniques to pretreat the assisted reproductive fluids. These two methods synergistically reduce the matrix effect of high concentrations of proteins, sugars, salts, amino acids, and buffer systems in the detection of organic acid salts in assisted reproductive fluids, protecting the detection instrument, improving the selectivity of the detection method, and enhancing the accuracy of qualitative and quantitative detection. During the pretreatment process, polyethylene glycol sterically excludes water molecules surrounding the protein, reducing the protein's solubility; polyvinylpyrrolidone enhances protein aggregation; and sodium chloride solution adjusts the ionic strength to assist in disrupting the protein hydration layer. These three factors synergistically precipitate and separate the protein from the assisted reproductive fluid, avoiding adverse effects such as high matrix effects on subsequent high-performance liquid chromatography (HPLC) detection. This improves the stability of the detection method, the reliability and accuracy of the detection results, and ensures effective control of the quality of the assisted reproductive fluids. Attached Figure Description

[0031] Figure 1 This is a chromatogram of the blank solution of Example 1 of the present invention.

[0032] Figure 2 This is the chromatogram of the reference solution from Example 1 of the present invention. Figure 2 In the diagram, A represents sodium citrate, B represents sodium pyruvate, and C represents sodium lactate.

[0033] Figure 3 This is the chromatogram of the test solution in Example 1 of the present invention. Figure 3 In the diagram, A represents sodium citrate, B represents sodium pyruvate, and C represents sodium lactate.

[0034] Figure 4 This is the chromatogram of the blank solution in Example 2 of the present invention.

[0035] Figure 5 This is the chromatogram of the reference solution from Example 2 of the present invention. Figure 5 In the diagram, A represents sodium citrate, B represents sodium pyruvate, and C represents sodium lactate.

[0036] Figure 6 This is the chromatogram of the test solution in Example 2 of the present invention. Figure 6 In the diagram, A represents sodium citrate, B represents sodium pyruvate, and C represents sodium lactate.

[0037] Figure 7 The chromatogram of the untreated test sample. Figure 7 In the diagram, A represents sodium citrate, B represents sodium pyruvate, and C represents sodium lactate. Detailed Implementation

[0038] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] Example 1

[0040] like Figure 1 , Figure 2 , Figure 3 The solid-phase extraction-liquid chromatography method for detecting organic acid salts in assisted reproductive fluids, as shown, includes the following steps:

[0041] Step (1): Accurately weigh 20 mg of sodium pyruvate, dissolve and dilute to 10 mL with 3.68 mmol / L sulfuric acid solution to obtain sodium pyruvate reference stock solution (sodium pyruvate concentration is 2 mg / mL); accurately weigh 30 mg of sodium citrate, dissolve and dilute to 10 mL with 3.68 mmol / L sulfuric acid solution to obtain sodium citrate reference stock solution (sodium citrate concentration is 3 mg / mL); accurately weigh 66 mg of sodium lactate, dissolve and dilute to 2 mL with 3.68 mmol / L sulfuric acid solution to obtain sodium lactate reference stock solution (sodium lactate concentration is 33 mg / mL).

[0042] Step (2): Take 5 μL of sodium pyruvate reference stock solution, 10 μL of sodium citrate reference stock solution, and 15 μL of sodium lactate reference stock solution and place them in a 15 mL centrifuge tube. Add 10 mL of 20% trifluoroacetic acid methanol solution and concentrate it to 0.3 mL under nitrogen blowing at 32 °C. Then, make up to 1 mL with 3.68 mmol / L sulfuric acid solution to obtain a solution with sodium pyruvate concentration of 10 μg / mL, sodium citrate concentration of 30 μg / mL, and sodium lactate concentration of 495 μg / mL, and obtain the reference solution.

[0043] Step (3): Pretreatment: Mix the assisted reproductive fluid and treatment agent, incubate at room temperature for 10 min, centrifuge, and take the supernatant to obtain the pretreated test sample; perform solid-phase extraction on the obtained pretreated test sample, specifically including the following steps:

[0044] Activation: Activate the Cleanert PWAX type column sequentially with 6 mL of methanol and 6 mL of water;

[0045] Sample loading: When the liquid surface flows over the upper sieve plate, take 1 mL of the pretreated sample and load it into the activated column, then discard the eluent;

[0046] Rinsing: When the liquid flows over the upper sieve plate, rinse the column with 5 mL of water and 5 mL of 50% methanol aqueous solution in sequence, discard the outflow, and wait until the liquid is dry. Then, use a vacuum pump to apply negative pressure for 12 minutes.

[0047] Elution: Connect the needle to the lower needle seat, elute the column with 10 mL of 20% trifluoroacetic acid methanol solution, collect the eluent in a 15 mL graduated centrifuge tube, and after the liquid has drained, use a vacuum pump to extract the remaining liquid in the column under negative pressure to obtain the eluent.

[0048] Concentration: The 15 mL centrifuge tube containing the eluent was concentrated to 0.3 mL by nitrogen blowing at 30 °C, and then brought to a final volume of 1 mL with 5.52 mmol / L sulfuric acid solution. The mixture was vortexed for 15 s to ensure thorough mixing. The liquid in the centrifuge tube was transferred to a syringe and filtered through a nylon filter membrane into a brown bottle to obtain the test solution. The ratio of the assisted reproductive fluid to the treatment agent was 105 mL: 25 mL. The treatment agent was a mixture of polyethylene glycol, polyvinylpyrrolidone, sodium chloride, and deionized water in a ratio of 2.4 g: 1.2 g: 0.8 g: 12 mL, stirred at 50 °C for 40 min, and cooled to room temperature.

[0049] Step (4): Accurately measure the blank solution, test solution, and reference solution, and inject them into the high-performance liquid chromatograph (HPLC) respectively. Test under chromatographic conditions and record the chromatograms. Calculate the peak area using the external standard method and record the results. The blank solution is a 5.52 mmol / L sulfuric acid solution. The chromatographic conditions include: an ion-type column (Rezex ROA-Organic Acid H+ (8%), 7.8*300 mm) with sulfonated polystyrene-divinylbenzene resin as the packing material; 5.52 mmol / L sulfuric acid solution as the mobile phase; a detection wavelength of 210 nm; a flow rate of 0.4 mL / min; a column temperature of 60 °C; and an injection volume of 10 μL.

[0050] The calculation results (three parallel copies) are shown in Table 1:

[0051] Table 1:

[0052]

[0053] Results Analysis: Based on GB / T 27417-2017 "Guideline for Conformity Assessment and Validation of Chemical Analysis Methods" and the "9101 Analytical Method Validation Guidelines" in Part IV of the 2025 edition of the Pharmacopoeia of the People's Republic of China, the minimum resolution between the main component and adjacent peaks in this embodiment was 1.6. The recoveries of sodium pyruvate, sodium citrate, and sodium lactate were all within the range of 80% to 115%, which met the relevant standard requirements. This indicates that the determination method provided by the technical solution of this application has good accuracy and does not cause loss of sodium pyruvate, sodium citrate, and sodium lactate during the determination process.

[0054] Example 2

[0055] like Figure 4 , Figure 5 , Figure 6 The solid-phase extraction-liquid chromatography method for detecting organic acid salts in assisted reproductive fluids, as shown, includes the following steps:

[0056] Step (1): Accurately weigh 20 mg of sodium pyruvate, dissolve and dilute to 10 mL with 3.68 mmol / L sulfuric acid solution to obtain sodium pyruvate reference stock solution (sodium pyruvate concentration is 2 mg / mL); accurately weigh 30 mg of sodium citrate, dissolve and dilute to 10 mL with 3.68 mmol / L sulfuric acid solution to obtain sodium citrate reference stock solution (sodium citrate concentration is 3 mg / mL); accurately weigh 66 mg of sodium lactate, dissolve and dilute to 2 mL with 3.68 mmol / L sulfuric acid solution to obtain sodium lactate reference stock solution (sodium lactate concentration is 33 mg / mL).

[0057] Step (2): Take 5 μL of sodium pyruvate reference stock solution, 10 μL of sodium citrate reference stock solution, and 15 μL of sodium lactate reference stock solution and place them in a 15 mL centrifuge tube. Add 10 mL of 20% formic acid methanol solution and concentrate to 0.3 mL under nitrogen blowing at 32 °C. Then, make up to 1 mL with 3.68 mmol / L sulfuric acid solution to obtain a solution with sodium pyruvate concentration of 10 μg / mL, sodium citrate concentration of 30 μg / mL, and sodium lactate concentration of 495 μg / mL, and obtain the reference solution.

[0058] Step (3): Pretreatment: Mix the assisted reproductive fluid and treatment agent, incubate at room temperature for 8 min, centrifuge, and take the supernatant to obtain the pretreated test sample; perform solid-phase extraction on the obtained pretreated test sample, specifically including the following steps:

[0059] Activation: Activate the Cleanert PAX type column sequentially with 6 mL of methanol and 6 mL of water;

[0060] Sample loading: When the liquid surface flows over the upper sieve plate, take 1 mL of the pretreated sample and load it into the activated column, then discard the eluent;

[0061] Rinsing: When the liquid flows over the upper sieve plate, rinse the column with 5 mL of water and 5 mL of 50% methanol aqueous solution in sequence, discard the outflow, and wait until the liquid is dry. Then, use a vacuum pump to apply negative pressure for 10 minutes.

[0062] Elution: Connect the needle to the lower needle seat, elute the column with 10 mL of 20% formic acid methanol solution, collect the effluent in a 15 mL graduated centrifuge tube, and after the liquid has drained, use a vacuum pump to extract the remaining liquid in the column under negative pressure to obtain the eluent.

[0063] Concentration: The 15 mL centrifuge tube containing the eluent was concentrated to 0.3 mL under nitrogen blowing at 30 °C. The volume was then adjusted to 1 mL with 3.68 mmol / L sulfuric acid solution. The mixture was vortexed for 10 seconds to ensure thorough mixing. The liquid was transferred to a syringe and filtered through a nylon membrane into a brown bottle to obtain the test solution. The ratio of the assisted reproductive fluid to the treatment agent was 100 mL: 20 mL. The treatment agent consisted of polyethylene glycol, polyvinylpyrrolidone, sodium chloride, and deionized water mixed in a ratio of 2 g: 1 g: 0.6 g: 10 mL, stirred at 45 °C for 30 min, and cooled to room temperature.

[0064] Step (4): Accurately measure the blank solution, test solution, and reference solution, and inject them into the high-performance liquid chromatograph (HPLC) respectively. Test under chromatographic conditions and record the chromatograms. Calculate the peak area using the external standard method and record the results. The blank solution is a 3.68 mmol / L sulfuric acid solution. The chromatographic conditions include: an ion-type column (Rezex ROA-Organic Acid H+ (8%), 7.8*300 mm) with sulfonated polystyrene-divinylbenzene resin as the packing material; 3.68 mmol / L sulfuric acid solution as the mobile phase; a detection wavelength of 210 nm; a flow rate of 0.4 mL / min; a column temperature of 60 °C; and an injection volume of 10 μL.

[0065] The calculation results (three parallel copies) are shown in Table 2:

[0066] Table 2:

[0067]

[0068] Results Analysis: Based on GB / T 27417-2017 "Guideline for Conformity Assessment and Validation of Chemical Analysis Methods" and the "9101 Analytical Method Validation Guidelines" in Part IV of the 2025 edition of the Pharmacopoeia of the People's Republic of China, the minimum resolution between the main component and adjacent peaks in this embodiment was 1.9. The recoveries of sodium citrate, sodium pyruvate, and sodium lactate were all within the range of 80% to 115%, which met the relevant standard requirements. This indicates that the determination method provided by the technical solution of this application has good accuracy and does not cause loss of sodium citrate, sodium pyruvate, and sodium lactate during the determination process.

[0069] Comparative Example 1

[0070] Compared with Example 1, the treatment agent used was replaced with polyvinylpyrrolidone, sodium chloride, and deionized water mixed in a ratio of 3.6g:0.8g:12mL, stirred at 50°C for 40min, and cooled to room temperature to obtain treatment agent-1. The rest was exactly the same as in Example 1. Solid-phase extraction-liquid chromatography was performed to detect organic acid salts in assisted reproductive fluids. The matrix effect of the detection method for organic acid salts in assisted reproductive fluids was enhanced, which adversely affected the actual content of organic acid salts, the stability and accuracy of the detection method.

[0071] Comparative Example 2

[0072] Compared with Example 1, the treatment agent used was replaced with polyethylene glycol, sodium chloride, and deionized water mixed in a ratio of 3.6g:0.8g:12mL, stirred at 50°C for 40min, and cooled to room temperature to obtain treatment agent-2. The rest was exactly the same as in Example 1. Solid-phase extraction-liquid chromatography was performed to detect organic acid salts in assisted reproductive fluids. The matrix effect of the detection method for organic acid salts in assisted reproductive fluids was enhanced, which adversely affected the actual content of organic acid salts, the stability and accuracy of the detection method.

[0073] Comparative Example 3

[0074] Compared with Example 1, the treatment agent used was replaced with polyethylene glycol, polyvinylpyrrolidone, and deionized water mixed in a ratio of 2.8g:1.6g:12mL, stirred at 50°C for 40min, and cooled to room temperature to obtain treatment agent-3. The rest was exactly the same as in Example 1. Solid-phase extraction-liquid chromatography was performed to detect organic acid salts in assisted reproductive fluids. The matrix effect of the detection method for organic acid salts in assisted reproductive fluids was enhanced, which adversely affected the actual content of organic acid salts, the stability and accuracy of the detection method.

[0075] Comparative Example 4

[0076] Compared with Example 1, the method for detecting organic acid salts in assisted reproductive fluids without solid-phase extraction has a large matrix effect and is not suitable for detecting organic acid salts in assisted reproductive fluids.

[0077] Comparative Example 5

[0078] like Figure 7 The solid-phase extraction-liquid chromatography method for detecting organic acid salts in assisted reproductive fluids, as shown in Example 1, does not involve pretreatment to obtain the test solution. The chromatogram of the obtained test solution shows that the matrix effect caused by high concentrations of proteins, etc., is significant, making it unsuitable for detecting organic acid salts in assisted reproductive fluids.

[0079] The above description is merely an example and illustration of the concept of the present invention. Those skilled in the art can make various modifications or additions to the specific embodiments described or use similar methods to replace them, as long as they do not deviate from the concept of the invention or exceed the scope defined in the claims, they should all fall within the protection scope of the present invention.

Claims

1. A solid-phase extraction-liquid chromatography method for the detection of organic acid salts in assisted reproductive fluids, characterized in that: Includes the following steps: Step (1): Take organic acid salts, dissolve and dilute them with the mobile phase to prepare solutions, thus obtaining the reference stock solution; Step (2): Take the reference standard stock solution and place it in a centrifuge tube. Add eluent and concentrate it under nitrogen blowing at 30-32℃. Then, make up to volume with the mobile phase to obtain the reference standard solution. Step (3): Pretreatment: Mix the assisted reproductive fluid and treatment agent, incubate at room temperature for 8-10 min, centrifuge, and take the supernatant to obtain the pretreated test sample; perform solid-phase extraction on the obtained pretreated test sample, specifically including the following steps: Activation: The SPE column was activated sequentially with methanol and water; Sample loading: When the liquid surface flows over the upper sieve plate, the pretreated sample is drawn up and loaded into the activated column, and the effluent is discarded; Rinsing: When the liquid flows over the upper sieve plate, rinse the column with water and then with a 50% methanol aqueous solution in sequence. Discard the outflow. Wait until the liquid is dry, then use a vacuum pump to apply negative pressure for 10-12 minutes. Elution: Connect the needle to the lower needle seat, elute the column with eluent, connect the graduated centrifuge tube to the outflow liquid, and after the liquid has drained, use a vacuum pump to extract the remaining liquid in the column under negative pressure to obtain the eluent. Concentration: The centrifuge tube containing the eluent is concentrated by nitrogen blowing at 30°C, and the volume is brought up with the mobile phase. After vortexing, the liquid in the centrifuge tube is transferred to a syringe and filtered into a brown bottle to obtain the test solution. Step (4): Accurately measure blank solution, test solution, and reference solution, and inject them into the high performance liquid chromatograph. Test under chromatographic conditions and record the chromatograms. Calculate the peak areas using the external standard method and record the results. In step (1), the organic acid salt is selected from at least one of sodium pyruvate, sodium citrate, and sodium lactate; The treatment agent in step (3) includes polyethylene glycol, polyvinylpyrrolidone, sodium chloride, and deionized water in a ratio of 2-2.4g:1-1.2g:0.6-0.8g:10-12mL.

2. The solid-phase extraction-liquid chromatography method for detecting organic acid salts in assisted reproductive fluids according to claim 1, characterized in that: The concentrations of each organic acid salt used in the reference stock solution in step (1) should be as follows: sodium pyruvate concentration of 2-2.5 mg / mL, sodium citrate concentration of 3-3.5 mg / mL, and sodium lactate concentration of 33-33.5 mg / mL.

3. The solid-phase extraction-liquid chromatography method for detecting organic acid salts in assisted reproductive fluids according to claim 1, characterized in that: The concentrations of each organic acid salt used in the reference solution in step (2) should be prepared as follows: sodium pyruvate at 10 μg / mL, sodium citrate at 30 μg / mL, and sodium lactate at 495 μg / mL.

4. The solid-phase extraction-liquid chromatography method for detecting organic acid salts in assisted reproductive fluids according to claim 1, characterized in that: The eluents mentioned in steps (2) and (3) are both 10-30% by mass trifluoroacetic acid methanol solution or 10-30% by mass formic acid methanol solution.

5. The solid-phase extraction-liquid chromatography method for detecting organic acid salts in assisted reproductive fluids according to claim 1, characterized in that: The preparation method of the treatment agent in step (3) is to mix polyethylene glycol, polyvinylpyrrolidone, sodium chloride and deionized water, stir at 45-50℃ for 30-40 min, and cool to room temperature.

6. The solid-phase extraction-liquid chromatography method for detecting organic acid salts in assisted reproductive fluids according to claim 1, characterized in that: The chromatographic conditions in step (4) include: an ion-type chromatographic column with sulfonated polystyrene-divinylbenzene resin as the packing material; a mobile phase; a detection wavelength of 210 nm; a flow rate of 0.4 mL / min; a column temperature of 60 °C; and an injection volume of 10 μL. The mobile phase in the chromatographic conditions is the same as the mobile phase in steps (1), (2), and (3) as a sulfuric acid solution of 1.84-7.36 mmol / L.

7. The solid-phase extraction-liquid chromatography method for detecting organic acid salts in assisted reproductive fluids according to claim 1, characterized in that: The elution order of the reference solution in the chromatogram is sodium citrate, sodium pyruvate, and sodium lactate, and the resolution between sodium citrate and sodium pyruvate should be no less than 1.5.

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