Synchronous detection method for L-asparagine monohydrate and L-alanyl-L-glutamine in assisted reproduction culture solution

Abstract

The invention discloses a method for synchronously detecting L-asparagine monohydrate and L-alanyl-L-glutamine in an assisted reproduction culture solution. The L-asparagine monohydrate and the L-alanyl-L-glutamine in the assisted reproduction culture solution are used as a detection reagent. According to the method, a pretreatment mode of combining protein precipitation with diluted hydrochloric acid extraction is adopted, a target object is separated through ion exchange chromatography, and a ninhydrin post-column derivatization technology is adopted to establish an external standard method quantitative analysis system. The method has the following advantages: (1) synchronous and efficient separation and accurate quantification of two amino acid substances are realized; (2) methodology verification shows that good repeatability (RSD (Relative Standard Deviation) is 1.05) and high sensitivity (the detection results of L-asparagine monohydrate and L-alanyl-L-glutamine are 0.00038 mg / mL and 0.00036 mg / mL respectively); (3) the operation is simple and convenient, and the single analysis time is about 60 minutes; and (4) the recovery rate is stabilized in a range of 93%-105%. The method provides a reliable analysis means for quality control of the assisted reproduction culture solution, and is particularly suitable for quantitative determination of key amino acid components in the assisted reproduction culture solution.

Description

Technical Field

[0001] This invention belongs to the field of amino acid detection technology in culture medium, specifically a method for the simultaneous detection of L-asparagine monohydrate and L-alanyl-L-glutamine in assisted reproductive culture medium. Background Technology

[0002] In assisted reproductive technology (ART), the quality of the embryo culture medium has a significant impact on embryo development and pregnancy success rates. L-Asparagine monohydrate and L-Alanyl-L-glutamine are commonly used key amino acid components in the culture medium. The former participates in energy metabolism and protein synthesis, while the latter, as a stable dipeptide form of glutamine, provides nitrogen to the embryo and maintains cellular antioxidant capacity. Because embryos are highly sensitive to the culture environment, changes in the concentration of these amino acids can directly affect their developmental potential; therefore, accurate monitoring of their content is crucial. Currently, methods for amino acid detection mainly include high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS / MS), and amino acid analyzers.

[0003] However, traditional methods for assisted reproductive culture media have complex compositions, containing various interfering substances such as proteins, inorganic salts, and vitamins. Traditional methods may suffer from cumbersome pretreatment, insufficient separation, or high detection limits. In particular, L-alanyl-L-glutamine is easily hydrolyzed under high temperature or acidic conditions, while L-asparagine is unstable in alkaline environments. Therefore, a mild yet highly selective analytical method is needed. Among existing technologies, ion exchange chromatography combined with post-column derivatization has unique advantages in amino acid analysis due to its excellent separation ability and high sensitivity for polar substances. However, methods for the simultaneous detection of these two specific amino acids in assisted reproductive culture media have not yet been reported. Furthermore, current pharmacopoeias and industry standards (such as USP and EP) are increasingly stringent in their quality control requirements for embryo culture media.

[0004] Therefore, there is an urgent need to establish an analytical method that is highly specific, accurate, and suitable for routine quality control. This invention solves technical challenges such as interference from complex matrices and stability at low concentrations by optimizing sample pretreatment, chromatographic conditions, and derivatization reactions, providing a reliable analytical tool for the quality assessment of assisted reproductive culture media. Summary of the Invention

[0005] The purpose of this invention is to provide a method for the simultaneous detection of L-asparagine monohydrate and L-alanyl-L-glutamine 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 L-asparagine monohydrate and L-alanyl-L-glutamine in assisted reproductive culture medium, the method comprising the following steps:

[0007] S1: Prepare the instrument, specifically the amino acid analyzer;

[0008] S2: Prepare the solutions, specifically including the following solutions:

[0009] Test solution: Accurately transfer 1 mL of sample solution into a 10 mL centrifuge tube using a pipette, add 1 mL of sulfosalicylic acid and 1 mL of 0.02 mol / L HCl into the 10 mL centrifuge tube, vortex for 30 s, centrifuge at 7000 rpm for 1 min, and filter through a 0.22 μm microporous membrane to obtain the sample test solution.

[0010] Blank sample solution: Prepared without adding any sample, following the same procedure as the test sample solution.

[0011] Standard stock solutions: L-asparagine monohydrate, L-alanyl-L-glutamine standard stock solutions: Accurately weigh 16.99 mg of L-asparagine (purity 99.4%) and 18.24 mg of L-alanyl-L-glutamine standard (purity 99.9%), and dilute to 50 mL with 0.02 mol / L HCl to prepare standard stock solutions with an L-asparagine monohydrate concentration of 0.38383 mg / mL and an L-alanyl-L-glutamine concentration of 0.36444 mg / mL.

[0012] Linear solutions: Transfer appropriate amounts of L-asparagine monohydrate and L-alanyl-L-glutamine standard stock solution into 10 mL centrifuge tubes, add 0.02 mol / L HCl to prepare standard curve solutions with 6 concentration gradients, denoted as STD1, STD2, STD3, STD4, STD5, and STD6;

[0013] Detection limit solution: Accurately transfer 1 mL of L-asparagine monohydrate and L-alanyl-L-glutamine standard solution STD2 into a 10 mL centrifuge tube, and prepare detection limit solutions with concentrations of 0.00038 mg / mL and 0.00036 mg / mL respectively using 0.02 mol / L HCl;

[0014] Limit of quantitation solutions: Accurately transfer 1 mL of L-asparagine monohydrate and L-alanyl-L-glutamine mixed standard solution STD3 into a 10 mL centrifuge tube, and prepare limit of quantitation solutions with concentrations of 0.00077 mg / mL and 0.00073 mg / mL respectively using 0.02 mol / L HCl;

[0015] Repeatable solutions: Prepare six parallel solutions by adding standard concentration solutions according to accuracy.

[0016] Accuracy solution: The sample spiking method is used. Take 1 mL of sample, add 1 mL of sulfosalicylic acid, and then add the following three standard solutions of different concentrations respectively. Prepare three parallel solutions to obtain low, medium, and high concentration spiking solutions.

[0017] S3: The detection method was validated using an amino acid analyzer;

[0018] S4: The contents of L-asparagine monohydrate and L-alanyl-L-glutamine in the sample were determined by an amino acid analyzer.

[0019] In a preferred embodiment, in step S1, the instrument preparation involves adjusting various parameters to the working state, using an LCAK07 / Li column with a column temperature gradient of 30℃-70℃; flow rate: elution pump 0.45mL / min + derivatization pump 0.25mL / min; injection volume: 50μL; mobile phase: lithium citrate A = pH 2.90; B = pH 4.20; C = pH 8.00; detection wavelength: 570nm + 440nm.

[0020] In a preferred embodiment, in step S1, the initial temperature is 30°C, held for 0 min, then increased to 220°C at a rate of 20°C / min, held for 1 min, and then increased to 300°C at a rate of 10°C / min, held for 1 min.

[0021] In a preferred embodiment, in step S3, the methodology validation items include specificity, limit of detection, limit of quantitation, linearity and range, repeatability, and accuracy.

[0022] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0023] This invention features a low detection limit, simple pretreatment, high repeatability, and good accuracy. It provides a feasible method for detecting the content of L-asparagine monohydrate and L-alanyl-L-glutamine in assisted reproductive products. Attached Figure Description

[0024] Figure 1 The spectra of blank sample solutions of L-asparagine monohydrate and L-alanyl-L-glutamine are shown.

[0025] Figure 2 The spectral data of L-asparagine monohydrate and L-alanyl-L-glutamine samples are shown.

[0026] Figure 3Spectra of L-asparagine monohydrate and L-alanyl-L-glutamine test sample solutions;

[0027] Figure 4 This is a schematic diagram of the standard curve for L-asparagine monohydrate;

[0028] Figure 5 This is a schematic diagram of the L-propionamide-L-glutamine standard curve. Detailed Implementation

[0029] 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.

[0030] Reference Figure 1-5 ,

[0031] Example:

[0032] A method for simultaneous detection of L-asparagine monohydrate and L-alanyl-L-glutamine in assisted reproductive culture medium.

[0033] Instruments and reagents:

[0034] Amino acid analyzer (SYKAM, S-433D), electronic balance (accuracy 0.0001g, Sartorius), L-asparagine monohydrate standard (99.4%, 100mg / bottle, Bepure), L-alanyl-L-glutamine (99.9%, Tanmo Quality Inspection Technology Co., Ltd.), sulfosalicylic acid (100g / bottle, Tianjin Damao Chemical Reagent Factory), hydrochloric acid, 500mL / bottle / GR, Chengdu Kelong Chemical Co., Ltd.

[0035] Instrument conditions:

[0036] The instrument parameter settings for L-asparagine monohydrate and L-alanyl-L-glutamine gas chromatographs are shown in the table below:

[0037] Instrument conditions for amino acid analyzer

[0038]

[0039] Solution preparation:

[0040] 1) Test solution: Accurately transfer 1 mL of sample solution into a 10 mL centrifuge tube using a pipette, add 1 mL of sulfosalicylic acid and 1 mL of 0.02 mol / L HCl into the 10 mL centrifuge tube, vortex for 30 s, centrifuge at 7000 rpm for 1 min, and filter through a 0.22 μm microporous membrane to obtain the sample test solution.

[0041] 2) Blank sample solution: Prepared without adding any sample, following the same procedure as the test sample solution.

[0042] 3) Standard stock solutions: L-asparagine monohydrate and L-alanyl-L-glutamine standard stock solutions: Accurately weigh 16.99 mg of L-asparagine (purity 99.4%) and 18.24 mg of L-alanyl-L-glutamine standard (purity 99.9%), and dilute to 50 mL with 0.02 mol / L HCl to prepare standard stock solutions with L-asparagine monohydrate concentration of 0.38383 mg / mL and L-alanyl-L-glutamine concentration of 0.36444 mg / mL.

[0043] 4) Linear solutions: According to the table below, transfer appropriate amounts of L-asparagine monohydrate and L-alanyl-L-glutamine standard stock solution into 10 mL centrifuge tubes, add 0.02 mol / L HCl to prepare standard curve solutions with 6 concentration gradients, denoted as STD1, STD2, STD3, STD4, STD5, and STD6.

[0044] linear solutions

[0045]

[0046] 5) Detection limit solution: Accurately transfer 1 mL of L-asparagine monohydrate and L-alanyl-L-glutamine standard solution STD2 into a 10 mL centrifuge tube, and prepare detection limit solutions with concentrations of 0.00038 mg / mL and 0.00036 mg / mL respectively using 0.02 mol / L HCl.

[0047] 6) Limit of quantitation solution: Accurately transfer 1 mL of L-asparagine monohydrate and L-alanyl-L-glutamine mixed standard solution STD3 into a 10 mL centrifuge tube, and prepare limit of quantitation solutions with concentrations of 0.00077 mg / mL and 0.00073 mg / mL respectively with 0.02 mol / L HCl.

[0048] 7) Repeatability solution: Prepare six parallel solutions using standard concentrations according to accuracy standards. 8) Accuracy solution: Use the sample spiking method. Take 1 mL of sample, add 1 mL of sulfosalicylic acid, and then add the following three concentrations of standard solution respectively. Prepare three parallel solutions for each concentration to obtain low, medium, and high concentration spiking solutions. The spiking concentrations are shown in the table below:

[0049] Asparagine monohydrate spiking concentration

[0050]

[0051] Alanyl-L-glutamine spiking concentration

[0052]

[0053] The methodological validation results for the determination of L-asparagine monohydrate and L-alanyl-L-glutamine content in an assisted reproductive product are as follows: Specificity: Blank sample solution, test solution, and spiked sample solution were tested separately, and chromatograms were recorded. Figure 1 - Figure 3 It can be seen that the resolution of L-asparagine monohydrate in this method is 2.5, the resolution of L-alanyl-L-glutamine and other amino acids is 17.2, and there are no interfering peaks at the target peak position in the blank sample solution, which meets the method validation evaluation criteria.

[0054] 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.

[0055] Asparagine monohydrate standard curve test results

[0056]

[0057] Alanyl-L-glutamine standard curve test results

[0058] 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 ≤ 8%. The test results are shown in the table below. The results indicate that: for L-asparagine monohydrate standard solution with a concentration of 0.00038 mg / mL and L-alanyl-L-glutamine standard solution with a concentration of 0.00036 mg / mL, the SNR was > 3:1; for L-asparagine monohydrate standard solution with a concentration of 0.00077 mg / mL and L-alanyl-L-glutamine standard solution with a concentration of 0.00073 mg / mL, the SNR was > 10:1. The RSD of the peak areas of the three LOQ solutions was < 8%, meeting the method validation evaluation criteria.

[0059] Limit of detection and limit of quantitation for asparagine monohydrate

[0060]

[0061] Limits of detection and limits of quantitation for alanyl-L-glutamine

[0062]

[0063] 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 area should be ≤4%. The test results are shown in the table below. The RSD of the determination results of L-asparagine monohydrate and L-alanyl-L-glutamine is <4%. The results show that the method has good repeatability and meets the method validation evaluation criteria.

[0064] Repeatability test results of asparagine monohydrate

[0065]

[0066] Repeatability test results of alanyl-L-glutamine

[0067]

[0068] Accuracy: Accuracy solutions were tested on the instrument. According to Guideline 9101 of the 2020 edition of the Chinese Pharmacopoeia, the recovery rate for low concentrations should be between 75% and 120%, the recovery rate for medium concentrations should be between 80% and 115%, and the recovery rate for high concentrations should be between 85% and 110%. The test results are shown in the table below. The results show that the accuracy of the target analyte content determination is good. The spiked recovery rate of L-asparagine monohydrate is in the range of 99% to 105%, and the spiked recovery rate of L-alanyl-L-glutamine is in the range of 93% to 104%, which meets the requirements of the spiked recovery rate for method validation and meets the evaluation criteria for method validation.

[0069] Asparagine Monohydrate Accuracy Test Results

[0070]

[0071] Alanyl-L-glutamine accuracy test results

[0072]

[0073] Note: Spiked recovery % = (Actual test concentration - Target analyte concentration in sample) / Theoretical spiked concentration * 100%

[0074] Test results of L-asparagine monohydrate and L-alanyl-L-glutamine content

[0075] The contents (mg / mL) of L-asparagine monohydrate and L-alanyl-L-glutamine were determined using an amino acid analyzer and calculated according to the following formula:

[0076] X = (C S -C0)×f;

[0077] In the formula:

[0078] X — The content of amino acids in the sample, in mg / mL;

[0079] Cs—Concentration of the sample test solution (mg / mL);

[0080] Co – Concentration of blank test solution (mg / mL);

[0081] f—Dilution factor;

[0082] The test solution was tested under the above chromatographic conditions. According to formula (1), the content of L-asparagine monohydrate in the sample with batch number J000006812 was calculated to be 0.0115 mg / mL and the content of L-alanyl-L-glutamine was 0.0230 mg / mL.

[0083] As described above, this invention employs an amino acid analyzer to determine the content of L-asparagine monohydrate and L-alanyl-L-glutamine in assisted reproductive products, and has conducted detailed methodological validation. This method features low detection limits, simple pretreatment, high repeatability, and good accuracy. It provides a feasible method for detecting the content of L-asparagine monohydrate and L-alanyl-L-glutamine in assisted reproductive products.

[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 the simultaneous detection of L-asparagine monohydrate and L-alanyl-L-glutamine in an assisted reproduction culture medium, characterized in that: The method comprises the following steps: S1: instrument preparation, prepare the amino acid analyzer; S2: solution preparation, specifically including the following solutions: Test sample solution: accurately pipette 1 mL of sample solution into a 10 mL centrifuge tube, add 1 mL of sulfosalicylic acid, 1 mL of 0.02 mol / L HCL into a 10 mL centrifuge tube, vortex for 30 s, centrifuge at 7000 rpm for 1 min, filter through a 0.22 μm microporous filter to obtain the sample test solution; Blank sample solution: without sample, according to the preparation of the test sample solution, it is obtained; Standard stock solution: L-asparagine monohydrate, L-alanyl-L-glutamine standard stock solution: accurately weigh L-asparagine 16.99 mg (purity 99.4%) and L-alanyl-L-glutamine standard 18.24 mg (purity 99.9%), and dilute to 50 mL with 0.02 mol / L HCL to prepare a standard stock solution with a concentration of 0.38383 mg / mL of L-asparagine monohydrate and 0.36444 mg / mL of L-alanyl-L-glutamine; Linear solution: remove an appropriate amount of L-asparagine monohydrate and L-alanyl-L-glutamine standard stock solution from a 10 mL centrifuge tube, and add 0.02 mol / L HCL to prepare 6 concentration gradient standard curve solutions, denoted as STD1, STD2, STD3, STD4, STD5, and STD6; Detection limit solution: accurately pipette 1 mL of L-asparagine monohydrate and L-alanyl-L-glutamine standard solution STD2 into a 10 mL centrifuge tube, and dilute to 0.02 mol / L HCL to prepare a detection limit solution with a concentration of 0.00038 mg / mL and 0.00036 mg / mL, respectively; Quantitative limit solution: accurately pipette 1 mL of L-asparagine monohydrate and L-alanyl-L-glutamine mixed standard solution STD3 into a 10 mL centrifuge tube, and dilute to 0.02 mol / L HCL to prepare a quantitative limit solution with a concentration of 0.00077 mg / mL and 0.00073 mg / mL, respectively; Repeatability solution: prepare 6 solutions in parallel with the standard concentration solution; Accuracy solution: use the sample spiking method; take 1 mL of sample, add 1 mL of sulfosalicylic acid, and then add standard solutions with the following three concentrations, respectively, to prepare three solutions in parallel to obtain low, medium, and high concentration spiked solutions; S3: method validation of the detection method using the amino acid analyzer; S4: analyze and determine the content of L-asparagine monohydrate and L-alanyl-L-glutamine in the sample using the amino acid analyzer.

2. The method for simultaneous detection of L-asparagine monohydrate and L-alanyl-L-glutamine in an assisted reproduction culture medium according to claim 1, characterized in that: In the step S1, the instrument is prepared for adjusting parameters to working condition, the chromatographic column is LCAK07 / Li, the column temperature is 30-70℃ gradient temperature rising; the flow rate is 0.45 mL / min for the elution pump and 0.25 mL / min for the derivative pump; the sample injection amount is 50 μL; the mobile phase is lithium citrate A=pH 2.90, B=pH 4.20 and C=pH 8.00; the detection wavelength is 570 nm+440 nm.

3. The method for simultaneous detection of L-asparagine monohydrate and L-alanyl-L-glutamine in an assisted reproduction culture medium according to claim 1, characterized in that: In the step S1, the initial temperature is 30℃, the temperature is kept for 0 min, the temperature is raised to 220℃ at a rate of 20℃ / min, the temperature is kept for 1 min, the temperature is raised to 300℃ at a rate of 10℃ / min, and the temperature is kept for 1 min.

4. The method for simultaneous detection of L-asparagine monohydrate and L-alanyl-L-glutamine in an assisted reproduction culture medium according to claim 1, characterized in that: In the step S3, the method validation items include specificity, detection limit, quantification limit, linearity and range, repeatability and accuracy.

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