A method for detecting the total amount of guanidines in body fluids of patients with renal failure

Abstract

The invention discloses a method for detecting the total amount of guanidines in body fluids of patients with renal failure, comprising the following steps: S1: sample pretreatment and preparation of a test solution: S11: collecting and processing sample 1: taking 5 ml of blood from a patient with renal failure, centrifuging at 3000 r / min for 10 minutes, filtering the supernatant through a 0.45 μm filter membrane, and placing it in a 2-8°C refrigerator for standby use; S12: collecting and processing sample 2: taking 5 ml of urine and peritoneal dialysis waste fluid from a patient with renal failure, filtering the supernatant through a 0.45 μm filter membrane, and placing it in a 2-8°C refrigerator for standby use; S13: preparing a test stock solution: accurately measuring sample 1 or sample 2, 3 ml of which is placed in a 10 ml volumetric flask, diluting with water to the scale, and shaking well. The invention establishes a method for determining the total amount of guanidines in body fluids of patients with renal failure, solves the problem that the determination solution is easily discolored, and realizes the accurate detection of guanidine groups in the body fluids of patients with renal failure; the determination method is simple, easy to operate, and short in time; not only improves the detection efficiency, but also improves the quality and safety of the product.

Description

Technical Field

[0001] The invention belongs to the technical field of medical testing, and particularly relates to a method for detecting the total amount of guanidines in body fluids of patients with renal failure. Background Art

[0002] Patients with renal failure have lost some or all of their kidneys' excretory function. Many of the final metabolites of proteins in the body cannot be excreted and accumulate, causing a range of toxic symptoms. These substances are therefore called toxic substances. In addition to urea and creatinine, guanidines are also major toxic substances. Therefore, strictly controlling the levels of guanidines in patients and accurately quantifying them is of paramount importance.

[0003] Currently, no method for determining the total amount of guanidines in the body fluids of patients with renal failure has been included in any standards or documents. Only some methods for determining methylguanidine have been included, most of which use gas chromatography or fluorescence methods, and require derivatization treatment before determination, greatly affecting the accuracy of the measurement results. Body fluids (blood, urine) are complex, containing various proteins, organic and inorganic small molecules, and a large number of metabolites cannot contain only one methylguanidine. The total amount of guanidines directly affects the toxin levels in the patient's body. Therefore, in order to better understand the accumulation of toxins in the patient's body, a method for determining the total amount of guanidines is particularly important. For this reason, the present invention proposes a method for detecting the total amount of guanidines in the body fluids of patients with renal failure. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for detecting the total amount of guanidines in the body fluids of patients with renal failure, so as to solve the problems raised in the above background technology.

[0005] To achieve the above object, the present invention provides the following technical solution: a method for detecting the total amount of guanidines in the body fluids of patients with renal failure, comprising the following steps: S1: sample pretreatment and preparation of a test solution:

[0006] S11: Sample collection and processing 1: 5 ml of blood from a patient with renal failure was collected and centrifuged at 3000 rpm for 10 minutes. The supernatant was filtered through a 0.45 μm filter membrane and stored in a refrigerator at 2-8°C until use.

[0007] S12: Sample collection and processing 2: 5 ml of urine and peritoneal dialysis waste fluid from a patient with renal failure were collected, the supernatant was filtered through a 0.45 μm filter membrane, and then stored in a refrigerator at 2-8°C for later use;

[0008] S13: Prepare the test sample stock solution: accurately measure 3 ml of sample 1 or sample 2 into a 10 ml volumetric flask, dilute to the mark with water, and shake well;

[0009] S14: Prepare the test solution: accurately measure 5 ml of the test sample stock solution and place it in a colorimetric tube. Add 1 ml each of 0.1% 8-hydroxyquinoline ethanol solution and 10% sodium hydroxide solution, mix well, add 4 ml of sodium hypobromite solution, mix well, let stand for 5 minutes, then add 2 ml of 10% guanidine hydrochloride aqueous solution and mix well to prepare the test solution.

[0010] S2: Preparation of reference solution:

[0011] Take 2-guanidinosuccinic acid reference substance, accurately weigh it, dissolve it in water and dilute it to make 300 μg of 2-guanidinosuccinic acid per 1 ml;

[0012] S3: Preparation of reference linear solution:

[0013] Linearity reference solution: Accurately measure 0.5ml, 1ml, 2ml, 3ml and 4ml of the reference solution and place them in colorimetric tubes respectively. Add 4.5ml, 4ml, 3ml, 2ml and 1ml of water in that order. Add 1ml each of 0.1% 8-hydroxyquinoline ethanol solution and 10% sodium hydroxide solution, mix well. Add 4ml of sodium hypobromite solution, mix well and let stand for 5 minutes. Then add 2ml of 10% guanidine hydrochloride aqueous solution and mix well. These are used as linearity reference solutions 1 / 2 / 3 / 4 / 5 respectively.

[0014] S4: Preparation of blank solution:

[0015] Accurately measure 5 ml of water and place it in a colorimetric tube. Add 1 ml each of 0.1% 8-hydroxyquinoline ethanol solution and 10% sodium hydroxide solution, mix well. Add 4 ml of sodium hypobromite solution, mix well, let stand for 5 minutes, then add 2 ml of 10% guanidine hydrochloride aqueous solution and mix well.

[0016] S5: taking each of the above solutions and testing them by UV-visible spectrophotometry;

[0017] Calculation formula: Guanidine content in wastewater

[0018] Where: 58 is the molecular weight of guanidine group;

[0019] 175 is: molecular weight of 2-guanidinosuccinic acid;

[0020] 10 / 3 is the dilution factor of the test sample stock solution;

[0021] A is: the absorbance value of the test solution after deducting the blank;

[0022] b is: the intercept of the linear equation;

[0023] s is: the slope of the linear equation;

[0024] 13 is: total volume of test solution, ml;

[0025] 5 is: the sampling volume of the test sample stock solution, ml.

[0026] Preferably, the measurement conditions of step S5 are: wavelength of 500 nm, integration time of 0.2 s.

[0027] Preferably, the sodium hypobromite should be of analytical grade or higher; and the 8-hydroxyquinoline should be of analytical grade or higher.

[0028] Preferably, the sodium hydroxide should be no less than analytical grade; the guanidine hydrochloride should be no less than analytical grade; and the ethanol should be no less than 95% analytical grade ethanol.

[0029] Preferably, the sample 1 and sample 2 processed in step S1 need to be sealed and stored in a refrigerator.

[0030] Compared with the existing technology, the beneficial effects of the present invention are: the present invention establishes a method for measuring the total amount of guanidines in the body fluids of patients with renal failure, solves the problem of easy fading of the measurement solution, and realizes the accurate detection of guanidine groups in the body fluids of patients with renal failure; the measurement method is simple, easy to operate and time-saving; it not only improves the detection efficiency, but also improves the quality and safety of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a linear diagram for verifying the guanidine content determination method of the present invention; wherein the linear equation for verifying the guanidine content method is: Y=0.0063x+0.0122; correlation coefficient r=0.998;

[0032] Figure 2 This is a full wavelength scanning diagram of the guanidine content determination method of the present invention. DETAILED DESCRIPTION

[0033] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0034] The present invention provides a technical solution:

[0035] Example

[0036] The method for detecting the guanidine content in peritoneal dialysis wastewater of this embodiment comprises the following steps:

[0037] Step 1: Treatment and preparation of test solution:

[0038] Sample 1: Take 5 ml of blood from a patient with renal failure, centrifuge at 3000 rpm for 10 minutes, filter the supernatant through a 0.45 μm filter membrane, and store in a refrigerator at 2-8°C until use.

[0039] Sample 2: Take 5 ml of urine and peritoneal dialysis waste fluid from a patient with renal failure, filter the supernatant through a 0.45 μm filter membrane, and place it in a refrigerator at 2-8°C for later use.

[0040] Test sample stock solution: Accurately measure 3 ml of sample 1 or sample 2 and place it in a 10 ml volumetric flask, add water to dilute to the scale, and shake well.

[0041] Test solution: Accurately measure 5 ml of the test sample stock solution and place it in a colorimetric tube. Add 1 ml each of 0.1% 8-hydroxyquinoline ethanol solution and 10% sodium hydroxide solution, mix well, add 4 ml of sodium hypobromite solution, mix well and let stand for 5 minutes, then add 2 ml of 10% guanidine hydrochloride aqueous solution and mix well to prepare the test solution.

[0042] Step 2: Preparation of reference solution:

[0043] Preparation of reference stock solution: Take an appropriate amount of 2-guanidinosuccinic acid reference substance, accurately weigh it, dissolve it in water and dilute it to make it contain 300 μg of 2-guanidinosuccinic acid per 1 ml.

[0044] Step 3: Preparation of reference linear solution:

[0045] Accurately measure 0.5 ml, 1 ml, 2 ml, 3 ml, and 4 ml of the reference stock solution (300 μg / ml) and place them in a 25 ml colorimetric tube. Add 4.5 ml, 4 ml, 3 ml, 2 ml, and 1 ml of water in that order, add 1 ml each of 0.1% 8-hydroxyquinoline ethanol solution and 10% sodium hydroxide solution, mix well, add 4 ml of sodium hypobromite solution, mix well, and let stand for 5 minutes. Then, add 2 ml of 10% guanidine hydrochloride aqueous solution and mix well. These solutions were used as linear reference solutions 1 / 2 / 3 / 4 / 5, respectively.

[0046] Step 4: Preparation of blank solution:

[0047] Accurately measure 5 ml of water and place it in a colorimetric tube. Add 1 ml each of 0.1% 8-hydroxyquinoline ethanol solution and 10% sodium hydroxide solution, mix well. Add 4 ml of sodium hypobromite solution, mix well, let stand for 5 minutes, then add 2 ml of 10% guanidine hydrochloride aqueous solution and mix well.

[0048] Step 5: UV-visible spectrophotometry detection:

[0049] Measurement conditions: wavelength: 500 nm; integration time: 0.2 s.

[0050] The method of this embodiment was verified by methodology:

[0051] 1. Exclusivity

[0052] 1.1 Determination of wavelength

[0053] Accurately weigh 15 mg of 2-guanidinosuccinic acid reference substance into a 50 ml volumetric flask, dissolve it in water and dilute to the mark, shake well, and use it as the reference substance stock solution.

[0054] Accurately measure 1 ml of the reference stock solution, add 4 ml of water, 1 ml each of 0.1% 8-hydroxyquinoline ethanol solution and 10% sodium hydroxide solution, mix well, add 4 ml of sodium hypobromite solution, mix well and let stand for 5 minutes, then add 2 ml of 10% guanidine hydrochloride aqueous solution and mix well to prepare the reference solution.

[0055] Place the reference solution in a cuvette and perform a full wavelength scan. Record the chromatogram.

[0056] 1.2 Exclusivity

[0057] Test solution: Accurately measure 5 ml of the test sample stock solution, add 1 ml each of 0.1% 8-hydroxyquinoline ethanol solution and 10% sodium hydroxide solution, mix well, add 4 ml of sodium hypobromite solution, mix well and let stand for 5 minutes, then add 2 ml of 10% guanidine hydrochloride aqueous solution and mix well to prepare the test solution.

[0058] Blank solution (negative control): Accurately measure 5 ml of water and place it in a colorimetric tube. Add 1 ml each of 0.1% 8-hydroxyquinoline ethanol solution and 10% sodium hydroxide solution, mix well. Add 4 ml of sodium hypobromite solution, mix well, let stand for 5 minutes, then add 2 ml of 10% guanidine hydrochloride aqueous solution and mix well.

[0059] Take the test solution, blank solution and the reference solution (positive control) under 1.1, and measure the absorbance at the determined wavelength by UV-visible spectrophotometry.

[0060] The specificity test results are shown in the following table:

[0061] Guanidine content determination specificity test result record

[0062]

[0063] Conclusion: The experimental results show that the measured values ​​of the positive control and the test solution are much greater than those of the negative control, indicating that the negative control has no interference with the sample and the method has good specificity.

[0064] 2. Linear range

[0065] Linear stock solution: Accurately weigh 15 mg of 2-guanidinosuccinic acid reference substance, place it in a 50 ml volumetric flask, dissolve it in water and dilute to the scale, shake well, so that each 1 ml contains 300 μg of 2-guanidinosuccinic acid.

[0066] Linear solution: Accurately measure 0.5 ml, 1 ml, 2 ml, 3 ml, and 4 ml of the linear stock solution (300 μg / ml) and place them in a 25 ml colorimetric tube. Add 4.5 ml, 4 ml, 3 ml, 2 ml, and 1 ml of water in that order, add 1 ml each of 0.1% 8-hydroxyquinoline ethanol solution and 10% sodium hydroxide solution, mix well, add 4 ml of sodium hypobromite solution, mix well, let stand for 5 minutes, then add 2 ml of 10% guanidine hydrochloride aqueous solution and mix well. These are used as linear reference solutions 1 / 2 / 3 / 4 / 5, respectively.

[0067] Blank solution: Accurately measure 5 ml of water and place it in a colorimetric tube. Add 1 ml each of 0.1% 8-hydroxyquinoline ethanol solution and 10% sodium hydroxide solution, mix well. Add 4 ml of sodium hypobromite solution, mix well, let stand for 5 minutes, then add 2 ml of 10% guanidine hydrochloride aqueous solution and mix well.

[0068] Take the above blank solution and linear solution, measure them at a wavelength of 500nm by ultraviolet spectrophotometry, record the absorbance value, and perform linear regression analysis with concentration as the horizontal axis and absorbance value as the vertical axis.

[0069] The results are shown in the table below:

[0070] Guanidine content determination-linear range result recording

[0071]

[0072] Conclusion: The experimental results show that in the range of 33%-267%, the linear correlation coefficient is 0.998, which is greater than 0.995, indicating that the method has good linearity.

[0073] 3. Recovery rate

[0074] The recovery test of this product was a blank spike recovery method, and the test concentrations were designed to be 67%, 100%, and 133% of the reference concentration (34.62 μg / ml) in this product.

[0075] Preparation of Accuracy Stock Solution Take an appropriate amount of 2-guanidinosuccinic acid reference substance, accurately weigh it, dissolve it in water and dilute it to make it contain 300 μg of 2-guanidinosuccinic acid per 1 ml.

[0076] Preparation of the accuracy solution: Accurately measure 1ml, 1.5ml, and 2ml of the accuracy stock solution into colorimetric tubes, respectively. Add 4ml, 3.5ml, and 3ml of water, followed by 1ml each of 0.1% 8-hydroxyquinoline ethanol solution and 1ml of 10% sodium hydroxide solution, and mix thoroughly. Add 4ml of sodium hypobromite solution, mix thoroughly, and let stand for 5 minutes. Then, add 2ml of 10% guanidine hydrochloride aqueous solution and mix thoroughly. These will be accuracy solutions 1, 2, and 3, respectively. (Prepare three replicates for each concentration.)

[0077] Determination: Take the blank solution and the accuracy solution and measure them by UV-visible spectrophotometry at a wavelength of 500 nm. Record the absorbance values ​​and calculate the recovery rate using the standard curve method according to the following formula.

[0078] formula:

[0079] Where A1 is the absorbance value of the accuracy solution after deducting the blank;

[0080] b is the intercept of the linear equation of the standard curve method;

[0081] s is the slope of the linear equation of the standard curve method;

[0082] C 理论 is the theoretical concentration of the accuracy solution.

[0083] The recovery results are shown in the table below:

[0084] Guanidine content determination - accuracy result record:

[0085]

[0086] Conclusion: The experimental results showed that the recovery rate was 100.5% and the RSD of 9 solutions was 0.71%, which proved that the method had good accuracy.

[0087] 4. Durability

[0088] 4.1 Solution stability

[0089] Take blank solution, reference linear solution and test solution, and measure according to the law at 0h, 0.5h and 1h respectively (test solution, blank solution and reference linear solution are prepared according to the law), record the absorbance value, calculate the total amount of guanidine groups by standard curve method, and examine its stability.

[0090] The results are shown in the table below:

[0091] Total guanidine group determination-solution stability result recording

[0092]

[0093] Conclusion: The experimental results show that the determination results at 0h, 0.5h and 1h are basically consistent, and the RSD value is 0.13%, which is less than 2.0%, proving that the solution stability of this method is good.

[0094] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting the total amount of guanidines in body fluids of patients with renal failure, characterized in that: The following steps are involved: S1: Sample pretreatment and preparation of test solution: S11: Sample collection and processing 1: 5 ml of blood from a patient with renal failure was collected and centrifuged at 3000 rpm for 10 minutes. The supernatant was filtered through a 0.45 μm filter membrane and stored in a refrigerator at 2-8°C until use. S12: Sample collection and processing 2: 5 ml of urine and peritoneal dialysis waste fluid from a patient with renal failure were collected, the supernatant was filtered through a 0.45 μm filter membrane, and then stored in a refrigerator at 2-8°C for later use; S13: Prepare the test sample stock solution: accurately measure 3 ml of sample 1 or sample 2 into a 10 ml volumetric flask, dilute to the mark with water, and shake well; S14: Prepare the test solution: accurately measure 5 ml of the test sample stock solution and place it in a colorimetric tube. Add 1 ml each of 0.1% 8-hydroxyquinoline ethanol solution and 10% sodium hydroxide solution, mix well, add 4 ml of sodium hypobromite solution, mix well, let stand for 5 minutes, then add 2 ml of 10% guanidine hydrochloride aqueous solution and mix well to prepare the test solution. S2: Preparation of reference solution: Take 2-guanidinosuccinic acid reference substance, accurately weigh it, dissolve it in water and dilute it to make 300 μg of 2-guanidinosuccinic acid per 1 ml; S3: Preparation of reference linear solution: Linearity reference solution: Accurately measure 0.5ml, 1ml, 2ml, 3ml and 4ml of the reference solution and place them in colorimetric tubes respectively. Add 4.5ml, 4ml, 3ml, 2ml and 1ml of water in that order. Add 1ml each of 0.1% 8-hydroxyquinoline ethanol solution and 10% sodium hydroxide solution, mix well. Add 4ml of sodium hypobromite solution, mix well and let stand for 5 minutes. Then add 2ml of 10% guanidine hydrochloride aqueous solution and mix well. These are used as linearity reference solutions 1 / 2 / 3 / 4 / 5 respectively. S4: Preparation of blank solution: Accurately measure 5 ml of water and place it in a colorimetric tube. Add 1 ml each of 0.1% 8-hydroxyquinoline ethanol solution and 10% sodium hydroxide solution, mix well. Add 4 ml of sodium hypobromite solution, mix well, let stand for 5 minutes, then add 2 ml of 10% guanidine hydrochloride aqueous solution and mix well. S5: taking each of the above solutions and testing them by UV-visible spectrophotometry; Calculation formula: Where: 58 is the molecular weight of guanidine group; 175 is: molecular weight of 2-guanidinosuccinic acid; 10 / 3 is the dilution factor of the test sample stock solution; A is: the absorbance value of the test solution after deducting the blank; b is: the intercept of the linear equation; s is: the slope of the linear equation; 13 is: total volume of test solution, ml; 5 is: the sampling volume of the test sample stock solution, ml.

2. The method for detecting the total amount of guanidines in body fluids of patients with renal failure according to claim 1, wherein: The measurement conditions of step S5 are: wavelength of 500 nm, integration time of 0.2 s.

3. The method for detecting the total amount of guanidines in body fluids of patients with renal failure according to claim 1, wherein: The sodium hypobromite should be no less than analytical grade; the 8-hydroxyquinoline should be no less than analytical grade.

4. The method for detecting the total amount of guanidines in body fluids of patients with renal failure according to claim 1, wherein: The sodium hydroxide should be no less than analytical grade; the guanidine hydrochloride should be no less than analytical grade; and the ethanol should be no less than 95% analytical grade.

5. The method for detecting the total amount of guanidines in body fluids of patients with renal failure according to claim 1, wherein: The samples 1 and 2 processed in step S1 need to be sealed and stored in a refrigerator.

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