A method for synchronously detecting sodium pyruvate in an assisted reproductive culture solution
The detection of sodium pyruvate in assisted reproductive culture medium by high performance liquid chromatography (HPLC) overcomes the shortcomings of existing detection methods, achieving efficient and convenient detection results, and supporting the assessment of embryonic developmental potential and the control of multiple pregnancy risks.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-04-02
- Publication Date
- 2026-07-10
AI Technical Summary
The lack of efficient, stable and economical methods in the current technology for detecting sodium pyruvate content in assisted reproductive culture media affects the assessment of embryonic developmental potential and the control of multiple pregnancy risks.
Simultaneous detection of sodium pyruvate in assisted reproductive culture medium was performed using high performance liquid chromatography (HPLC). By preparing test solutions, standard solutions, and linear solutions, and combining specific flow rates and detection wavelengths, an efficient and simple detection method was achieved.
It achieves detection with low detection limits, good repeatability and accuracy, supports quality control of assisted reproductive culture media, reduces the risk of multiple pregnancies and improves pregnancy success rate.
Smart Images
Figure CN122361647A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of culture medium component detection technology, specifically a method for simultaneous detection of sodium pyruvate in assisted reproductive culture medium. Background Technology
[0002] Since the birth of the world's first test-tube baby in 1978, Assisted Reproductive Technology (ART) has been widely recognized globally and applied in the clinical treatment of infertility, considered one of the most important achievements in 20th-century medicine. In vitro fertilization-embryo transfer (IVF-ET) technology occupies a core position within this technological system. During early embryonic development, energy metabolism is a key factor affecting embryonic developmental potential. Studies have shown that pyruvate is the primary energy source for the embryo during early cleavage, while glucose is not converted into a primary energy source until the blastocyst stage. Pyruvate not only participates in embryonic energy metabolism but also prevents developmental arrest and participates in cellular antioxidant stress processes. Pyruvate deficiency can cause developmental arrest in the embryo before reaching the 8-cell stage, severely affecting its ability to continue developing. Therefore, accurate determination of sodium pyruvate content in ART culture media is of crucial clinical significance for assessing culture medium quality and predicting embryonic developmental potential. By detecting the consumption of pyruvate in the embryo culture medium, embryonic viability and growth potential can be non-invasively assessed, providing a basis for selective single embryo transfer, thereby reducing the risk of multiple pregnancies and improving pregnancy success rates. However, current quality control documents or technical standards for in vitro reproductive culture media do not clearly specify the indicators and detection methods for component content. Industry standard YY / T 1695-2020 only covers the detection methods for amino acids in culture media, but does not address the detection of energy substances, which poses a challenge to the quality control and standardization of culture media.
[0003] In the existing technology, there is a method for detecting three indicator components, namely glucose, sodium lactate, and sodium pyruvate, in in vitro reproductive culture medium using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-MS / MS). However, this method requires highly specialized operation, which limits its application in routine quality control environments.
[0004] Therefore, there is an urgent need to establish an analytical method that is highly specific, efficient, stable, economical, and applicable to routine quality control. Summary of the Invention
[0005] The purpose of this invention is to provide a method for the simultaneous detection of sodium pyruvate in assisted reproductive culture medium in order to solve the problems mentioned above.
[0006] The technical solution adopted in this invention is as follows: A method for simultaneous detection of sodium pyruvate in assisted reproductive culture medium, comprising the following steps:
[0007] S1: Prepare the instrument, specifically the high-performance liquid chromatograph;
[0008] S2: Prepare solutions, including: test solution, blank sample solution, standard stock solution, linear solution, limit of detection solution, limit of quantitation solution, repeatability solution, and accuracy solution;
[0009] S3: The detection method was validated using high performance liquid chromatography.
[0010] S4: The content of sodium pyruvate in the sample was determined by high performance liquid chromatography.
[0011] In a preferred embodiment, in step S1, the instrument preparation involves adjusting various parameters to the working state, using an Agilent C18 column (250 mm * 4.6 mm * 5 µm) at a column temperature of 30 °C; a flow rate of 1.0 mL / min; an injection volume of 20 μL; a mobile phase of 0.04 mol / L tetraethylammonium bromide solution (95) and methanol (5); and a detection wavelength of 230 nm.
[0012] In a preferred embodiment, the preparation method of the test solution in step S2 includes: taking an appropriate amount of sample into an ultrafiltration centrifuge tube (30KD), centrifuging at 9500 rpm for 10 min, and filtering the filtrate through a 0.22 μm microporous membrane to obtain the sample test solution.
[0013] In a preferred embodiment, the blank sample solution in step S2 is prepared by: without adding any sample, according to the test sample solution.
[0014] In a preferred embodiment, the method for preparing the standard stock solution in step S2 includes: taking 20.72 mg of sodium pyruvate standard (purity 99.1%), and diluting it to 50 mL with primary water to prepare a standard stock solution with sodium pyruvate concentration of 0.4107 mg / mL.
[0015] In a preferred embodiment, the method for preparing the linear solution in step S2 includes: transferring an appropriate amount of sodium pyruvate standard stock solution into a 10 mL volumetric flask, adding primary water to prepare standard curve solutions with five concentration gradients, denoted as STD1, STD2, STD3, STD4, and STD5.
[0016] In a preferred embodiment, step S2, the method for preparing the detection limit solution includes: accurately transferring 0.25 mL of sodium pyruvate standard solution STD5 into a 10 mL volumetric flask, and preparing a detection limit solution with a concentration of 1.027 μg / mL using primary water.
[0017] In a preferred embodiment, step S2, the method for preparing the limit of quantitation solution includes: accurately transferring 1 mL of sodium pyruvate standard solution STD5 into a 10 mL volumetric flask, and preparing a limit of quantitation solution with a concentration of 4.107 μg / mL using primary water.
[0018] In a preferred embodiment, in step S2, the method for preparing the repeatable solution includes: preparing 6 solutions in parallel by adding a standard concentration solution according to accuracy;
[0019] The preparation method of the accuracy solution includes: taking 200 μL, 400 μL and 750 μL of standard stock solution with a concentration of 410.7 mg / L respectively, diluting it with the sample solution diluted 5 times to 10 mL, transferring it to an ultrafiltration centrifuge tube, and centrifuging at 9500 rpm for 10 min to obtain low, medium and high concentration spiked solutions.
[0020] In a preferred embodiment, the methodology validation items in step S3 include specificity, limit of detection, limit of quantitation, linearity and range, repeatability, and accuracy.
[0021] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:
[0022] This invention achieves a detection limit as low as 1.027 μg / mL, accurately capturing trace amounts of sodium pyruvate in samples. Pretreatment requires only ultrafiltration centrifugation and membrane filtration, making the operation simple and each analysis taking approximately 15 minutes. The method exhibits excellent repeatability with an RSD of 2.2%, and multiple detections show consistent results. The recovery rate remains stable at 92.4%–108.6%, demonstrating good accuracy and accurately reflecting the actual sodium pyruvate content in the sample. This method provides an efficient, reliable, and feasible solution for detecting sodium pyruvate content in assisted reproductive products, strongly supporting the quality control of assisted reproductive culture media. Attached Figure Description
[0023] Figure 1 This is a schematic diagram of the process principle of the present invention.
[0024] Figure 2 The spectrum of a blank sodium pyruvate sample solution;
[0025] Figure 3 The spectrum of sodium pyruvate standard;
[0026] Figure 4 The spectrum of the sodium pyruvate test solution;
[0027] Figure 5 This is a schematic diagram of the sodium pyruvate standard curve. Detailed Implementation
[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.
[0029] Example: Refer to Figure 1-5 A method for simultaneous detection of sodium pyruvate in assisted reproductive culture medium, comprising the following instruments and reagents:
[0030] High performance liquid chromatograph (Agilent 1260 Infinity II), electronic balance (accuracy 0.0001g, XSR105DU / A), sodium pyruvate standard (99.1%, 100mg / bottle, Tanmo Quality Inspection Technology Co., Ltd.), tetraethylammonium bromide (50 g / bottle, Shanghai Maclean Biochemical Technology Co., Ltd.), methanol (4 L / bottle, TEDIA Reagent Company, USA).
[0031] Instrument conditions:
[0032] The instrument parameter settings for sodium pyruvate high-performance liquid chromatography are shown in the table below:
[0033] Instrument conditions for high performance liquid chromatography
[0034]
[0035] Solution preparation:
[0036] 1) Test solution: Take an appropriate amount of sample into an ultrafiltration centrifuge tube (30KD), centrifuge at 9500 rpm for 10 min, take the filtrate to obtain the sample test solution, and filter it through a 0.22μm microporous membrane to obtain the sample test solution;
[0037] 2) Blank sample solution: Prepared without adding any sample, following the same procedure as the test sample solution.
[0038] 3) Standard stock solution: 20.72 mg of sodium pyruvate standard (purity 99.1%) was diluted to 50 mL with primary water to prepare a standard stock solution with sodium pyruvate concentration of 0.4107 mg / mL.
[0039] 4) Linear solutions: According to the table below, transfer an appropriate amount of sodium pyruvate standard stock solution into a 10 mL volumetric flask, add primary water to prepare standard curve solutions with 5 concentration gradients, and denoted as STD1, STD2, STD3, STD4, and STD5.
[0040] linear solutions
[0041]
[0042] 5) Detection limit solution: Accurately transfer 0.25 mL of sodium pyruvate standard solution STD 5 into a 10 mL volumetric flask, and prepare a detection limit solution with a concentration of 1.027 μg / mL using primary water;
[0043] 6) Limit of quantitation solution: Accurately transfer 1 mL of sodium pyruvate standard solution STD5 into a 10 mL volumetric flask and prepare a limit of quantitation solution with a concentration of 4.107 μg / mL using primary water;
[0044] 7) Reproducible solution: Prepare 6 parallel solutions by adding standard concentration solution according to accuracy.
[0045] 8) Accuracy solutions: Take 200 μL, 400 μL, and 750 μL of standard stock solution with a concentration of 410.7 mg / L, respectively, and dilute to 10 mL with the sample solution diluted 5 times. Transfer to ultrafiltration centrifuge tubes and centrifuge at 9500 rpm for 10 min to obtain low, medium, and high concentration spiked solutions; the spiked concentrations are shown in the table below:
[0046] Sodium pyruvate spiking concentration
[0047] name Concentration of added standard solution (μg / mL) Volume of standard solution added (μL) Add to the theoretical test concentration (µg / mL) Accuracy Solution 1 410.7 200 8.214 Accuracy Solution 2 410.7 400 16.428 Accuracy Solution 3 410.7 750 30.802
[0048] The methodological validation results for testing the sodium pyruvate content in one assisted reproductive product are as follows:
[0049] Specificity: The blank sample solution, test solution, and spiked sample solution were tested separately, and the chromatograms were recorded. Figure 2 - Figure 4 It can be seen that the sodium pyruvate resolution in this method is 2.5, and there are no interfering peaks at the target peak position in the blank sample solution, which meets the method validation evaluation criteria.
[0050] Linearity: The standard curve solution was tested on the instrument, and the test results are shown in the table below. The results show that the target peak has a good linear relationship in the range from the lowest concentration point to the highest concentration point, with a correlation coefficient R > 0.99, which meets the method validation evaluation criteria.
[0051] Sodium pyruvate standard curve test results
[0052]
[0053] Limit of Detection (LOD) and Limit of Quantification (LOQ): LOD and LOQ solutions were tested under the specified conditions. According to Guideline 9101 of the 2020 edition of the Chinese Pharmacopoeia, Part IV, the LOD was defined as the concentration of a solution with a signal-to-noise ratio (SNR) ≥ 3:1, and the LOQ was defined as the concentration of a solution with a SNR ≥ 10:1. The RSD of the peak area of the LOQ solution should be ≤ 6%. The test results are shown in the table below. The results indicate that when the sodium pyruvate standard solution concentration was 1.027 μg / mL, the SNR was > 3:1; when the sodium pyruvate standard solution concentration was 4.107 μg / mL, the SNR was > 10:1. The RSD of the peak areas of the three LOQ solutions was < 6%, which meets the method validation evaluation criteria.
[0054] Limit of detection and limit of quantitation for sodium pyruvate
[0055]
[0056] Repeatability: The repeatability solution was tested on the instrument. According to the guidance 9101 of the 2020 edition of the Chinese Pharmacopoeia, the RSD of the target peak concentration should be ≤4%. The test results are shown in the table below. The RSD of the determination of sodium pyruvate is <4%, which shows that the method has good repeatability and meets the method validation evaluation criteria.
[0057] Repeatability test results of sodium pyruvate
[0058]
[0059] Accuracy: The accuracy solution was tested on the instrument. According to the guidance 9101 of the 2020 edition of the Chinese Pharmacopoeia, the recovery rate should be between 85% and 110%. The test results are shown in the table below. The results show that the accuracy of the target content determination is good, and the recovery rate of sodium pyruvate spiked in the range of 92.4% to 108.6% meets the requirements of the spiked recovery rate for method validation and meets the evaluation criteria for method validation.
[0060] Sodium pyruvate accuracy test results
[0061]
[0062] Note: Spiked recovery rate % = (Actual test concentration - Target analyte concentration in sample) / Theoretical spiked concentration * 100%
[0063] Sodium pyruvate content test results
[0064] The content of sodium pyruvate was tested using high performance liquid chromatography (HPLC). μ (g / mL) is calculated using the following formula:
[0065]
[0066] In the formula:
[0067] X — The content of sodium pyruvate in the sample, in units of μ g / mL;
[0068] Cs — Concentration of the sample test solution ( μ g / mL);
[0069] Co — Concentration of blank test solution ( μ g / mL);
[0070] f—Dilution factor;
[0071] The test solution was tested under the above chromatographic conditions. According to formula (1), the sodium pyruvate content in the sample with batch number J000006812 was calculated to be 24.62 μg / mL.
[0072] As described above, this invention employs high-performance liquid chromatography (HPLC) to determine the sodium pyruvate content in assisted reproductive products, and the methodology has been validated in detail. This method features a low detection limit, simple pretreatment, high repeatability, and good accuracy, providing a feasible approach for the detection of sodium pyruvate content in assisted reproductive products.
[0073] It should be noted that, in this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the term "comprising" or any other variations thereof is intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.
[0074] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. A method for simultaneous detection of sodium pyruvate in assisted reproductive culture medium, characterized in that: The method includes the following steps: S1: Prepare the instrument, specifically the high-performance liquid chromatograph; S2: Prepare solutions, including: test solution, blank sample solution, standard stock solution, linear solution, limit of detection solution, limit of quantitation solution, repeatability solution, and accuracy solution; S3: The detection method was validated using high performance liquid chromatography; S4: The content of sodium pyruvate in the sample was determined by high performance liquid chromatography.
2. The method for simultaneous detection of sodium pyruvate in assisted reproductive culture medium as described in claim 1, characterized in that: In step S1, the instrument preparation involves adjusting various parameters to the working state. The chromatographic column is an Agilent C18, 250mm*4.6mm*5µm, with a column temperature of 30℃; the flow rate is 1.0mL / min; the injection volume is 20μL; the mobile phase is 0.04mol / L tetraethylammonium bromide solution (95): methanol (5); and the detection wavelength is 230nm.
3. The method for simultaneous detection of sodium pyruvate in assisted reproductive culture medium as described in claim 1, characterized in that: In step S2, the preparation method of the test solution includes: taking an appropriate amount of sample, placing it in an ultrafiltration centrifuge tube (30KD), centrifuging at 9500 rpm for 10 min, and filtering the filtrate through a 0.22 μm microporous membrane to obtain the sample test solution.
4. The method for simultaneous detection of sodium pyruvate in assisted reproductive culture medium as described in claim 1, characterized in that: In step S2, the blank sample solution is prepared by: without adding any sample, according to the method for preparing the test sample solution.
5. The method for simultaneous detection of sodium pyruvate in assisted reproductive culture medium as described in claim 1, characterized in that: In step S2, the preparation method of the standard stock solution includes: 20.72 mg of sodium pyruvate standard (purity 99.1%), diluted to 50 mL with primary water to prepare a standard stock solution with sodium pyruvate concentration of 0.4107 mg / mL.
6. The method for simultaneous detection of sodium pyruvate in assisted reproductive culture medium as described in claim 1, characterized in that: In step S2, the method for preparing the linear solution includes: transferring an appropriate amount of sodium pyruvate standard stock solution into a 10 mL volumetric flask, adding primary water to prepare standard curve solutions with 5 concentration gradients, denoted as STD1, STD2, STD3, STD4, and STD5.
7. The method for simultaneous detection of sodium pyruvate in assisted reproductive culture medium as described in claim 1, characterized in that: In step S2, the method for preparing the detection limit solution includes: accurately transferring 0.25 mL of sodium pyruvate standard solution STD5 into a 10 mL volumetric flask, and preparing a detection limit solution with a concentration of 1.027 μg / mL using primary water.
8. The method for simultaneous detection of sodium pyruvate in assisted reproductive culture medium as described in claim 1, characterized in that: In step S2, the method for preparing the limit of quantitation solution includes: accurately transferring 1 mL of sodium pyruvate standard solution STD5 into a 10 mL volumetric flask, and preparing a limit of quantitation solution with a concentration of 4.107 μg / mL using primary water.
9. The method for simultaneous detection of sodium pyruvate in assisted reproductive culture medium as described in claim 1, characterized in that: In step S2, the method for preparing the repeatable solution includes: preparing 6 solutions in parallel by adding a standard concentration solution according to accuracy; The preparation method of the accuracy solution includes: taking 200 μL, 400 μL and 750 μL of standard stock solution with a concentration of 410.7 mg / L respectively, diluting it with the sample solution diluted 5 times to 10 mL, transferring it to an ultrafiltration centrifuge tube, and centrifuging at 9500 rpm for 10 min to obtain low, medium and high concentration spiked solutions.
10. The method for simultaneous detection of sodium pyruvate in assisted reproductive culture medium as described in claim 1, characterized in that: In step S3, the methodology validation items include specificity, limit of detection, limit of quantitation, linearity and range, repeatability, and accuracy.