Method for detecting protein binding uremic toxins and use thereof

Abstract

The application provides a method and application for detecting protein-bound uremic toxins, which comprises the following steps: mixing a blood sample and a protein precipitation extractant, centrifugally separating to obtain supernatant, mixing the supernatant and the protein precipitation extractant, centrifugally separating, filtering the treated supernatant to obtain a sample solution to be detected; preparing a standard solution of each sample to be detected, mixing the standard solutions of all the samples to be detected, and preparing a plurality of mixed standard solutions with different concentrations of the samples to be detected; performing high performance liquid chromatography on the mixed standard solutions to draw a standard curve between the concentration and the peak area of each sample to be detected, performing high performance liquid chromatography on the sample solution to be detected to obtain the determination peak area of each sample to be detected, and calculating the content of each sample to be detected according to the corresponding standard curve. The application solves the problems of low detection sensitivity and difficulty in accurate detection of the existing detection methods.

Description

Technical Field

[0001] This invention relates to the field of protein-bound uremic toxin detection technology, and more specifically, to a method for detecting protein-bound uremic toxins and its application. Background Technology

[0002] Chronic kidney disease (CKD) is a clinical syndrome characterized by a series of symptoms and metabolic disorders resulting from the progressive and irreversible decline in kidney function due to various kidney diseases, eventually leading to loss of function. The end-stage of chronic kidney disease is commonly known as uremia, which is a syndrome comprised of a series of clinical manifestations that occur when chronic kidney failure reaches its terminal stage.

[0003] Protein-bound uremic toxins (PBUTs) are an important class of uremic toxins. They readily bind to serum proteins. In healthy individuals, serum PBUT concentrations remain low. However, in patients with chronic renal failure (CKD), as renal function declines, serum PBUT concentrations gradually increase, peaking at stage 5. Furthermore, due to competition for albumin binding sites, the free concentration of these toxins in plasma increases, causing damage to various organs and triggering numerous diseases. Studies have shown that PBUT levels are a valuable diagnostic indicator for CKD and its various complications, particularly closely related to cardiovascular events, the leading cause of death in CKD patients. Therefore, developing a method for detecting PBUTs is of great significance for the diagnosis and treatment of kidney diseases and their related cardiovascular complications.

[0004] Protein-bound uremic toxins are mainly classified into: (1) phenols, such as hydroquinone and phenol; (2) indoles, such as indole-3-acetic acid, indolesulfonate, and melatonin; (3) polyamines, such as tetramethylene diamine and spermine; (4) advanced glycation end products, such as 3-deoxyglucuronide, glyoxal, and pentosine; (5) peptides, such as leptin and retinol-binding protein; and (6) hippurates, such as hippuric acid and p-hydroxyhippuric acid. Among these, indoles and hippurates are typical representatives of protein-bound uremic toxins.

[0005] Currently, most of the protein-bound uremic toxin detection methods reported domestically and internationally employ enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CIA), ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS), and liquid chromatography. ELISA and CIA have the following drawbacks: (1) low sensitivity; when sample concentration is very low, a large sample volume is required for enrichment and purification; (2) poor applicability; antibodies to the analyte are prone to cross-reaction with structurally similar endogenous substances or metabolites, leading to false positive results; (3) low analytical efficiency; primarily targeting single substances, simultaneous detection of multiple substances requires different reagents, resulting in complex sample processing steps. UHPLC-MS / MS can simultaneously detect different protein-bound uremic toxins, but the detection time is long, and the equipment cost is high, preventing its widespread adoption. Liquid chromatography, due to its limited detection limit, is insensitive for low-concentration toxin detection; and the complex composition of plasma / serum means that impurities can severely affect the accuracy of detection results during quantitative analysis, preventing its widespread clinical application. Summary of the Invention

[0006] The present invention aims to solve the problems of low detection sensitivity and difficulty in accurate detection of existing protein-bound uremic toxin detection methods.

[0007] To address the above problems, the first aspect of the present invention provides a method for detecting protein-bound uremic toxins, comprising:

[0008] After mixing the blood sample and the protein precipitation extractant, centrifuge to obtain the supernatant. Mix the supernatant and the protein precipitation extractant, centrifuge to obtain the treated supernatant. Filter the treated supernatant to obtain the sample solution to be tested.

[0009] Prepare standard solutions for each type of test sample separately, mix all the standard solutions of the test samples to prepare multiple mixed standard solutions with different concentrations of the test samples, wherein the test samples include hippuric acid, indophenol sulfate and indole-3-acetic acid;

[0010] The mixed standard solution is detected by high performance liquid chromatography (HPLC), and a standard curve is plotted between the concentration and peak area of ​​each test sample. The test sample solution is detected by HPLC to obtain the measured peak area of ​​each test sample. Based on the measured peak area of ​​each test sample and its corresponding standard curve, the content of each test sample in the test sample solution is calculated.

[0011] Furthermore, the protein precipitation extractant used for precipitation extraction of the blood sample is different from the protein precipitation extractant used for precipitation extraction of the supernatant.

[0012] Furthermore, the protein precipitation extractant is methanol or acetonitrile.

[0013] Furthermore, the protein precipitation extractant used for precipitation extraction of the blood sample is methanol, and the protein precipitation extractant used for precipitation extraction of the supernatant is acetonitrile.

[0014] Furthermore, the volume ratio of the blood sample to the protein precipitation extractant is 1:1, and the volume ratio of the supernatant to the protein precipitation extractant is 1:1.

[0015] Further, the step of preparing standard solutions for each type of sample to be tested separately, and mixing all the standard solutions of the samples to be tested to prepare multiple mixed standard solutions with different concentrations of the samples to be tested, includes:

[0016] A standard solution for each of the test samples was prepared using methanol. All the standard solutions for the test samples were mixed evenly. Then, mixed standard solutions with different concentration gradients were prepared using blank plasma to obtain multiple mixed standard solutions with different concentrations of the test samples.

[0017] Furthermore, in the high-performance liquid chromatography (HPLC) detection, the chromatographic conditions are as follows: octadecylsilane-bonded silica gel is used as the packing material, mobile phase A is a mixed solution of methanol and ammonium formate, mobile phase B is a mixed solution of methanol and water, the chromatographic column is an Inertsil ODS column, and the column temperature is 25℃.

[0018] Furthermore, the volume ratio of methanol to ammonium formate in mobile phase A is 20:80, and the volume ratio of methanol to water in mobile phase B is 90:10.

[0019] A second aspect of the present invention provides a kit for detecting protein-bound uremic toxins, which uses the method for detecting protein-bound uremic toxins described in the first aspect.

[0020] Furthermore, the kit includes a protein precipitation extractant, a mixed standard solution, mobile phase A, and mobile phase B.

[0021] The method for detecting protein-bound uremic toxins described in this invention optimizes blood sample pretreatment and high-performance liquid chromatography (HPLC) conditions. This method enables the simultaneous detection of three protein-bound uremic toxins in blood samples: indophenol sulfate (IS), indole-3-acetic acid (IAA), and hippuric acid (HA). It allows for precise qualitative and quantitative analysis of the detected substances and exhibits high detection sensitivity. This method overcomes the problems of insensitivity and inaccurate qualitative and quantitative analysis in existing technologies when the concentration of these three protein-bound uremic toxins in blood samples is low. Furthermore, this invention employs a two-stage protein precipitation extraction process, followed by filtration of the supernatant to obtain the test sample solution. This effectively removes plasma proteins, reduces matrix effects, and prevents incomplete protein precipitation that could affect peak elution during HPLC analysis, leading to deviations in detection results. It also avoids clogging of the HPLC system and reduced instrument lifespan.

[0022] The kit for detecting protein-bound uremic toxins described in this invention can simultaneously detect the content of hippuric acid, indole-3-acetic acid, and indolesulfonate. When applied to practical operations, this kit has good operability and practical value, and provides great convenience for the simultaneous detection of the content of hippuric acid, indole-3-acetic acid, and indolesulfonate. Attached Figure Description

[0023] Figure 1 A process flow diagram of the method for detecting protein-bound uremic toxins provided in an embodiment of the present invention;

[0024] Figure 2 This is a peak elution result diagram of experimental group 1 in Example 2 of the present invention;

[0025] Figure 3 This is a peak elution result diagram of experimental group 2 in Example 2 of the present invention;

[0026] Figure 4 This is a peak elution result diagram of experimental group 3 in Example 2 of the present invention;

[0027] Figure 5 This is a peak elution result diagram of experimental group 4 in Example 2 of the present invention;

[0028] Figure 6 This is a peak elution result diagram of experimental group 5 in Example 2 of the present invention;

[0029] Figure 7 This is a peak time diagram of hippuric acid (HA), indophenol sulfate (IS), and indole-3-acetic acid (IAA) in Example 3 of the present invention. Detailed Implementation

[0030] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0031] Furthermore, the terms "comprising," "including," "containing," and "having" are non-restrictive and can refer to the addition of other steps and components that do not affect the results. Unless otherwise specified, all materials, equipment, and reagents are commercially available.

[0032] Furthermore, although the present invention describes each step in the preparation process in the form of S1, S2, S3, etc., this description is only for ease of understanding. The form of S1, S2, S3, etc. does not indicate a limitation on the order of each step.

[0033] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0034] Combination Figure 1 As shown, the first aspect of this invention provides a method for detecting protein-bound uremic toxins, comprising:

[0035] Step S1: Mix the blood sample and protein precipitation extractant, centrifuge to obtain the supernatant after separation, mix the supernatant and protein precipitation extractant, centrifuge to obtain the treated supernatant, filter the treated supernatant to obtain the sample solution to be tested.

[0036] Step S2: Prepare standard solutions for each type of sample to be tested separately, mix all the standard solutions of the samples to be tested, and prepare multiple mixed standard solutions with different concentrations of the samples to be tested. The samples to be tested include hippuric acid, indophenol sulfate and indole-3-acetic acid.

[0037] Step S3: Perform high performance liquid chromatography (HPLC) on the mixed standard solution, plot a standard curve between the concentration and peak area of ​​each test sample, perform HPLC on the test sample solution to obtain the peak area of ​​each test sample, and calculate the content of each test sample in the test sample solution based on the peak area of ​​each test sample and its corresponding standard curve.

[0038] The method for detecting protein-bound uremic toxins provided in this embodiment optimizes blood sample pretreatment and high-performance liquid chromatography (HPLC) conditions. This method enables the simultaneous detection of three protein-bound uremic toxins in blood samples: indophenol sulfate (IS), indole-3-acetic acid (IAA), and hippuric acid (HA). It allows for precise qualitative and quantitative analysis of the detected substances and exhibits high detection sensitivity. This addresses the problems of insensitivity and inaccurate qualitative and quantitative analysis in existing technologies when the concentration of these three protein-bound uremic toxins in blood samples is low. Furthermore, this embodiment employs a two-stage protein precipitation extraction process, followed by filtration of the supernatant to obtain the test sample solution. This effectively removes plasma proteins, reduces matrix effects, and prevents incomplete protein precipitation that could affect peak elution during HPLC analysis, leading to deviations in detection results. It also avoids clogging of the HPLC system and reduced instrument lifespan.

[0039] In this embodiment, the blood sample is a plasma sample or a serum sample.

[0040] In this embodiment, different protein precipitation extractants are used for the two precipitation extractions; specifically, the protein precipitation extractant used for the blood sample precipitation extraction is different from the protein precipitation extractant used for the supernatant precipitation extraction. The protein precipitation extractant works by altering the dielectric constant of plasma, causing the biomacromolecules with surface water layers to dehydrate, aggregate, and precipitate. Since the blood sample and protein precipitation extractant are mixed, and the supernatant is obtained after centrifugation, the blood sample and supernatant have different compositions. Using different protein precipitation extractants allows for more targeted precipitation extraction of both the blood sample and the supernatant, improving the protein precipitation extraction effect. This facilitates effective removal of plasma proteins, reduces matrix effects, and makes protein precipitation more thorough.

[0041] Based on the above embodiments, the protein precipitation extractant is methanol or acetonitrile. The protein precipitation extractants used in the two precipitation extractions are different: methanol is used in the first extraction, and acetonitrile is used in the second. Specifically, the blood sample is mixed with methanol, centrifuged, and the resulting supernatant is obtained. The supernatant is then mixed with acetonitrile, centrifuged, and the treated supernatant is obtained. The treated supernatant is then filtered to obtain the sample solution to be tested. Therefore, using methanol to precipitate and extract the blood sample first, followed by acetonitrile to precipitate and extract the supernatant, can more effectively remove plasma proteins from both the blood sample and the supernatant, further improving the clarity of the supernatant and ensuring more thorough protein precipitation, thus improving the accuracy of the test results.

[0042] Based on the above embodiments, the volume ratio of blood sample to protein precipitation extractant is 1:1, and the volume ratio of supernatant to protein precipitation extractant is 1:1. Therefore, a volume ratio of 1:1 for both blood sample and protein precipitation extractant, and a volume ratio of 1:1 for supernatant to protein precipitation extractant, can avoid the following: if the proportion of protein precipitation extractant is too low, protein precipitation will be incomplete, and impurities will affect the peak elution effect, thus causing deviations in the detection results. It can also avoid the following: if the proportion of protein precipitation extractant is too high, excessive dilution of the blood sample will reduce the detection limit and affect the sensitivity of the detection.

[0043] In this embodiment, the supernatant after filtration is obtained by using a 0.45µm nylon 66 membrane or a 0.45µm polytetrafluoroethylene filter membrane to obtain the sample solution to be tested.

[0044] In this embodiment, step S2 involves preparing standard solutions for each type of sample to be tested, mixing all the standard solutions for the samples to be tested, and preparing multiple mixed standard solutions with different concentrations of the samples to be tested. This includes: preparing standard solutions for each type of sample to be tested separately using methanol, mixing all the standard solutions for the samples to be tested evenly, and then preparing mixed standard solutions with different concentration gradients using blank plasma to obtain multiple mixed standard solutions with different concentrations of the samples to be tested. Specifically, after preparing the standard solutions for each type of sample to be tested, a certain amount of all the standard solutions for the samples to be tested can be taken and diluted with blank plasma to different factors to form mixed standard solutions.

[0045] The concentrations of the hippuric acid standard solution were 14 mg / mL, the indolesulfonate standard solution were 2 mg / mL, and the indole-3-acetic acid standard solution were 2 mg / mL.

[0046] In this embodiment, in step S3, the chromatographic conditions for high-performance liquid chromatography (HPLC) detection are as follows: octadecylsilane-bonded silica gel is used as the packing material; mobile phase A is a mixed solution of methanol and ammonium formate; mobile phase B is a mixed solution of methanol and water; the detection wavelength is 229 nm; and the chromatographic column is an Inertsil ODS column (4.6 × 250 mm, 5 μm). The flow rate was 1.0 ml / min, the elution method was isocratic elution, the injection volume was 10 μL, and the column temperature was 25 °C.

[0047] Based on the above embodiments, the volume ratio of methanol to ammonium formate in mobile phase A is 20:80, and the volume ratio of methanol to water in mobile phase B is 90:10. Therefore, by using the above mobile phases A and B, the interference of substances in plasma on the detection of hippuric acid, indolesulfonate, and indole-3-acetic acid can be avoided, and the effective separation and content detection of these three substances can be achieved.

[0048] In this embodiment, the elution order of the analytes in high-performance liquid chromatography (HPLC) detection is as follows: hippuric acid, indolesulfonate, and indole-3-acetic acid, with a elution time interval of 1 minute or more. Specifically, the elution time of hippuric acid is approximately 10 minutes, that of indolesulfonate is approximately 13 minutes, and that of indole-3-acetic acid is approximately 14 minutes. This avoids interference between the elution times of the substances being too close together.

[0049] A second aspect of the present invention provides a kit for detecting protein-bound uremic toxins, wherein the kit employs the method for detecting protein-bound uremic toxins described in the first aspect.

[0050] Specifically, the kit includes a protein precipitation extractant, a mixed standard solution, mobile phase A, and mobile phase B. The protein precipitation extractant, the mixed standard solution, mobile phase A, and mobile phase B are the same as those in the first aspect, and will not be described again here.

[0051] To provide a more detailed description of the present invention, specific embodiments will be used to further illustrate the invention below. Unless otherwise specified, the experimental methods used in the embodiments of the present invention are conventional methods; unless otherwise specified, the materials and reagents used in the embodiments of the present invention are commercially available. In this embodiment, the blank plasma is plasma from healthy individuals obtained from a blood bank.

[0052] Example 1

[0053] This embodiment provides a method for determining a protein precipitation extractant, specifically employing the following method:

[0054] Experimental Group 1: Take 0.7 ml of plasma sample, add 0.7 ml of acetonitrile, vortex for 60 s, centrifuge (13000 rpm, 20 min), take the supernatant and filter it through a 0.45 μm filter membrane to obtain the test sample solution, and measure the turbidity of the test sample solution.

[0055] Experimental Group 2: Take 0.7 ml of plasma sample, add 0.7 ml of methanol, vortex for 60 s, centrifuge (13000 rpm, 20 min), take the supernatant and filter it through a 0.45 μm filter membrane to obtain the test sample solution, and measure the turbidity of the test sample solution.

[0056] Experimental Group 3: Take 0.7 ml of plasma sample, add 0.7 ml of methanol, vortex for 60 s, centrifuge (13000 rpm, 5 min), take 0.7 ml of supernatant, add 0.7 ml of methanol, vortex for 60 s, centrifuge (13000 rpm, 5 min), take the supernatant and filter through a 0.45 μm filter membrane to obtain the test sample solution, and measure the turbidity of the test sample solution;

[0057] Experimental Group 4: Take 0.7 ml of plasma sample, add 0.7 ml of methanol, vortex for 60 s, centrifuge (13000 rpm, 5 min), take 0.7 ml of supernatant, add 0.7 ml of acetonitrile, vortex for 60 s, centrifuge (13000 rpm, 5 min), take the supernatant and filter through a 0.45 μm filter membrane to obtain the test sample solution, and measure the turbidity of the test sample solution;

[0058] Experimental Group 5: Take 0.7 ml of plasma sample, add 0.7 ml of acetonitrile, vortex for 60 s, centrifuge (13000 rpm, 5 min), take 0.7 ml of supernatant, add 0.7 ml of methanol, vortex for 60 s, centrifuge (13000 rpm, 5 min), take the supernatant and filter through a 0.45 μm filter membrane to obtain the test sample solution, and measure the turbidity of the test sample solution;

[0059] Experimental Group 6: Take 0.7 ml of plasma sample, add 0.7 ml of acetonitrile, vortex for 60 s, centrifuge (13000 rpm, 5 min), take 0.7 ml of supernatant, add 0.7 ml of acetonitrile, vortex for 60 s, centrifuge (13000 rpm, 5 min), take the supernatant and filter through a 0.45 μm filter membrane to obtain the test sample solution, and measure the turbidity of the test sample solution.

[0060] Experimental Group 7: Take 0.7 ml of plasma sample and add 0.7 ml of methanol and acetonitrile mixed solution (the two can be mixed in a 1:1 ratio, the same below). Vortex for 60 s and centrifuge (13000 rpm, 5 min). Take 0.7 ml of supernatant, add 0.7 ml of methanol and acetonitrile, vortex for 60 s and centrifuge (13000 rpm, 5 min). Filter the supernatant through a 0.45 μm filter membrane to obtain the test sample solution. Measure the turbidity of the test sample solution.

[0061] Table 1. Turbidity after precipitation with different protein precipitating agents

[0062] Group Protein precipitation extractant type Turbidity Experimental group 1 Acetonitrile 1.88 Experimental group 2 methanol 2.47 Experimental group 3 Methanol + Methanol 1.29 Experimental group 4 Methanol + Acetonitrile 0.12 Experimental group 5 Acetonitrile + Methanol 0.41 Experimental group 6 Acetonitrile + Acetonitrile 1.00 Experimental group 7 Methanol and acetonitrile + Methanol and acetonitrile 0.53

[0063] It should be noted that the turbidity calculation is performed by establishing a standard curve using standard solutions and their corresponding turbidities, measuring the OD (absorbance) value of the sample to be tested, and then substituting it into the formula to obtain the result.

[0064] As shown in Table 1, the sample with the lowest turbidity was obtained by performing two centrifugation operations, first using methanol as the protein precipitation extractant and then using acetonitrile as the protein precipitation extractant. This indicates that using methanol to precipitate and extract the blood sample first, followed by acetonitrile to precipitate and extract the supernatant, allows for more targeted precipitation and extraction of both the blood sample and the supernatant, improving the efficiency of protein precipitation and extraction. This effectively removes plasma proteins, reduces matrix effects, and improves the accuracy of the test results. Furthermore, a 1:1 volume ratio of blood sample to protein precipitation extractant and a 1:1 volume ratio of supernatant to protein precipitation extractant in both precipitation and extraction operations further enhances the effective removal of plasma proteins and reduces matrix effects.

[0065] Example 2

[0066] This embodiment provides a method for determining chromatographic conditions, wherein the flow rate and wavelength remain constant in each group of experimental examples, and only the mobile phase and its ratio are changed. The mobile phase in each group of experimental examples is as follows:

[0067] Experimental Group 1: Mobile phase A consisted of a mixture of acetonitrile and 0.1% TFA aqueous solution. The experiments were conducted with acetonitrile to TFA aqueous solution volume ratios of 1.4:8.6, 2:8, 3:7, 4:6, and 5:5. The peak elution was observed, and the results are shown below. Figure 2 The peak elution results for the acetonitrile and TFA aqueous solution with a volume ratio of 1.4:8.6 are shown in the figure. Figure 2 (a) The peak elution results for an acetonitrile and TFA aqueous solution with a volume ratio of 2:8 are shown in the figure. Figure 2 (b) The peak elution results for an acetonitrile and TFA aqueous solution with a volume ratio of 3:7 are shown in the figure. Figure 2 (c) The peak elution results for an acetonitrile and TFA aqueous solution with a volume ratio of 4:6 are shown in the figure. Figure 2 (d) The peak elution results for an acetonitrile and TFA aqueous solution with a volume ratio of 5:5 are shown in the figure. Figure 2 (e).

[0068] Experimental Group 2: Mobile phase A consisted of a mixture of acetonitrile and 50 mmol / L ammonium formate aqueous solution. The mobile phase was developed at volume ratios of acetonitrile to 50 mmol / L ammonium formate aqueous solution of 2:8, 3:7, and 4:6. The peak elution was observed, and the results are shown below. Figure 3 The peak results for the acetonitrile and 50 mmol / L ammonium formate aqueous solution at a volume ratio of 2:8 are shown in the figure. Figure 3(a) The peak elution results for acetonitrile and 50 mmol / L ammonium formate aqueous solution in a volume ratio of 3:7 are shown in the figure. Figure 3 (b) The peak results for the volume ratio of acetonitrile and 50 mmol / L ammonium formate aqueous solution of 4:6 are shown in the figure. Figure 3 (c)

[0069] Experimental Group 3: Mobile phase A consisted of a mixture of methanol and 0.1% TFA aqueous solution. The mobile phase was developed at volume ratios of 2:8, 3:7, and 4:6. The peak elution was observed, and the results are shown below. Figure 4 The peak elution results for methanol and 0.1% TFA aqueous solution at a volume ratio of 2:8 are shown in the figure. Figure 4 (a) The peak elution results for methanol and 0.1% TFA aqueous solution at a volume ratio of 3:7 are shown in the figure. Figure 4 (b) The peak elution results for methanol and 0.1% TFA aqueous solution at a volume ratio of 4:6 are shown in the figure. Figure 4 (c)

[0070] Experimental Group 4: Mobile phase A consisted of a mixture of methanol and 50 mmol / L ammonium formate aqueous solution. The mobile phase was developed at volume ratios of 2:8, 3:7, and 4:6. The peak elution was observed, and the results are shown below. Figure 5 The peak elution results for methanol and 50 mmol / L ammonium formate aqueous solution at a volume ratio of 2:8 are shown in the figure. Figure 5 (a) The peak elution results for methanol and 50 mmol / L ammonium formate aqueous solution in a volume ratio of 3:7 are shown in the figure. Figure 5 (b) The peak elution results for methanol and 50 mmol / L ammonium formate aqueous solution at a volume ratio of 4:6 are shown in the figure. Figure 5 (c)

[0071] Experimental Group 5: Mobile phase A consisted of a mixture of methanol and 50 mmol / L ammonium formate aqueous solution. The prepared mixed standard solution and real plasma were mixed at volume ratios of 2:8 and 3:7, respectively. The chromatogram peaks were observed, and the results are shown in [Figure 5]. Figure 6 The volume ratio of methanol to 50 mmol / L ammonium formate aqueous solution was 3:7. The peak elution results of the mixture with the standard solution are shown in the figure. Figure 6 (a); The volume ratio of methanol and 50 mmol / L ammonium formate aqueous solution was 2:8. The peak elution results of the mixture with the standard solution are shown in the figure. Figure 6 (b); The volume ratio of methanol and 50 mmol / L ammonium formate aqueous solution was 3:7. The peak results of the mixture with real plasma are shown in the figure. Figure 6(c); The volume ratio of methanol and 50 mmol / L ammonium formate aqueous solution was 2:8. The peak results of the mixture with real plasma are shown in the figure. Figure 6 (d)

[0072] Depend on Figure 2 It can be seen that using a mixed solution of acetonitrile and 0.1% TFA aqueous solution as mobile phase A, regardless of the mixing ratio, it is impossible to effectively separate and detect the content of hippuric acid, indophenol sulfate, and indole-3-acetic acid.

[0073] Depend on Figure 3 It can be seen that using a mixed solution of acetonitrile and 50 mmol / L ammonium formate aqueous solution as mobile phase A, regardless of the mixing ratio, it is impossible to effectively separate and detect the content of hippuric acid, indophenol sulfate, and indole-3-acetic acid.

[0074] Depend on Figure 4 It can be seen that using a mixed solution of methanol and 0.1% TFA aqueous solution as mobile phase A, regardless of the mixing ratio, it is impossible to effectively separate and detect the content of hippuric acid, indophenol sulfate, and indole-3-acetic acid.

[0075] Depend on Figure 5 It can be seen that when a mixed solution of methanol and 50 mmol / L ammonium formate aqueous solution is used as mobile phase A, the effective separation and content detection of hippuric acid, indophenol sulfate, and indole-3-acetic acid can be achieved when the volume ratio of methanol to 50 mmol / L ammonium formate aqueous solution is 2:8 and 3:7, respectively. However, when the volume ratio of methanol to 50 mmol / L ammonium formate aqueous solution is 4:6, the effective separation and content detection of these three substances cannot be achieved.

[0076] Depend on Figure 6 It can be seen that when a mixed solution of methanol and 50 mmol / L ammonium formate aqueous solution is used as mobile phase A, and the volume ratio of methanol to 50 mmol / L ammonium formate aqueous solution is 2:8, the substances in the plasma will not interfere with the detection of hippuric acid, indophenol sulfate, and indole-3-acetic acid.

[0077] Example 3

[0078] This embodiment provides a method for detecting protein-bound uremic toxins, including the following steps:

[0079] (1) Plasma pretreatment: Take 0.7 ml of anticoagulated plasma into a 1.5 ml EP tube, add 0.7 ml of chromatographic methanol at a ratio of 1:1 (volume ratio), vortex for 60 s, and centrifuge at 13000 rpm / min for 5 min; take 0.7 mL of supernatant, add 0.7 ml of chromatographic acetonitrile at a ratio of 1:1 (volume ratio), vortex for 60 s, and centrifuge at 13000 rpm / min for 5 min to obtain the treated supernatant, and filter the treated supernatant through a 0.45 μm nylon 66 membrane or a 0.45 μm polytetrafluoroethylene filter membrane to obtain the sample solution to be tested.

[0080] (2) Preparation of standard solutions for the test samples: Accurately weigh 140 mg of hippuric acid standard and dissolve it in 10 mL of methanol to prepare a hippuric acid standard solution with a concentration of 14 mg / mL; accurately weigh 20 mg of indolesulfonate and dissolve it in 10 mL of methanol to prepare an indolesulfonate standard solution with a concentration of 2 mg / mL; accurately weigh 20 mg of indole-3-acetic acid standard and dissolve it in 10 mL of methanol to prepare an indole-3-acetic acid standard solution with a concentration of 2 mg / mL. Mix the hippuric acid standard solution, indolesulfonate standard solution, and indole-3-acetic acid standard solution thoroughly, and then dilute them with blank plasma to prepare a series of mixed standard solutions with different concentrations, thus obtaining multiple mixed standard solutions with different concentrations of the test samples.

[0081] (3) Setting up liquid chromatography conditions: Set up the relevant parameters according to the following chromatographic conditions, place mobile phase A, replace the corresponding column, purge the tubing, and equilibrate the column with the prepared mobile phase A for about 20 minutes. After the baseline and pressure stabilize, load the set chromatographic method file, inject the sample for analysis, and the detection conditions are as follows:

[0082] Detection wavelength: 229nm;

[0083] Chromatographic column: Inertsil ODS column, 4.6 × 250 mm, 5 μm.

[0084] Flow rate: 1.0 ml / min;

[0085] Elution method: isocratic elution;

[0086] Mobile phase A: A mixed solution of methanol and 50 mmol / L ammonium formate (HCOONH4), with a volume ratio of methanol to ammonium formate of 20:80. The ammonium formate solution is 50 mmol / L ammonium formate dissolved in water for injection, diluted to 1 L, and filtered through a 0.45 μm filter membrane.

[0087] Mobile phase B: A mixed solution of methanol and water, with a volume ratio of methanol to water of 90:10;

[0088] Injection volume: 10 μL;

[0089] Column temperature: 25 degrees Celsius (room temperature);

[0090] Reverse chromatography column.

[0091] (4) Injection and Data Processing: After injection analysis, the chromatographic data were integrated. The elution times of hippuric acid, indolesulfonate, and indole-3-acetic acid were approximately 10 min, 13 min, and 14 min, respectively. See [link to relevant documentation]. Figure 7 By plotting a standard curve between the concentration and peak area of ​​each sample, a correlation (R²) between peak area (ordinate) and concentration (radix) is established. 2 >0.999), thus the concentration of protein-bound toxoids in the sample can be calculated from the peak area of ​​the sample, and quantitative analysis can be completed.

[0092] (5) After the sample injection analysis is completed, use mobile phase B to flush the column at a flow rate of 1 ml / min for at least 30 minutes, until the UV response baseline and column pressure stabilize. After flushing, disconnect the software and instrument, and turn off the instrument power and computer.

[0093] Example 4

[0094] This embodiment provides the procedure for determining the linearity and detection limit of indophenol sulfate, indole-3-acetic acid, and hippuric acid in plasma using a method for detecting protein-bound uremic toxins. The specific steps include the following:

[0095] Hippuric acid standard solution, indolesulfonate standard solution, and indole-3-acetic acid standard solution were mixed thoroughly and then diluted with blank plasma to prepare a series of mixed standard solutions of different concentrations. The preparation was repeated three times, followed by pretreatment (i.e., protein precipitation treatment of the blank plasma before measurement). The concentration of the analyte added to the blank plasma was plotted as the x-axis, and the peak area of ​​the analyte in the plasma minus the peak area of ​​the analyte in the blank plasma was plotted as the y-axis to create a calibration curve. The detection limit was determined based on the standard deviation of the response value and the slope of the standard curve. The R0 values ​​for each analyte were calculated. 2 The standard curves, linear ranges, and detection limits for the three measurements of each analyte are shown in Table 2.

[0096] Table 2 Standard curves and detection limits for each analyte

[0097]

[0098]

[0099] As can be seen from Table 2, the linear relationships of each analyte in this embodiment are good within the corresponding linear range, and the standard curves of each analyte meet the standard requirements R. 2≥0.99 indicates that the detection method of this embodiment meets the relevant requirements according to national standards and the data is reliable. In addition, the detection limit of hippuric acid is 0.41ug / ml, the detection limit of indole-3-acetic acid is 0.2ug / ml, and the detection limit of indophenol sulfate is 1.06ug / ml, indicating that the detection limit of each analyte in the detection method of this embodiment is low and the detection sensitivity is high.

[0100] Example 5

[0101] This embodiment provides the procedure for determining the precision of indophenol sulfate, indole-3-acetic acid, and hippuric acid in plasma in a method for detecting protein-bound uremic toxins, specifically including the following steps:

[0102] Precision was assessed using real plasma samples. Hippuric acid standard solution, indophenol sulfate standard solution, and indole-3-acetic acid standard solution were mixed evenly and then diluted with blank plasma to prepare mixed standard solutions of protein-binding toxins at high, medium, and low concentrations. After sample pretreatment, each concentration was measured in triplicate. Recovery rate and coefficient of variation were performed, and the results are shown in Table 3.

[0103] The recovery rate and coefficient of variation were analyzed in accordance with GB / T27417-2017 Guidelines for Conformity Assessment of Chemical Analysis Methods. The recovery rate and coefficient of variation were calculated using the following formulas:

[0104] R = 100% × C1 / C2;

[0105] Where R is the recovery rate, C1 is the measured concentration, and C2 is the theoretical concentration.

[0106] CV = 100% × Standard Deviation / Mean

[0107] Wherein, CV is the coefficient of variation, standard deviation refers to the standard deviation of three measurements, and mean refers to the average of three measurements.

[0108] Table 3. Recovery rates and coefficients of variation for each analyte.

[0109]

[0110]

[0111] As shown in Table 3, the recovery rates of each analyte are all within the range of 93.94% to 108.72%, indicating that the detection method of this embodiment has good accuracy. The coefficients of variation of each analyte are all less than 5.28%, indicating that the detection method of this embodiment has good precision.

[0112] Example 6

[0113] This embodiment provides an application of the method for detecting protein-bound uremic toxins. Specifically, the reagent kit for detecting protein-bound uremic toxins was used in volunteers. The reagent kit was tested using the method for detecting protein-bound uremic toxins described in the above embodiment. The detection results for each substance are shown in Table 4.

[0114] Table 4. Detection results of each substance measured by the kit.

[0115] serial number hippuric acid ug / ml Indole-3-acetic acid ug / ml Indophenol sulfate ug / ml Volunteer 1 19.65 1.28 30.93 Volunteer 2 8.93 0.58 21.02 Volunteer 3 35.12 1.42 37.24 Volunteer 4 15.84 1.42 29.86 Volunteer 5 49.58 1.34 33.27 Volunteer 6 17.94 0.81 21.11 Volunteer 7 14.95 1.52 23.66 Volunteer 8 6.40 1.37 16.90

[0116] As shown in Table 4, the kit provided in this embodiment can simultaneously detect the content of hippuric acid, indole-3-acetic acid, and indolesulfonate, indicating that the kit provided in this embodiment has good operability and can be used in actual operation to detect hippuric acid, indole-3-acetic acid, and indolesulfonate in patients, and has good practical value and convenience.

[0117] While the disclosure is as stated above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of this disclosure, and all such changes and modifications will fall within the protection scope of this invention.

Claims

1. A method for detecting protein-bound uremic toxins, characterized in that, include: After mixing the blood sample and the protein precipitation extractant, centrifuge to obtain the supernatant. Mix the supernatant and the protein precipitation extractant, centrifuge to obtain the treated supernatant. Filter the treated supernatant to obtain the sample solution to be tested. Prepare standard solutions for each type of test sample separately, mix all the standard solutions of the test samples to prepare multiple mixed standard solutions with different concentrations of the test samples, wherein the test samples include hippuric acid, indophenol sulfate and indole-3-acetic acid; The mixed standard solution is detected by high performance liquid chromatography (HPLC) to plot a standard curve between the concentration and peak area of ​​each test sample. The test sample solution is detected by HPLC to obtain the peak area of ​​each test sample. Based on the peak area of ​​each test sample and its corresponding standard curve, the content of each test sample in the test sample solution is calculated. The protein precipitation extractant used for precipitating and extracting the blood sample is different from the protein precipitation extractant used for precipitating and extracting the supernatant. The protein precipitation extractant used for precipitation extraction of the blood sample is methanol, and the protein precipitation extractant used for precipitation extraction of the supernatant is acetonitrile; In high-performance liquid chromatography (HPLC) detection, the chromatographic conditions are as follows: The detection wavelength is 229nm; Octadecylsilane-bonded silica gel is used as a filler; The elution method is isocratic elution; Mobile phase A is a mixed solution of methanol and ammonium formate, and mobile phase B is a mixed solution of methanol and water. The volume ratio of methanol to ammonium formate in mobile phase A is 20:80, and the volume ratio of methanol to water in mobile phase B is 90:

10.

2. The method for detecting protein-bound uremic toxins according to claim 1, characterized in that, The volume ratio of the blood sample to the protein precipitation extractant is 1:1, and the volume ratio of the supernatant to the protein precipitation extractant is 1:

1.

3. The method for detecting protein-bound uremic toxins according to claim 1, characterized in that, The preparation of standard solutions for each type of test sample, and the mixing of all the standard solutions for the test samples to prepare multiple mixed standard solutions with different concentrations of the test samples, includes: A standard solution for each of the test samples was prepared using methanol. All the standard solutions for the test samples were mixed evenly. Then, mixed standard solutions with different concentration gradients were prepared using blank plasma to obtain multiple mixed standard solutions with different concentrations of the test samples.

4. The method for detecting protein-bound uremic toxins according to claim 1, characterized in that, Chromatographic conditions also included: an Inertsil ODS column and a column temperature of 25°C.

5. A kit for detecting protein-bound uremic toxins, characterized in that, The detection is performed using the method for detecting protein-bound uremic toxins as described in any one of claims 1 to 4.

6. The kit for detecting protein-bound uremic toxins according to claim 5, characterized in that, The kit includes a protein precipitation extractant, a mixed standard solution, mobile phase A, and mobile phase B.

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