Liquid chromatography determination method for mixed solution of glyoxylic acid, glycollic acid and oxalic acid and application of liquid chromatography determination method

Abstract

The invention discloses a liquid chromatography determination method for a mixed solution of glyoxylic acid, glycollic acid and oxalic acid and application. The method comprises the following steps: (1) preparing standard solutions with different concentrations; (2) detecting the standard solutions with different concentrations by using high performance liquid chromatography; (3) establishing a linear regression equation of each acid according to the relationship between the concentration of the standard solution and the corresponding peak area; (4) pre-treating a sample to be detected, detecting under the same condition as that in the step (2) by using high performance liquid chromatography, and recording the peak area of each acid in the sample to be detected; and (5) substituting the peak area of each acid in the to-be-detected sample into the corresponding linear regression equation, and calculating to obtain the concentrations of glyoxylic acid, glycollic acid and oxalic acid in the to-be-detected sample. The method is a liquid chromatographic determination method which is simple and convenient to operate, excellent in separation effect, mild in chromatographic condition and compatible with various detectors, can accurately quantify three strong-polarity mixed acids of glyoxylic acid, glycollic acid and oxalic acid, and has important practical application value.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry technology, specifically relating to a liquid chromatography method and application for the determination of a mixture of glyoxylic acid, glycolic acid, and oxalic acid. In particular, it relates to a method for the simultaneous separation and detection of three strongly polar small-molecule organic acids—glyoxylic acid, glycolic acid, and oxalic acid—based on high-performance liquid chromatography (HPLC). This method is applicable to the quantitative analysis of the three acids in a mixture in scenarios such as the electrolytic synthesis of glyoxylic acid from oxalic acid, providing technical support for quality control and process optimization in chemical production processes. Background Technology

[0002] Glyoxylic acid, as an important chemical raw material, is widely used in pharmaceutical synthesis (such as the synthesis of penicillin and cephalosporin drug intermediates), food additives (such as flavor enhancers and preservative precursors), and the paper industry (such as the preparation of paper strengthening agents). In the key synthetic route of glyoxylic acid—the electrolytic synthesis process of oxalic acid—glyoxylic acid, unreacted oxalic acid, and the byproduct glycolic acid are present in the reaction system simultaneously. The content of these three acids directly determines the purity of the product and the reaction efficiency. Therefore, accurate quantification of these three acids in the mixture is crucial.

[0003] Currently, the main analytical methods for the above three mixed acids include chemical titration, potentiometric titration, spectrophotometry, and gas chromatography, but these methods generally have significant drawbacks:

[0004] High operational complexity: Most methods require derivatization pretreatment (such as gas chromatography which requires converting organic acids into ester derivatives), which is cumbersome, time-consuming, and the purity and amount of derivatization reagents are prone to errors.

[0005] Highly susceptible to interference: Chemical titration and potentiometric titration rely on acid-base reactions or electrode responses. Other ions in the mixed system (such as metal ions introduced during electrolysis) can interfere with endpoint determination, leading to inaccurate quantitative results.

[0006] Limited applicability: Spectrophotometry is limited by the overlapping ultraviolet absorption characteristics of the three acids, making it difficult to effectively distinguish them. It can only be used to determine a single component or the total amount.

[0007] Liquid chromatography (LC) has become the mainstream technique for organic acid analysis in recent years due to its ease of operation and lack of derivatization requirements. However, glyoxylic acid, glycolic acid, and oxalic acid all exhibit high polarity, strong water solubility, and similar molecular structures, resulting in extremely weak retention on traditional reversed-phase columns and making baseline separation difficult. Existing studies, such as Lu Lihong et al. using an acetonitrile-water mixture (volume ratio 1.6:98.4) as the mobile phase, and Li Lanting et al. using a reversed-phase-weak anion exchange mixed-mode column with 10% acetonitrile-prepared phosphate buffer as the mobile phase, have achieved some separation. However, both studies suffer from an excessively high aqueous mobile phase ratio (>90%). Prolonged use of a high aqueous mobile phase leads to swelling of the stationary phase and loss of the bonded phase, significantly shortening the column's lifespan (typically only 1 / 3 to 1 / 2 of that under normal operating conditions). Furthermore, the high aqueous system has poor compatibility with mass spectrometry detectors, preventing coupled analysis and limiting the method's broader applications.

[0008] Therefore, in view of the above-mentioned technical problems, it is necessary to provide a liquid chromatography method and application for the determination of a mixture of glyoxylic acid, glycolic acid and oxalic acid. Summary of the Invention

[0009] The purpose of this invention is to provide a liquid chromatography method and application for the determination of a mixture of glyoxylic acid, glycolic acid and oxalic acid.

[0010] To achieve the above objectives, a specific embodiment of the present invention provides the following technical solution:

[0011] A liquid chromatography method for the determination of a mixture of glyoxylic acid, glycolic acid, and oxalic acid, including:

[0012] (1) Prepare standard solutions of different concentrations. The standard solutions are mixed standard solutions containing glyoxylic acid, glycolic acid and oxalic acid.

[0013] (2) Use high performance liquid chromatography to detect standard solutions of different concentrations and record the peak area corresponding to each acid;

[0014] (3) Based on the relationship between the concentration of the standard solution and the corresponding peak area, establish a linear regression equation for each acid;

[0015] (4) After pretreatment of the sample to be tested, high performance liquid chromatography is used to detect it under the same conditions as in step (2), and the peak area of ​​each acid in the sample to be tested is recorded.

[0016] (5) Substitute the peak area of ​​each acid in the sample into the corresponding linear regression equation to calculate the concentration of glyoxylic acid, glycolic acid and oxalic acid in the sample.

[0017] The conditions for liquid chromatography are as follows: the chromatographic column is a strongly polar stationary phase column, the mobile phase is a mixture of acetonitrile and acidic aqueous solution, the flow rate is 0.1-1.2 mL / min, the volume percentage of acetonitrile is 10%-100%, the column temperature is 20-45℃, the detection wavelength is 200-300 nm, and the injection volume is 1-25 μL.

[0018] In one or more embodiments of the present invention, the detection wavelength is 200-260nm, preferably 210nm.

[0019] In one or more embodiments of the present invention, the injection volume is 2-20 μL, preferably 5 μL.

[0020] In one or more embodiments of the present invention, both the standard solution and the solution of the test sample are filtered through a 0.22 μm nylon filter. Preferably, both the standard solution and the solution of the test sample are in a solvent of 50% acetonitrile-50% water (volume ratio), and are filtered through a 0.22 μm nylon filter.

[0021] In one or more embodiments of the present invention, the chromatographic column is a Shim-pack Scepter Diol-HILIC-120 column with dimensions of 4.6 × 150 mm and 5 μm.

[0022] In one or more embodiments of the present invention, the acid in the acidic aqueous solution constituting the mobile phase is selected from: phosphoric acid, trifluoroacetic acid, acetic acid, formic acid, potassium dihydrogen phosphate, sodium dihydrogen phosphate, ammonium acetate, and ammonium formate.

[0023] In one or more embodiments of the present invention, the concentration of acid in the acidic aqueous solution constituting the mobile phase is 0.01%-1%. Preferably, the concentration of the acidic aqueous solution is 0.01%-0.5%, more preferably 0.05%.

[0024] In one or more embodiments of the present invention, the volume percentage of acetonitrile in the mobile phase is 50%-100%.

[0025] In one or more embodiments of the present invention, the volume percentage of acetonitrile in the mobile phase is 50%, and the concentration of the acidic aqueous solution is 0.05%.

[0026] In one or more embodiments of the present invention, the volume percentage of acetonitrile in the mobile phase is 50%, and the concentration of the aqueous acetic acid solution is 0.05%.

[0027] In one or more embodiments of the present invention, the column temperature is 25-35°C, preferably 35°C.

[0028] In one or more embodiments of the present invention, the flow rate is 0.2-1 mL / min. Preferably, it is 1 mL / min.

[0029] In one or more embodiments of the present invention, the liquid chromatography method is applied to the detection of a mixture of glyoxylic acid, glycolic acid, and oxalic acid.

[0030] In one or more embodiments of the present invention, the conditions for liquid chromatography are as follows: the chromatographic column used for liquid chromatography determination is a Shim-pack Scepter Diol-HILIC-120 column with specifications of 4.6 × 150 mm and 5 μm;

[0031] The mobile phase used in the liquid chromatography determination was an acetonitrile-acetic acid aqueous solution system;

[0032] The concentration of the acidic aqueous solution in the mobile phase used for liquid chromatography determination is 0.05%;

[0033] The flow rate used for liquid chromatography determination was 1 ml / min;

[0034] The organic phase acetonitrile ratio used in liquid chromatography was 50%.

[0035] The column temperature used for liquid chromatography determination was 35℃;

[0036] The detection wavelength used in liquid chromatography was 210 nm.

[0037] The injection volume used for liquid chromatography determination is 5 μL.

[0038] Compared with existing technologies, the liquid chromatography method and application for the determination of glyoxylic acid, glycolic acid, and oxalic acid mixtures of the present invention eliminates the need for derivatization pretreatment. By controlling the liquid chromatography conditions, it directly achieves the analysis of three strongly polar small-molecule mixed organic acids: glyoxylic acid, glycolic acid, and oxalic acid. Furthermore, the high organic phase ratio is beneficial to column life, and the LC-MS compatibility is high, overcoming the drawback of the current demanding conditions for simultaneous detection of these three acids using liquid chromatography. The separation, sensitivity, accuracy, and precision of glyoxylic acid, glycolic acid, and oxalic acid are all excellent, and the linear regression equation for glyoxylic acid R0 is satisfactory. 2 The value reached 0.9999, and the linear regression equation for glycolic acid R... 2 The linear regression equation for oxalate reached 0.9999, R0. 2 The recovery rate is 0.9997, and the recovery rate is high, with an average recovery rate of 99.6% and a relative standard deviation (RSD) of 0.15%, enabling very accurate quantification of glyoxylic acid, glycolic acid, and oxalic acid in the sample. Attached Figure Description

[0039] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0040] Figure 1 The liquid chromatography chromatograms of a mixed standard solution of glyoxylic acid, glycolic acid, and oxalic acid show the following retention times: glyoxylic acid 4.2 min, glycolic acid 5.8 min, and oxalic acid 7.5 min, respectively. The peaks of the three acids are symmetrical and do not overlap.

[0041] Figure 2 Example 1: Linear relationship between glyoxylic acid concentration and peak area, with the horizontal axis representing concentration (mg / mL) and the vertical axis representing peak area, and the linear fit R² = 0.9999;

[0042] Figure 3 Linear relationship between glycolic acid concentration and peak area in Example 1, R² = 0.9999;

[0043] Figure 4 Linear relationship between oxalic acid concentration and peak area in Example 1, R² = 0.9997;

[0044] Figure 5 Linear relationship between glyoxylic acid concentration and peak area in Example 2, R² = 0.9999;

[0045] Figure 6 Linear relationship between glycolic acid concentration and peak area in Example 2, R² = 0.9999;

[0046] Figure 7 Linear relationship between oxalic acid concentration and peak area in Example 2, R² = 0.9999. Detailed Implementation

[0047] To enable those skilled in the art to better understand the technical solutions in this disclosure, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments disclosed herein. Obviously, the described embodiments are only some embodiments of this disclosure, and not all embodiments. Based on the embodiments in this disclosure, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this disclosure.

[0048] To address the shortcomings of existing analytical methods, such as complex operation, strong interference, and harsh chromatographic conditions (high aqueous mobile phase), this invention provides a liquid chromatography method for the determination of a mixture of glyoxylic acid, glycolic acid, and oxalic acid. By optimizing the selection of chromatographic column and mobile phase system, the method achieves efficient separation and accurate quantification of the three acids, while extending the service life of the chromatographic column and improving the compatibility of the method with detectors such as mass spectrometers.

[0049] The liquid chromatography determination method provided by this invention can be specifically implemented using the following steps:

[0050] Preparation of standard solutions: Accurately weigh glyoxylic acid, glycolic acid, and oxalic acid reference standards respectively, and prepare standard stock solutions of the three acids using 50% acetonitrile-50% water (volume ratio) as solvent; then dilute the stock solutions stepwise to obtain at least 5 mixed standard solutions with different concentration gradients (covering the expected concentration range of the three acids in the sample to be tested).

[0051] Standard curve plotting: High performance liquid chromatography (HPLC) was used to detect mixed standard solutions of different concentrations, and the chromatographic peak area of ​​each acid at the corresponding concentration was recorded; linear regression analysis was performed with the standard solution concentration as the abscissa (X) and the peak area as the ordinate (Y) to obtain the linear regression equation and correlation coefficient (R²) for each acid.

[0052] Sample pretreatment: Accurately weigh the sample to be tested (such as the reaction solution for the electrolytic synthesis of glyoxylic acid from oxalic acid), place it in a volumetric flask, dissolve it with 50% acetonitrile-50% water and dilute to the mark, filter it through a 0.22μm nylon filter to remove particulate impurities, and obtain the sample solution to be tested.

[0053] Test sample detection: Under the same chromatographic conditions as those for plotting the standard curve, inject the test sample solution into the high performance liquid chromatograph and record the peak areas of glyoxylic acid, glycolic acid, and oxalic acid.

[0054] Quantitative calculation: Substitute the peak areas of the three acids in the sample into the corresponding linear regression equations to calculate the concentrations of glyoxylic acid, glycolic acid, and oxalic acid in the sample.

[0055] The key chromatographic conditions are shown in the table below, including the basic range and preferred scheme:

[0056]

[0057] Example 1

[0058] The liquid chromatography method for determining glyoxylic acid, glycolic acid, and oxalic acid—three strongly polar mixed acids—in this embodiment includes:

[0059] (1) Chromatographic conditions

[0060] The high-performance liquid chromatograph was a Shimadzu LC-2060C3D model from Japan, with a detection wavelength of 210 nm. A highly polar stationary phase column was used, specifically a Shim-pack Scepter Diol-HILIC-120 column with dimensions of 4.6 × 150 mm and 5 μm. The mobile phase was an acetonitrile-0.05% acetic acid aqueous solution system, with a flow rate of 1 ml / min, an acetonitrile content of 50%, a column temperature of 35 °C, and an injection volume of 5 μL.

[0061] (2) Plotting the standard curve

[0062] Accurately weigh 1.1291 g of glyoxylic acid reference standard, 1.0203 g of glycolic acid reference standard, and 0.1183 g of oxalic acid reference standard, and place them in 100 ml volumetric flasks. Dissolve them in 50% acetonitrile-50% water as solvent and dilute to the mark. This solution is used as the standard stock solution. Then, the standard stock solution is serially diluted to prepare glyoxylic acid standard solutions of 1.1291, 2.8228, 3.7637, 5.6455, and 11.2910 mg / ml; glycolic acid standard solutions of 1.0203, 2.55075, 3.4010, 5.1015, and 10.2030 mg / ml; and oxalic acid standard solutions of 0.1183, 0.2958, 0.3943, 0.5915, and 1.1830 mg / ml.

[0063] The standard solutions of different concentrations were filtered through a 0.22 μm nylon filter and injected into a liquid chromatogram under the above chromatographic conditions. A linear curve was plotted with the standard solution concentration as the x-axis and the peak area corresponding to each concentration as Y, yielding the linear regression equation Y. 乙醛酸 =332693X-26347.1, Relevance R 2 乙醛酸 =0.9999, Y 乙醇酸 =408993X-2922.07, Relevance R 2 乙醇酸 =0.9999, Y 草酸 =2284970X + 34364, Relevance R 2 草酸 =0.9997.

[0064] The above results indicate that the peak area of ​​glyoxylic acid (1.1291-11.2910 mg / ml), glycolic acid (1.0203-10.2030 mg / ml), and oxalic acid (0.1183-1.1830 mg / ml) showed a good linear relationship with the concentration.

[0065] (3) Determination of the contents of glyoxylic acid, glycolic acid and oxalic acid in the sample to be tested

[0066] Accurately weigh the sample to be tested and place it in a 100 ml volumetric flask. Dissolve it in 50% acetonitrile-50% water as a solvent and dilute to the mark. Filter the solution through a 0.22 μm nylon filter and inject it into a liquid chromatograph under the above chromatographic conditions. Obtain the peak areas of glyoxylic acid, glycolic acid, and oxalic acid in the sample to be tested and the standard solution under the same chromatographic conditions. Calculate the content of glyoxylic acid, glycolic acid, and oxalic acid in the sample to be tested according to the linear regression equation.

[0067] (4) Precision determination

[0068] The glyoxylic acid peak area data obtained from six consecutive injections of the same sample are shown in the table below:

[0069]

[0070] The relative standard deviation (RSD) of the glyoxylic acid peak area in the above 6 injections was 0.82%, indicating that the liquid chromatography determination method of the present invention has good precision and is reliable.

[0071] The peak area data of glycolic acid obtained from six consecutive injections of the same sample are shown in the table below:

[0072]

[0073] The relative standard deviation (RSD) of the peak area of ​​glycolic acid in the above 6 injections was 0.05%, indicating that the liquid chromatography determination method of the present invention has good precision and is reliable.

[0074] The peak area data of oxalic acid obtained from six consecutive injections of the same sample are shown in the table below:

[0075]

[0076] The relative standard deviation (RSD) of the oxalic acid peak area in the above six injections was 0.33%, indicating that the liquid chromatography determination method of the present invention has good precision and is reliable.

[0077] (5) Recovery rate determination

[0078] Three different gradients of glyoxylic acid, glycolic acid, and oxalic acid standard solutions were precisely added to the sample with known content, and the concentrations of glyoxylic acid, glycolic acid, and oxalic acid were controlled within their respective linear curve ranges. The solution was filtered through a 0.22 μm nylon filter and injected into a liquid chromatogram under the above chromatographic conditions. Each gradient was injected in parallel five times, and the average spiked recovery rate was calculated to be 99.6%, with a relative standard deviation (RSD) of 0.15%, indicating that the spiked recovery rate of the liquid chromatography method of the present invention is good.

[0079] Example 2

[0080] The liquid chromatography method for determining glyoxylic acid, glycolic acid, and oxalic acid—three strongly polar mixed acids—in this embodiment includes:

[0081] (1) Chromatographic conditions

[0082] The high-performance liquid chromatograph (HPLC) used was a Shimadzu LC-2060C3D model from Japan, with a detection wavelength of 220 nm. A highly polar stationary phase column was used, specifically a Shim-pack Scepter Diol-HILIC-120 column with dimensions of 4.6 × 250 mm and 5 μm. The mobile phase was acetonitrile-0.05% phosphoric acid aqueous solution, with a flow rate of 0.5 ml / min, an acetonitrile content of 80%, a column temperature of 30 °C, and an injection volume of 10 μL.

[0083] (2) Plotting the standard curve

[0084] Accurately weigh 1.2351 g of glyoxylic acid reference standard, 1.1278 g of glycolic acid reference standard, and 0.1276 g of oxalic acid reference standard, and place them in 100 ml volumetric flasks. Dissolve them in 50% acetonitrile-50% water as solvent and dilute to the mark. This solution is used as the standard stock solution. Then, the standard stock solution is serially diluted to prepare glyoxylic acid standard solutions of 1.2351, 3.0878, 4.117, 6.1755, and 12.3510 mg / ml; glycolic acid standard solutions of 1.1279, 2.8196, 3.7595, 5.6393, and 11.2780 mg / ml; and oxalic acid standard solutions of 0.1276, 0.3190, 0.4253, 0.6380, and 1.2760 mg / ml.

[0085] The standard solutions of different concentrations were filtered through a 0.22 μm nylon filter and injected into a liquid chromatogram under the above chromatographic conditions. A linear curve was plotted with the standard solution concentration as the x-axis and the peak area corresponding to each concentration as Y, yielding the linear regression equation Y. 乙醛酸 =293675X + 2299.5, Relevance R 2 乙醛酸 =0.9999, Y 乙醇酸 =409712X + 129.89, Relevance R 2 乙醇酸 =0.9999, Y 草酸 =2E+06X-507.12, Relevance R 2 草酸 =0.9999.

[0086] The above results indicate that the peak area of ​​glyoxylic acid in the concentration range of 1.2351-12.3510 mg / ml, glycolic acid in the concentration range of 1.1279-11.2780 mg / ml, and oxalic acid in the concentration range of 0.1276-1.2760 mg / ml showed a good linear relationship with the concentration.

[0087] (3) Determination of the contents of glyoxylic acid, glycolic acid and oxalic acid in the sample to be tested

[0088] Accurately weigh the sample to be tested and place it in a 100 ml volumetric flask. Dissolve it in 50% acetonitrile-50% water as a solvent and dilute to the mark. Filter the solution through a 0.22 μm nylon filter and inject it into a liquid chromatograph under the above chromatographic conditions. Obtain the peak areas of glyoxylic acid, glycolic acid, and oxalic acid in the sample to be tested and the standard solution under the same chromatographic conditions. Calculate the content of glyoxylic acid, glycolic acid, and oxalic acid in the sample to be tested according to the linear regression equation.

[0089] (4) Precision determination

[0090] The glyoxylic acid peak area data obtained from six consecutive injections of the same sample are shown in the table below:

[0091]

[0092] The relative standard deviation (RSD) of the glyoxylic acid peak area in the above 6 injections was 0.41%, indicating that the liquid chromatography determination method of the present invention has good precision and is reliable.

[0093] The peak area data of glycolic acid obtained from six consecutive injections of the same sample are shown in the table below:

[0094]

[0095] The relative standard deviation (RSD) of the peak area of ​​glycolic acid in the above 6 injections was 0.65%, indicating that the liquid chromatography determination method of the present invention has good precision and is reliable.

[0096] The peak area data of oxalic acid obtained from six consecutive injections of the same sample are shown in the table below:

[0097]

[0098] The relative standard deviation (RSD) of the oxalic acid peak area in the above 6 injections was 0.18%, indicating that the liquid chromatography determination method of the present invention has good precision and is reliable.

[0099] (5) Recovery rate determination

[0100] Three different gradients of glyoxylic acid, glycolic acid, and oxalic acid standard solutions were precisely added to the sample with known content, and the concentrations of glyoxylic acid, glycolic acid, and oxalic acid were controlled within their respective linear curve ranges. The solution was filtered through a 0.22 μm nylon filter and injected into a liquid chromatogram under the above chromatographic conditions. Each gradient was injected in parallel five times, and the average spiked recovery rate was calculated to be 99.7%, with a relative standard deviation (RSD) of 0.45%, indicating that the spiked recovery rate of the liquid chromatography method of the present invention is good.

[0101] Example 3

[0102] The liquid chromatography method for determining glyoxylic acid, glycolic acid, and oxalic acid—three strongly polar mixed acids—in this embodiment includes:

[0103] (1) Chromatographic conditions

[0104] The high-performance liquid chromatograph (HPLC) used was a Shimadzu LC-2060C3D model from Japan, with a detection wavelength of 200 nm. A highly polar stationary phase column was used, specifically a Shim-pack Scepter Diol-HILIC-120 column with dimensions of 4.6 × 250 mm and 5 μm. The mobile phase was acetonitrile-0.05% trifluoroacetic acid aqueous solution, with a flow rate of 0.6 ml / min, an acetonitrile content of 85%, a column temperature of 40 °C, and an injection volume of 2 μL.

[0105] (2) Plotting the standard curve

[0106] Accurately weigh 1.2881 g of glyoxylic acid reference standard, 1.1193 g of glycolic acid reference standard, and 0.1143 g of oxalic acid reference standard, and place them in 100 ml volumetric flasks. Dissolve them in 50% acetonitrile-50% water as solvent and dilute to the mark. This solution is used as the standard stock solution. Then, the standard stock solution is serially diluted to prepare glyoxylic acid standard solutions of 1.2881, 3.2203, 4.2937, 6.4405, and 12.8810 mg / ml; glycolic acid standard solutions of 1.1193, 2.7984, 3.7311, 5.5967, and 11.1930 mg / ml; and oxalic acid standard solutions of 0.1143, 0.2858, 0.3810, 0.5715, and 1.1430 mg / ml.

[0107] The standard solutions of different concentrations were filtered through a 0.22 μm nylon filter and injected into a liquid chromatogram under the above chromatographic conditions. A linear curve was plotted with the standard solution concentration as the x-axis and the peak area corresponding to each concentration as Y, yielding the linear regression equation Y. 乙醛酸 =113007X-3482.4, Relevance R 2 乙醛酸 =0.9999, Y乙醇酸 =166516X-20983, Relevance R 2 乙醇酸 =0.9999, Y 草酸 =1E+06X-20901, Relevance R 2 草酸 =0.9996.

[0108] The above results indicate that the peak area of ​​glyoxylic acid in the concentration range of 1.2881-12.8810 mg / ml, glycolic acid in the concentration range of 1.1193-11.1930 mg / ml, and oxalic acid in the concentration range of 0.1143-1.1430 mg / ml showed a good linear relationship with the concentration.

[0109] (3) Determination of the contents of glyoxylic acid, glycolic acid and oxalic acid in the sample to be tested

[0110] Accurately weigh the sample to be tested and place it in a 100 ml volumetric flask. Dissolve it in 50% acetonitrile-50% water as a solvent and dilute to the mark. Filter the solution through a 0.22 μm nylon filter and inject it into a liquid chromatograph under the above chromatographic conditions. Obtain the peak areas of glyoxylic acid, glycolic acid, and oxalic acid in the sample to be tested and the standard solution under the same chromatographic conditions. Calculate the content of glyoxylic acid, glycolic acid, and oxalic acid in the sample to be tested according to the linear regression equation.

[0111] (4) Precision determination

[0112] The glyoxylic acid peak area data obtained from six consecutive injections of the same sample are shown in the table below:

[0113]

[0114] The relative standard deviation (RSD) of the glyoxylic acid peak area in the above six injections was 0.94%, indicating that the liquid chromatography determination method of the present invention has good precision and is reliable.

[0115] The peak area data of glycolic acid obtained from six consecutive injections of the same sample are shown in the table below:

[0116]

[0117] The relative standard deviation (RSD) of the peak area of ​​glycolic acid in the above 6 injections was 0.55%, indicating that the liquid chromatography determination method of the present invention has good precision and is reliable.

[0118] The peak area data of oxalic acid obtained from six consecutive injections of the same sample are shown in the table below:

[0119]

[0120] The relative standard deviation (RSD) of the oxalic acid peak area in the above six injections was 0.74%, indicating that the liquid chromatography determination method of the present invention has good precision and is reliable.

[0121] (5) Recovery rate determination

[0122] Three different gradients of glyoxylic acid, glycolic acid, and oxalic acid standard solutions were precisely added to the sample with known content, and the concentrations of glyoxylic acid, glycolic acid, and oxalic acid were controlled within their respective linear curve ranges. The solution was filtered through a 0.22 μm nylon filter and injected into a liquid chromatogram under the above chromatographic conditions. Each gradient was injected in parallel five times, and the average spiked recovery rate was calculated to be 99.3%, with a relative standard deviation (RSD) of 0.78%, indicating that the spiked recovery rate of the liquid chromatography method of the present invention is good.

[0123] Example 4

[0124] The liquid chromatography method for determining glyoxylic acid, glycolic acid, and oxalic acid—three strongly polar mixed acids—in this embodiment includes:

[0125] (1) Chromatographic conditions

[0126] The high-performance liquid chromatograph (HPLC) used was a Shimadzu LC-2060C3D model from Japan, with a detection wavelength of 230 nm. A highly polar stationary phase column was used, specifically a Shim-pack Scepter Diol-HILIC-120 column with dimensions of 4.6 × 250 mm and 5 μm. The mobile phase was acetonitrile-0.04% phosphoric acid aqueous solution, with a flow rate of 0.3 ml / min, an acetonitrile content of 95%, a column temperature of 25 °C, and an injection volume of 20 μL.

[0127] (2) Plotting the standard curve

[0128] Accurately weigh 1.2136 g of glyoxylic acid reference standard, 1.1232 g of glycolic acid reference standard, and 0.1032 g of oxalic acid reference standard, and place them in 100 ml volumetric flasks. Dissolve them in 50% acetonitrile-50% water as solvent and dilute to the mark. This solution is used as the standard stock solution. Then, the standard stock solution is serially diluted to prepare glyoxylic acid standard solutions of 1.2137, 3.0341, 4.0457, 6.0684, and 12.1360 mg / ml; glycolic acid standard solutions of 1.1232, 2.8080, 3.7440, 5.6160, and 11.2320 mg / ml; and oxalic acid standard solutions of 0.1032, 0.2580, 0.3440, 0.5160, and 1.0320 mg / ml.

[0129] The standard solutions of different concentrations were filtered through a 0.22 μm nylon filter and injected into a liquid chromatogram under the above chromatographic conditions. A linear curve was plotted with the standard solution concentration as the x-axis and the peak area corresponding to each concentration as Y, yielding the linear regression equation Y. 乙醛酸 =1E+06X-166502, Relevance R 2 乙醛酸 =0.9995, Y 乙醇酸 =1E+06X-189137, Relevance R 2 乙醇酸 =0.9999, Y 草酸 =1E+07X-186016, Relevance R 2 草酸 =0.9994.

[0130] The above results indicate that the peak area of ​​glyoxylic acid in the concentration range of 1.2137-12.1360 mg / ml, glycolic acid in the concentration range of 1.1232-11.2320 mg / ml, and oxalic acid in the concentration range of 0.1032-1.0320 mg / ml showed a good linear relationship with the concentration.

[0131] (3) Determination of the contents of glyoxylic acid, glycolic acid and oxalic acid in the sample to be tested

[0132] Accurately weigh the sample to be tested and place it in a 100 ml volumetric flask. Dissolve it in 50% acetonitrile-50% water as a solvent and dilute to the mark. Filter the solution through a 0.22 μm nylon filter and inject it into a liquid chromatograph under the above chromatographic conditions. Obtain the peak areas of glyoxylic acid, glycolic acid, and oxalic acid in the sample to be tested and the standard solution under the same chromatographic conditions. Calculate the content of glyoxylic acid, glycolic acid, and oxalic acid in the sample to be tested according to the linear regression equation.

[0133] (4) Precision determination

[0134] The glyoxylic acid peak area data obtained from six consecutive injections of the same sample are shown in the table below:

[0135]

[0136] The relative standard deviation (RSD) of the glyoxylic acid peak area in the above six injections was 0.46%, indicating that the liquid chromatography determination method of the present invention has good precision and is reliable.

[0137] The peak area data of glycolic acid obtained from six consecutive injections of the same sample are shown in the table below:

[0138]

[0139] The relative standard deviation (RSD) of the peak area of ​​glycolic acid in the above 6 injections was 0.26%, indicating that the liquid chromatography determination method of the present invention has good precision and is reliable.

[0140] The peak area data of oxalic acid obtained from six consecutive injections of the same sample are shown in the table below:

[0141]

[0142] The relative standard deviation (RSD) of the oxalic acid peak area in the above six injections was 0.61%, indicating that the liquid chromatography determination method of the present invention has good precision and is reliable.

[0143] (5) Recovery rate determination

[0144] Three different gradients of glyoxylic acid, glycolic acid, and oxalic acid standard solutions were precisely added to the sample with known content, and the concentrations of glyoxylic acid, glycolic acid, and oxalic acid were controlled within their respective linear curve ranges. The solution was filtered through a 0.22 μm nylon filter and injected into a liquid chromatogram under the above chromatographic conditions. Each gradient was injected in parallel five times, and the average spiked recovery rate was calculated to be 99.1%, with a relative standard deviation (RSD) of 0.97%, indicating that the spiked recovery rate of the liquid chromatography method of the present invention is good.

[0145] Example 5:

[0146] The only difference between this embodiment and Example 1 is that the mobile phase is changed to acetonitrile-0.05% phosphoric acid aqueous solution (50:50, v / v).

[0147] Precision and recovery validation:

[0148] Precision: For the same sample, in 6 consecutive injections, the RSD of the peak areas of the three acids was <1.0%;

[0149] Recovery rate: When standard solutions of low, medium and high concentrations were added to samples of known concentrations, the average recovery rate was 99.7% and the RSD was 0.47.

[0150] Example 6:

[0151] The only difference between this embodiment and Example 1 is that the mobile phase is changed to acetonitrile-0.05% trifluoroacetic acid aqueous solution (50:50, v / v).

[0152] Precision and recovery validation:

[0153] Precision: For the same sample, in 6 consecutive injections, the RSD of the peak areas of the three acids was <1.0%;

[0154] Recovery rate: When standard solutions of low, medium and high concentrations were added to samples of known concentrations, the average recovery rate was 99.5% and the RSD was 0.34%.

[0155] Example 7:

[0156] The only difference between this embodiment and Example 1 is that the mobile phase is changed to an aqueous solution of acetonitrile-0.05% acetic acid (80:20, v / v).

[0157] Precision and recovery validation:

[0158] Precision: For the same sample, in 6 consecutive injections, the RSD of the peak areas of the three acids was <1.0%;

[0159] Recovery rate: When standard solutions of low, medium and high concentrations were added to samples of known concentrations, the average recovery rate was 99.2% and the RSD was 0.66%.

[0160] Example 8:

[0161] The only difference between this embodiment and Embodiment 1 is that the mobile phase is changed to acetonitrile.

[0162] Precision and recovery validation:

[0163] Precision: For the same sample, in 6 consecutive injections, the RSD of the peak areas of the three acids was <1.0%;

[0164] Recovery rate: When standard solutions of low, medium and high concentrations were added to samples of known concentrations, the average recovery rate was 99.1% and the RSD was 0.96%.

[0165] Example 9:

[0166] The only difference between this embodiment and Example 1 is that the mobile phase is changed to acetonitrile-0.01% acetic acid aqueous solution (50:50, v / v).

[0167] Precision and recovery validation:

[0168] Precision: For the same sample, in 6 consecutive injections, the RSD of the peak areas of the three acids was <1.0%;

[0169] Recovery rate: When standard solutions of low, medium and high concentrations were added to samples of known concentrations, the average recovery rate was 99.1% and the RSD was 0.98%.

[0170] Example 10:

[0171] The only difference between this embodiment and Example 1 is that the mobile phase is changed to acetonitrile-0.5% acetic acid aqueous solution (50:50, v / v).

[0172] Precision and recovery validation:

[0173] Precision: For the same sample, in 6 consecutive injections, the RSD of the peak areas of the three acids was <1.0%;

[0174] Recovery rate: When standard solutions of low, medium and high concentrations were added to samples of known concentrations, the average recovery rate was 99.3% and the RSD was 0.77%.

[0175] Example 11:

[0176] The only difference between this embodiment and Example 1 is that the mobile phase is changed to acetonitrile-0.1% acetic acid aqueous solution (50:50, v / v).

[0177] Precision and recovery validation:

[0178] Precision: For the same sample, in 6 consecutive injections, the RSD of the peak areas of the three acids was <1.0%;

[0179] Recovery rate: When standard solutions of low, medium and high concentrations were added to samples of known concentrations, the average recovery rate was 99.8% with an RSD of 0.21%.

[0180] Example 12:

[0181] The only difference between this embodiment and Embodiment 1 is that the column temperature is 25°C.

[0182] Precision and recovery validation:

[0183] Precision: For the same sample, in 6 consecutive injections, the RSD of the peak areas of the three acids was <1.0%;

[0184] Recovery rate: When standard solutions of low, medium and high concentrations were added to samples of known concentrations, the average recovery rate was 99.6% and the RSD was 0.14%.

[0185] Example 13:

[0186] The only difference between this embodiment and Embodiment 1 is that the column temperature is 30°C.

[0187] Precision and recovery validation:

[0188] Precision: For the same sample, in 6 consecutive injections, the RSD of the peak areas of the three acids was <1.0%;

[0189] Recovery rate: When standard solutions of low, medium and high concentrations were added to samples of known concentrations, the average recovery rate was 99.6% and the RSD was 0.17%.

[0190] Example 14:

[0191] The only difference between this embodiment and Embodiment 1 is that the column temperature is 45°C.

[0192] Precision and recovery validation:

[0193] Precision: For the same sample, in 6 consecutive injections, the RSD of the peak areas of the three acids was <1.0%;

[0194] Recovery rate: When standard solutions of low, medium and high concentrations were added to samples of known concentrations, the average recovery rate was 99.5% and the RSD was 0.22%.

[0195] Example 15:

[0196] The only difference between this embodiment and Embodiment 1 is that the flow rate is 0.6 mL / min.

[0197] Precision and recovery validation:

[0198] Precision: For the same sample, in 6 consecutive injections, the RSD of the peak areas of the three acids was <1.0%;

[0199] Recovery rate: When standard solutions of low, medium and high concentrations were added to samples of known concentrations, the average recovery rate was 99.8% with an RSD of 0.16%.

[0200] Example 16:

[0201] The only difference between this embodiment and Embodiment 1 is that the flow rate is 0.2 mL / min.

[0202] Precision and recovery validation:

[0203] Precision: For the same sample, in 6 consecutive injections, the RSD of the peak areas of the three acids was <1.0%;

[0204] Recovery rate: When standard solutions of low, medium and high concentrations were added to samples of known concentrations, the average recovery rate was 99.0% and the RSD was 0.79%.

[0205] Example 17:

[0206] The only difference between this embodiment and Embodiment 1 is that the detection wavelength is 200nm.

[0207] Precision and recovery validation:

[0208] Precision: For the same sample, in 6 consecutive injections, the RSD of the peak areas of the three acids was <1.0%;

[0209] Recovery rate: When standard solutions of low, medium and high concentrations were added to samples of known concentrations, the average recovery rate was 98.6% and the RSD was 1.20%.

[0210] Example 18:

[0211] The only difference between this embodiment and Embodiment 1 is that the detection wavelength is 260nm.

[0212] Precision and recovery validation:

[0213] Precision: For the same sample, in 6 consecutive injections, the RSD of the peak areas of the three acids was <1.0%;

[0214] Recovery rate: When standard solutions of low, medium and high concentrations were added to samples of known concentrations, the average recovery rate was 98.5% and the RSD was 1.51%.

[0215] Example 19:

[0216] The only difference between this embodiment and Example 1 is that the mobile phase is changed to acetonitrile-0.8% acetic acid aqueous solution (50:50, v / v).

[0217] Precision and recovery validation:

[0218] Precision: For the same sample, in 6 consecutive injections, the RSD of the peak areas of the three acids was <1.0%;

[0219] Recovery rate: When standard solutions of low, medium and high concentrations were added to samples of known concentrations, the average recovery rate was 98.2% and the RSD was 1.40%.

[0220] Example 20:

[0221] The only difference between this embodiment and Embodiment 1 is that the column temperature is 20°C.

[0222] Precision and recovery validation:

[0223] Precision: For the same sample, in 6 consecutive injections, the RSD of the peak areas of the three acids was <1.0%;

[0224] Recovery rate: When standard solutions of low, medium and high concentrations were added to samples of known concentrations, the average recovery rate was 98.9% and the RSD was 0.98%.

[0225] Comparative Example 1:

[0226] The only difference between this comparative example and Example 1 is that the mobile phase was changed to an aqueous solution of acetonitrile-0.05% acetic acid (5:95, v / v).

[0227] Precision and recovery verification: Separation could not be achieved.

[0228] Comparative Example 2:

[0229] The only difference between this comparative example and Example 1 is that the mobile phase was changed to an aqueous solution of acetonitrile-0.008% acetic acid (50:50, v / v).

[0230] Precision and recovery validation:

[0231] Precision: For the same sample, in 6 consecutive injections, the RSD of the peak areas of the three acids was <1.0%;

[0232] Recovery rate: When standard solutions of low, medium and high concentrations were added to samples of known concentrations, the average recovery rate was 97.3% and the RSD was 1.67%.

[0233] Based on the above comparison, the significant advantages of the present invention compared with the prior art are as follows:

[0234] No derivatization required, simple operation: the mixture is directly filtered and then detected, eliminating the derivatization step and reducing operational errors and analysis time (single analysis cycle <15min).

[0235] Excellent separation performance: By using a highly polar stationary phase column (such as Shim-pack Scepter Diol-HILIC-120) and an optimized acetonitrile-acidic aqueous solution system, the resolution of the three acids is >1.5 (meeting the requirements of high performance liquid chromatography quantitative analysis), with no mutual interference;

[0236] Mild chromatographic conditions extend column life: The mobile phase contains acetonitrile at a ratio of ≥50% (preferred ratio is 50%), avoiding damage to the stationary phase of the chromatographic column by high aqueous phase, and the column life can be extended to 2-3 times under conventional high aqueous phase conditions;

[0237] Excellent detection performance:

[0238] Good linearity: The linear regression equations for glyoxylic acid and glycolic acid both reached R² of 0.9999, and for oxalic acid, R² reached 0.9997. The linear range covered 1.0-12.0 mg / mL (glyoxylic acid and glycolic acid) and 0.1-1.2 mg / mL (oxalic acid), meeting the detection requirements of samples with different concentrations.

[0239] High precision: For the same sample, the relative standard deviation (RSD) of the peak areas of the three acids was <1.0% for six consecutive injections (glyoxylic acid RSD=0.82%, glycolic acid RSD=0.05%, oxalic acid RSD=0.33%).

[0240] High accuracy: The average recovery rate of spiked samples reached 99.6%, with an RSD of 0.15%, indicating reliable quantitative results;

[0241] High compatibility: The high proportion of acetonitrile mobile phase has excellent compatibility with mass spectrometry detectors (such as ESI-MS), and can be extended to qualitative confirmation and trace analysis of three acids.

[0242] It will be apparent to those skilled in the art that this disclosure is not limited to the details of the exemplary embodiments described above, and that this disclosure can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of this disclosure is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within this disclosure. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0243] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A liquid chromatography method for the determination of a mixture of glyoxylic acid, glycolic acid, and oxalic acid, characterized in that, The method includes: (1) Prepare standard solutions of different concentrations, wherein the standard solutions are mixed standard solutions containing glyoxylic acid, glycolic acid and oxalic acid; (2) Use high performance liquid chromatography to detect standard solutions of different concentrations and record the peak area corresponding to each acid; (3) Based on the relationship between the concentration of the standard solution and the corresponding peak area, establish a linear regression equation for each acid; (4) After pretreatment of the sample to be tested, high performance liquid chromatography is used to detect it under the same conditions as in step (2), and the peak area of ​​each acid in the sample to be tested is recorded. (5) Substitute the peak area of ​​each acid in the sample into the corresponding linear regression equation to calculate the concentration of glyoxylic acid, glycolic acid and oxalic acid in the sample. The conditions for the liquid chromatography are as follows: the chromatographic column is a strongly polar stationary phase column, the mobile phase is a mixture of acetonitrile and acidic aqueous solution, the flow rate is 0.1-1.2 mL / min, the volume percentage of acetonitrile is 10%-100%, the column temperature is 20-45℃, the detection wavelength is 200-300 nm, and the injection volume is 1-25 μL.

2. The liquid chromatography determination method according to claim 1, characterized in that, The chromatographic column was a Shim-packScepter Diol-HILIC-120 column with dimensions of 4.6 × 150 mm and 5 μm.

3. The liquid chromatography determination method according to claim 1, characterized in that, The acid in the acidic aqueous solution constituting the mobile phase is selected from: phosphoric acid, trifluoroacetic acid, acetic acid, formic acid, potassium dihydrogen phosphate, sodium dihydrogen phosphate, ammonium acetate, and ammonium formate.

4. The liquid chromatography determination method according to claim 3, characterized in that, The concentration of acid in the acidic aqueous solution constituting the mobile phase is 0.01%-1%.

5. The liquid chromatography determination method according to claim 4, characterized in that, The volume percentage of acetonitrile in the mobile phase is 50%-100%.

6. The liquid chromatography determination method according to claim 5, characterized in that, The mobile phase contains 50% acetonitrile by volume and 0.05% acidic aqueous solution.

7. The liquid chromatography determination method according to claim 6, characterized in that, The mobile phase contains 50% acetonitrile by volume and 0.05% acetic acid aqueous solution.

8. The liquid chromatography determination method according to claim 1, characterized in that, The column temperature is 25-35℃.

9. The liquid chromatography determination method according to claim 1, characterized in that, The flow rate is 0.2-1 mL / min.

10. The application of the liquid chromatography determination method according to any one of claims 1-9 in the detection of a mixture of glyoxylic acid, glycolic acid, and oxalic acid.

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