Method for measuring glucose content in embryo culture medium

Abstract

This invention discloses a method for determining glucose content in embryo culture medium. In this invention, proteins and amino acids are filtered from the sample using ultrafiltration centrifuge tubes. High-performance liquid chromatography (HPLC) is used as the detector, and an external standard curve method is employed to detect the glucose content in the embryo culture medium. The pretreatment step of ultrafiltration centrifuge tube filtration eliminates interference from human serum albumin and some amino acids in the embryo culture medium on the determination of the target component, glucose. An amino-based column is specifically selected; the hydroxyl groups in glucose form hydrogen bonds with the amino groups on the stationary phase of the amino-based column, separating them from other components in the culture medium under the action of the mobile phase, thus achieving separation. The method uses a differential detector to establish an external standard curve for quantitative determination, which is characterized by rapid and simple operation, good repeatability, and accurate results, making it suitable for determining glucose content in embryo culture medium.

Description

Technical Field

[0001] This invention belongs to the field of embryo culture medium detection technology, specifically a method for determining the glucose content in embryo culture medium. Background Technology

[0002] In embryo culture media, glucose is a key component that plays different functions at different stages of embryonic development. Its most direct role is that the embryo produces glucose through metabolism to meet the needs of cell division, growth, and development. In the early cleavage stage, embryonic energy metabolism mainly relies on oxidative phosphorylation through the tricarboxylic acid cycle, resulting in a lower demand for glucose; excessively high glucose levels may be detrimental. In the late blastocyst stage, embryos primarily rely on glucose metabolism to meet the needs of synthesizing cell membrane macromolecules. Furthermore, glucose is also an important component in maintaining a stable osmotic pressure in the culture medium. The accurate concentration of glucose, balanced with other nutrients such as amino acids, is one of the core techniques in in vitro embryo culture and blastocyst culture, directly affecting the success rate of IVF. Embryo culture media contains amino acids such as taurine, sodium pyruvate, D-(+)-glucose, gentamicin sulfate, calcium lactate pentahydrate, human serum albumin, folic acid (vitamin B9), cobalamin (vitamin B12), and L-alanyl-L-glutamine.

[0003] However, the complex composition of the culture medium causes significant interference with glucose content determination in existing technologies.

[0004] Therefore, there is an urgent need to develop a fast, convenient, efficient, sensitive, reproducible, and accurate method for detecting glucose content to meet the quality control requirements of embryo culture medium. Summary of the Invention

[0005] The purpose of this invention is to provide a method for determining the glucose content in embryo culture medium in order to solve the problems mentioned above.

[0006] The technical solution adopted in this invention is as follows: a method for determining the glucose content in embryo culture medium, the method comprising the following steps:

[0007] S1. Sample pretreatment: Take 10 mL of sample and filter it into an ultrafiltration centrifuge tube (3KD). Then centrifuge at 9500 rpm for 10 min. Take the supernatant and filter it through a 0.22 μm microporous membrane as the sample test solution. Prepare a blank solution using the same method without adding sample.

[0008] S2. Solution preparation: This specifically includes the following solutions:

[0009] (1) D-(+)-glucose standard stock solution: Accurately weigh 100.72 mg of standard D-(+)-glucose, place it in a 25 mL volumetric flask, dissolve it in 85% acetonitrile and dilute to the mark to prepare a standard stock solution with a concentration of 3.948 mg / mL;

[0010] (2) D-(+)-glucose standard curve solution: Transfer 0.050 mL, 0.125 mL, and...

[0011] 0.250 mL, 0.500 mL, 1.000 mL, and 1.500 mL of L-(+)- glucose standard stock solution were dissolved and diluted to volume with 80% acetonitrile to prepare a series of standard working solutions with concentrations of 0.02 mg / mL, 0.049 mg / mL, 0.099 mg / mL, 0.20 mg / mL, 0.39 mg / mL, and 0.59 mg / mL, respectively, and labeled as STD-1, STD-2, STD-3, STD-4, STD-5, and STD-6.

[0012] (3) System suitability solution: Take 0.250 mL of LD-(+)- glucose standard stock solution into a 10 mL volumetric flask, dissolve and dilute with 80% acetonitrile, and shake well to obtain 6 parallel solutions;

[0013] (4) Specific solutions: Take blank solution, STD-3 standard working solution and sample test solution;

[0014] (5) Detection limit solution: Accurately transfer 0.025 mL of D-(+)-glucose standard stock solution into a 10 mL volumetric flask, dissolve and dilute to the mark with 80% acetonitrile to prepare a detection limit solution with a concentration of 0.00987 mg / mL;

[0015] (6) Limit of quantitation solution: Accurately transfer 0.050 mL of D-(+)-glucose standard stock solution into a 10 mL volumetric flask, dissolve and dilute to the mark with 80% acetonitrile to prepare a limit of quantitation solution with a concentration of 0.01974 mg / mL;

[0016] (7) Repeatable solution: Take a 10 mL volumetric flask, add 0.250 mL L-(+)- glucose standard stock solution, and dilute to the mark with sample test solution; shake well to obtain the solution. Prepare 6 parallel solutions.

[0017] (8) Accuracy solutions: Using the sample spiking method, add 0.125 mL, 0.250 mL, and 0.375 mL of L-(+)- glucose standard stock solution to a 10 mL volumetric flask, respectively, and dilute to 10 mL with sample test solution to obtain three accuracy solutions of low, medium, and high concentration levels. Prepare three parallel solutions for each concentration level.

[0018] S3. The method for determining glucose content was validated using a high-performance liquid chromatograph (differential detector). The validation included system suitability, specificity, limit of detection, limit of quantitation, linear range, repeatability, and accuracy.

[0019] S4. The glucose content in the embryo culture medium was determined using a high-performance liquid chromatograph (differential detector).

[0020] In a preferred embodiment, in step S1, the ultrafiltration centrifuge tube is made of a modified polyethersulfone membrane material with 3K. Before use, add enough primary water to submerge the membrane, place it in an ice bath for 10 minutes, and then pour out the water for later use.

[0021] In a preferred embodiment, in step S3, linear regression analysis is performed on the standard curve solution to obtain a linear regression equation and establish a standard curve. The correlation coefficient of the curve equation should be ≥0.99.

[0022] In a preferred embodiment, in step S3, the specific solution is tested and analyzed. The resolution of the target peak (glucose) in the sample solution should be greater than 1.5, and the blank solution should be free of interference or have a response value less than 1 / 3 of the limit of quantitation.

[0023] In a preferred embodiment, in step S3, the detection limit solution is injected three times repeatedly, and the signal-to-noise ratio should be >3:1 for each injection.

[0024] In a preferred embodiment, in step S3, the limit of quantitation solution is injected three times repeatedly, and the signal-to-noise ratio should be >10:1 for each injection.

[0025] In a preferred embodiment, in step S3, the repeatability solution is analyzed to obtain repeatability test results, and the RSD should be ≤3%.

[0026] In a preferred embodiment, in step S3, the accuracy solution is analyzed to obtain an accuracy result, and the recovery rate should be between 90% and 108%.

[0027] In a preferred embodiment, the chromatographic conditions in step S4 are as follows: detector: RID differential detector; column: Agilent Polaris NH2 (250mm*4.6mm, 5um); column temperature: 40℃; injection volume: 20μL; flow rate: 1.0mL / min; mobile phase: acetonitrile:10mM ammonium formate 80:20.

[0028] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0029] This invention establishes a method for determining glucose content in embryo culture medium. The method involves filtration in ultrafiltration centrifuge tubes (3KD) followed by centrifugation at 9500 rpm. The procedure is simple, provides good separation, and exhibits high sensitivity and accuracy, meeting routine testing requirements. High-performance liquid chromatography (HPLC) with a differential detector was employed. The method's linearity, limit of detection, limit of quantitation, repeatability, and accuracy were comprehensively validated to ensure the reliability and reproducibility of the results, thereby improving the accuracy of the detection. Attached Figure Description

[0030] Figure 1 This is a chromatogram of glucose standard in an embodiment of the present invention;

[0031] Figure 2 This is a chromatogram of the embryo culture medium test solution in an embodiment of the present invention;

[0032] Figure 3 This is a chromatogram of the blank reagent solution in an embodiment of the present invention;

[0033] Figure 4 This is a chromatogram of the spiked embryo culture medium sample in an embodiment of the present invention;

[0034] Figure 5 This is a schematic diagram of the glucose standard curve in an embodiment of the present invention;

[0035] Figure 6 This is a schematic diagram of the process principle in this invention. Detailed Implementation

[0036] 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.

[0037] Reference Figure 1-6 A method for determining the glucose content in an embryo culture medium, comprising the following steps:

[0038] 1. Sample pretreatment: Take 10 mL of embryo culture medium and filter it into an ultrafiltration centrifuge tube (3KD). Then centrifuge at 9500 rpm for 10 min. Take the supernatant and filter it through a 0.22 μm microporous membrane as the sample test solution. Prepare a blank solution using the same method without adding sample.

[0039] 2. Solution preparation: This specifically includes the following solutions:

[0040] (1) D-(+)-glucose standard stock solution: Accurately weigh 100.72 mg of standard D-(+)-glucose, place it in a 25 mL volumetric flask, dissolve it in 85% acetonitrile and dilute to the mark to prepare a standard stock solution with a concentration of 3.948 mg / mL;

[0041] (2) D-(+)-glucose standard curve solution: Transfer 0.050 mL, 0.125 mL, 0.250 mL, 0.500 mL, 1.000 mL, and 1.500 mL of D-(+)-glucose standard stock solution into six 10 mL volumetric flasks, dissolve and dilute with 80% acetonitrile to prepare a series of standard working solutions, which are labeled as STD-1, STD-2, STD-3, STD-4, STD-5, and STD-6, respectively. The specific concentrations are shown in Table 1 below:

[0042] Table 1. D-(+)-Glucose Standard Curve Solution

[0043]

[0044] (3) System suitability solution: Take 0.250 mL of L-(+)- glucose standard stock solution into a 10 mL volumetric flask, dissolve and dilute with 80% acetonitrile, and shake well to obtain 6 parallel solutions.

[0045] (4) Specific solutions: Take blank solution, STD-3 standard working solution and sample test solution;

[0046] (5) Detection limit solution: Accurately transfer 0.025 mL of D-(+)-glucose standard stock solution into a 10 mL container.

[0047] Dissolve the sample in 80% acetonitrile and dilute to the mark in a volumetric flask to prepare a detection limit solution with a concentration of 0.00987 mg / mL.

[0048] (6) Limit of quantitation solution: Accurately transfer 0.050 mL of D-(+)-glucose standard stock solution into a 10 mL volumetric flask, dissolve and dilute to the mark with 80% acetonitrile to prepare a limit of quantitation solution with a concentration of 0.01974 mg / mL.

[0049] (7) Repeatable solution: Take a 10 mL volumetric flask, add 0.250 mL of L-(+)- glucose standard stock solution, and dilute to the mark with test solution; prepare 6 parallel solutions.

[0050] (8) Accuracy solutions: Using the sample spiking method, 0.125 mL, 0.250 mL, and 0.375 mL of L-(+)- glucose standard stock solution were added to 10 mL volumetric flasks, respectively. The solutions were then diluted to 10 mL with sample test solution to obtain low, medium, and high concentration accuracy solutions. Three replicates were prepared for each concentration level. The spiking concentrations are shown in Table 2 below:

[0051] Table 2 Spiked Concentration

[0052]

[0053] 3. Establish the test method: Analyze each solution using a high-performance liquid chromatograph under the set conditions and record the peak area; instrument conditions are shown in Table 3 below:

[0054] Table 3 Test conditions for D-(+)-glucose

[0055]

[0056] 4. Method validation data analysis:

[0057] (1) Specificity: Take the reagent blank solution, test solution and sample spiked solution respectively for instrument testing, record their chromatograms, and then... Figure 1 - Figure 4 It can be seen that there are no interfering peaks near the glucose target in the sample solution, which meets the requirements of the method validation and evaluation standard.

[0058] (2) System suitability: Take the system suitability solution and test it 6 times consecutively according to the instrument conditions in Table 3. The RSD of the target peak area should be less than 4%. The test results are shown in Table 4. The results show that the RSD is 1.3%, which meets the requirements of the methodology validation evaluation standard.

[0059] Table 4 System Suitability Test Results

[0060]

[0061] (3) Linear range: Linear regression analysis was performed on the standard curve solution to obtain the linear regression equation and establish the standard curve. The results of the linear equation are shown in Table 5. The results in the table 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.

[0062] Table 5 Standard Curve Test Results

[0063]

[0064] (4) Limit of detection and limit of quantitation: The limit of detection and limit of quantitation solutions were tested according to the instrument conditions in Table 3. The test results are shown in Table 6. The results show that: when the concentration of the standard solution is 0.00987 mg / mL, the signal-to-noise ratio of D-(+)-glucose is >3:1; when the concentration of the standard solution is 0.01974 mg / mL, the signal-to-noise ratio is >10:1. The RSD of the peak areas of the three limit of quantitation solutions is <15%, which meets the method validation evaluation criteria.

[0065] Table 6 Limit of Detection and Limit of Quantification

[0066]

[0067] (5) Accuracy: The accuracy solution was tested on the instrument according to the instrument conditions in Table 3. The recovery rate of D-(+)-glucose in each accuracy solution should be between 90% and 108%. The test results are shown in Table 7. The results show that the recovery rate ranges from 101.52% to 104.19%. The accuracy of this method is good and meets the method validation evaluation criteria.

[0068] Table 7 Accuracy Test Results

[0069]

[0070] Note: Recovery rate % = (actual test concentration - background concentration) / theoretical spiked concentration * 100%.

[0071] 5. Quantitative analysis:

[0072] The glucose content in the embryo culture medium was analyzed using the above method, and the glucose content of the test sample was calculated according to the following formula. The glucose content in the embryo culture medium was 0.0571 mg / mL:

[0073] x=(cs-c0)×f

[0074] In the formula:

[0075] X represents the content of D-(+)-glucose in the sample, in mg / mL.

[0076] Cs represents the concentration of the sample test solution (mg / mL).

[0077] C0 represents the concentration of the blank test solution (mg / mL).

[0078] f represents the dilution factor of the sample.

[0079] As can be seen from the above, this invention can accurately detect the glucose content in embryo culture medium. Through rigorous experimental design and verification procedures, the accuracy and reliability of the detection results are ensured. The detection method of this invention has good repeatability, high sensitivity, and is simple to operate, making it suitable for the detection of glucose content in embryo culture medium and for product quality control.

[0080] In this invention, ultrafiltration centrifuge tubes (3KD) are used to filter proteins and amino acids in the sample, and high performance liquid chromatography (using a differential detector (RID) as the detector) with external standard curve method is used to detect the glucose content in the embryo culture medium. Features of this invention: (1) After the pretreatment operation of ultrafiltration centrifuge tubes (3KD), the interference of human serum albumin and some amino acid components in the embryo culture medium on the determination of the target component glucose content can be eliminated;

[0081] In this invention, an amino chromatography column is specifically selected. The hydroxyl groups in glucose form hydrogen bonds with the amino groups in the stationary phase of the amino chromatography column, and under the action of the mobile phase, they are separated from other components in the culture medium, thereby achieving the separation effect.

[0082] In this invention, a differential detector is used to establish an external standard curve quantification method. Results show that it exhibits good linearity within the concentration range of 0.02 mg / mL to 0.6 mg / mL, with a recovery rate in the range of 101.5% to 104.2%. The method is quick and easy to operate. The detection method of this invention has good repeatability, is simple and rapid, and provides accurate results, making it suitable for determining the glucose content in embryo culture media.

[0083] As described above, this invention establishes a method for determining glucose content in embryo culture medium. The method involves filtration in ultrafiltration centrifuge tubes (3KD) followed by centrifugation at 9500 rpm. The procedure is simple, provides good separation, and exhibits high sensitivity and accuracy, meeting routine testing requirements. High-performance liquid chromatography (HPLC) and a differential detector were used to comprehensively validate the method's linearity, limit of detection, limit of quantitation, repeatability, and accuracy, ensuring the reliability and reproducibility of the results and thus improving the accuracy of the detection.

[0084] 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.

[0085] 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 determining the glucose content in an embryo culture medium, characterized in that: The method includes the following steps: S1. Sample pretreatment: Take 10 mL of sample and filter it into a 3 KD ultrafiltration centrifuge tube. Then centrifuge at 9500 rpm for 10 min. Take the supernatant and filter it through a 0.22 μm microporous membrane as the sample test solution. Prepare a blank solution in the same way without adding sample. S2. Solution preparation: This specifically includes the following solutions: (1) D-(+)-glucose standard stock solution: Accurately weigh 100.72 mg of standard D-(+)-glucose, place it in a 25 mL volumetric flask, dissolve it in 85% acetonitrile and dilute to the mark to prepare a standard stock solution with a concentration of 3.948 mg / mL; (2) D-(+)-glucose standard curve solution: Transfer 0.050 mL, 0.125 mL, 0.250 mL, 0.500 mL, 1.000 mL, and 1.500 mL of D-(+)-glucose standard stock solution into six 10 mL volumetric flasks, dissolve and dilute with 80% acetonitrile to prepare a series of standard working solutions with concentrations of 0.02 mg / mL, 0.049 mg / mL, 0.099 mg / mL, 0.20 mg / mL, 0.39 mg / mL, and 0.59 mg / mL, respectively, and label them as STD-1, STD-2, STD-3, STD-4, STD-5, and STD-6; (3) System suitability solution: Take 0.250 mL of LD-(+)- glucose standard stock solution into a 10 mL volumetric flask, dissolve and dilute with 80% acetonitrile, and shake well to obtain 6 parallel solutions; (4) Specific solutions: Take blank solution, STD-3 standard working solution and sample test solution; (5) Detection limit solution: Accurately transfer 0.025 mL of D-(+)-glucose standard stock solution into a 10 mL volumetric flask, dissolve and dilute to the mark with 80% acetonitrile to prepare a detection limit solution with a concentration of 0.00987 mg / mL; (6) Limit of quantitation solution: Accurately transfer 0.050 mL of D-(+)-glucose standard stock solution into a 10 mL volumetric flask, dissolve and dilute to the mark with 80% acetonitrile to prepare a limit of quantitation solution with a concentration of 0.01974 mg / mL; (7) Repeatable solution: Take a 10 mL volumetric flask, add 0.250 mL L-(+)- glucose standard stock solution, and dilute to the mark with sample test solution; shake well to obtain the solution. Prepare 6 parallel solutions. (8) Accuracy solutions: Using the sample spiking method, add 0.125 mL, 0.250 mL, and 0.375 mL of L-(+)- glucose standard stock solution to a 10 mL volumetric flask, respectively, and dilute to 10 mL with sample test solution to obtain three accuracy solutions of low, medium, and high concentration levels. Prepare three parallel solutions for each concentration level. S3. The method for determining glucose content was validated using high performance liquid chromatography. The validation included system suitability, specificity, limit of detection, limit of quantitation, linear range, repeatability, and accuracy. S4. The glucose content in the embryo culture medium was determined using high performance liquid chromatography.

2. The method for determining glucose content in embryo culture medium as described in claim 1, characterized in that: In step S1, the ultrafiltration centrifuge tube is made of modified polyethersulfone membrane material with 3K. Before use, add enough primary water to submerge the membrane, and after ice bath for 10 minutes, pour out the water for later use.

3. The method for determining glucose content in embryo culture medium as described in claim 1, characterized in that: In step S3, linear regression analysis is performed on the standard curve solution to obtain the linear regression equation and establish the standard curve. The correlation coefficient of the curve equation should be ≥0.

99.

4. The method for determining glucose content in embryo culture medium as described in claim 1, characterized in that: In step S3, the specific solution is tested and analyzed. The resolution of the glucose target peak in the sample solution should be greater than 1.5, and the blank solution should be free of interference or have a response value less than 1 / 3 of the limit of quantitation.

5. The method for determining glucose content in embryo culture medium as described in claim 1, characterized in that: In step S3, the detection limit solution is injected three times repeatedly, and the signal-to-noise ratio should be >3:1 for each injection.

6. The method for determining glucose content in embryo culture medium as described in claim 1, characterized in that: In step S3, the quantitation limit solution is injected three times repeatedly, and the signal-to-noise ratio should be >10:1 for each injection.

7. The method for determining glucose content in embryo culture medium as described in claim 1, characterized in that: In step S3, the repeatability solution is analyzed to obtain repeatability test results, and the RSD should be ≤3%.

8. The method for determining glucose content in embryo culture medium as described in claim 1, characterized in that: In step S3, the accuracy solution is analyzed to obtain the accuracy result, and the recovery rate should be between 90% and 108%.

9. The method for determining glucose content in embryo culture medium as described in claim 1, characterized in that: In step S4, the chromatographic conditions are: detector: RID differential detector; Chromatographic column: Agilent Polaris NH2; column temperature: 40℃; injection volume: 20μL; flow rate: 1.0mL / min; Mobile phase: Acetonitrile: 10mM ammonium formate 80: 20.

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