Method for simultaneously detecting organic acids and anion metabolites in human body

Abstract

The invention provides a method for simultaneously detecting organic acids and anions in human urine. By adjusting and optimizing pretreatment conditions and elution procedures, matrix interference in a urine sample is effectively removed, and effective separation of 10 organic acids and 4 anions in the sample is realized. The method is suitable for simultaneously detecting anion components in urine samples of various people such as healthy people, diabetics and autism patients, the pretreatment operation is simple and convenient, the sensitivity of the detection method is high, 14 anions in the samples can be simultaneously detected, and the detection accuracy is high. The detection efficiency of a plurality of organic and inorganic anions in a biological sample is greatly improved.

Description

Technical Field

[0001] The invention relates to the technical field of organic acid and anion analysis; in particular, the invention relates to a method for detecting organic acids and anions in human urine. Background Art

[0002] Organic acids are common small molecule polar metabolites and are byproducts of metabolic pathways such as amino acid metabolism, glutathione metabolism, glycolysis, homocysteine ​​metabolism, ketone body synthesis, lipid metabolism, tricarboxylic acid (TCA) cycle, urea cycle, and neurotransmitter turnover in the human body. Studies have shown that citric acid, hydroxypropionic acid, dihydroxypropionic acid, and tartaric acid are produced by gastrointestinal yeast and bacteria, succinic acid, fumaric acid, malic acid, 2-ketoglutaric acid, aconitic acid, and citric acid are related to mitochondrial function and are considered to be potential biomarkers for neurological disorders and autism; α-ketoglutaric acid is a key intermediate in the TCA cycle, and is also an energy source and antioxidant in mammalian cells. Its concentration difference is significantly reflected in cardiovascular diseases and liver cancer; succinic acid is an important intermediate in the TCA cycle and a driving factor for obesity-induced inflammation. It is related to cardiovascular diseases and aortic diseases such as aortic dissection and aortic aneurysm.

[0003] Urine is the excretion waste of daily metabolism of organisms. It contains rich inorganic anions and organic acids. The detection of organic acid types and contents can reflect the metabolic status of the human body and is the preferred diagnostic tool for many metabolic diseases. Since urine metabolite levels are not affected by the body's homeostatic mechanism, they may have more significant changes than blood. Therefore, studying the levels and changing patterns of organic acids and anion metabolites involved in glycolysis, TCA cycle, etc. in urine can be used to pre-diagnose metabolic disorders and assist in monitoring the efficacy of disease patients. It is of great significance for exploring changes in the physiological state of the human body.

[0004] Based on the current research status, the determination methods of organic acids mainly include colorimetry, high performance liquid chromatography, gas chromatography, solid phase microextraction, capillary electrophoresis, ion chromatography and other methods. Among them, colorimetry is convenient and fast, suitable for batch detection, but there are problems such as high detection limit and large interference; high performance liquid chromatography and gas chromatography detect a wide variety of organic acids, and there are problems such as cumbersome pre-treatment derivatization operations and poor repeatability. There is no literature report on the ion chromatography detection of metabolism-related organic acids and anions in human urine, and the previous methods are difficult to take into account simplicity, speed, efficiency and sensitivity. Ion exchange chromatography is widely used in the separation of inorganic and organic anions, amino acids, carbohydrate compounds and other substances, and the pre-treatment is simple, the detection is efficient and sensitive, and it is a detection method suitable for the determination of target components in urine samples. Therefore, the present invention aims to study and establish a method for the simultaneous determination of multiple organic acids in urine based on ion exchange chromatography that is efficient, stable and easy to operate. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for detecting metabolites in urine samples with high sensitivity and simplicity in sample processing, with high detection limit, large interference and complicated and time-consuming derivatization required for the existing determination of organic acids in urine.

[0006] The present invention is achieved through the following technical solutions:

[0007] A method for efficiently and quickly determining the content of organic acids in human urine comprises the following process steps:

[0008] S1. Preparation of standard stock solutions and mixed standard solutions of organic acids commonly found in human urine

[0009] Accurately weigh the set amount of organic acid standard, dissolve it with ultrapure water, and make up to 50 mL to obtain a 1.0 g / L organic acid standard stock solution, which is stored at 4°C away from light. Accurately weigh the set amount of the above organic acid standard stock solution, dissolve it with ultrapure water, and prepare a 0.8-8.0 mg / L organic acid mixed standard solution;

[0010] The method of anion exchange chromatography-conductivity detection (AEC-CD) is used to establish the conditions for determining the organic acid standard; the corresponding organic acid concentration-peak area standard curve is established according to the concentration and peak area of ​​the organic acid standard solution; the peak area value of each organic acid in the urine to be tested is obtained by using the above-determined method conditions for determining the organic acid standard, and the concentration of each organic acid in the urine to be tested is calculated by the organic acid concentration-peak area standard curve;

[0011] Wherein, the organic acid standard is glycolic acid, lactic acid, 2-hydroxybutyric acid, pyruvic acid, oxalic acid, α-ketoglutaric acid, isocitric acid, cis-aconitic acid, and trans-aconitic acid;

[0012] S2. Determination of urine creatinine content

[0013] Take 0, 20, 50, 100, 150, 200 μL of 10 mmol / L creatinine standard respectively in a 15 mL centrifuge tube, add ultrapure water to 10 mL, and prepare 0, 20, 50, 100, 150, 200 μmol / L creatinine standard solutions. Take 100-200 μL of creatinine standard solutions of each concentration and add them to a 96-well ELISA plate, then add 25-75 μL of creatinine colorimetric solution and 25-75 μL of creatinine assay buffer to each well in turn;

[0014] Take 5-15μL of urine sample in a 2mL centrifuge tube, add 800-1000μL of ultrapure water, take 8-12μL of diluted urine sample and add it to a 96-well ELISA plate, then add 20-80μL of creatinine colorimetric solution and 20-80μL of creatinine assay buffer to each well in turn. Repeat 3 times and take the average value of the results;

[0015] All the above solutions were placed at room temperature for 10-30 min to fully react, and then placed in an ELISA reader to measure the absorbance at 510 nm (OD510). The measurement result was the average of three repeated tests.

[0016] S3. Urine sample purification and stability treatment

[0017] Take 250-500 μL of urine to be tested, add 250-500 μL of protein precipitant, let stand at 4°C for 1-2 hours, centrifuge at 5000-10000 r / min for 10-30 minutes, separate and collect the supernatant, mix the sample with ultrapure water at a mass volume ratio of 1:5-1:10, perform water bath ultrasound for 25-50 minutes, pass through a needle filter and pre-treatment column, and obtain the purified test sample;

[0018] S4. The conditions for the determination of organic acid standards using high performance ion exchange chromatography are as follows:

[0019] Ion chromatography conditions:

[0020] Ion chromatograph: Dionex ICS-6000; chromatographic column: Dionex IonPacTM AG15 guard column (50×4mm) and Dionex IonPacTM AS15 chromatographic column (250×4mm) or other chromatographic columns with equivalent performance; injection volume: 25-120μL; column temperature: 25-35℃; flow rate: 0.8-1.2mL / min; mobile phase A is ultrapure water, and mobile phase B is 500mmol / L NaOH aqueous solution; gradient elution, the specific gradient elution program is shown in the table below (Table 1).

[0021] Table 1 Ion chromatography multi-stage gradient elution conditions

[0022]

[0023] In the above technical solution, the common organic acids in urine include glycolic acid, lactic acid, 2-hydroxybutyric acid, pyruvic acid, oxalic acid, α-ketoglutaric acid, isocitric acid, cis-aconitic acid, and trans-aconitic acid.

[0024] Preferably, in the above steps S2 and S3, the organic acid mixed standard solution and the urine sample to be tested are filtered through a 0.22 μm nylon needle filter membrane.

[0025] Preferably, in the above step S3, ethanol is used as a protein precipitant in the urine sample to be tested.

[0026] Preferably, in step S3, the urine sample pretreatment column is Dionex InGuard TM Na / HRP cartridge.

[0027] Optionally, in the above step S4, the suppressor is a Dionex ADRS 600 (4 mm) anion suppressor, and the suppression current during the detection process is 28 mA.

[0028] Optionally, in the above step S4, the detection cell temperature of the conductivity detector during use is 35°C.

[0029] Optionally, the method is used to simultaneously determine glycolic acid, lactic acid, 2-hydroxybutyric acid, pyruvic acid, oxalic acid, α-ketoglutaric acid, isocitric acid, cis-aconitic acid, trans-aconitic acid, chloride ion (Cl - ), nitrate ion (NO 3 - ), sulfate ion (SO 4 2- ), phosphate ion (PO 4 3- ), qualitative analysis was performed based on retention time, and quantitative analysis was performed based on peak area using external standard method.

[0030] Compared with the prior art, the present invention has the following significant beneficial effects:

[0031] This chapter optimizes the urine pretreatment method, and establishes the AEC-CD method for detecting organic acids and anions in urine by optimizing the chromatographic detection conditions, realizing the separation and detection of difficult-to-separate organic acids. The pretreatment is simple and convenient, and the test results are accurate and reliable, providing important help for the exploration of the metabolic laws of organic acids in human urine and disease diagnosis.

[0032] The invention establishes an AEC-CD detection method, and 10 organic acids and 4 inorganic anions have good linearity within a certain mass concentration range, and the linear correlation coefficients are all greater than or equal to 0.999; the detection limit range of the method is 0.01-0.10 mg / L, the quantitative limit range is 0.015-0.30 mg / L, the spiked recovery rate is between 97.0-105%, the accuracy is good, the sensitivity is high, and the simultaneous detection of 14 anion components in urine samples of biological bodies can be achieved.

[0033] The present invention realizes effective separation and detection of 10 organic acids and 4 anions in urine samples. The urine sample contains many interfering matrices and low content of the analyte. The present invention realizes effective separation of multi-component anion groups by optimizing the multi-stage gradient elution procedure, and the separation degree is not less than 1.5, which effectively improves the sensitivity and accuracy of the detection.

[0034] The present invention requires a simple processing method for the urine sample to be tested, does not involve complex solvents and instruments, and only requires the addition of a protein precipitant and a pre-treatment column to remove most of the matrix proteins and other impurities, effectively reducing the damage of the sample matrix to the ion chromatograph and significantly reducing the detection cost.

[0035] The present invention proposes a detection method for human urine, which is sensitive, convenient, efficient, accurate and highly practical. It can be applied to the detection of organic acids in the urine of healthy people and disease patients, and provides more reference detection data for targeted analysis of human urine and pre-diagnosis of diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The detection spectrum of 14 component standards is shown; among them: 1. glycolic acid; 2. lactic acid; 3. 2-hydroxybutyric acid; 4. pyruvic acid; 5. Cl - ; 6.SO 4 2- ; 7. Oxalic acid; 8. NO 3 - ; 9.α-ketoglutarate; 10.PO 4 3- ; 11. Citric acid; 12. Isocitrate; 13. Cis-aconitic acid; 14. Trans-aconitic acid.

[0037] Figure 2 The detection spectrum of organic and inorganic anions in the urine of healthy people is shown; among them: 1. glycolic acid; 2. lactic acid; 3. 2-hydroxybutyric acid; 4. pyruvic acid; 5. Cl - ; 6.SO 4 2- ; 7. Oxalic acid; 8. NO 3 - ; 9.α-ketoglutarate; 10.PO 4 3- ; 11. Citric acid; 12. Isocitrate; 13. Cis-aconitic acid; 14. Trans-aconitic acid.

[0038] Figure 3 The detection spectrum of organic and inorganic anions in the urine of diabetic patients is shown; among them: 1. glycolic acid; 2. lactic acid; 3. 2-hydroxybutyric acid; 4. pyruvic acid; 5. Cl - ; 6.SO 4 2- ; 7. Oxalic acid; 8. NO 3 - ; 9.α-ketoglutarate; 10.PO 4 3- ; 11. Citric acid; 12. Isocitrate; 13. Cis-aconitic acid; 14. Trans-aconitic acid. Specific Embodiments

[0039] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which the embodiments of the present invention belong. If the definitions stated in this part are contrary to or otherwise inconsistent with the definitions stated in the patents, patent applications, published patent applications and other publications incorporated herein by reference, the definitions listed in this part shall prevail over the definitions incorporated herein by reference.

[0041] Example 1 Methodological Verification of the Method of the Present Invention

[0042] 1. Specificity and System Suitability Investigation

[0043] Inject the blank solution and the standard solution once each to investigate the interference situation and the resolution of the ions to be measured. The blank solution is an ethanol solution with a volume fraction of 50%. The concentrations of each ion in the standard solution are as follows: glycolic acid 2.00 mg / L, lactic acid 0.89 mg / L, 2-hydroxybutyric acid 1.30 mg / L, pyruvic acid 7.72 mg / L, Cl - 1.00 mg / L, SO 4 2- 1.00 mg / L, oxalic acid 0.50 mg / L, NO 3 - 0.50 mg / L, α-ketoglutaric acid 1.00 mg / L, PO 4 3- 5.00 mg / L, citric acid 5.00 mg / L, isocitric acid 5.00 mg / L, cis-aconitic acid 5.00 mg / L, trans-aconitic acid 7.00 mg / L. Detection is carried out according to the above gradient elution conditions, and the resolution results of each ion are shown in Table 2. As can be seen from Table 2, except for NO 3 - and α-ketoglutaric acid, the resolution of the chromatographic peaks of other components is not less than 1.5, meeting the conditions for complete separation, indicating that the method has strong specificity.

[0044] Table 2 Resolution Results

[0045]

[0046] 2. Detection Limit and Quantitation Limit

[0047] The standard solution was gradually diluted and tested under the above gradient elution conditions. When the signal-to-noise ratio S / N was about 3, the concentration was the detection limit, and three injections were made; when the S / N was about 10, the concentration was the quantitative limit, and 6 injections were made. The results are shown in Table 3. As can be seen from Table 3, the detection limits of the 14 components were between 0.004-0.100 mg / L. The method of the present invention is sensitive, accurate, and has a low detection limit.

[0048] Table 3 Detection limit and quantification limit

[0049]

[0050] 3. Linearity and range

[0051] Mixed standard solutions of different concentrations were prepared to investigate the linearity of the peak area of ​​each component. Six points were taken for each ion, and the concentrations of each point were as follows: glycolic acid: 0.5, 3.33, 13.3, 20.0, 33.3, 50.0 mg / L; lactic acid 0.02, 1.19, 4.77, 7.16, 11.9, 17.9 mg / L; 2-hydroxybutyric acid 0.13, 0.87, 3.47, 5.20, 8.67, 13.0 mg / L; pyruvic acid 0.20, 1.03, 4.12, 6.18, 10.3, 15.4 mg / L; oxalic acid 0.20, 0.67, 2.67, 4.00, 6.67, 10 .0mg / L; α-ketoglutaric acid 0.30, 0.67, 2.67, 4.00, 6.67, 10.0mg / L; citric acid 0.25, 2.00, 8.00, 12.0, 20.0mg / L; isocitric acid 0.20, 0.67, 2.67, 4.00, 6.67, 10.0mg / L; cis-aconitic acid 0.21, 0.56, 2.24, 3.36, 5.60, 8.40mg / L; trans-aconitic acid 0.14, 0.88, 3.55, 5.32, 8.87, 13.3mg / L; Cl - 5.00, 33.3, 133, 200, 333, 500mg / L; SO 4 2- 2.50, 16.7, 66.7, 100, 166.7, 250mg / L; NO 3 - 0.20, 3.33, 13.3, 20.0, 33.3, 50.0mg / L; PO 4 3- 0.20, 20.0, 80.0, 120, 200, 300 mg / L. A linear fit was performed with the peak area (μS*min) as the ordinate and the corresponding mass concentration (mg / L) of each component as the abscissa to establish the mass concentration-peak area standard curve of each component.3 - The separation degree with α-ketoglutaric acid is less than 1.5, and the vertical axis is set as the peak height (μS) and the horizontal axis is set as the mass concentration (mg / L) to establish a standard curve. The linear regression equation and linear correlation coefficient (r) of each component are shown in Table 4. As can be seen from Table 4, the linear correlation coefficient of each component is greater than 0.9990, and the peak area of ​​each component shows a good linear relationship with the concentration change within the prepared concentration range.

[0052] Table 4 Regression equation and linear correlation coefficient of standard curve of each component

[0053]

[0054] 4. Precision

[0055] Prepare 1.00mg / L glycolate, 0.89mg / L lactate, 1.30mg / L 2-hydroxybutyrate, 7.72mg / L pyruvate, 1.00mg / L oxalate, 2.00mg / L α-ketoglutarate, 5.00mg / L citrate, 5.00mg / L isocitrate, 6.50mg / L cis-aconitate, 5.00mg / L trans-aconitate, Cl - 1.00mg / L, SO 4 2- 1.00mg / L, NO 3 - 1.00mg / L, PO 4 3- 5.00mg / L mixed standard solution was sampled and tested according to the above ion chromatography conditions, and the RSD of the peak height of NO3- and α-ketoglutaric acid and the RSD of the peak area of ​​the remaining components were calculated. The specific data are shown in Table 5. As can be seen from Table 5, the RSD of the peak response of each component for 6 consecutive injections is 0.51-2.26%, indicating that the instrument has good stability and high precision.

[0056] Table 5 Ion chromatography precision (n=6)

[0057]

[0058]

[0059] 5. Repeatability

[0060] Take 250-500 μL of the same sample, add 250-500 μL of protein precipitant, stand at 4°C for 1-2h, centrifuge at 5000-10000 r / min for 10-30min, separate and collect the supernatant, mix the sample with ultrapure water at a mass volume ratio of 1:5-1:10, perform water bath ultrasound for 25-50min, pass through a needle filter and pretreatment column, and perform parallel treatment 6 times. The 6 parallel solutions after treatment are sampled for ion chromatography detection to obtain the peak response values ​​of each component in the sample. The results are shown in Table 6. As can be seen from Table 6, the RSD of the peak response of each component after 6 parallel treatments is 0.42-2.94%, the method has good repeatability, and the detection results are stable and reliable.

[0061] Table 6 Method repeatability (n=6)

[0062]

[0063] 6. Spike recovery rate

[0064] Take urine samples with known content of each component, add 50% content of each component standard, inject the obtained sample into ion chromatography for detection, and calculate the spike recovery rate according to the background amount and the spiked amount. The results are shown in Table 7. As shown in Table 7, the average spiked recovery rate of each component in 6 parallel treatments is between 95.81% and 103.62%, and the RSD of the spiked recovery rate is 0.13-3.11%. The recovery rate is reasonable and the accuracy is good, which can meet the detection requirements.

[0065] Table 7 Spiked recovery (n=6)

[0066]

[0067]

[0068]

[0069] 7. Stability

[0070] The stability of the standard solution at room temperature and 4°C and the stability of the urine sample solution at room temperature, 4°C and -80°C were investigated respectively; mixed standard solution and treated urine sample solution of a certain concentration were placed at room temperature for 0, 6, 12, 18, 24 hours and at 4°C for 48 hours, and then sampled for detection; urine samples were stored at -80°C for 30 days, taken out and treated, and then sampled for ion chromatography detection. The stability results of the mixed standard solution are shown in Table 8, and the stability results of the urine sample are shown in Table 9. As shown in Tables 8 and 9, the stability of each component of the standard and urine sample is good.

[0071] Table 8 Stability of mixed standard solution

[0072]

[0073]

[0074]

[0075] Table 9 Urine sample solution stability

[0076]

[0077]

[0078]

[0079] Example 2 Simultaneous detection of organic and inorganic anions in urine samples of healthy people

[0080] 1. Standard solution configuration

[0081] Inorganic anions were purchased from the National Center for Standardization of Materials. - ), nitrate ion (NO 3 - ), sulfate ion (SO 4 2- ), phosphate ion (PO 4 3- ) standard solutions, all with an ion concentration of 1000 mg / L, were stored at 4°C and prepared into standard working solutions of corresponding concentrations according to experimental needs; organic acids were purchased high-purity glycolic acid, lactic acid, 2-hydroxybutyric acid, pyruvic acid, oxalic acid, α-ketoglutaric acid, isocitric acid, cis-aconitic acid and trans-aconitic acid standard substances, prepared into standard stock solutions of 1000 mg / L using ultrapure water, stored at 4°C and prepared into standard working solutions of corresponding concentrations according to experimental needs.

[0082] 2. Determination of organic acid and inorganic anion content in urine

[0083] Take 5-15μL of urine sample from healthy people in a 2mL centrifuge tube, add 800-1000μL of ultrapure water, take 8-12μL of diluted urine sample and add it to a 96-well ELISA plate, then add 20-80μL of creatinine colorimetric solution and 20-80μL of creatinine assay buffer to each well in turn, place at room temperature for 10-30min to fully react, place in an ELISA instrument to measure the absorbance at 510nm (OD510), and take the average value of 3 repeated tests as the measurement result to obtain the content of organic acids and inorganic anions in urine of healthy people.

[0084] 3. Pretreatment of urine samples from healthy people

[0085] Take 250-500μL of urine from healthy people to be tested, add 250-500μL of ethanol, let it stand at 4℃ for 1-2h, centrifuge at 5000-10000r / min for 10-30min, separate and collect the supernatant, mix the sample with ultrapure water at a mass volume ratio of 1:5-1:10, perform water bath ultrasound for 25-50min, filter through a 0.22μm nylon needle filter and Dionex InGuard TM Na / HRP cartridge was used to obtain urine samples from healthy people after purification.

[0086] 4. Ion chromatography conditions

[0087] Ion chromatograph: Dionex ICS-6000; chromatographic columns: Dionex IonPacTM AG15 guard column (50×4 mm) and Dionex IonPacTM AS15 chromatographic column (250×4 mm); injection volume: 25-120 μL; column temperature: 25-35°C; flow rate: 0.8-1.2 mL / min; mobile phase A was ultrapure water, and mobile phase B was 500 mmol / L NaOH aqueous solution; gradient elution, the specific gradient elution program is shown in Table 1 above.

[0088] 5. Test results

[0089] The results are shown in Table 10. The method of the present invention can effectively detect Cl in urine of healthy people at the same time. - 、NO 3 - 、SO 4 2- ,PO 4 3- , glycolic acid, lactic acid, 2-hydroxybutyric acid, pyruvic acid, oxalic acid, α-ketoglutaric acid, isocitric acid, cis-aconitic acid, trans-aconitic acid, etc., among which Cl - 、SO 4 2- ,PO 4 3- , citric acid, lactic acid and glycolic acid content.

[0090] Table 10 Test results of organic acid and inorganic anion content in urine of healthy people

[0091]

[0092] Example 3 Simultaneous detection of organic and inorganic anions in urine samples of diabetic patients

[0093] 1. Standard solution configuration

[0094] Inorganic anions were purchased from the National Center for Standardization of Materials. -), nitrate ion (NO 3 - ), sulfate ion (SO 4 2- ), phosphate ion (PO 4 3- ) standard solutions, all with an ion concentration of 1000 mg / L, were stored at 4°C and prepared into standard working solutions of corresponding concentrations according to experimental needs; organic acids were purchased high-purity glycolic acid, lactic acid, 2-hydroxybutyric acid, pyruvic acid, oxalic acid, α-ketoglutaric acid, isocitric acid, cis-aconitic acid and trans-aconitic acid standard substances, prepared into standard stock solutions of 1000 mg / L using ultrapure water, stored at 4°C and prepared into standard working solutions of corresponding concentrations according to experimental needs.

[0095] 2. Determination of organic acid and inorganic anion content in urine

[0096] Take 5-15 μL of urine sample from a diabetic patient in a 2 mL centrifuge tube, add 800-1000 μL of ultrapure water, take 8-12 μL of the diluted urine sample and add it to a 96-well ELISA plate, then add 20-80 μL of creatinine colorimetric solution and 20-80 μL of creatinine assay buffer to each well in turn, place at room temperature for 10-30 min to fully react, place in an ELISA instrument to measure the absorbance at 510 nm (OD510), and take the average value of 3 repeated tests as the measurement result to obtain the content of organic acids and inorganic anions in the urine of diabetic patients.

[0097] 3. Pretreatment of urine samples from diabetic patients

[0098] Take 250-500 μL of urine from the diabetic patient to be tested, add 250-500 μL of ethanol, let it stand at 4°C for 1-2 hours, centrifuge at 5000-10000 r / min for 10-30 minutes, separate and collect the supernatant, mix the sample with ultrapure water at a mass volume ratio of 1:5-1:10, perform water bath ultrasound for 25-50 minutes, filter through a 0.22 μm nylon needle filter and Dionex InGuard TM Na / HRP column was used to obtain the purified urine sample from diabetic patients.

[0099] 4. Ion chromatography conditions

[0100] Ion chromatograph: Dionex ICS-6000; chromatographic columns: Dionex IonPacTM AG15 guard column (50×4mm) and Dionex IonPacTM AS15 chromatographic column (250×4mm); injection volume: 25-120μL; column temperature: 25-35℃; flow rate: 0.8-1.2mL / min; mobile phase A is ultrapure water, and mobile phase B is 500mmol / L NaOH aqueous solution; gradient elution, the specific gradient elution program is shown in Table 11.

[0101] 5. Test results

[0102] The results are shown in Table 11. The method of the present invention can effectively detect Cl in the urine of diabetic patients at the same time. - 、NO 3 - 、SO 4 2- ,PO 4 3- , glycolic acid, lactic acid, 2-hydroxybutyric acid, pyruvic acid, oxalic acid, α-ketoglutaric acid, isocitric acid, cis-aconitic acid, trans-aconitic acid, etc.; SO 4 2- , Cl - , transaconitic acid, glycolic acid and other components are higher than those in healthy people, NO 3 - , α-ketoglutaric acid, oxalic acid and other components are lower than those in healthy people.

[0103] Table 11 Test results of organic acid and inorganic anion content in urine of diabetic patients

[0104]

[0105] Example 4 Simultaneous detection of organic and inorganic anions in urine samples of autistic patients

[0106] 1. Standard solution configuration

[0107] Inorganic anions were purchased from the National Center for Standardization of Materials. - ), nitrate ion (NO 3 - ), sulfate ion (SO 4 2- ), phosphate ion (PO 4 3-) standard solutions, all with an ion concentration of 1000 mg / L, were stored at 4°C and prepared into standard working solutions of corresponding concentrations according to experimental needs; organic acids were purchased high-purity glycolic acid, lactic acid, 2-hydroxybutyric acid, pyruvic acid, oxalic acid, α-ketoglutaric acid, isocitric acid, cis-aconitic acid and trans-aconitic acid standard substances, prepared into standard stock solutions of 1000 mg / L using ultrapure water, stored at 4°C and prepared into standard working solutions of corresponding concentrations according to experimental needs.

[0108] 2. Determination of organic acid and inorganic anion content in urine

[0109] Take 5-15 μL of urine sample from autistic patients in a 2 mL centrifuge tube, add 800-1000 μL of ultrapure water, take 8-12 μL of diluted urine sample and add it to a 96-well ELISA plate, then add 20-80 μL of creatinine colorimetric solution and 20-80 μL of creatinine assay buffer to each well in turn, place at room temperature for 10-30 min to fully react, place in an ELISA instrument to measure the absorbance at 510 nm (OD510), and take the average value of 3 repeated tests as the measurement result, that is, the content of organic acid and inorganic anion in urine of autistic patients.

[0110] 3. Pretreatment of urine samples from autistic patients

[0111] Take 250-500 μL of urine from autistic patients, add 250-500 μL of ethanol, let stand at 4°C for 1-2 hours, centrifuge at 5000-10000 r / min for 10-30 minutes, separate and collect the supernatant, mix the sample with ultrapure water at a mass volume ratio of 1:5-1:10, perform water bath ultrasound for 25-50 minutes, filter through a 0.22 μm nylon needle filter and Dionex InGuard TM Na / HRP column to obtain the purified urine sample from autistic patients.

[0112] 4. Ion chromatography conditions

[0113] Ion chromatograph: Dionex ICS-6000; chromatographic columns: Dionex IonPacTM AG15 guard column (50×4 mm) and Dionex IonPacTM AS15 chromatographic column (250×4 mm); injection volume: 25-120 μL; column temperature: 25-35°C; flow rate: 0.8-1.2 mL / min; mobile phase A was ultrapure water, and mobile phase B was 500 mmol / L NaOH aqueous solution; gradient elution, the specific gradient elution program is shown in Table 1 above.

[0114] 5. Test results

[0115] The results are shown in Table 12. The method of the present invention can effectively detect Cl in the urine of autistic patients at the same time.- 、NO 3 - 、SO 4 2- ,PO 4 3- , glycolic acid, lactic acid, 2-hydroxybutyric acid, pyruvic acid, oxalic acid, α-ketoglutaric acid, isocitric acid, cis-aconitic acid, and trans-aconitic acid; the levels of glycolic acid, citric acid, and oxalic acid in autistic patients are higher than those in healthy people.

[0116] Table 12. Test results of organic acid and inorganic anion content in urine of autistic patients

[0117]

[0118] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above description of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

[0119] The disclosed embodiments of the present invention are intended to illustrate the core concepts thereof, to demonstrate the scope and potential of the present invention, and do not limit its application to the specific manner described. By selecting and describing these embodiments in detail, this specification is intended to clearly explain the working principle of the present invention and its benefits in practical applications, so that professionals in the technical field can fully understand and effectively utilize the present invention. The scope of protection of the present invention is strictly defined in accordance with the claims and all possible equivalent embodiments covered therein.

Claims

1. A method for detecting multiple organic acids in human urine, characterized in that: The method comprises the following steps: 1) Prepare standard stock solutions and mixed standard solutions of organic acids; 2) Prepare a standard working solution of creatinine and measure the content of creatinine in urine; 3) Load urine sample for ion chromatography; 4) performing multi-step gradient elution on the loaded ion chromatography column; 5) receiving and analyzing anions in the eluent; The multi-stage gradient elution in step 4) is to elute the ion chromatography column with eluent and water respectively; the eluent is an aqueous solution of alkali. The method conditions for determining the organic acid standard using a high-performance ion exchange chromatograph are as follows: Ion chromatograph: Dionex ICS-6000; chromatographic column: Dionex IonPacTM AG15 guard column (50×4mm) and Dionex IonPacTM AS15 chromatographic column (250×4mm) or other chromatographic columns with equivalent performance; injection volume: 25-120μL; column temperature: 25-35℃; flow rate: 0.8-1.2mL / min; mobile phase A is ultrapure water, and mobile phase B is 500mmol / L NaOH aqueous solution; gradient elution, the specific gradient elution program is shown in the table below.

2. The detection method according to claim 1, characterized in that The urine samples are urine from healthy people, urine from diabetic patients, and urine from autistic patients.

3. The detection method according to claim 1, characterized in that The anions are glycolate, lactate, 2-hydroxybutyrate, pyruvate, oxalate, α-ketoglutarate, isocitrate, aconitate, transaconitate, chloride (Cl - ), nitrate ion (NO3 - ), sulfate ion (SO4 2- ), phosphate ion (PO4 3- ) among them; preferably 2 of them; more preferably 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 of them; most preferably all 14 of them.

4. The detection method according to claim 1, characterized in that Before step 3), the urine sample needs to be treated before purification.

5. The detection method according to claim 4, characterized in that: The pre-treatment comprises the following steps: 1) Adding a protein precipitant to the urine sample; 2) centrifuging the sample treated with the protein precipitant to obtain a supernatant; 3) diluting the obtained supernatant and ultrasonicating it in a water bath for 10-30 minutes; 4) The diluted and ultrasonicated sample was filtered through a 0.22 μm nylon needle filter and a Dionex InGuard TM Na / HRP cartridge.

6. The detection method according to claim 1, characterized in that The ion chromatography columns described in step 4) are Dionex IonPacTM AG15 guard column (50×4 mm) and Dionex IonPacTM AS15 chromatography column (250×4 mm).

7. The detection method according to claim 1, characterized in that The water is ultrapure water.

8. The detection method according to claim 1, characterized in that The eluent is a sodium hydroxide aqueous solution with a concentration of 500 mmol / L.

9. The detection method according to claim 7 or 8, characterized in that: The multi-stage gradient elution described in step 4) is as follows:

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