A method for determining the concentrations of indophenol sulfate and p-cresol sulfate in plasma

Abstract

This invention relates to the field of indophenol sulfate and p-cresol sulfate technology, and particularly to a method for determining the concentrations of indophenol sulfate and p-cresol sulfate in plasma. The method involves pretreating the sample, followed by determination of the concentrations of indophenol sulfate and p-cresol sulfate in the sample using liquid chromatography-mass spectrometry and an internal standard method. The sample includes plasma, and the pretreatment involves protein precipitation. This method exhibits good linearity, a low limit of quantitation, strong anti-interference capabilities, and high precision and accuracy.

Description

Technical Field

[0001] This invention relates to the field of indophenol sulfate and p-cresol sulfate, and in particular to a method for determining the concentration of indophenol sulfate and p-cresol sulfate in plasma. Background Technology

[0002] Cardiorenal syndrome is a common pathological condition in which the heart and kidneys influence each other; acute or chronic dysfunction in either organ can lead to acute or chronic dysfunction in the other, creating a vicious cycle that ultimately results in the failure of both organs. Indoxyl sulfate (ISP) and p-cresylsulfate (PCS) are compounds with potential therapeutic effects on cardiorenal syndrome due to their antioxidant, anti-inflammatory, and antiplatelet aggregation activities.

[0003] Despite the rapid advancements in the diagnosis and treatment of cardiovascular and renal diseases in recent years, adverse clinical events, particularly those leading to cardiorenal syndrome, continue to rise steadily, posing a significant public health concern. Chronic kidney disease (CKD) and cardiovascular disease are closely linked. Compared to the general population, cardiovascular disease has a higher prevalence and poorer prognosis in CKD patients. Conversely, cardiovascular disease is a leading cause of death among dialysis patients, accounting for approximately 40-50% of all deaths in the dialysis population. Current research considers CKD a major risk factor for cardiovascular disease, with decreased glomerular filtration rate and proteinuria being independent risk factors. Animal studies have also demonstrated that renal insufficiency can promote the development of heart failure and atherosclerosis in vivo. These studies indicate that CKD can promote the development of cardiovascular diseases such as heart failure and coronary heart disease, but the specific mechanisms remain unclear.

[0004] Indophenol sulfate and p-cresol sulfate are both protein-bound uremic toxins derived from intestinal dietary proteins. In patients with chronic kidney disease (CHD), ISP and PCS accumulate in the body as renal function declines, causing damage to the body. Serum concentrations of both ISP and PCS in CHD patients are associated with CHD progression, major adverse cardiovascular events, cardiovascular mortality, and all-cause mortality. Therefore, ISP and PCS may be important factors promoting the progression of CHD and cardiovascular disease, but their specific mechanisms remain not fully understood.

[0005] Indophenol sulfate, also known as sodium 3-(2H-gamma-3-yl)2-hydroxypropionate, is a natural compound widely found in traditional Chinese medicine and plants such as bell pepper. Studies have shown that indophenol sulfate has potential therapeutic effects on cardiorenal syndrome. Its main mechanism of action is through antioxidant reactions, preventing oxidative damage and reducing the risk of developing cardiorenal syndrome. Indophenol sulfate can also reduce inflammation by decreasing neutrophil infiltration, thus alleviating the severity of cardiorenal syndrome. Furthermore, indophenol sulfate also has antiplatelet aggregation and opsonizing effects, regulating cardiovascular function and increasing renal blood perfusion.

[0006] p-Cresol sulfate, also known as sodium methyl sulfate, is a synthetic small molecule compound and is also considered a potential drug for treating cardiorenal syndrome. Its main mechanism of action is to reduce oxidative stress and prevent the development of cardiorenal syndrome by acting as an antioxidant. In addition, p-cresol sulfate can reduce the activity of the renin-angiotensin-aldosterone system and promote nitric oxide synthesis, thus playing a role in coordinating cardiorenal function. The antioxidant capacity of p-cresol sulfate can also improve kidney function by lowering serum creatinine levels, thereby alleviating the symptoms of cardiorenal syndrome.

[0007] In summary, indophenol sulfate and p-cresol sulfate, as compounds, show great potential in the treatment of cardiorenal syndrome. They possess various biological activities, such as antioxidant, anti-inflammatory, antiplatelet aggregation, and cardiovascular function regulation, making them potentially effective therapeutic agents. However, these drugs also have certain side effects; therefore, careful monitoring and evaluation of the patient's condition and physical response are necessary during use, and they must be used under the guidance of a physician.

[0008] Currently, there are analytical methods available on the market that use liquid chromatography-mass spectrometry (LC-MS) to determine the concentration, but their drawbacks are that the LLOQ is 5 ng / mL and the limit of quantitation is relatively high. Summary of the Invention

[0009] The purpose of this invention is to provide a method for determining the concentrations of indophenol sulfate and p-cresol sulfate in plasma, using indophenol sulfate... 13 C6 and p-cresol sulfate-D7 were used as internal standards. Extraction was performed by protein precipitation. The limits of quantification for indophenol sulfate and p-cresol sulfate in plasma were both 1.000 ng / mL, and the linear range was good within the range of 1.000 ng / mL to 100.000 ng / mL.

[0010] To achieve the above and other related objectives, the present invention provides the following technical solutions:

[0011] In a first aspect, the present invention provides a method for determining the concentrations of indophenol sulfate and p-cresol sulfate in plasma, which involves pretreating the sample and then determining the concentrations of indophenol sulfate and p-cresol sulfate in the sample by liquid chromatography-mass spectrometry and internal standard method.

[0012] The sample includes a plasma sample, the pretreatment method is protein precipitation, and the internal standard for the internal standard method is indole sulfate- 13 C6 and p-cresol sulfuric acid-D7.

[0013] In one embodiment of the present invention, more detailed content is provided, including the following steps:

[0014] Firstly, sample preparation:

[0015] (1) Prepare stock solutions of indophenol sulfate and p-cresol sulfate by dissolving in methanol. 13 C6 and p-cresol sulfuric acid-D7 internal standard stock solutions;

[0016] (2) Prepare standard curve working solutions and quality control working solutions by diluting indolesulfonate and p-cresol sulfuric acid stock solutions with a diluent (methanol:water, volume ratio, 1:1); prepare standard curve working solutions and quality control working solutions by diluting indolesulfonate- p-cresol sulfuric acid stock solutions with a diluent (methanol:water, volume ratio, 1:1). 13 Prepare internal standard working solution using C6 and p-cresol sulfuric acid-D7 internal standard stock solution;

[0017] (3) Prepare different concentrations of accompanying standard curve samples, quality control samples, system suitability samples, and test samples using blank plasma;

[0018] (4) Sample pretreatment: Mix the above-prepared standard curve sample, quality control sample, system suitability sample and test sample, add methanol as precipitant, mix well, centrifuge, take the supernatant, and add reconstitution solution (methanol:water, volume ratio, 1:1).

[0019] (5) Sample analysis: The concentrations of indophenol sulfate and p-cresol sulfate in the sample were determined by liquid chromatography-mass spectrometry and internal standard method. This process also includes standard curve method and quality control samples.

[0020] Secondly, the specific parameters of the detection method:

[0021]

[0022] parameter API6500 parameter API6500 CUR 35 Gas1 55 CAD 9 Gas2 55 IS -4500 EP -10 TEM 550 CXP -15

[0023] Chromatographic column: Shim-pack GIST C18-AQ (3μm, 2.1×100mm);

[0024] Mobile phase A: 5 mM ammonium acetate aqueous solution;

[0025] Mobile phase B: Methanol;

[0026] Injection washing solution: methanol: acetonitrile: isopropanol: water (volume ratio, 1:1:1:1);

[0027] Injection volume: 2 μL;

[0028] Flow rate: 0.4 mL / min;

[0029] Column temperature: 40℃;

[0030] Gradient elution: The elution procedure is as follows:

[0031] time A％ B％ 0.5 80 20 2.0 20 80 3.5 20 80 3.6 80 20 5.0 80 20 5.0 STOP

[0032] The beneficial effects of this invention are:

[0033] (1) This invention uses indolesulfonate- 13 C6 and p-cresol sulfate-D7 were used as internal standards. Plasma was extracted by protein precipitation. The lower limit of quantification for both indophenol sulfate and p-cresol sulfate in plasma was 1.000 ng / mL, and the linear relationship was good within the range of 1.000 ng / mL to 100.000 ng / mL.

[0034] (2) The present invention also screened plasma anticoagulants, compared the storage stability of heparin sodium, heparin lithium and EDTA, and preferred EDTA as the anticoagulant for testing plasma.

[0035] (3) The present invention also screened chromatographic columns, compared ACQUITY BEH C18 (2.1×100mm, 1.7μm) and Shim-pack GIST C18-AQ (3μm, 2.1×100mm), and preferred Shim-pack GIST C18-AQ (3μm, 2.1×100mm) as the chromatographic column of the present method.

[0036] (4) The present invention also underwent verification experiments. 1) By continuously running three analytical batches at four concentrations (QC) (1.000, 3.000, 30.000, 75.000 ng / mL), the intra-batch and inter-batch precision and accuracy were evaluated. Among them, the intra-batch %RE of ISP was -6.0% to 4.2%, the intra-batch %CV was 1.6% to 11.2%, the inter-batch %RE was -4.3% to 0.9%, and the inter-batch %CV was 4.3% to 11.2%; the intra-batch %RE of PCS was -5.7% to 3.5%, the intra-batch %CV was 1.8% to 10.3%, the inter-batch %RE was -2.8% to 0.3.5%, and the inter-batch %CV was 6.1% to 10.3%. In summary, the method of the present invention has good linearity, low limit of quantitation, strong anti-interference ability, and high precision and accuracy. Attached Figure Description

[0037] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0038] Figure 1 This is a schematic diagram of the fitting curve for the standard sample of this invention;

[0039] Figure 2 The spectrum of the blank quality control sample (QCO) containing only internal standard in this invention;

[0040] Figure 3 The image shows the LLOQ sample (1.000 ng / mL) of this invention.

[0041] Among them, indoxyl sulfate (ISP) and p-cresyl sulfate (PCS) are used. Detailed Implementation

[0042] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0043] Unless otherwise specified, the instruments and reagents used in the examples are readily available from commercial companies, and some of the instruments, reagents and samples are shown in Tables 1 and 2 below.

[0044] Table 1

[0045]

[0046] Table 2

[0047]

[0048] The experiments in this invention follow the guidelines for the validation of quantitative analysis methods for biological samples in General Chapter 9012 of the 2020 edition of the Chinese Pharmacopoeia, Part IV, and ICH-M10, "Validation of Bioanalytical Methods and Analysis of Samples". The English terms used in this invention are well-known in the field and therefore will not be explained individually.

[0049] Example 1

[0050] A method for determining the concentrations of indophenol sulfate and p-cresol sulfate in plasma includes the following steps: 1. Preparing the mobile phase, syringe wash solution, diluent, precipitant, and reconstitution solution:

[0051] (1) Mobile phase A (5mM ammonium acetate aqueous solution): Weigh about 385mg of ammonium acetate into a reagent bottle of appropriate volume, add 1000mL of purified water to dissolve, mix well, and let stand at room temperature.

[0052] (2) Mobile phase B (methanol): Transfer 1000 mL of methanol to a reagent bottle of appropriate volume and place at room temperature.

[0053] (3) Needle washing solution (methanol:acetonitrile:isopropanol:water, 1:1:1:1, volume ratio): Transfer 250mL methanol, 250mL acetonitrile, 250mL isopropanol and 250mL purified water to a reagent bottle of appropriate volume, mix well and place at room temperature.

[0054] (4) Diluent (methanol:water mixture, 50:50, v / v): Transfer 500 mL of methanol and 500 mL of purified water to a reagent bottle of appropriate volume, mix well, and place at room temperature.

[0055] (5) Precipitant (methanol): Transfer 1000 mL of methanol to a reagent bottle of appropriate volume and place at room temperature.

[0056] (6) Reconstituted solution (methanol:water mixture, 50:50, v / v): Transfer 500 mL of methanol and 500 mL of purified water to a reagent bottle of appropriate volume, mix well, and place at room temperature.

[0057] 2. Prepare the following samples and solutions in 1.5 mL polypropylene centrifuge tubes at room temperature:

[0058] (1) Preparation of stock solutions of indophenol sulfate and p-cresol sulfate: Weigh approximately 30 mg of indophenol sulfate and p-cresol sulfate reference standards accurately, calculate the mass based on the content, dissolve in DMSO to prepare a stock solution of indophenol sulfate and p-cresol sulfate with a concentration of 4.000 mg / mL, and store at -20℃ for later use.

[0059] (2) Preparation of standard curve working solution and quality control working solution: Accurately transfer a certain volume of indophenol sulfate and p-cresol sulfate stock solution, and dilute with diluent to obtain standard curve working solutions with concentrations of 2000 μg / mL, 1600 μg / mL, 1000 μg / mL, 400 μg / mL, 200 μg / mL, 80 μg / mL, 40 μg / mL, and 20 μg / mL, and quality control working solutions with concentrations of 1500 μg / mL, 600 μg / mL, 60 μg / mL, and 20 μg / mL, and store at -20℃ for later use.

[0060] (3) Indophenol sulfate 13 Preparation of C6 and p-cresol sulfate-D7 internal standard stock solution: Accurately weigh indophenol sulfate- 13 C6 and 2 mg of p-cresol sulfate-D7 standard were dissolved in methanol to prepare indophenol sulfate with a concentration of 1.000 mg / mL. 13 C6 and p-cresol sulfate-D7 internal standard stock solutions should be stored at -20°C for future use.

[0061] (4) Indophenol sulfate 13 Preparation of C6 and p-cresol sulfuric acid-D7 internal standard working solution: Take indophenol sulfuric acid- 13 Take 500 μL of C6 internal standard stock solution, add 400 μL of p-cresol sulfate-D7 internal standard stock solution, and add 19100 μL of diluent. Mix well to obtain indophenol sulfate with a concentration of 25.000 / 20.000 μg / mL. 13 C6 and p-cresol sulfuric acid-D7 internal standard working solutions should be stored at -20℃ for later use. (5) For system suitability, preparation of standard curve samples and quality control samples, refer to Tables 3 and 4:

[0062] Table 3 Standard Curve Samples

[0063]

[0064] Table 4 Quality Control Samples

[0065]

[0066] 3. Sample pretreatment:

[0067] (1) Vortex the prepared system suitability sample, standard curve sample and quality control sample on a multi-tube vortexer for about 30 seconds.

[0068] (2) Take 50 μL of the above plasma sample into a 96-well plate with a circular deep well, add 20 μL of internal standard working solution and 500 μL of methanol, vortex the 96-well plate at 1000 rpm for 5 min and centrifuge at 2000 g for 5 min (10℃).

[0069] (3) Transfer 100 μL of supernatant to a new circular deep-well 96-well plate, add 500 μL of reconstitution solution, and vortex at 1000 rpm for 5 min.

[0070] (4) Transfer 100 μL of supernatant to a new circular deep-well 96-well plate, add 500 μL of reconstitution solution, vortex at 1000 rpm for 5 min, and use for sample injection analysis.

[0071] 4. Verify the results of the investigation.

[0072] (1) System applicability assessment

[0073] The system suitability test results of three analytical batches during the validation process of this method showed that the coefficients of variation (%CV) of the retention times of the analyte and internal standard were all less than 15%, and the coefficients of variation (%CV) of the peak area ratio were all less than 15%, which met the acceptance criteria for the validation of this method, indicating that the system suitability of this method is good. The specific results are shown in Table 5.

[0074] Table 5: System Applicability

[0075] Table 5-1: Suitability of the Indophenol Sulfate System

[0076]

[0077] Table 5-2: Suitability of p-cresol sulfate system

[0078]

[0079] (2) Residue

[0080] After the sample analysis was completed using the standard curve to quantify the upper limit, a blank plasma sample was immediately analyzed, and the residual effects of this method were found to be within the acceptable range. Specific data are shown in Table 6.

[0081] Table 6: Residues

[0082] Table 6-1: Residues in the analytical batch of indophenol sulfate

[0083]

[0084] Table 6-2: Residues in p-cresol sulfate analytical batches

[0085]

[0086] (3) Matrix effect

[0087] The normal plasma matrix effect, hemolytic plasma matrix effect, and hyperlipidemic plasma matrix effect of indophenol sulfate and p-cresol sulfate at various concentration levels in this method met the corresponding requirements. Specific data are shown in Table 7.

[0088] Table 7: Matrix Effect

[0089] Table 7-1: Matrix Effects of Indophenol Sulfate

[0090]

[0091]

[0092] Table 7-2: Matrix effect of p-cresol sulfate

[0093]

[0094]

[0095] (4) Standard Curve

[0096] The linearity investigation ranges for indophenol sulfate and p-cresol sulfate in this study were determined to be 1.000–100.000 μg / mL, with specific investigation points at 1.000, 2.000, 4.000, 10.000, 20.000, 50.000, 80.000, and 100.000 μg / mL. Using the analyte concentration as x and the analyte-to-internal standard peak area ratio as y, a weighted least squares linear regression was performed with a weighting coefficient of 1 / x². The accuracy deviation of the back-calculated values ​​at each calibration point and the correlation coefficient of the standard curve both met the acceptable criteria for validation of this method. Specific data are shown in Table 8.

[0097] Table 8: Standard Curve

[0098] Table 8-1: Standard Curve of Indophenol Sulfate

[0099]

[0100]

[0101] Table 8-2: Standard Curve for p-Cresol Sulfate

[0102]

[0103] (5) Accuracy and precision

[0104] The accuracy and precision of this method for detecting indophenol sulfate and p-cresol sulfate at various concentration levels in both intra-batch and inter-batch samples meet the acceptance criteria for method validation, indicating that the method has good precision and accuracy. Specific data are shown in Table 9.

[0105] Table 9: Accuracy and Precision

[0106] Table 9-1: Accuracy and Precision of Indophenol Sulfate

[0107]

[0108]

[0109] Table 9-2: Accuracy and Precision of p-Cresol Sulfate

[0110]

[0111]

[0112] (6) Lower limit of quantitation

[0113] In this method, the limits of quantification for both indophenol sulfate and p-cresol sulfate are 1.000 μg / mL. The coefficients of variation (%CV) for intra-assay and inter-assay precision of LLOQ are both less than 20%, and the accuracy deviations are all within ±20%, meeting the acceptance criteria for validation of this method. Specific data are shown in Table 9.

[0114] (7) Extraction recovery rate

[0115] The overall recovery rate of indophenol sulfate after treatment by this method was 91.5%, with an overall %CV of 3.5%, and the average recovery rate of the internal standard was 98.9%, with a %CV of 0.2%. The overall recovery rate of p-cresol sulfate was 93.5%, with an overall %CV of 2.8%, which met the acceptable criteria for validation of this method. Specific data are shown in Table 10.

[0116] Table 10: Extraction Recovery Rate

[0117] Table 10-1: Recovery of Indophenol Sulfate Extraction

[0118]

[0119] Table 10-2: Recovery rate of p-cresol extracted with sulfuric acid

[0120]

[0121]

[0122] (8) Stability of plasma samples at room temperature

[0123] Plasma samples met the stability requirements after being placed at room temperature for 27 hours. Specific data are shown in Table 11.

[0124] Table 11: Room Temperature Stability

[0125] Table 11-1: Room temperature stability of indophenol sulfate

[0126]

[0127] Table 11-2: Room temperature stability of p-cresol sulfuric acid

[0128]

[0129] 5. Conclusion

[0130] The above experimental results demonstrate that the LC-MS / MS method established in this study for the quantitative analysis of indophenol sulfate and p-cresol sulfate concentrations in human plasma exhibits the following advantages: simple blood sample pretreatment, good linearity in quantitative analysis, high recovery and sensitivity, good selectivity, good accuracy and precision, and good sample stability under specified conditions. All validation items and data comply with the relevant requirements of the "Guidelines for Validation of Quantitative Analysis Methods for Biological Samples" in the Pharmacopoeia of the People's Republic of China (2020 Edition, Part IV, 9012) and the ICH "M10: Validation of Bioanalytical Methods and Sample Analysis" guidelines, and can be used to determine the concentrations of indophenol sulfate and p-cresol sulfate in human plasma.

[0131] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A method for measuring concentrations of indoxyl sulfate (ISP) and p-cresyl sulfate (PCS) in blood plasma, characterized by, After sample pretreatment, the concentrations of indoxyl sulfate (ISP) and p-cresyl sulfate (PCS) in the samples were determined by liquid chromatography-mass spectrometry (LC-MS) and internal standard method. The samples included plasma samples; the pretreatment method was protein precipitation; and the internal standard for the internal standard method was indoxyl sulfate. 13 Two substances: C6 and p-cresol sulfuric acid-D7.

2. The method for measuring concentrations of indoxyl sulfate and p-cresyl sulfate in blood plasma according to claim 1, characterized by, The liquid chromatography-mass spectrometry (LC-MS) conditions include: Chromatographic column: Shim-pack GIST C18-AQ (3μm, 2.1×100mm); Mobile phase A: 5 mM ammonium acetate aqueous solution; Mobile phase B: Methanol; Injection washing solution: methanol: acetonitrile: isopropanol: water (volume ratio, 1:1:1:1); Injection volume: 2 μL; Flow rate: 0.4 mL / min; Column temperature: 40℃; Gradient elution: The elution procedure is as follows:

3. The method for determining concentrations of indoxyl sulfate and p-cresyl sulfate in blood plasma according to claim 1, characterized by, The mass spectrometry conditions for the liquid chromatography-mass spectrometry system include:

4. The method for determining the concentrations of indophenol sulfate and p-cresol sulfate in plasma according to claim 1, characterized in that, The plasma sample contained K2EDTA as an anticoagulant.

5. The method for determining concentrations of indoxyl sulfate and p-cresyl sulfate in blood plasma according to claim 1, characterized by, The protein precipitant used for protein precipitation is methanol.

6. The method for determining concentrations of indoxyl sulfate and p-cresyl sulfate in blood plasma according to claim 1, characterized by, Includes the following steps: (1) Indoxylsulfuric acid and p-cresolsulfuric acid stock solutions were prepared in methanol. Indoxylsulfuric acid was prepared in methanol- 13 C6 and p-cresol sulfuric acid-D7 internal standard stock solutions; (2) Indoxylsulfonic acid and p-cresolsulfonic acid stock solutions were prepared into standard working solution and quality control working solution with diluent (methanol: water, volume ratio, 1:1); Indoxylsulfonic acid and p-cresolsulfonic acid stock solutions were prepared into standard working solution and quality control working solution with diluent (methanol: water, volume ratio, 1:1); 13 C6 and p-cresolsulfonic acid-D7 internal standard stock solutions were prepared into internal standard working solution; (3) Prepare different concentrations of accompanying standard curve samples, quality control samples, system suitability samples, and test samples using blank plasma; (4) Sample pretreatment: Mix the above-prepared standard curve sample, quality control sample, system suitability sample and test sample, add methanol as precipitant, mix well, centrifuge, take the supernatant, and add reconstitution solution (methanol:water, volume ratio, 1:1). (5) Sample analysis: The concentrations of indophenol sulfate and p-cresol sulfate in the sample were determined by liquid chromatography-mass spectrometry and internal standard method.

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