Method for combined detection of multiple protein binding toxoid and application

Abstract

The invention provides a method for combined detection of multiple protein binding toxoid and application, the method for combined detection of multiple protein binding toxoid comprises the following steps: mixing a blood sample and a protein precipitation extraction agent, carrying out centrifugal separation, and taking the supernatant as a test solution; the method comprises the following steps: preparing mixed standard substance solutions, diluting the mixed standard substance solutions step by step to obtain a series of mixed standard substance solutions, and enabling the mixed standard substance solutions to contain hippuric acid, indole-3-acetic acid, indol sulfate and p-cresol sulfate; carrying out high performance liquid chromatography determination on the series of mixed standard substance solutions, and respectively drawing standard curves between the concentrations and chromatographic peak areas of hippuric acid, indole-3-acetic acid, indol sulfate and p-cresol sulfate; and carrying out high performance liquid chromatography determination on the test sample solution, and respectively calculating the contents of hippuric acid, indole-3-acetic acid, indol sulfate and p-cresol sulfate in the test sample through the standard curve according to the chromatographic peak area. The detection method is simple and efficient.

Description

Technical Field

[0001] The present invention relates to the technical field of protein-bound toxin detection, and in particular to a method and application for jointly detecting multiple protein-bound toxins. Background Art

[0002] Protein-bound uremic toxins are an important class of substances in uremic toxins. They are small molecules (molecular weight < 500 Da). In the human body, such substances are easily bound to human serum proteins to form medium and large molecules. Protein-bound uremic toxins are mainly divided into the following categories: (1) Phenols: such as hydroquinone and phenol; (2) Indoles: such as indole-3-acetic acid, indoxyl sulfate and melatonin; (3) Polyamines: such as tetramethylethylenediamine and spermine; (4) Advanced glycation end products: such as 3-deoxyglucosone, glyoxal and pentosidine; (5) Peptides: such as leptin and retinol binding protein; (6) Hippurates: such as hippuric acid and para-hydroxyhippuric acid. Among them, indoles and hippurates are typical representatives of protein-bound uremic toxins.

[0003] Studies have shown that the level of protein-bound uremic toxins is a valuable diagnostic indicator for chronic kidney disease (CKD) and its various complications, especially closely related to cardiovascular events, the primary cause of death in CKD patients. Therefore, monitoring and managing the content of protein-bound uremic toxins is of great significance for the diagnosis of kidney disease and its cardiovascular complications and other related diseases and for clinical optimization of patient treatment plans. In the prior art, protein-bound uremic toxins are mainly detected by enzyme-linked immunosorbent assay, chemiimmunoluminescence, ultra-high performance liquid chromatography-tandem mass spectrometry, liquid chromatography, etc. However, the existing liquid chromatography method is used for detection, and the detection type is relatively single. If it involves the need to detect multiple toxins in a blood sample, multiple injections are required for detection, and the sample preparation method and detection conditions need to be changed each time. Not only is the operation cumbersome, but the detection time is also relatively long, which greatly reduces the detection efficiency. Summary of the invention

[0004] The present invention aims to solve the problem that the existing protein-bound uremic toxin detection method cannot simultaneously detect four protein-bound uremic toxins in a blood sample, making the detection method cumbersome and reducing the detection efficiency.

[0005] To solve the above problems, the present invention provides a method for jointly detecting multiple protein-bound toxoids in a first aspect, comprising the following steps:

[0006] After mixing the blood sample and the protein precipitation extractant, centrifugation is performed, and the supernatant is taken as the test solution, wherein the protein precipitation extractant is methanol, and the volume ratio of the blood sample to the protein precipitation extractant is 1:1 to 1:3;

[0007] A mixed standard solution is prepared, and the mixed standard solution is diluted step by step to obtain a series of mixed standard solutions, wherein the mixed standard solution contains hippuric acid, indole-3-acetic acid, indoxyl sulfate and p-cresol sulfate;

[0008] The series of mixed standard solutions are subjected to high performance liquid chromatography determination, and standard curves between the concentrations of hippuric acid, indole-3-acetic acid, indoxyl sulfate and p-cresol sulfate and the chromatographic peak areas are drawn respectively;

[0009] The test solution is subjected to high performance liquid chromatography to obtain the chromatographic peak areas of hippuric acid, indole-3-acetic acid, indoxyl sulfate and p-cresol sulfate, and the contents of hippuric acid, indole-3-acetic acid, indoxyl sulfate and p-cresol sulfate in the test sample are calculated respectively through the hippuric acid standard curve, indole-3-acetic acid standard curve, indoxyl sulfate standard curve and p-cresol sulfate standard curve.

[0010] The second aspect of the present invention provides a kit for the combined detection of multiple protein-bound toxins, characterized in that the multiple protein-bound toxins are detected simultaneously using the method for the combined detection of multiple protein-bound toxins as described in the first aspect.

[0011] The method and kit for jointly detecting multiple protein-bound toxins described in the present invention, by optimizing the blood sample pretreatment method and high-efficiency chromatography conditions, establish a method that can simultaneously detect four protein-bound uremic toxins, namely, hippuric acid, indole-3-acetic acid, indoxyl sulfate and p-cresol sulfate, in a blood sample, and can perform accurate qualitative and quantitative analysis, and the method can simultaneously detect the contents of four protein-bound uremic toxins in a short time, with high detection efficiency, simple operation, high detection accuracy, and batch detection. In this embodiment, methanol is used as a protein precipitation extractant, and by setting the ratio of the protein precipitation extractant to the blood sample, effective removal of plasma proteins can be achieved, matrix effects can be reduced, incomplete protein precipitation can be avoided, the peak effect during chromatographic detection can be affected, the accuracy of the detection results can be affected, and subsequent clogging of the liquid phase system can be avoided, affecting the service life of the instrument. At the same time, acetonitrile and ammonium formate buffer are used as mobile phases during high-performance liquid chromatography detection, which can improve the peak effect of various protein-bound uremic toxins, so that each protein-bound uremic toxin has a good peak shape, which can effectively improve the detection quality and improve the detection efficiency.

[0012] The present invention performs methodological validation on the detection method provided by the present invention according to the Chinese Pharmacopoeia 2020 edition 9012 "Guiding Principles for Validation of Quantitative Analysis Methods for Biological Samples" (hereinafter referred to as "Principle 9012") and the Chinese Pharmacopoeia 2020 edition 9101 "Guiding Principles for Validation of Analytical Methods" (hereinafter referred to as "Principle 9101"). The validation contents of "Principle 9012" include selectivity, residue, linear range, lower limit of quantification, accuracy, precision, dilution reliability, matrix effect, and stability. The validation contents of "Principle 9101" include specificity. With comprehensive reference to the range requirements for accuracy and precision in "Principle 9012" and "Principle 9101", the validation results of the detection method provided by the present invention can meet the requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 is the high performance liquid chromatogram in Example 1 of the present invention;

[0014] Figure 2 is the high performance liquid chromatogram in Example 2 of the present invention;

[0015] Figure 3 is the high performance liquid chromatogram in Example 3 of the present invention;

[0016] Figure 4 is the high performance liquid chromatogram in Example 4 of the present invention;

[0017] Figure 5 It is the high performance liquid chromatogram in Example 5 of the present invention. DETAILED DESCRIPTION

[0018] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0019] In addition, the terms "comprising", "including", "containing", and "having" are not restrictive, that is, other steps and other components that do not affect the results can be added. Unless otherwise specified, materials, equipment, and reagents are commercially available.

[0020] In addition, although the present invention describes the steps in the preparation in the form of S110, S120, S130, etc., this description is only for ease of understanding, and the form of S110, S120, S130, etc. does not limit the sequence of the steps.

[0021] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0022] A first aspect of an embodiment of the present invention provides a method for jointly detecting multiple protein-bound toxins, comprising the following steps:

[0023] Step S110, after mixing the blood sample and the protein precipitation extractant, centrifuge and separate, take the supernatant as the test solution, the protein precipitation extractant is methanol, and the volume ratio of the blood sample to the protein precipitation extractant is 1:1 to 1:3.

[0024] Specifically, the blood sample and the protein precipitation extractant were mixed, vortexed for 60 seconds, and centrifuged at a speed of 13000 rpm / min for 20 minutes. The supernatant was filtered to obtain a test solution. The protein precipitation extractant was methanol, and the volume ratio of the blood sample to the protein precipitation extractant was 1:1 to 1:3.

[0025] The blood sample is plasma or serum. Serum is generally used for testing, which is more convenient.

[0026] In this embodiment, methanol is used as a protein precipitation extractant, which can remove plasma proteins in the blood sample, improve the clarity of the supernatant to reduce the interference of impurities, and enable each protein-bound toxin to have a good peak shape.

[0027] In the present embodiment, the volume ratio of the blood sample and the protein precipitation extractant is 1:1 to 1:3. Thus, the volume ratio of the blood sample and the protein precipitation extractant is within the above range, which can not only avoid the protein precipitation extractant being too low, the ability to remove plasma proteins being low, resulting in turbidity of the supernatant, clogging the chromatographic column, and affecting the accuracy of the test results, but also avoid the protein precipitation extractant being too high in proportion, diluting the content of the test sample in the supernatant, reducing the peak area corresponding to the chromatogram, and resulting in unqualified recovery of low concentrations. On the basis of the above embodiment, as a preferred embodiment, the volume ratio of the blood sample and the protein precipitation extractant is 1:2. Thus, the blood sample and the protein precipitation extractant are mixed at this volume ratio, which is conducive to improving the accuracy of the test results and making the recovery rate of low concentrations meet the standard, so as to improve the accuracy of the detection method.

[0028] In this embodiment, the supernatant is filtered through a polytetrafluoroethylene filter membrane with a pore size of 0.45 μm, which is beneficial to further improve the clarity of the supernatant.

[0029] Step S120, preparing a mixed standard solution, diluting the mixed standard solution step by step to obtain a series of mixed standard solutions, wherein the mixed standard solution contains hippuric acid, indole-3-acetic acid, indoxyl sulfate and p-cresol sulfate.

[0030] Specifically, a hippuric acid standard solution, an indole-3-acetic acid standard solution, an indoxyl sulfate standard solution and a p-cresol sulfate standard solution are prepared with methanol respectively; the indole-3-acetic acid standard solution, the indoxyl sulfate standard solution and the p-cresol sulfate standard solution are diluted with distilled water to obtain an indole-3-acetic acid standard intermediate solution, an indoxyl sulfate standard intermediate solution and a p-cresol sulfate standard intermediate solution respectively; the hippuric acid standard solution, the indole-3-acetic acid standard intermediate solution, the indoxyl sulfate standard intermediate solution and the p-cresol sulfate standard intermediate solution are mixed evenly, and then diluted step by step with blank plasma to obtain a series of mixed standard solutions.

[0031] More specifically, 14 mg of hippuric acid (HA) standard was accurately weighed, dissolved in 10 ml of methanol, and prepared into a hippuric acid standard solution with a concentration of 1.4 mg / mL; 10 mg of indoxyl sulfate (IS) standard was accurately weighed, dissolved in 10 ml of methanol, and prepared into an indoxyl sulfate standard solution with a concentration of 1 mg / mL; 10 mg of indole-3-acetic acid (IAA) standard was accurately weighed, dissolved in 10 ml of methanol, and prepared into a 1 mg / mL indole-3-acetic acid standard solution; 10 mg of p-cresol sulfate (PCS) standard was accurately weighed, dissolved in 10 ml of methanol, and prepared into a 1 mg / mL p-cresol sulfate standard solution, which was stored at 4°C for later use;

[0032] Dilute the 1 mg / mL indoxyl sulfate standard solution with distilled water to obtain a 0.4 mg / mL indoxyl sulfate standard intermediate solution; dilute the 1 mg / mL p-cresol sulfate standard solution with distilled water to obtain a 0.4 mg / mL p-cresol sulfate standard intermediate solution; dilute the 1 mg / mL indole-3-acetic acid standard solution with distilled water to obtain a 0.4 mg / mL indole-3-acetic acid standard intermediate solution I, and then dilute the 0.4 mg / mL indole-3-acetic acid standard intermediate solution I with distilled water to obtain a 0.04 mg / mL indole-3-acetic acid standard intermediate solution II, and store at 4°C for later use;

[0033] 140 μL of hippuric acid standard solution, indole-3-acetic acid standard intermediate solution II, indoxyl sulfate standard intermediate solution and p-cresol sulfate standard intermediate solution were taken respectively, diluted with 840 μL of blank plasma to prepare mixed standard solution No. 1, and then mixed standard solution No. 1 was diluted with blank plasma to obtain mixed standard solution No. 2, and then diluted step by step with blank plasma to obtain mixed standard solution No. 3, mixed standard solution No. 4, mixed standard solution No. 5 and mixed standard solution No. 6. The concentrations of hippuric acid, indole-3-acetic acid, indoxyl sulfate and p-cresol sulfate in the six mixed standard solutions are shown in Table 1.

[0034] Table 1

[0035]

[0036] It should be noted that the concentration of p-cresol sulfate is not shown in the mixed standard solution No. 6 in Table 1 because its concentration is low and its recovery rate does not meet the standard.

[0037] Step S130, performing high performance liquid chromatography on the series of mixed standard solutions, and drawing standard curves between the concentrations and chromatographic peak areas of hippuric acid, indole-3-acetic acid, indoxyl sulfate and p-cresol sulfate, respectively.

[0038] Specifically, a series of mixed standard solutions were taken for high performance liquid chromatography determination, and the peak areas of hippuric acid, indole-3-acetic acid, indoxyl sulfate and p-cresol sulfate in the series of mixed standard solutions were recorded. The concentration of each substance was used as the horizontal coordinate, and the chromatographic peak area measured at the corresponding concentration was used as the vertical coordinate to draw standard curves between the concentration and chromatographic peak area of ​​hippuric acid, indole-3-acetic acid, indoxyl sulfate and p-cresol sulfate.

[0039] In this embodiment, a DAD detector is used for high performance liquid chromatography determination, and the chromatographic conditions of the high performance liquid chromatography determination are:

[0040] The chromatographic column was an InertsilODS-3 column (particle size 5 μm, inner diameter 4.6 nm × length 250 mm);

[0041] Mobile phase A was ammonium formate buffer, and mobile phase B was acetonitrile;

[0042] Isocratic elution, the elution flow rate is 0.8 mL / min to 1.2 mL / min, preferably, the elution flow rate is 1 mL / min;

[0043] The injection volume was 20 μL;

[0044] The detection wavelengths are 265nm and 277nm, of which the detection wavelengths of HA and PCS are 265nm; the detection wavelengths of IS and IAA are 277nm;

[0045] The column temperature is 25°C to 35°C, preferably, the column temperature is 25°C.

[0046] On the basis of the above-described embodiment, as a preferred embodiment, the volume ratio of acetonitrile and ammonium formate buffer in the mobile phase is 12:88, and the concentration of ammonium formate buffer is 50mmol / L. Thus, acetonitrile and ammonium formate buffer are used as mobile phases, and the two are mixed in the above ratio, which can reduce the interference of impurities, make each protein-bound toxin have a good peak shape, and be conducive to the separation of hippuric acid, indole-3-acetic acid, indoxyl sulfate and p-cresol sulfate, thereby improving the accuracy of the test results.

[0047] On the basis of the above embodiment, as a preferred embodiment, the ammonium formate buffer solution is prepared by the following method: dissolving ammonium formate in water for injection, fixing the volume, filtering through a filter membrane, and obtaining the ammonium formate buffer solution. Specifically, 3.153 g of ammonium formate is dissolved in water for injection, fixed the volume to 1 L, and filtered through a 0.45 μm filter membrane to obtain the ammonium formate buffer solution. Thus, the ammonium formate buffer solution prepared by the above method is conducive to reducing impurities in the mobile phase, thereby further reducing the interference of impurities in the mobile phase.

[0048] In this embodiment, the peak time of hippuric acid is about 6 minutes, the peak time of indole-3-acetic acid is about 10 minutes, the peak time of indoxyl sulfate is about 20 minutes, and the peak time of p-cresol sulfate is about 35 minutes. The time intervals between the peaks of each substance are greater than 1 minute, the intervals are relatively long, and they do not interfere with each other.

[0049] Step S140, performing high performance liquid chromatography on the test solution to obtain the chromatographic peak areas of hippuric acid, indole-3-acetic acid, indoxyl sulfate and p-cresol sulfate, and calculating the contents of hippuric acid, indole-3-acetic acid, indoxyl sulfate and p-cresol sulfate in the test solution respectively through the hippuric acid standard curve, indole-3-acetic acid standard curve, indoxyl sulfate standard curve and p-cresol sulfate standard curve.

[0050] Specifically, the test solution is subjected to high performance liquid chromatography using the chromatographic conditions in step S130 to obtain the chromatographic peak areas of hippuric acid, indole-3-acetic acid, indoxyl sulfate and p-cresol sulfate, respectively. The chromatographic peak area of ​​hippuric acid is substituted into the hippuric acid standard curve to calculate the content of hippuric acid in the test solution. The chromatographic peak area of ​​indole-3-acetic acid is substituted into the indole-3-acetic acid standard curve to calculate the content of indole-3-acetic acid in the test solution. The chromatographic peak area of ​​indoxyl sulfate is substituted into the indoxyl sulfate standard curve to calculate the content of indoxyl sulfate in the test solution. The chromatographic peak area of ​​p-cresol sulfate is substituted into the p-cresol sulfate standard curve to calculate the content of p-cresol sulfate in the test solution.

[0051] The method for jointly detecting multiple protein-bound toxins provided in this embodiment is to establish a method for simultaneously detecting four protein-bound uremic toxins, namely, hippuric acid, indole-3-acetic acid, indoxyl sulfate and p-cresol sulfate, in a blood sample by optimizing the blood sample pretreatment method and high-efficiency chromatography conditions, and to perform accurate qualitative and quantitative analysis. The method is capable of simultaneously detecting the contents of four protein-bound uremic toxins in a short time, has high detection efficiency, is easy to operate, has high detection accuracy, and can perform batch detection. In this embodiment, methanol is used as a protein precipitation extractant, and by setting the ratio of the protein precipitation extractant to the blood sample, effective removal of plasma proteins can be achieved, matrix effects can be reduced, incomplete protein precipitation can be avoided, the peaking effect during chromatographic detection can be affected, the accuracy of the detection results can be affected, and subsequent clogging of the liquid phase system can be avoided, affecting the service life of the instrument. At the same time, acetonitrile and ammonium formate buffer are used as mobile phases during high-performance liquid chromatography detection, which can improve the peaking effect of various protein-bound uremic toxins, so that each protein-bound uremic toxin has a good peak shape, which can effectively improve the detection quality and improve the detection efficiency.

[0052] This example performs methodological validation on the detection method provided in this example in accordance with the Chinese Pharmacopoeia 2020 edition 9012 "Guiding Principles for Validation of Quantitative Analysis Methods for Biological Samples" (hereinafter referred to as "Principle 9012") and the Chinese Pharmacopoeia 2020 edition 9101 "Guiding Principles for Validation of Analytical Methods" (hereinafter referred to as "Principle 9101"). The validation contents of "Principle 9012" include selectivity, residue, linear range, lower limit of quantification, accuracy, precision, dilution reliability, matrix effect, and stability. The validation contents of "Principle 9101" include specificity. With comprehensive reference to the range requirements for accuracy and precision in "Principle 9012" and "Principle 9101", the validation results of the detection method provided in this example can meet the requirements.

[0053] Among them, this embodiment mainly verifies the detection method provided in this embodiment based on "Principle 9012", which is a specific document for the detection and verification of drug concentrations in targeted biological matrices (such as whole blood, serum, plasma, and urine); and "Principle 9101" has more rigorous and detailed requirements for the range of accuracy and precision. The results of the detection method provided in this embodiment are verified with comprehensive reference to the range requirements of accuracy and precision in "Principle 9012" and "Principle 9101".

[0054] The second aspect of this embodiment provides a kit for the combined detection of multiple protein-bound toxins, which uses the method for the combined detection of multiple protein-bound toxins as described in the first aspect to simultaneously detect multiple protein-bound toxins.

[0055] Specifically, the kit includes a protein precipitation extractant, a mixed standard solution and a mobile phase, wherein the specific types of the protein precipitation extractant, the mixed standard solution and the mobile phase are the same as those in the first aspect and will not be repeated here.

[0056] The kit for the combined detection of multiple protein-bound toxins provided in this embodiment can simultaneously detect the contents of four protein-bound uremic toxins, namely hippuric acid, indole-3-acetic acid, indoxyl sulfate and p-cresol sulfate, in blood samples, with high detection efficiency, simple operation and high detection accuracy.

[0057] In order to further explain the present invention in detail, the present invention will be further explained in conjunction with specific examples. The experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified; the materials, reagents, etc. used in the examples of the present invention are all purchased from the market unless otherwise specified, wherein the blank plasma in this example refers to the plasma of healthy people obtained from a blood station.

[0058] Example 1

[0059] This embodiment provides a method for jointly detecting multiple protein-bound toxins, comprising the following steps:

[0060] (1) Plasma pretreatment: 0.5 mL of plasma sample was placed in an EP tube, 1 mL of chromatographic methanol was added according to the volume ratio of plasma sample to methanol of 1:2, vortexed for 1 min, and then centrifuged at 13,000 rpm for 20 min. The supernatant was then taken and filtered through a 0.45 μm polytetrafluoroethylene filter membrane to obtain the test solution;

[0061] (2) Prepare mixed standard solution: accurately weigh 14 mg of hippuric acid standard, dissolve it in 10 ml of methanol to prepare a hippuric acid standard solution with a concentration of 1.4 mg / mL; accurately weigh 10 mg of indoxyl sulfate standard, dissolve it in 10 ml of methanol to prepare a 1 mg / mL indoxyl sulfate standard solution; accurately weigh 10 mg of indole-3-acetic acid standard, dissolve it in 10 ml of methanol to prepare a 1 mg / mL indole-3-acetic acid standard solution; accurately weigh 10 mg of p-cresol sulfate standard, dissolve it in 10 ml of methanol to prepare a 1 mg / mL p-cresol sulfate standard solution, and store it at 4°C for future use; then dilute the indoxyl sulfate standard solution and sulfuric acid with distilled water respectively. p-cresol standard solution and indole-3-acetic acid standard solution to obtain indoxyl sulfate standard intermediate solution, p-cresol sulfate standard intermediate solution, indole-3-acetic acid standard intermediate solution I and indole-3-acetic acid standard intermediate solution II, which are stored at 4°C for later use; 140 μL of hippuric acid standard solution, indole-3-acetic acid standard intermediate solution II, indoxyl sulfate standard intermediate solution and p-cresol sulfate standard intermediate solution are respectively taken and diluted with 840 μL of blank plasma to prepare mixed standard solution No. 1, which is then diluted stepwise with blank plasma to obtain mixed standard solution No. 2, mixed standard solution No. 3, mixed standard solution No. 4, mixed standard solution No. 5 and mixed standard solution No. 6 in Table 1, respectively.

[0062] (3) Setting relevant parameters according to the following HPLC conditions, taking the series of mixed standard solutions in step (2) for HPLC determination respectively, the chromatographic conditions of HPLC determination are:

[0063] DAD detector;

[0064] The chromatographic column was an Inertsil ODS-3 column (particle size 5 μm, inner diameter 4.6 nm × length 250 mm);

[0065] Mobile phase A was 50 mmol / L ammonium formate buffer, mobile phase B was acetonitrile, and the volume ratio of acetonitrile to ammonium formate buffer was 12:88;

[0066] Isocratic elution, elution flow rate is 1mL / min;

[0067] The injection volume was 20 μL;

[0068] The detection wavelengths are 265nm and 277nm, of which the detection wavelengths of HA and PCS are 265nm; the detection wavelengths of IS and IAA are 277nm;

[0069] The column temperature was 25°C.

[0070] After the sample injection analysis is completed, the chromatogram is as follows: Figure 1 As shown, the chromatographic data are integrated, and the peak time of hippuric acid is about 6 minutes, the peak time of indole-3-acetic acid is about 10 minutes, the peak time of indoxyl sulfate is about 20 minutes, and the peak time of p-cresol sulfate is about 35 minutes. By drawing the standard curve between the concentration and chromatographic peak area of ​​hippuric acid, indole-3-acetic acid, indoxyl sulfate and p-cresol sulfate, the corresponding relationship between the peak area (ordinate) and the concentration (abscissa) is established.

[0071] (4) using the chromatographic conditions in step (3) to perform high performance liquid chromatography on the test solution in step (1), respectively obtaining the chromatographic peak areas of hippuric acid, indole-3-acetic acid, indoxyl sulfate and p-cresol sulfate, substituting the chromatographic peak area of ​​hippuric acid into the hippuric acid standard curve to calculate the content of hippuric acid in the test solution, substituting the chromatographic peak area of ​​indole-3-acetic acid into the indole-3-acetic acid standard curve to calculate the content of indole-3-acetic acid in the test solution, substituting the chromatographic peak area of ​​indoxyl sulfate into the indoxyl sulfate standard curve to calculate the content of indoxyl sulfate in the test solution, substituting the chromatographic peak area of ​​p-cresol sulfate into the p-cresol sulfate standard curve to calculate the content of p-cresol sulfate in the test solution, thereby completing quantitative analysis.

[0072] Example 2

[0073] This example provides a method for determining a preferred protein precipitation extractant. The chromatographic conditions of each experimental group are the same as those in Example 1, and only the type of protein precipitation extractant is changed. Specifically:

[0074] Experimental group 1: 0.5 mL of plasma sample was added to 0.5 mL of acetonitrile, mixed, vortexed for 60 s, and centrifuged at a speed of 13000 rpm / min for 20 min. The supernatant was filtered and then filtered through a 0.45 μm filter membrane to obtain a test solution I. The test solution I was subjected to high performance liquid chromatography. The chromatogram is shown in FIG. Figure 2 (A);

[0075] Experimental group 2: The operation method is the same as that in experimental group 1, except that the protein precipitation extractant is perchloric acid, the volume ratio of plasma sample to perchloric acid is 1:1, and the test solution II is obtained. The test solution II is subjected to high performance liquid chromatography. The chromatogram is shown in Figure 2 (B);

[0076] Experimental group 3: The operation method is the same as that in experimental group 1, except that the protein precipitation extractant is methanol, and the volume ratio of plasma sample to methanol is 1:2. The test solution III is obtained and the test solution III is subjected to high performance liquid chromatography. The chromatogram is shown in Figure 2 (C).

[0077] Depend on Figure 2 It can be seen that the use of different protein precipitation extractants and different volume ratios of blood samples and protein precipitation extractants have an impact on the chromatographic response value. Although different protein precipitation extractants and different volume ratios of blood samples and protein precipitation extractants were used in experimental groups 1 to 3, the plasma proteins in the blood samples were removed to obtain clear supernatants. However, when experimental groups 1 to 3 were subsequently tested by high performance liquid chromatography, the peak effects of the tests were different. Among them, methanol in experimental group 3 was used as the protein precipitation extractant, and the volume ratio of blood samples to methanol was 1:2. The peak effect of high performance liquid chromatography detection of the test solution was the best, and the peaks of each substance were sharp and symmetrical, which could make each protein-bound uremic toxin have a good peak shape, indicating that the use of the protein precipitation extractant in experimental group 3 is conducive to further reducing the interference of impurities and improving the accuracy of detection.

[0078] Example 3

[0079] This example provides a method for determining a preferred mobile phase. The chromatographic conditions of each experimental group are the same as those in Example 1, and only the mobile phase is changed. Specifically:

[0080] Experimental Group 1: When conducting HPLC determination, trifluoroacetic acid was used as the mobile phase. The chromatogram is shown in Figure 3 (A);

[0081] Experimental Group 2: When conducting HPLC determination, a mixture of formic acid and ammonia was used as the mobile phase. The chromatogram is shown in Figure 3 (B), wherein the mixture of formic acid and aqueous ammonia is prepared by dissolving 2.5 mL of formic acid in 1000 mL of distilled water and then adding aqueous ammonia to adjust the pH to 4.0;

[0082] Experimental group 3: When conducting HPLC determination, acetonitrile and ammonium formate buffer were used as mobile phases. The chromatogram is shown in Figure 1 , wherein the volume ratio of acetonitrile to ammonium formate buffer is 12:88.

[0083] Depend on Figure 1 and 3 It can be seen that when acetonitrile and ammonium formate buffer are used as mobile phases, the peak elution effect is the best during HPLC detection, with fewer interfering peaks, and various protein-bound uremic toxins can be effectively separated. The peaks of each substance are sharp and symmetrical, and each protein-bound uremic toxin has a good peak shape, indicating that using acetonitrile and ammonium formate buffer as mobile phases is beneficial to reducing the interference of impurities and improving the accuracy of detection.

[0084] Example 4

[0085] This example provides a method for determining a preferred mobile phase. The chromatographic conditions of each experimental group are the same as those in Example 1, and only the mobile phase is changed. Specifically:

[0086] Experimental group 1: In the HPLC determination, an Inertsil ODS-3 column (particle size 5 μm, inner diameter 4.6 nm × length 250 mm) was used as the chromatographic column, acetonitrile and 50 mmol / L ammonium formate buffer were used as the mobile phase, and the volume ratio of acetonitrile to ammonium formate buffer was 20:80. The chromatogram is shown in Figure 4 (A);

[0087] Experimental group 2: In the HPLC determination, an Inertsil ODS-3 column (particle size 5 μm, inner diameter 4.6 nm × length 250 mm) was used as the chromatographic column, acetonitrile and 50 mmol / L ammonium formate buffer were used as the mobile phase, and the volume ratio of acetonitrile to ammonium formate buffer was 15:85. The chromatogram is shown in Figure 4 (B);

[0088] Experimental group 3: In the HPLC determination, an Inertsil ODS-3 column (particle size 5 μm, inner diameter 4.6 nm × length 250 mm) was used as the chromatographic column, acetonitrile and 50 mmol / L ammonium formate buffer were used as the mobile phase, and the volume ratio of acetonitrile to ammonium formate buffer was 12:88. The chromatogram is shown in Figure 1 .

[0089] Depend on Figure 1 and Figure 4 It can be seen that the volume ratio of acetonitrile and ammonium formate buffer will affect the peak time of hippuric acid, and the earlier the peak time of hippuric acid is, the greater the impact of impurity peaks in the blood sample will be. When the volume ratio of acetonitrile and ammonium formate buffer is 12:88, the peak time of hippuric acid is 6 minutes, which is not only less affected by impurity peaks but also helps to improve detection efficiency.

[0090] Example 5

[0091] This example provides a method for determining a preferred chromatographic column. The chromatographic conditions of each experimental group are the same as those in Example 1, and only the chromatographic column is changed. Specifically:

[0092] Experimental Group 1: When conducting HPLC determination, UltimateXB-C18 column (particle size 5 μm, inner diameter 4.6 nm × length 250 mm) was used as the chromatographic column. The chromatogram is shown in Figure 5 ;

[0093] Experimental Group 2: In the HPLC determination, an Inertsil ODS-3 column (particle size 5 μm, inner diameter 4.6 nm × length 250 mm) was used as the chromatographic column. The chromatogram is shown in Figure 1 .

[0094] Depend on Figure 1 and Figure 5 It can be seen that when the Ultimate XB-C18 column is used for HPLC determination, the chromatographic peaks of hippuric acid and impurities are mixed together and difficult to separate, which affects the accuracy of the detection. However, when the Inertsil ODS-3 column is used for HPLC determination, each substance can be effectively separated from the impurities, and each protein-bound uremic toxin has a good peak shape, and the elution time of each protein-bound uremic toxin is appropriate, which is conducive to improving the accuracy and efficiency of the detection.

[0095] Example 6

[0096] This example provides a method for determining a better elution mode. The chromatographic conditions of each experimental group are the same as those in Example 1, and only the elution mode is changed. Specifically:

[0097] Experimental group 1: When performing high performance liquid chromatography determination, an Inertsil ODS-3 column (particle size 5 μm, inner diameter 4.6 nm × length 250 mm) was used as the chromatographic column, 50 mmol / L ammonium formate buffer was used as the mobile phase A, acetonitrile was used as the mobile phase B, and gradient elution was performed as follows:

[0098] 0-10min, mobile phase A:mobile phase B is 88:12;

[0099] 10-15 min, mobile phase A:mobile phase B changed from 88:12 to 80:20;

[0100] 15-20 min, mobile phase A:mobile phase B changed from 80:20 to 88:12;

[0101] From 20 to 28 minutes, the ratio of mobile phase A to mobile phase B was maintained at 88:12.

[0102] Experimental Group 2: When performing HPLC determination, an Inertsil ODS-3 column (particle size 5 μm, inner diameter 4.6 nm × length 250 mm) was used as the chromatographic column, 50 mmol / L ammonium formate buffer was used as the mobile phase A, acetonitrile was used as the mobile phase B, the volume ratio of acetonitrile to ammonium formate buffer was 12:88, and isocratic elution was adopted.

[0103] The same mixed standard solution was injected multiple times for high performance liquid chromatography determination to detect the peak time of p-cresol sulfate. The results are shown in Table 2.

[0104] Table 2

[0105]

[0106] It can be seen from Table 2 that when performing HPLC determination, different injection modes have different effects on the peak time of the same protein-bound uremic toxin. The HPLC determination of p-cresol sulfate by gradient elution can shorten the single sample running time, but the time interval between the two peaks of injection sequence 1 and injection sequence 10 is greater than 1 min, and the peak time fluctuates greatly. The HPLC determination of p-cresol sulfate by isocratic elution increases the single sample running time, but the time interval between the two peaks of injection sequence 1 and injection sequence 10 is less than 0.1 min, and the peak time fluctuates less. This shows that the use of isocratic elution in this embodiment is beneficial to improving the stability of the peak time of protein-bound uremic toxins.

[0107] Example 7

[0108] This example provides an operation method for determining the linearity and detection limit of hippuric acid, indole-3-acetic acid, indoxyl sulfate and p-cresol sulfate in a method for joint detection of multiple protein-bound toxins, which specifically comprises the following steps:

[0109] According to Table 1, a series of hippuric acid standard solutions with mass concentrations of 140 μg / ml, 70 μg / ml, 35 μg / ml, 17.5 μg / ml, 8.75 μg / ml, and 4.375 μg / ml were prepared using blank plasma;

[0110] A series of indole-3-acetic acid standard solutions with mass concentrations of 4 μg / ml, 2 μg / ml, 1 μg / ml, 0.5 μg / ml, 0.25 μg / ml, and 0.125 μg / ml were prepared using blank plasma;

[0111] Use blank plasma to prepare indoxyl sulfate standard solutions with mass concentrations of 40μg / ml, 20μg / ml, 10μg / ml, 5μg / ml, 2.5μg / ml, and 1.25μg / ml respectively;

[0112] A series of p-cresol sulfate standard solutions with mass concentrations of 40μg / ml, 20μg / ml, 10μg / ml, 5μg / ml and 2.5μg / ml were prepared using blank plasma.

[0113] The preparation was repeated three times, and each protein-bound uremic toxin standard solution was subjected to HPLC detection. The chromatographic conditions were the same as those in Example 1. The peak area of ​​each protein-bound uremic toxin standard solution was used as the ordinate (y), and the concentration of each protein-bound uremic toxin standard solution was used as the abscissa (x). A calibration curve was drawn, and the detection limit was determined based on the standard deviation of the response value and the slope of the standard curve. The results of the standard curve, linear range and detection limit of the three measurements of each protein-bound uremic toxin are shown in Table 3.

[0114] Table 3

[0115]

[0116] As can be seen from Table 3, the linear relationship of each protein-bound uremic toxin in this embodiment is good within the corresponding linear range, and the standard curve of each protein-bound uremic toxin meets the standard requirements. 2 ≥0.99, indicating that the detection method of this embodiment is verified to meet the relevant requirements according to national standards, and the data is reliable; in addition, the detection limit of hippuric acid is 0.411μg / ml, the detection limit of indole-3-acetic acid is 0.042μg / ml, the detection limit of indoxyl sulfate is 0.07μg / ml, and the detection limit of p-cresol sulfate is 0.1μg / ml, indicating that the detection method provided in this embodiment has high detection sensitivity of each protein-bound uremic toxin, which is beneficial to improve the accuracy of detection.

[0117] Example 8

[0118] This example provides a system suitability test for determining a method for combined detection of multiple protein-bound toxins, comprising the following steps:

[0119] A standard solution of hippuric acid with a mass concentration of 4.375 μg / ml, a standard solution of indole-3-acetic acid with a mass concentration of 0.125 μg / ml, a standard solution of indole sulfate with a mass concentration of 1.25 μg / ml, and a standard solution of p-cresol sulfate with a mass concentration of 2.5 μg / ml were prepared with blank plasma, respectively, as system suitability test solutions, and high performance liquid chromatography was performed according to the chromatographic conditions in Example 1, and the chromatogram was recorded, and the peak order was: hippuric acid, indole-3-acetic acid, indole sulfate, and p-cresol sulfate. Under the chromatographic conditions, each protein-bound uremic toxin can be effectively separated, with less interference, and the separation degree of each protein-bound uremic toxin is shown in Table 4.

[0120] Table 4

[0121] Protein-bound uremic toxins Separation Hippuric acid 2.24 Indole-3-acetic acid 5.76 Indoxyl sulfate 17.28 p-Cresol Sulfate 10.05

[0122] It can be seen from Table 4 that the separation degree of each protein-bound uremic toxin is greater than 1.5, and there is no interfering substance near each protein-bound uremic toxin, and its specificity meets the requirements of "Chinese Pharmacopoeia 9101".

[0123] Example 9

[0124] This example provides a method for determining the accuracy and precision of a combined detection of multiple protein-bound toxoids, comprising the following steps:

[0125] Use blank plasma to prepare hippuric acid standard solutions with mass concentrations of 70 μg / ml, 35 μg / ml, and 8.75 μg / ml, respectively, as high, medium, and low concentration hippuric acid standard solutions;

[0126] Blank plasma was used to prepare indole-3-acetic acid standard solutions with mass concentrations of 2 μg / ml, 1 μg / ml, and 0.5 μg / ml, respectively, as high, medium, and low concentration indole-3-acetic acid standard solutions;

[0127] Blank plasma was used to prepare indoxyl sulfate standard solutions with mass concentrations of 20 μg / ml, 10 μg / ml, and 5 μg / ml, respectively, as high, medium, and low concentrations of indoxyl sulfate standard solutions;

[0128] Blank plasma was used to prepare p-cresol sulfate standard solutions with mass concentrations of 20 μg / ml, 10 μg / ml and 2.5 μg / ml, which were used as high, medium and low concentrations of p-cresol sulfate standard solutions, respectively.

[0129] After the sample pretreatment, each standard solution was measured 5 times in parallel at each concentration, and 3 batches were measured at each concentration. The recovery rate and relative standard deviation (RSD) of the detection method of Example 1 were calculated, and the results are shown in Tables 5 to 8.

[0130] Table 5

[0131]

[0132] Table 6

[0133]

[0134]

[0135] Table 7

[0136]

[0137] Table 8

[0138]

[0139] According to the range requirements of "Principle 9012" (recovery rate: ±15%, lower limit of quantification ±20%; coefficient of variation: ±15%; lower limit of quantification ±20%) and "Principle 9101" (recovery rate: 100ppm 85%~110%, 10ppm80%~115%, 1ppm 75%~120%; coefficient of variation: 100ppm 4%, 10ppm 6%, 1ppm 8%).

[0140] It can be seen from Table 5 that the recovery rate and RSD of hippuric acid are within the above range, indicating that the accuracy and precision of the detection method of this embodiment meet the requirements of 9012 and 9101 of Part IV of the Chinese Pharmacopoeia 2020 Edition.

[0141] It can be seen from Table 6 that the recovery rate and RSD of indole-3-acetic acid are within the above ranges, indicating that the accuracy and precision of the detection method of this embodiment meet the requirements of 9012 and 9101 of Part IV of the Chinese Pharmacopoeia 2020 Edition.

[0142] It can be seen from Table 7 that the recovery rate and RSD of indoxyl sulfate are within the above ranges, indicating that the accuracy and precision of the detection method of this embodiment meet the requirements of 9012 and 9101 of Part IV of the Chinese Pharmacopoeia 2020 Edition.

[0143] It can be seen from Table 8 that the recovery rate and RSD of p-cresol sulfate are within the above range, indicating that the accuracy and precision of the detection method of this embodiment meet the requirements of 9012 and 9101 of Part IV of the Chinese Pharmacopoeia 2020 Edition.

[0144] The test results in Tables 5 to 8 illustrate that the detection method provided in this embodiment has good accuracy and precision, and the test results are accurate and reliable.

[0145] Although the disclosure is disclosed as above, the protection scope of the disclosure is not limited thereto. Those skilled in the art may make various changes and modifications without departing from the spirit and scope of the disclosure, and these changes and modifications will fall within the protection scope of the present invention.

Claims

1. A method for the combined detection of multiple protein-bound toxoids, characterized in that: The steps include: After mixing the blood sample and the protein precipitation extractant, centrifugation is performed, and the supernatant is taken as the test solution, wherein the protein precipitation extractant is methanol, and the volume ratio of the blood sample to the protein precipitation extractant is 1:1 to 1:3; A mixed standard solution is prepared, and the mixed standard solution is diluted step by step to obtain a series of mixed standard solutions, wherein the mixed standard solution contains hippuric acid, indole-3-acetic acid, indoxyl sulfate and p-cresol sulfate; The series of mixed standard solutions are subjected to high performance liquid chromatography determination, and standard curves between the concentrations of hippuric acid, indole-3-acetic acid, indoxyl sulfate and p-cresol sulfate and the chromatographic peak areas are drawn respectively; The test solution is subjected to high performance liquid chromatography to obtain the chromatographic peak areas of hippuric acid, indole-3-acetic acid, indoxyl sulfate and p-cresol sulfate, and the contents of hippuric acid, indole-3-acetic acid, indoxyl sulfate and p-cresol sulfate in the test sample are calculated respectively through the hippuric acid standard curve, indole-3-acetic acid standard curve, indoxyl sulfate standard curve and p-cresol sulfate standard curve.

2. The method for joint detection of multiple protein-bound toxoids according to claim 1, characterized in that: The volume ratio of the blood sample to the protein precipitation extractant is 1:

2.

3. The method for joint detection of multiple protein-bound toxoids according to claim 1, characterized in that: The mixed standard solution is prepared, and the mixed standard solution is diluted step by step to obtain a series of mixed standard solutions, including: Use methanol to prepare hippuric acid standard solution, indole-3-acetic acid standard solution, indoxyl sulfate standard solution and p-cresol sulfate standard solution respectively; After diluting the indole-3-acetic acid standard solution, the indoxyl sulfate standard solution and the p-cresol sulfate standard solution with distilled water, an indole-3-acetic acid standard intermediate solution, an indoxyl sulfate standard intermediate solution and a p-cresol sulfate standard intermediate solution are obtained respectively; The hippuric acid standard solution, indole-3-acetic acid standard intermediate solution, indoxyl sulfate standard intermediate solution and p-cresol sulfate standard intermediate solution were mixed evenly, and then diluted stepwise with blank plasma to obtain a series of mixed standard solutions.

4. The method for joint detection of multiple protein-bound toxoids according to claim 1, characterized in that: The concentration of hippuric acid in the series of mixed standard solutions is 4.375 μg / mL to 140 μg / mL, the concentration of indole-3-acetic acid is 0.125 μg / mL to 4 μg / mL, the concentration of indole sulfate is 1.25 μg / mL to 40 μg / mL, and the concentration of p-cresol sulfate is 2.5 μg / mL to 40 μg / mL.

5. The method for joint detection of multiple protein-bound toxoids according to claim 1, characterized in that: In the HPLC determination, the chromatographic conditions are: The chromatographic column is an Inertsil ODS-3 column; Mobile phase A was ammonium formate buffer, and mobile phase B was acetonitrile; Isocratic elution, elution flow rate 0.8mL / min to 1.2mL / min; The injection volume was 20 μL; The detection wavelengths are 265nm and 277nm; The column temperature was 25°C to 35°C.

6. The method for joint detection of multiple protein-bound toxoids according to claim 5, characterized in that: The volume ratio of acetonitrile to ammonium formate buffer in the mobile phase is 12:88, and the concentration of the ammonium formate buffer is 50 mmol / L.

7. The method for joint detection of multiple protein-bound toxoids according to claim 5, characterized in that: The ammonium formate buffer solution is prepared by the following method: Dissolve ammonium formate in water for injection, make up to volume, and filter through a 0.45 μm filter membrane to obtain ammonium formate buffer solution.

8. The method for joint detection of multiple protein-bound toxoids according to claim 1, characterized in that: The blood sample is plasma or serum.

9. A kit for the combined detection of multiple protein-bound toxins, characterized in that: The method for jointly detecting multiple protein-bound toxins as described in any one of claims 1 to 8 is used to simultaneously detect multiple protein-bound toxins.

10. The kit for joint detection of multiple protein-bound toxoids according to claim 9, characterized in that: The kit comprises a protein precipitation extractant, a mixed standard solution and a mobile phase.

Images