Method for synchronously and quantitatively detecting urotoxin p-cresol sulfate and indoxyl sulfate based on LC-MS / MS (liquid chromatography-tandem mass spectrometry) of alternative analyte method and application

Abstract

The invention belongs to the technical field of biomarker detection, and particularly relates to a method for synchronously and quantitatively detecting urotoxin p-cresol sulfate and indoxyl sulfate based on LC-MS / MS of an alternative analyte method and application. According to the method, stable isotope labeled pCS-d4 and INDS-d4 are creatively adopted as alternative analytes, pCS-d7 and INDS-13C6 are adopted as internal labels, a protein binding state target object is released through serum protein precipitation, a free state target object is obtained through an ultrafiltration method, a universal reversed-phase C18 chromatographic column is adopted for gradient elution and separation, and the detection result is accurate. An organic solvent containing ammonium acetate, formic acid aqueous solution-methanol and the like is used as a mobile phase, and a dual specific detection system for the total content and the free state content of the target object in the serum is established through a multi-reaction monitoring (MRM) mode of LC-MS / MS. The problem of accurate quantification of the nephropathy biomarker in a biological sample under the condition of no blank matrix is solved in a breakthrough manner, and key data support is provided for evaluating pathological toxicity risk of urotoxin.

Description

Technical Field

[0001] This invention belongs to the field of biomarker detection technology, specifically relating to a method and application of simultaneous quantitative detection of uremic toxin p-cresol sulfate and indophenol sulfate by LC-MS / MS based on the alternative analyte method. Background Technology

[0002] Uremic toxins (UTs) are substances that accumulate in the bodies of patients with end-stage renal failure and can adversely interact with the body's biological functions. Protein-bound uremic toxins (PBUTs) exist in the bloodstream in both free solute and protein-bound forms. During hemodialysis in uremic patients, protein-bound uremic toxins are almost impossible to remove. The accumulation of PBUTs in the body is clinically highly correlated with renal, cardiovascular, and skeletal toxicity.

[0003] Indophenol sulfate (INDS) and p-cresol sulfate (pCS) are sensitive biomarkers for renal function and cardiovascular events. Accurate detection of different forms of INDS and pCS in the blood is crucial for clinical diagnosis and treatment, and they should be continuously monitored to protect patients from disease progression and its adverse consequences. In recent decades, there have been reports on the quantification of INDS and pCS using LC-UV, LC-FL (ultraviolet-fluorescence), LC-MS / MS, and LC-QTOF-MS (quadrupole time-of-flight mass spectrometry). However, due to the lack of blank matrices for preparing calibration and quality control (QS) samples, accurate quantitative analysis of INDS and pCS in biological matrices is not possible. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a method and application for the simultaneous quantitative detection of uremic toxin p-cresol sulfate and indophenol sulfate using LC-MS / MS based on the surrogate analyte method. This invention uses stable isotopes as surrogate analytes and internal standards, and uses a real matrix to quantify total INDS and pCS in serum. This overcomes the difficulty of analyzing endogenous substances without a blank matrix and eliminates the matrix effect in biological sample analysis, thus making the quantitative analysis of pCS and INDS accurate and reliable, providing valuable data support for clinical practice.

[0005] To achieve the objectives of this invention, the following technical solution is adopted: The first aspect of the present invention provides a method for simultaneous quantitative detection of uremic toxin p-cresol sulfate and indophenol sulfate by LC-MS / MS based on the alternative analyte method, comprising the following steps: (1) Establish standard curves for biomatrix alternative analytes pCS-d4 and INDS-d4; (2) Mix the sample to be tested with methanol solution, then mix with acetonitrile solution containing internal standard, centrifuge, take the supernatant and mix with distilled water to obtain sample solution; (3) Take the sample solution for LC-MS / MS detection, and calculate the concentration of p-cresol sulfate and indophenol sulfate in the sample based on the detection results and the standard curve; The internal standards are pCS-d7 and INDS- 13 C6.

[0006] As endogenous analytes, the quantification of INDS and pCS is more complex from both analytical and validation perspectives. Due to the lack of truly blank matrices with sufficiently low analyte concentrations, standard addition methods often struggle to prepare calibration or QC samples because it is difficult to obtain blank biological matrix samples free of the analyte or samples with accurately known analyte concentrations. Using alternative matrices and real analyte quantification methods often leads to matrix effects, which can be either ion suppression or ion enhancement. The accurate concentrations of INDS and pCS are directly affected by matrix effects, especially when matrix effects cause differences in mass spectrometric behavior between the analyte and the internal standard (IS). Therefore, accurate quantification of INDS and pCS in biological matrices remains a significant challenge for technical personnel.

[0007] In the above method, this invention creatively employs stable isotope-labeled p-cresol sulfate-d4 and indophenol sulfate-d4 as alternative analytes, combined with simple sample pretreatment and optimized chromatographic and mass spectrometric conditions, to establish a dual quantitative detection system. This breakthrough solves the challenge of quantifying nephropathy biomarkers in biological samples without a blank matrix, achieving a detection sensitivity of 0.01 μM for free targets, a spiked recovery rate of 77.3%-104.3%, and intra- and inter-batch precision (RSD < 15%). It can accurately quantify the concentrations of two forms of toxins in the serum of CKD patients, providing crucial data support for assessing the pathological toxicity risk of uremic toxins.

[0008] In some specific embodiments of the present invention, step (1), establishing the biomatrix standard curves for the alternative analytes pCS-d4 and INDS-d4, includes: a. Preparation of working solutions for a series of concentration standard curves: Take pCS-d4 and INDS-d4 standard substances, dissolve and dilute them in 30% methanol aqueous solution to obtain a series of concentrations; take the solutions of the series of concentrations and add them to the biological matrix and internal standard solution, mix well, precipitate, take the supernatant, and mix it with distilled water at a volume ratio of 1:2 to obtain the solution. b. Inject the working solutions of the series of concentration standard curves into the LC-MS / MS instrument, and determine the surrogate analytes pCS-d4 and INDS-d4, and the internal standards pCS-d7 and INDS-d4. 13The C6 peak area ratio was used to obtain the standard curves for pCS-d4 and INDS-d4.

[0009] The preparation of the internal standard solution includes: taking pCS-d7 and INDS- 13 C6 standard substance, diluted with acetonitrile, yields the internal standard solution.

[0010] In some embodiments of the present invention, the biological matrix includes serum and serum filtrate.

[0011] Serum was used to quantitatively analyze the total pCS and INDS content in serum, including free uremic toxins and protein-bound uremic toxins; serum filtration solution was used to quantitatively analyze free pCS and INDS in serum.

[0012] Further, the method for preparing the serum filtrate is as follows: add one serum sample to a Millipore Amicon Ultra-4 Centrifugal Filter 10 kDa MWCO ultrafiltration centrifuge tube, centrifuge at 7500×g, and the serum filtrate sample is obtained.

[0013] In some embodiments of the present invention, the volume ratio of the series of concentration solutions, biological matrix, and acetonitrile solution containing internal standard in step a is 5:50:100; the volume ratio of the supernatant obtained by centrifugation to distilled water is 1:2; the concentrations of pCS-d4 and INDS-d4 in the serum of the series of concentration solutions are 0.025~50 μM, and the concentrations in the serum filtrate are 0.025~50 μM.

[0014] In some embodiments of the present invention, in step (2), the volume ratio of the sample to be tested, the methanol solution and the acetonitrile solution containing the internal standard is (5~15):1:(10~30), and the methanol solution is a 30% methanol aqueous solution.

[0015] In some embodiments of the present invention, in step (2), the concentration of the internal standard in the acetonitrile solution containing the internal standard is 0.5~1.5 μM.

[0016] In some embodiments of the present invention, in step (2), the sample to be tested is mixed with the methanol solution under a vortex for a duration of 20-40 seconds, and then placed at room temperature for 1-3 minutes after vortexing.

[0017] In some embodiments of the present invention, in step (2), the mixing with the acetonitrile solution containing the internal standard is carried out under a vortex for a period of 1 to 3 minutes.

[0018] The volume ratio of the test sample, methanol solution, and acetonitrile solution containing the internal standard can be 10:1:10, 10:1:11, 10:1:12, 10:1:13, 10:1:14, 10:1:15, 10:1:16, 10:1:17, 10:1:18, 10:1:19, 10:1:20, or 10:1:30, etc. The concentration of the internal standard in the acetonitrile solution can be 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0 μM, 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, or 1.5 μM. μM, etc., the vortexing time for mixing the sample to be tested with the methanol solution in step (2) can be 20s, 21s, 22s, 23s, 24s, 25s, 26s, 27s, 28s, 29s, 30s, 31s, 32s, 33s, 34s, 35s, 36s, 37s, 38s, 39s or 40s, etc., and the time for placing at room temperature after vortexing can be 1min, 2min or 3min, etc., and the vortexing time for mixing with the acetonitrile solution containing the internal standard can be 1min, 2min or 3min, etc., but is not limited to the values ​​listed above. Other unlisted values ​​within the above range are also applicable.

[0019] Furthermore, in step (3) during the concentration determination process, each batch of samples includes a certain amount of quality control samples. The specific steps are as follows: following the method in step a, prepare quality control serum and serum filtrate samples with low, medium, and high concentrations of surrogate analytes, take the supernatant and inject it into the LC-MS / MS instrument, and monitor the accuracy of the determination process for each batch of samples. The number of quality control samples is approximately 6% of the number of samples in each batch, and the low, medium, and high concentrations are evenly distributed in each batch of samples.

[0020] In some embodiments of the present invention, in step (3), the liquid phase conditions of the LC-MS / MS include: an injection volume of 1~10 μL; a Thermo Hypersil Gold™ C18 column, 2.1×150 mm, 5 μm; a column temperature of 35~45℃; a mobile phase consisting of mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution of formic acid-ammonium acetate and mobile phase B is a methanol-isopropanol solution; and a flow rate of 0.40~0.55 mL / min.

[0021] In some embodiments of the present invention, the mobile phase A is a 5 mM ammonium acetate solution containing 0.1% formic acid, and the mobile phase B is 90% methanol-10% isopropanol.

[0022] The injection volume can be 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, or 10 μL, etc.; the column temperature can be 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, 41℃, 42℃, 43℃, 44℃, or 45℃, etc.; and the flow rate can be 0.40 mL / min, 0.41 mL / min, 0.42 mL / min, 0.43 mL / min, 0.44 mL / min, 0.45 mL / min, 0.46 mL / min, 0.47 mL / min, 0.48 mL / min, 0.49 mL / min, 0.50 mL / min, 0.51 mL / min, 0.52 mL / min, 0.53 mL / min, 0.54 mL / min, or 0.55 mL / min. mL / min, etc., but not limited to the values ​​listed above. Other unlisted values ​​within the above range are also applicable.

[0023] In some embodiments of the present invention, in the LC-MS / MS of step (3), the elution method of the liquid phase is gradient elution.

[0024] In some embodiments of the present invention, the mass spectrometry conditions of the LC-MS / MS include: Ion source: electrospray ionization source; mass spectrometry detection mode: multi-ion reaction monitoring negative ion mode; scan time: 9 min; ion source voltage: -3.0 kV; ion source temperature: 550°C; curtain gas, nebulizer gas, and auxiliary device: 25 psi, 65 psi, and 75 psi, respectively; declustering voltage: -45 V.

[0025] In some embodiments of the present invention, the sample to be tested is serum or serum filtrate.

[0026] A second aspect of the invention provides the application of the method for simultaneous quantitative detection of uremic toxin p-cresol sulfate and indophenol sulfate by LC-MS / MS based on the alternative analyte method in detecting the content of p-cresol sulfate and indophenol sulfate in kidney, cardiovascular and bone toxicity-related diseases.

[0027] Compared with the prior art, the present invention has the following beneficial effects: (1) In view of the defects in the existing technology of endogenous uremic toxin detection, such as severe matrix interference and the inability to obtain blank matrix, which makes it impossible to obtain the absolute content of analytes in biological matrix, this invention innovatively uses stable isotope-labeled p-cresol sulfate-d4 and indophenol sulfate-d4 as alternative analytes, and establishes a dual quantitative detection system by combining simple sample pretreatment and optimized chromatographic and mass spectrometric conditions.

[0028] (2) This invention has made a breakthrough in solving the problem of quantifying nephropathy biomarkers in biological samples without blank matrix, and has made the detection sensitivity of free target 0.01 μM, spiked recovery rate 77.3%~104.3%, intra-assay and inter-assay precision RSD<15%, which can accurately quantify the concentration of two forms of toxins in the serum of CKD patients, and provide key data support for assessing the pathological toxicity risk of uremic toxins. Attached Figure Description

[0029] Figure 1 Here are typical mass chromatograms of human serum with MRM as determined in this embodiment of the invention; (A) blank serum; (B) blank serum with added surrogate analytes (INDS-d4 and pCS-d4, LLOQ: 0.025 μM) and internal standard (INDS-). 13 (C) C6 and pCS-d7, 1 μM); (C) Human serum sample-4 with internal standard (INDS-) 13 C6 and pCS-d7, 1 μM).

[0030] Figure 2 Here are typical mass chromatograms of MRM human serum filtrate determined in embodiments of the present invention; (A) blank serum filtrate; (B) blank serum filtrate with added surrogate analytes (INDS-d4 and pCS-d4, 0.025 μM) and internal standard (INDS-). 13 C6 and pCS-d7, 1 μM); (C) Human serum filtrate sample-4 with internal standard (INDS-) 13 C6 and pCS-d7, 1 μM). Detailed Implementation

[0031] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0032] Example 1: Establishment of a method for determining pCS and INDS levels in serum (I) Solution Preparation Standard stock solution: Weigh appropriate amounts of pCS, pCS-d4, INDS, and INDS-d4 standard substances into polypropylene tubes, and add 30% methanol aqueous solution to prepare a standard stock solution with a concentration of 10 mM. Internal standard solution / precipitant: Weigh pCS-d7, INDS- 13 An appropriate amount of C6 standard material was placed in a polypropylene tube and prepared into a 4 mM standard stock solution with methanol. An appropriate amount of pCS-d7 and INDS-13C6 internal standard stock solutions were prepared into a solution containing 1 μM internal standard with acetonitrile, which was also used as a precipitant for sample processing.

[0033] (II) Establishment of serum standard curve a. Preparation of working solutions for a series of serum standard curves: Take pCS-d4 and INDS-d4 standard stock solutions and dilute them with 30% methanol aqueous solution to prepare a series of mixed concentration standard curve solutions with concentrations of 0.25 μM, 1 μM, 2.5 μM, 5 μM, 25 μM, 50 μM, 100 μM, 250 μM and 500 μM. Take 5 μL of each concentration solution, add 50 μL of blank matrix human serum and mix well, then add 100 μL of internal standard solution, vortex mix for 2 min, centrifuge at 14000 rmp for 10 min at 4℃, take 50 μL of supernatant, add 100 μL of distilled water and mix well to obtain serum standard curve working solutions with concentrations of 0.025 μM, 0.1 μM, 0.25 μM, 0.5 μM, 2.5 μM, 5 μM, 10 μM, 25 μM and 50 μM. b. Preparation of quality control working solution: Take pCS-d4 and INDS-d4 standard stock solutions and dilute them with 30% methanol aqueous solution to prepare a series of mixed quality control solutions with concentrations of 0.5 μM (low, QL), 20 μM (medium QM), and 40 μM (high QH). Take 5 μL of each quality control solution, add 50 μL of blank matrix human serum and mix well, then add 100 μL of internal standard solution, vortex mix for 2 min, centrifuge at 14000 rmp for 10 min at 4℃, take 50 μL of supernatant, add 100 μL of distilled water and mix well to obtain serum quality control working solutions with concentrations of 0.05 μM, 2.0 μM, and 40 μM. c. Inject working solutions of serum standard curves at various concentrations into the LC-MS / MS instrument to determine the surrogate analytes pCS-d4 and INDS-d4, and the internal standards pCS-d7 and INDS-d4. 13 The peak area of ​​C6 was plotted with the concentration of the working solution of the series of serum standard curves on the x-axis (X) and the ratio of the peak areas of pCS-d4, INDS-d4 and the internal standard on the y-axis (Y), using the weighted least squares method (W=1 / X). 2 Regression calculations were performed to obtain a linear regression equation, which became the serum standard curve replacing the analytes pCS-d4 and INDS-d4. This standard curve was subsequently used to calculate the serum concentrations of the analytes pCS and INDS.

[0034] The chromatographic conditions are as follows: The chromatographic column was Thermo Hypersil Gold. TMC182.1*50mm5 μm; injection volume 5mL; column temperature 40°C; mobile phase A was 0.1% formic acid-5mM ammonium acetate aqueous solution, and mobile phase B was 90% methanol-10% isopropanol. The gradient elution program is shown in Table 1.

[0035] Mass spectrometry conditions: Ion source: electrospray ionization source; Mass spectrometry detection mode: multi-ion reaction monitoring negative ion mode; Scan time: 9 min; Ion source voltage: -3.0 kV; Ion source temperature: 550°C; Curtain gas, nebulizer gas, and auxiliary gas: 25 psi, 65 psi, and 75 psi, respectively; Declustering voltage: -45 kV. V; Alternative analyte pCS-d4: Quantitative detection ion pair m / z 190.9→m / z 79.9, Qualitative detection ion pair m / z 190.9→m / z 111; Corresponding internal standard pCS-d7: Quantitative detection ion pair m / z 193.9→m / z 79.9, Qualitative detection ion pair m / z 193.9→m / z 114, Collision voltage -21V; Alternative analyte INDS-d4: Quantitative detection ion pair m / z 215.9→m / z 79.9, Qualitative detection ion pair m / z 215.9→m / z 136; Corresponding internal standard INDS- 13 C6 quantitative detection of ion pairs m / z 217.9→m / z 79.9, qualitative detection of ion pairs m / z 217.9→m / z 138, collision voltage -27V.

[0036] (III) Determination of pCS and INDS content in serum samples Serum sample and blank sample processing: Take 50 μL of serum and add 5 μL of 30% methanol solution, vortex to mix, then add 100 μL of internal standard solution, vortex to mix for 2 min, centrifuge at 14000 rmp for 10 min at 4℃, take 50 μL of supernatant, add 100 μL of distilled water, mix well to obtain serum sample. Take 50 μL of serum and add 5 μL of 30% methanol solution. Vortex to mix. Then add 100 μL of acetonitrile and vortex to mix for 2 min. Centrifuge at 14000 rpm for 10 min at 4℃. Take 50 μL of the supernatant and add 100 μL of distilled water. Mix well to obtain the serum blank sample (without surrogate analytes and internal standards).

[0037] Serum matrix effect, extraction recovery rate, sample preparation recovery rate: Matrix effect: Take 50 μL of distilled water and add 5 μL of low, medium, and high quality control solutions, vortex to mix, then add 100 μL of internal standard solution, vortex to mix for 2 min, centrifuge at 14000 rpm for 10 min at 4℃, take 50 μL of supernatant, add 100 μL of distilled water, mix well and inject the sample, and measure the ratio A of the peak area of ​​INDS-d4 and pCS-d4 to the corresponding internal standard. Take 50 μL of serum and add 100 μL of internal standard solution, vortex to mix for 2 min, centrifuge at 14000 rpm for 10 min at 4℃, take 125 μL of supernatant, add 5 μL of low, medium, and high series quality control solutions, vortex to mix, take 50 μL of supernatant, add 100 μL of distilled water, mix well and inject the sample, and measure the ratio B of the peak area of ​​INDS-d4 and pCS-d4 to the corresponding internal standard. The matrix effect of the substitute analyte is: (B / A*100)%.

[0038] Extraction recovery: Take 50 μL of serum and add 5 μL of low, medium and high quality control solutions, vortex to mix, then add 100 μL of internal standard solution, vortex to mix for 2 min, centrifuge at 14000 rmp for 10 min at 4℃, take 50 μL of supernatant, add 100 μL of distilled water, mix well and inject the sample, and measure the ratio D of the peak area of ​​INDS-d4 and pCS-d4 to the corresponding internal standard. The extraction recovery rate of the surrogate analyte is (D / B)*100%.

[0039] Recovery rate: Take 50 μL of serum and add 5 μL of 30% methanol solution, vortex to mix, then add 100 μL of internal standard solution, vortex to mix for 2 min, centrifuge at 14000 rpm for 10 min at 4℃, take 50 μL of supernatant, add 100 μL of distilled water, mix well and inject. Calculate the concentrations C1 of INDS and pCS using the serum standard curve prepared on the same day. Take 50 μL of the same serum sample and add 5 μL of INDS and pCS standard solutions with concentrations of 2.5 μM, 25 μM, and 250 μM, vortex to mix, then add 100 μL of internal standard solution, vortex to mix for 2 min, centrifuge at 14000 rpm for 10 min at 4℃, take 50 μL of supernatant, add 100 μL of distilled water... μL, mix and inject. The concentrations C2 of INDS and pCS were calculated using the serum standard curve prepared on the same day. The recovery rate of the analyte was: ((C2-C1) / C2)*100%.

[0040] Serum sample testing Take 50 μL of serum sample, process it according to the above method, and then take 5 μL. Inject 5 μL into an LC-MS / MS system to detect the peak areas of pCS and INDS and their corresponding internal standards pCS-d7 and INDS-. 13The C6 peak area ratio is substituted into the serum standard curves of the alternative analytes pCS-d4 and INDS-d4 obtained in step (II) to calculate the content of pCS and INDS in the serum sample.

[0041] The following experimental examples illustrate the beneficial effects of the present invention: Experimental Example 1: Validation of the selectivity, linearity, precision and accuracy, matrix effect, extraction recovery, and spiking recovery of this invention. 1. Main Reagent Information 1) Standard Product 1: APExBio, USA; Indoxyl sulfate (INDS): CAS: 2642-37-7, purity: 99.76%; p-Cresyl sulfate (pCS): CAS: 3237-58-3, purity: 98%.

[0042] 2) Standard 2: Toronto Research Chemicals, Canada pCS-d4: CAS: 91978-69-7, Purity: ≥95%; pCS-d7: Product No.: TRC-T536802, Purity: ≥95%.

[0043] 3) Standard Product 3: Sigma-Aldrich, USA; INDS-d4: CAS: 1346601-03-3, Purity: ≥97%; INDS- 13 C6: PubChem No.: 329769186, Purity: ≥99%.

[0044] 4) Key reagents: Fish Scientific, USA; Methanol, acetonitrile, and isopropanol LC / MS grade.

[0045] 2. Instrument parameters 1) Chromatographic conditions are shown in Table 1.

[0046] Table 1 Chromatographic conditions

[0047] 2) Refer to Table 2 for the gradient elution procedure.

[0048] Table 2 Elution Procedure

[0049] 3) Mass spectrometry conditions are shown in Table 3.

[0050] Table 3 Mass Spectrometry Conditions

[0051] Among them, the quantitative ion pair is Q1 / Q3, and the qualitative ion pair is Q1 / Q3*.

[0052] 3. Sample preparation 1) Preparation of serum standard curve and quality control samples: Empty serum samples and serum standard curve samples were prepared using serum matrix and standard working solutions: 0 μM, 0.025 μM, 0.1 μM, 0.25 μM, 0.5 μM, 2.5 μM, 5 μM, 10 μM, 25 μM, 50 μM; serum quality control samples: QC-L: 0.05 μM, QC-M: 2 μM, QC-H: 40 μM. 2) Sample and validation requirements for linearity, precision, and accuracy of serum standard curves. Take serum standard curve samples and prepare 3 serum standard curve samples (at least 2 days, prepared on the same day) according to the same method as in Example 1, containing at least 6 concentrations (excluding 0 concentration), to establish a standard curve. Plot the concentration (C) of the surrogate analyte in the biological sample on the x-axis (X) and the peak area ratio of the surrogate analyte to the internal standard on the y-axis (Y), and use the weighted least squares method (W=1 / X). 2 Regression calculations are performed to obtain the linear regression equation, which is the standard curve. The concentration of each standard curve sample is calculated using the regression equation, but blank and zero concentration samples are not included in the calculation. The deviation of their backcalculated concentrations should be within ±15% (±20% for LLOQ). 75% of the samples should meet the above requirements. Unqualified samples should be discarded, and regression calculations should be performed again.

[0053] Prepare six LLOQ, six low-, six medium-, and six high-concentration quality control samples (QC). Calculate the concentration of the QC samples using the standard curve for each batch, and use this to determine the precision and accuracy of the method (prepare and test at least three batches over at least two days). The intra-batch and inter-batch precision (%RSD) of the low-, medium-, and high-concentration QC samples should not exceed 15%, and the LLOQ should not exceed 20%. The accuracy (%Bias) of the average value of the low-, medium-, and high-concentration QC samples should be within ±15%, and the LLOQ should be within ±20%. 3) Validation requirements for serum matrix effect, extraction recovery rate, and sample loading recovery rate. Matrix effect and extraction recovery: Prepare six low-, six-, and six-high concentration quality control samples (prepared from six different matrix sources / batches). Calculate the concentration of the QC samples using the standard curve for each batch, and then determine the precision and accuracy of the method. The precision (%RSD) of the low-, medium-, and high-concentration QC samples should not exceed 15%, and the accuracy of the low-, medium-, and high-concentration QC samples should be within ±15% of the labeled concentration. Recovery rate: The concentration of INDS and pCS after being added to the sample matrix should be ≥75% of the labeled concentration.

[0054] 4. Results 1) Typical mass chromatograms are shown below. Figure 1 .

[0055] 2) The validation results of the serum standard curve linearity, precision, accuracy, matrix effect, recovery rate, etc. are shown in Tables 4, 5 and 6.

[0056] Table 4. Validation results of pCS-d4 serum standard curve (unit: μM, n=6)

[0057] Table 5. Validation results of INDS-d4 serum standard curve (unit: μM, n=6)

[0058] Table 6. Validation results of precision, accuracy, matrix effect, extraction recovery, and spiking recovery of pCS-d4 and INDS-d4.

[0059] Note: C* refers to the standard concentration; Ext-Rec: extraction recovery rate; Sam-Rec: sample recovery rate; where R INDS = ;R pCS = .

[0060] from Figure 1The results in Tables 4, 5, and 6 show that the surrogate analytes INDS-d4 and pCS-d4 in human serum exhibit a standard curve range of 0.025–50 μM, with a limit of quantitation (LOQ) of 0.025 μM and good linearity. The LQ, low, medium, and high concentrations for quality control precision and accuracy testing were 0.025 μM, 0.05 μM, 2 μM, and 40 μM, respectively, and all three batches passed the precision and accuracy tests. Matrix effects did not affect analytical stability. The extraction recovery rate met the validation requirements. The recoveries of the endogenous analytes INDS and pCS calculated using these surrogate analytes were ≥75% of the labeled concentrations, indicating good accuracy and reproducibility of the method. In summary, the method of this invention has high specificity, accuracy, and reproducibility, and can be used for the detection of INDS and pCS concentrations in serum, making it suitable for clinical biomarker monitoring research.

[0061] Example 2: Establishment of a method for determining pCS and INDS content in serum filtrate Serum filtrate preparation: Add one serum sample to a Millipore Amicon Ultra-4 CentrifugalFilter 10 kDa MWCO ultrafiltration centrifuge tube, centrifuge at 7500 ×g for 10 min, and then remove the filtered solution to obtain the serum filtrate sample.

[0062] (a) Solution preparation Standard stock solution: Prepared in the same way as the standard stock solution in Example 1.

[0063] (ii) Establishment of serum filtrate standard curve: same as the preparation of standard stock solution in Example 1.

[0064] a. Preparation of working solutions for standard curves of serum filtrate at various concentrations (same as in Example 1): Take pCS-d4 and INDS-d4 standard stock solutions and dilute them with 30% methanol aqueous solution to prepare a series of mixed concentration standard curve solutions with concentrations of 0.1 μM, 0.25 μM, 1 μM, 2.5 μM, 5 μM, 25 μM, 50 μM, 100 μM, 250 μM, and 500 μM. Take 5 μL of each concentration solution, add 50 μL of blank matrix human serum filtrate and mix well, then add 100 μL of internal standard solution, vortex mix for 2 min, centrifuge at 14000 rmp for 10 min at 4℃, take 50 μL of supernatant, add 100 μL of distilled water and mix well to obtain serum standard curve working solutions with concentrations of 0.01 μM, 0.025 μM, 0.1 μM, 0.25 μM, 0.5 μM, 2.5 μM, 5 μM, 10 μM, 25 μM, and 50 μM. b. Preparation of quality control working solution (same as in Example 1): c. Calculation method for serum filtrate concentrations of analytes pCS and INDS (same as in Example 1): Chromatographic and mass spectrometry conditions (same as in Example 1): (III) Determination of pCS and INDS content in serum filtrate samples This section includes: 1. Main formulation information; 2. Instrument parameters; 3. Sample preparation method is exactly the same as in Example 1, except that the serum sample is replaced with the corresponding serum filtrate sample.

[0065] Example 2: The selectivity, linearity, precision and accuracy, matrix effect, extraction recovery, and spiking recovery of this invention were verified. The implementation methods and requirements are the same as in Example 1. result 1) Typical mass chromatograms are shown below. Figure 2 .

[0066] 2) The validation results of the serum filtrate standard curve linearity, precision, accuracy, matrix effect, recovery rate, etc. are shown in Tables 7 and 8.

[0067] Table 7. Validation results of the pCS-d4 serum filtrate standard curve (unit: μM, n=6)

[0068] Table 8. Validation results of the INDS-d4 serum filtrate standard curve (unit: μM, n=6)

[0069] from Figure 2 As shown in Tables 4-5, the surrogate analytes INDS-d4 and pCS-d4 in human serum filtrate exhibit a standard curve range of 0.01–50 μM and a limit of quantitation (LOQ) of 0.01 μM, demonstrating good linearity. The LQ, low, medium, and high concentrations for quality control precision and accuracy testing were 0.01 μM, 0.05 μM, 2 μM, and 40 μM, respectively, and all three batches passed the precision and accuracy tests. Matrix effects did not affect analytical stability. The extraction recovery rate met the validation requirements. The recoveries of endogenous analytes INDS and pCS calculated using these surrogate analytes were ≥75% of the labeled concentrations, indicating good accuracy and reproducibility of the method. In summary, the method of this invention has high specificity, accuracy, and reproducibility, and can be used for the concentration detection of INDS and pCS in serum filtrate, making it suitable for clinical biomarker monitoring research.

[0070] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0071] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A method for simultaneous quantitative detection of uremic toxin p-cresol sulfate and indophenol sulfate by LC-MS / MS based on the substitution analyte method, characterized in that, Includes the following steps: (1) Establish standard curves for biomatrix alternative analytes pCS-d4 and INDS-d4; (2) Mix the sample to be tested with methanol solution, then mix with acetonitrile solution containing internal standard, centrifuge, take the supernatant and mix with distilled water to obtain sample solution; (3) Take the sample solution for LC-MS / MS detection, and calculate the concentration of p-cresol sulfate and indophenol sulfate in the sample based on the detection results and the standard curve; The internal standards are pCS-d7 and INDS- 13 C6.

2. The method for simultaneous quantitative detection of uremic toxin p-cresol sulfate and indophenol sulfate by LC-MS / MS based on the alternative analyte method as described in claim 1, characterized in that, The biological matrix includes serum and serum filtrate.

3. The method for simultaneous quantitative detection of uremic toxin p-cresol sulfate and indophenol sulfate by LC-MS / MS based on the alternative analyte method as described in claim 1, characterized in that, In step (2), the volume ratio of the sample to be tested, the methanol solution and the acetonitrile solution containing the internal standard is (5~15):1:(10~30), and the methanol solution is a 30% methanol aqueous solution.

4. The method for simultaneous quantitative detection of uremic toxin p-cresol sulfate and indophenol sulfate by LC-MS / MS based on the alternative analyte method as described in claim 1, characterized in that, In step (2), the concentration of the internal standard in the acetonitrile solution containing the internal standard is 0.5-1.5 μM.

5. The method for simultaneous quantitative detection of uremic toxin p-cresol sulfate and indophenol sulfate by LC-MS / MS based on the alternative analyte method as described in claim 1, characterized in that, In step (2), the sample to be tested is mixed with the methanol solution under a vortex for 20-40 seconds, and then placed at room temperature for 1-3 minutes after vortexing.

6. The method for simultaneous quantitative detection of uremic toxin p-cresol sulfate and indophenol sulfate by LC-MS / MS based on the alternative analyte method as described in claim 1, characterized in that, In step (2), the mixing with the acetonitrile solution containing the internal standard is carried out under a vortex, and the vortexing time is 1~3 min.

7. The method for simultaneous quantitative detection of uremic toxin p-cresol sulfate and indophenol sulfate by LC-MSMS based on the alternative analyte method as described in claim 1, characterized in that, The LC-MS / MS liquid phase conditions in step (3) include: injection volume of 1~10 μL; chromatographic column of Thermo Hypersil Gold™ C18 column, 2.1×150mm, 5 μm; column temperature of 35~45℃; mobile phase consisting of mobile phase A and mobile phase B, wherein mobile phase A is formic acid-ammonium acetate aqueous solution and mobile phase B is methanol-isopropanol solution; flow rate of 0.35~0.55 mL / min.

8. The method for simultaneous quantitative detection of uremic toxin p-cresol sulfate and indophenol sulfate by LC-MS / MS based on the alternative analyte method as described in claim 1, characterized in that, In step (3) of the LC-MS / MS, the elution method of the liquid phase is gradient elution, and the gradient elution process is as follows: 。 9. The method for simultaneous quantitative detection of uremic toxin p-cresol sulfate and indophenol sulfate by LC-MS / MS based on the alternative analyte method as described in claim 1, characterized in that, The mass spectrometry conditions for the LC-MS / MS include: Ion source: electrospray ionization source; mass spectrometry detection mode: multi-ion reaction monitoring negative ion mode; scan time: 9 min; ion source voltage: -3.0 kV; ion source temperature: 550°C; curtain gas, nebulizing gas, and auxiliary gas: 25 psi, 65 psi, and 75 psi, respectively; declustering voltage: -45 V.

10. The method of any one of claims 1-9 is used for detecting the content of cresol sulfate and indophenol sulfate in diseases related to kidney, cardiovascular and bone toxicity.

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