Method for determining glyceric acid in biological sample

Abstract

The invention belongs to the technical field of analysis and detection, and particularly relates to a method for determining glyceric acid in a biological sample, which comprises the following steps: mixing the biological sample with an extracting solution, and extracting to obtain supernate; evaporating and concentrating the supernate, re-dissolving the supernate in an acetonitrile aqueous solution, and filtering to obtain a solution to be detected; the extracting solution comprises an internal standard solution and a solvent, the internal standard solution comprises DL-glyceric acid-2, 3, 3-d3 calcium salt dihydrate and a methanol / water solution, and the solvent is acetonitrile; and testing the to-be-tested solution through liquid chromatography-tandem mass spectrometry, and obtaining the content of glyceric acid in the to-be-tested solution according to the standard curve. The determination steps are simple and convenient, and a large number of samples can be rapidly and simultaneously treated. By adopting an isotope internal standard method for quantification, the matrix effect can be greatly eliminated, and accurate quantification can be achieved. And reference can be provided for clinical early diagnosis of gestational diabetes mellitus.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of analytical detection, and particularly relates to a method for determining glyceric acid in a biological sample. BACKGROUND

[0002] Gestational diabetes mellitus (GDM) refers to impaired glucose tolerance (IGT) or diabetes mellitus first found during pregnancy. When blood glucose control of pregnant women with gestational diabetes mellitus is poor, excessive glucose will enter the fetal body through the placenta, leading to an increased risk of adverse pregnancy outcomes. OGTT (oral glucose tolerance test) is the gold standard and core method for diagnosing gestational diabetes mellitus (GDM). However, due to the lack of obvious early symptoms and large individual differences, the OGTT test method cannot screen pregnant women with abnormal glucose metabolism in the early stage, which limits the prediction and diagnosis of GDM.

[0003] Before the clinical symptoms of GDM appear, the metabolism in the body has often changed significantly, and metabolomics technology can capture these changes, so it has a significant advantage in finding early predictors. The biological samples for GDM detection by metabolomics include blood, urine, feces, milk, hair, amniotic fluid, placenta, umbilical cord blood and meconium. Although a large number of differential metabolites closely related to the pathophysiology of GDM have been found, such as amino acids, lipids and bile acid metabolism, their reliability and clinical application value have not been fully verified by large-scale and high-quality research.

[0004] Glyceric acid is a key intermediate product of glycerol metabolism and serine synthesis pathway, and its content directly reflects the rate of sugar metabolism and can be used as a marker for gestational diabetes mellitus. Due to the complexity of biological samples, many interference factors, and the characteristics of glyceric acid, such as large polarity, insolubility in water, and lack of conjugated groups, it is not easy to be detected in liquid chromatography-mass spectrometry, which limits the application of glyceric acid as a marker in the early diagnosis of gestational diabetes mellitus. SUMMARY

[0005] The purpose of the present application is to provide a method for determining glyceric acid in a biological sample, thereby overcoming the shortcomings of the prior art. The method uses DL-glyceric acid-2,3,3-d3 calcium salt dihydrate as an internal standard and analyzes the glyceric acid content in the biological sample by liquid chromatography-mass spectrometry. The method is simple, high-throughput, low-cost and fast, with an analysis time of only 6 minutes. It has guiding significance for the early diagnosis of gestational diabetes mellitus and is easy to popularize and popularize in clinical practice.

[0006] In order to achieve the above purpose, the technical scheme of the present application is as follows: The present application provides a method for determining glyceric acid in a biological sample, comprising the following steps: The biological sample is mixed with an extraction solution to obtain supernatant after extraction treatment; the supernatant is concentrated by evaporation, redissolved in an acetonitrile aqueous solution, and filtered to obtain a to-be-tested solution; wherein the extraction solution comprises an internal standard solution and a solvent, the internal standard solution comprises DL-glyceric acid-2,3,3-d3 calcium salt dihydrate and a methanol / water solution, and the solvent is acetonitrile; The to-be-tested solution is tested by liquid chromatography tandem mass spectrometry, and the content of glyceric acid in the to-be-tested solution is obtained according to a standard curve.

[0007] The present application uses DL-glyceric acid-2,3,3-d3 calcium salt dihydrate as an internal standard, replaces the matrix PBS or water, effectively separates precipitated proteins, removes impurities, reduces ion interference, reduces matrix effects, and at the same time can maximize the retention of glyceric acid, avoid its decomposition or oxidation, improve the extraction recovery rate, and thus detect the most real glyceric acid content. The glyceric acid content in the biological sample is analyzed by liquid chromatography tandem mass spectrometry, which has the characteristics of simple operation, high throughput, low cost and rapidity, and the analysis time is only 6 minutes. It has guiding significance for early diagnosis of gestational diabetes mellitus and is easy to popularize and popularize in clinical practice.

[0008] In some other embodiments, the biological sample is one of plasma, serum, blood, urine, feces and saliva; and the extraction treatment is vortexing, oscillation and centrifugation in sequence. The biological sample has a wide source and a simple pretreatment method.

[0009] In some other embodiments, the volume ratio of the biological sample to the extraction solution is 1:(3-5); and the concentration of the internal standard in the extraction solution is 1-2 μg / mL. Specifically, the volume ratio of the biological sample to the extraction solution is 1:3, 1:4 or 1:5; and the concentration of the internal standard in the extraction solution is 1, 1.5 or 2 μg / mL.

[0010] In some other embodiments, the volume ratio of the supernatant to the acetonitrile aqueous solution is (2-4):1; and the volume concentration of the acetonitrile aqueous solution is 35-45%. Specifically, the volume ratio of the supernatant to the acetonitrile aqueous solution is 2:1, 3:1 or 4:1; and the volume concentration of the acetonitrile aqueous solution is 35, 40 or 45%.

[0011] In some other embodiments, the volume ratio of the biological sample to the extraction solution is 1:4; the concentration of the extraction solution is 1.5 μg / mL; the volume ratio of the supernatant to the acetonitrile aqueous solution is 2.5:1; and the volume concentration of the acetonitrile aqueous solution is 40%.

[0012] The glyceric acid in the sample is sufficiently extracted in this range and can maximize the retention of glyceric acid, avoid its decomposition or oxidation, and thus detect the most real glyceric acid content.

[0013] In some other embodiments, the chromatographic column used in the liquid chromatography tandem mass spectrometry is Agilent RRHDEclipse Plus C18, the column temperature is 25-30℃, the flow rate is 0.25-0.35 mL / min, and the injection volume is 3-5 μL; The mobile phase is divided into mobile phase A and mobile phase B, the mobile phase A is one of formic acid-formic acid ammonium acetonitrile solution, formic acid-acetic acid ammonium acetonitrile solution and acetonitrile; The mobile phase B is one of formic acid-formic acid ammonium water solution, formic acid-acetic acid ammonium water solution and acetic acid ammonium water solution; In the mobile phase A and the mobile phase B, the volume concentration of formic acid is 0.01%-1%, and the concentration of formic acid ammonium or acetic acid ammonium is 1-15 mM.

[0014] In this chromatographic analysis, the addition of formic acid ammonium / acetic acid ammonium in the mobile phase is beneficial to improve the response of glyceric acid, increase the separation degree, and better separate glyceric acid from the interferents. The total running time can be 6.0 min or less than 6.0 min.

[0015] In some other embodiments, the column temperature used in the liquid chromatography is 25℃, the flow rate is 0.3 mL / min, the injection volume is 5 μL, the mobile phase A is acetonitrile, the mobile phase B is water containing 5 mM acetic acid ammonium, and the gradient elution program is as follows: 0-1 min, 10% A; 1-2 min, 10%-50% A; 2-2.5 min, 50%-90% A; 2.5-3 min, 90% A; 3-6 min, 90%-10% A.

[0016] Since the sample to be measured is a plasma sample, the endogenous substances are complex, and need to be extracted before being analyzed. Gradient elution is performed with A: acetonitrile and B: water containing 5 mM acetic acid ammonium as the mobile phase, which not only can reduce the interference of impurities existing in the plasma and improve the peak shape, but also can better separate the chromatographic peaks of glyceric acid and its interferents and reduce the retention time.

[0017] In some other embodiments, the liquid chromatography tandem mass spectrometry uses the negative ion electrospray ionization multi-ion reaction monitoring mode, the curtain gas is 35-45 kPa, the spray voltage is 4000-5000 V, the desolvation temperature is 450-550℃, GS1 is 50-60 kPa, GS2 is 45-55 kPa, and the ion pair used is one of 74.9 / 76.1 and 56.9 / 61.9.

[0018] In some other embodiments, in the mass spectrum, curtain gas: 40 kPa, spray voltage: 4500 V, desolvation temperature: 500 DEG C, GS1: 55 kPa, GS2: 50 kPa, the ion pair used is 74.9 / 76.1.

[0019] Advantages of the present application: (1) The present application uses DL-glycerate-2,3,3-d3 calcium salt dihydrate as an internal standard, effectively separates precipitated protein, removes impurities, reduces ion interference, reduces matrix effect, and at the same time can maximize the retention of glycerate, avoiding its decomposition or oxidation, so as to detect the most real glycerate content. The glycerate in the biological sample is quantitatively analyzed by liquid chromatography-mass spectrometry analysis, which has the characteristics of simple operation, high throughput, low cost and rapid analysis time of only 6 minutes, and has guiding significance for early diagnosis of gestational diabetes mellitus, and is easy to popularize and popularize in clinic.

[0020] (2) Since the sample to be measured is a biological sample such as plasma, the endogenous substances are complex, and need to be extracted before analysis. The detection conditions of chromatography and mass spectrometry analysis are optimized, and the response value change, peak shape, whether tailing, peak value, peak time and the like are investigated, and finally the chromatography and mass spectrometry conditions are selected. The results show that the method has high sensitivity, short analysis time, high throughput, good precision, and is suitable for large-scale clinical detection.

[0021] (3) The pre-treatment step of the present application is simple, can quickly and simultaneously process a large number of samples, and is suitable for investigation and analysis of glycerate in vivo metabolism of large-scale population. The use of isotope internal standard method for quantification can greatly eliminate matrix effect and achieve accurate quantification. It can provide a reference for early clinical diagnosis of gestational diabetes mellitus. BRIEF DESCRIPTION OF DRAWINGS

[0022] The drawings accompanying the specification of the present application form a part thereof, serve to provide further understanding of the present application, and together with the description of the exemplary embodiments of the present application and their description serve to explain the present application, and do not constitute an improper limitation of the present application.

[0023] Figure 1 The mass spectrum of the glycerate calcium salt dihydrate standard in Example 1 of the present application is shown in Figure 1. Figure 2 The internal standard mass spectrum of the glycerate calcium salt dihydrate in Example 1 of the present application is shown in Figure 2. Figure 3 The standard curve established in Example 1 of the present application is shown in Figure 3. DETAILED DESCRIPTION

[0024] Those skilled in the art will understand that the following examples are only for illustration of the present application and should not be considered as limiting the scope of the present application. The specific conditions are not specified in the examples, and the conventional conditions or the conditions recommended by the manufacturer are used. The components used are not specified by the manufacturer, and are all conventional products available on the market.

[0025] For the existing detection methods of organic acids, including ion chromatography, capillary electrophoresis, gas chromatography, high performance liquid chromatography, liquid chromatography tandem mass spectrometry, etc. Among them, the disadvantages of ion chromatography are long detection time, high requirement for instruments and high price. Capillary electrophoresis has short analysis time, but the preparation of buffer is relatively complicated, and a variety of solvents need to be adjusted according to certain proportions. The operation of gas chromatography for analyzing organic acids is complex, and the accuracy of the results is not high, because the pre-column treatment is not easy to quantify and cannot be qualitatively. Endogenous metabolites glyceric acid are difficult to be detected due to its characteristics: high polarity, insoluble in water, and no conjugated group. It can be seen that the above methods cannot provide a high-efficiency and accurate detection method for large-scale clinical detection. In addition to the above commonly used detection methods, there are some special methods for the detection of organic acids, such as nuclear magnetic resonance spectroscopy (NMR) and fluorescence spectroscopy. These methods are usually used for more in-depth structural analysis and trace analysis, and the detection process is relatively complex and the detection cost is relatively large.

[0026] Liquid chromatography-mass spectrometry technology has the advantages of high accuracy, high resolution, short detection time, etc., and can achieve simultaneous qualitative and quantitative analysis of multiple substances. In the prior art, liquid chromatography tandem mass spectrometry (LC-MS) combined with standard samples can accurately quantify the content of organic acids in samples. However, there is no method for detecting glyceric acid at present.

[0027] The method for determining glyceric acid in a biological sample of the present application is further described below in combination with specific examples: Example 1 A method for determining glyceric acid in a biological sample, comprising the following steps: (1) Sample pretreatment: Take 200 μL of sample (for example, plasma), add 800 μL of extraction solution, vortex for 5 min, centrifuge at 14000 rpm for 5 min, and prepare the supernatant. Take 900 μL of the supernatant, concentrate and dry, add 500 μL of 40% acetonitrile aqueous solution (ACN-H2O), vortex and shake, filter with a 0.2 μm filter membrane, and obtain the biological sample to be tested.

[0028] The preparation method of the extraction solution is as follows: DL-glyceric acid-2,3,3-d3 calcium salt dihydrate (product number: 616672, CAS: 1312713-87-3, mass spectrum as shown in Figure 2Isotopic internal standard (shown in Figure 1) was dissolved in methanol / water solution (1:1 by volume) to prepare an internal standard solution with a concentration of 1 mg / mL. The internal standard solution was diluted with acetonitrile to prepare a solution containing 1.5 μg / mL of internal standard as the extraction solution.

[0029] Glyceric acid is highly polar and viscous, and is not easy to quantify. Commercially available glyceric acid standard products include sodium glycerate and calcium glycerate dihydrate. However, the molecular weight of sodium glycerate is not clear, and it cannot be quantified. Therefore, the calcium glycerate dihydrate is selected as the standard product, and the DL-glyceric acid-2,3,3-d3 calcium salt dihydrate is selected as the internal standard.

[0030] (2) Preparation of standard solution: The glyceric acid standard solution (product number: ZTR-D253085, CAS: 6000-41-5, TRC, mass spectrum shown in Figure 1) was prepared with water as a stock solution, and then the standard solution was prepared with water and diluted in gradient to 7 concentrations (0.31, 0.625, 1.25, 2.5, 5, 10, 20 μg / mL). Figure 1

[0031] (3) Liquid chromatography tandem mass spectrometry (LC-MS) detection: The liquid chromatography tandem mass spectrometer used is AB Q-Trap 5500.

[0032] Liquid chromatography parameters: The chromatographic column type is Agilent RRHD Eclipse Plus C18, 3.0x100mm, 1.8um, the column temperature is 25℃, the flow rate is 0.3mL / min, the injection volume is 5μL, and the mobile phase is divided into mobile phase A and mobile phase B, wherein the mobile phase A is acetonitrile and the mobile phase B is water containing 5mM ammonium acetate; the gradient elution conditions are shown in Table 1: 0-1min, 10% A; 1-2min, 10%-50% A; 2-2.5min, 50%-90% A; 2.5-3min, 90% A; 3-6min, 90%-10% A.

[0033] Table 1 Gradient elution program

[0034] In this embodiment, the mobile phase A: acetonitrile and the mobile phase B: water containing 5mM ammonium acetate are used as the mobile phase for gradient elution, which not only reduces the interference of impurities present in the plasma and improves the peak shape, but also better separates the glyceric acid and its interfering substance chromatographic peaks, and reduces the retention time.

[0035] ​Mass spectrometry parameters: negative ion electrospray ionization, multiple ion reaction monitoring mode, mass spectrometry parameter settings include ion source parameter settings and MRM parameter settings (as shown in Table 2). Among them, the ion source parameters include: curtain gas: 40 kPa, spray voltage: 4500 V, desolvation temperature: 500 ℃, atomization gas (GS1) pressure: 55 kPa, auxiliary gas (GS2) pressure: 50 kPa.

[0036] Table 2 parent ion, daughter ion parameter table

[0037] From Table 2, by optimizing the mass spectrometry conditions, a total of 2 ion pairs can be selected, specifically: 74.9 / 76.1, 56.9 / 61.9 ion pairs.

[0038] (4) Preparation of standard curve: Take 200 μL of each of the 7 concentrations (0.31, 0.625, 1.25, 2.5, 5, 10, 20 μg / mL) of the standard solution prepared in step (2), and add 800 μL of the extraction solution, respectively. Process according to the sample pretreatment method in step (1) to prepare 7 concentrations of mixed standard working solution.

[0039] Using LC-MS to determine the peak area of each mixed standard working solution, taking the concentration of each mixed standard working solution as the x-axis and the peak area of each mixed standard working solution as the y-axis; the linear range, standard curve equation, linear correlation coefficient and detection limit are shown in Table 3, and the standard curve is shown in Figure 3 .

[0040] Table 3 Linear range and equation of glyceric acid

[0041] From Table 3, it can be seen that the glyceric acid curve has good correlation R>0.99, and the deviation is within ±15 %.

[0042] (5) Reproducibility The 20 μg / mL calcium glycerate dihydrate standard stock solution in step (2) was diluted horizontally into 6 1.25 μg / mL standard solutions, and 800 μL of acetonitrile containing internal standard solution was added as extraction solution. The other preparation methods are the same as in Example 1, and the sample was injected. The relative standard deviation (RSD) was 1.8 %, and the RSD was less than 15.0 %. The reproducibility test met the requirements.

[0043] (6) Spiked recovery rate The water solution was used to gradient dilute 20 μg / mL glyceric acid calcium salt dihydrate standard solution stock solution, and 7 standard solution with concentrations of 0.31, 0.625, 1.25, 2.5, 5, 10 and 20 μg / mL were prepared, and the standard curve was obtained by injection. The low-concentration clinical sample was selected as the base sample, the standard was added, and the low-concentration and high-concentration samples to be measured (2.5 and 16 μg / mL) were prepared. Each concentration sample was divided into 5 parts, the recovery rate was calculated according to formula (1), and the recovery rate should meet the requirements of 85%-115%. The analysis results are shown in Table 4.

[0044] Formula (1) R - recovery rate; C - the average measured concentration of the standard added in the low-concentration clinical sample, μg / mL; C 0 - the average measured concentration of the low-concentration clinical sample, μg / mL; C s - the amount of standard added, μg / mL.

[0045] Table 4 Recovery rate of glyceric acid at different concentrations

[0046] As shown in Table 4, the recovery rates at different concentrations are all within the range of 85%-115%, and the relative standard deviations of the 6 recovery rate data are less than 10.0%. The recovery rate meets the requirements.

[0047] (6) Quantification of glyceric acid in the sample to be measured: The peak area of the biological sample to be measured was substituted into the standard curve equation of glyceric acid in Table 3 to inversely calculate the concentration of glyceric acid in the biological sample.

[0048] (7) Clinical sample detection 20 serum samples of normal persons were selected, and the samples were detected by the mass spectrometer after the sample pretreatment according to step (1), and the data are shown in Table 5.

[0049] Table 5 Detection results of clinical samples

[0050] Comparative Example 1 A method for measuring glyceric acid in a biological sample, which is different from Example 1, is that the mobile phase used in the liquid chromatography-mass spectrometry (LC-MS) detection in step (3) is methanol and water, and the other processes are consistent with those of Example 1.

[0051] Since the sample to be measured is a plasma sample, the endogenous substances are complex, and need to be extracted and then analyzed. When methanol and water are used as the mobile phase, it is difficult to completely separate glyceric acid from the impurities present in the plasma.

[0052] Comparative Example 2 A method for determining glyceric acid in a biological sample, which is different from Example 1, is that in step (2), PBS, 2% BSA, and 5% BSA are respectively used as the replacement matrix in the preparation of the standard solution, and the other processes are consistent with Example 1.

[0053] It is found that, since glyceric acid is an endogenous metabolite, the use of BSA as the replacement matrix has an influence on the background. The use of PBS as the replacement matrix has a better effect, a low baseline, and almost no influence of matrix effect. However, considering the damage of long-term injection of salt solution to the instrument and the chromatographic column, water is selected as the replacement matrix in this embodiment. When water is used as the replacement matrix, the extraction recovery rate is between 50% and 60%. It is expected that, when PBS is used as the replacement matrix, the extraction recovery rate can be more than 80%.

[0054] Comparative Example 3 A method for determining glyceric acid in a biological sample, which is different from Example 1, is that in step (1), acetonitrile, methanol-acetonitrile (volume ratio 1:1), methanol, and 0.1% methanol-acetonitrile are respectively used as the extraction liquid, and the volume ratio of the biological sample to the extraction liquid and the response value of glyceric acid are shown in Table 6. The other processes are consistent with Example 1.

[0055] Table 6 Response value of glyceric acid prepared by different extraction liquids

[0056] It can be seen from Table 6 that, when the volume ratio of the biological sample to the extraction liquid in Example 1 is 1:4, the extraction efficiency of glyceric acid is the highest.

[0057] Comparative Example 4 A method for determining glyceric acid in a biological sample, which is different from Example 1, is that in step (3), Agilent RRHD Eclipse Plus C18, APCELL PAK ADME HR, PC HILIC, and Titank C18 are respectively selected as the chromatographic column, and the separation results are shown in Table 7.

[0058] Since glyceric acid has high polarity, it is difficult to have retention in the chromatographic column. By comparing the retention time and peak shape when different types of chromatographic columns are used for separation, it is finally determined that the Agilent RRHD Eclipse Plus C18 column is used. The effects of different types of chromatographic columns on different substances are shown in Table 7.

[0059] Table 7 Effects of different types of chromatographic columns on different substances

[0060] In Table 7, “√” represents good, and “x” represents poor.

[0061] In summary, the present application is pretreated by a simple liquid-liquid extraction method on the biological sample, and then enters chromatographic separation and mass spectrometry detection, selects a pair of qualitative ions and quantitative ions, uses the relative retention time of glyceric acid and the qualitative ion pair as the qualitative basis, and quantifies by preparing a standard curve with a standard product. Meanwhile, the present application uses two levels of quality control products to investigate the accuracy and effectiveness of the method, and avoids distortion of the detection results.

[0062] The present application first realizes the purpose of detecting glyceric acid in biological samples by liquid chromatography-mass spectrometry method, optimizes the chromatographic mass spectrometry conditions, and reduces the influence of interfering substances. The method is simple, rapid, and has a high throughput and low cost, and the analysis time can be 6 minutes or less than 6 minutes. At the same time, the internal standard method is used for quantification, which ensures the accuracy of the results and effectively monitors the glyceric acid level in the human body, has guiding significance for early diagnosis of gestational diabetes, and is easy to popularize and popularize in clinical practice. The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for determining glyceric acid in a biological sample, characterized in that: The following steps are involved: The biological sample is mixed with an extracting solution, and after extraction treatment, a supernatant is obtained; the supernatant is evaporated and concentrated, redissolved in an acetonitrile aqueous solution, and filtered to obtain a test solution; wherein the extracting solution comprises an internal standard solution and a solvent, the internal standard solution comprises DL-glyceric acid-2,3,3-d3 calcium salt dihydrate and methanol / water solution, and the solvent is acetonitrile; The solution to be tested is tested by liquid chromatography tandem mass spectrometry, and the content of glyceric acid in the solution to be tested is obtained according to the standard curve.

2. The method for determining glyceric acid in a biological sample according to claim 1, wherein: The biological sample is one of plasma, serum, blood, urine, feces and saliva.

3. The method for determining glyceric acid in a biological sample according to claim 1, wherein: The extraction process is to perform vortexing, shaking and centrifugation in sequence.

4. The method for determining glyceric acid in a biological sample according to claim 1, wherein: The mixing volume ratio of the biological sample to the extract is 1:(3-5); The concentration of DL-glyceric acid-2,3,3-d3 calcium salt dihydrate in the extract is 1-2 μg / mL.

5. The method for determining glyceric acid in a biological sample according to claim 1, wherein: The volume ratio of the supernatant to the acetonitrile aqueous solution is (2-4):1; The volume concentration of the acetonitrile aqueous solution is 35-45%.

6. The method for determining glyceric acid in a biological sample according to claim 1, wherein: The mixing volume ratio of the biological sample to the extract is 1:4; The concentration of DL-glyceric acid-2,3,3-d3 calcium salt dihydrate in the extract is 1.5 μg / mL; The volume ratio of the supernatant to the acetonitrile aqueous solution is 2.5:1; The volume concentration of the acetonitrile aqueous solution is 40%.

7. The method for determining glyceric acid in a biological sample according to claim 1, wherein: The chromatographic column used in liquid chromatography-tandem mass spectrometry was an Agilent RRHD Eclipse Plus C18, the column temperature was 25-30 °C, the flow rate was 0.25-0.35 mL / min, and the injection volume was 3-5 μL. The mobile phase is divided into mobile phase A and mobile phase B, and mobile phase A is one of formic acid-ammonium formate acetonitrile solution, formic acid-ammonium acetate acetonitrile solution and acetonitrile; Mobile phase B is one of an aqueous solution of formic acid-ammonium formate, an aqueous solution of formic acid-ammonium acetate, and an aqueous solution of ammonium acetate; In mobile phase A and mobile phase B, the volume concentration of formic acid is 0.01-1%, and the concentration of ammonium formate or ammonium acetate is 1-15 mM.

8. The method for determining glyceric acid in a biological sample according to claim 7, wherein: The column temperature used in liquid chromatography was 25°C; the flow rate was 0.3 mL / min, and the injection volume was 5 μL; mobile phase A was acetonitrile, and mobile phase B was water containing 5 mM ammonium acetate. The separation was performed using a gradient elution program: 0-1 min, 10% A; 1-2 min, 10%-50% A; 2-2.5 min, 50%-90% A; 2.5-3 min, 90%A; 3-6min, 90%-10%A.

9. The method for determining glyceric acid in a biological sample according to claim 1, wherein: Liquid chromatography tandem mass spectrometry employed negative ion electrospray ionization in multiple ion reaction monitoring mode, curtain gas: 35-45 kPa, spray voltage: 4000-5000 V, desolvation temperature: 450-550 °C, GS1: 50-60 kPa, GS2: 45-55 kPa, and one of the ion pairs used was 74.9 / 76.1 and 56.9 / 61.

9.

10. The method for determining glyceric acid in a biological sample according to claim 9, wherein: In the mass spectrometry, the curtain gas was 40 kPa, the spray voltage was 4500 V, the desolvation temperature was 500 °C, GS1 was 55 kPa, GS2 was 50 kPa, and the ion pair used was 74.9 / 76.1.

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