CN113029978A - Method for detecting phosphorus binding capacity of lanthanum-containing reagent - Google Patents

Abstract

The invention provides a method for detecting the phosphorus binding capacity of a lanthanum containing reagent, comprising the steps of: a) preparing an organic acid buffer: b) preparing a phosphate standard solution; c) preparing a sample solution and detecting: weighing a sample, and diluting with an organic acid buffer solution to obtain a sample solution; precisely adding a phosphate standard solution, shaking, taking a reaction solution, adding water to a constant volume to obtain a test solution, detecting the content of phosphorus by using a molybdenum-antimony resistance method, and measuring the absorbance; d) taking a phosphate standard solution with the same volume as that in the step c), adding water to fix the volume to the same volume as that in the step c), counting as a reference solution, and detecting the phosphorus content by using a molybdenum-antimony resistance method; e) the phosphorus binding capacity was calculated. The method is simple and convenient to operate, low in cost and similar to the environment of the body fluid of the digestive tract, and can accurately evaluate the phosphorus binding capacity of the lanthanum-containing reagent.

Description

A kind of method for detecting phosphorus binding ability of lanthanum-containing reagent

technical field

The invention belongs to the field of chemical detection, and relates to a method for detecting the phosphorus binding ability of a lanthanum-containing reagent.

Background technique

Hyperphosphatemia is a disease in which phosphate levels in the blood exceed normal levels due to abnormal renal metabolism; it is a common complication of chronic renal failure, especially in patients with end-stage renal disease, which can be seen in 80% of dialysis patients. A large number of data show that: hyperphosphatemia stimulates the parathyroid gland to secrete a large amount of parathyroid hormone (PTH), which is the cause of secondary hyperparathyroidism (SHPT), calcium and phosphorus product changes, vitamin D metabolism disorders, renal osteoarthritis. important factor in disease. Hyperphosphatemia is also closely related to serious cardiovascular complications such as coronary artery disease and heart valve calcification, which seriously reduces the quality of life of patients and is the main cause of increased mortality.

At present, the treatment measures for chronic renal failure hyperphosphatemia mainly include restricting phosphorus intake, adequate dialysis, using phosphorus binders, and parathyroidectomy if necessary. Because phosphorus is widely distributed in nature, it exists in almost all kinds of food, and those with high protein content also have high phosphorus content, so phosphorus restriction diet is not easy to achieve, clinical compliance is poor, and it is difficult to maintain adequate nutrition. Patients with end-stage renal disease require the use of phosphorus binders to reduce intestinal absorption of phosphorus to treat hyperphosphatemia.

An important quality indicator of phosphorus binders is phosphorus binding capacity. CN96193918.4 established a method for evaluating the phosphorus binding capacity of different crystalline water lanthanum carbonate samples, including the following operations:

a) Dissolve 13.75g anhydrous Na ₂ HPO ₄ and 8.5g NaCl in 1L deionized water to prepare a basic solution;

b) adding concentrated hydrochloric acid to adjust the 100ml base solution to pH3;

c) Take 5ml of the sample and pass it through a 0.02μm filter to obtain a sample at time 0. It was analyzed with the delta diagnostic colorimetric phosphorus kit;

d) Add 5ml of fresh basal solution to make it reach 100ml again, and adjust the pH value to about 3 again;

e) adding a certain amount of dry powdered lanthanum carbonate hydrate to make lanthanum more than twice the moles of phosphate, and stirring at room temperature;

f) Sampling is carried out at intervals of 0.5-10 minutes, and the percentage of phosphate is determined as in c) above.

The subsequent patent documents CN103127042A, CN 103127041A, and TW201613560A basically use this method to detect phosphorus binding capacity. The method as disclosed in CN103127042A comprises the following steps:

1) Preparation of phosphate solution: Weigh 0.2197g of anhydrous potassium dihydrogen phosphate and dissolve it in water, transfer it into a 1000ml volumetric flask, add 5ml of sulfuric acid solution, dilute with water to the mark, and obtain a phosphate containing 50.0μg/ml of phosphorus. Phosphate solution;

2) Combination with phosphorus: Weigh 13.9 mg of lanthanum carbonate, lanthanum lactate hemihydrate, lanthanum lactate trihydrate, lanthanum lactate tetrahydrate, and lanthanum lactate hexahydrate in the amount of lanthanum, and add to 1~ No. 6 beaker containing 100 ml of the phosphate solution obtained in step 1) was fully combined with phosphorus for 60 minutes under stirring.

3) Determination of content: Calculate the concentration of No. 1 to No. 6 before adding lanthanum-containing compounds, dilute to 5.0 μg/ml in proportion, add 5 ml to a 25 ml volumetric flask, add 10 ml of water to dilute, and then add to the volumetric flask. 1ml 10% ascorbic acid solution, mix well, add 2ml molybdate solution after 30 seconds, mix well, stand for 15 minutes, centrifuge, take the supernatant and measure it by molybdenum antimony antibody method, and further obtain the remaining water in each water sample. Phosphorus content.

However, phosphorus-binding capacity is an important evaluation index for the effectiveness of phosphorus-binding agents, which must be studied and evaluated. It is not clear whether the existing technology can be applied to the determination of lanthanum polystyrene sulfonate, so it is necessary to further develop a new detection method for lanthanum-containing phosphorus binders.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a method for detecting the phosphorus-binding ability of a lanthanum-containing reagent. After repeated tests, the inventor has developed a measurement method using an organic acid buffer as a standard solution and a sample preparation system. Under the buffer system provided by the present invention, the combination of lanthanum-containing reagents and phosphorus can be more accurately detected, and the amount of phosphorus removed by calculation can be more accurately detected. quality to assess the ability of lanthanum-containing reagents to bind phosphorus. The detection method of the invention uses a buffer with a certain pH value as a standard solution and a sample preparation system. It has been verified that compared with the prior art, the phosphorus binding capacity can be more accurately determined, and it is also more suitable for the characteristics of lanthanum polystyrene sulfonate samples. .

In one aspect, the present invention provides a method for detecting the binding ability of a lanthanum-containing reagent to phosphorus, the method comprising the following steps:

a) Preparation of organic acid buffer:

b) Preparation of phosphate standard solution: the phosphate standard solution is obtained by configuring the organic acid buffer in step a);

c) configure the lanthanum-containing sample solution with the organic acid buffer in step a), add the phosphate standard solution, count it as the test solution, and use the molybdenum-antimony antibody method to detect the phosphorus content;

d) take the phosphate standard solution equal to step c), dilute to an equal volume with step c) with water, count it as a reference solution, and use the molybdenum antimony antibody method to detect the phosphorus content;

e) The ability of a unit amount of lanthanum to bind phosphorus is calculated by the reduction in phosphorus content.

In a preferred embodiment, the method comprises the steps of:

a) Preparation of organic acid buffer: Weigh metal salt or ammonium salt of organic acid, add water to dissolve it, and adjust pH value with acid to obtain organic acid buffer;

b) preparing a phosphate standard solution; weighing phosphate, and diluting it with the organic acid buffer of step a) to obtain a phosphate standard solution;

c) Preparation of sample solution and detection: Weigh the lanthanum-containing reagent sample and dilute it with an organic acid buffer to obtain a lanthanum-containing reagent sample solution; accurately add the phosphate standard solution, shake it, take the reaction solution, and dilute to a certain volume with water , counted as the test solution, use the molybdenum antimony method to measure the absorbance;

d) take the phosphate standard solution equal to step c), dilute to an equal volume with step c) with water, count it as a reference solution, and use the molybdenum antimony antibody method to detect the phosphorus content;

e) The ability of a unit amount of lanthanum to bind phosphorus is calculated by the reduction in phosphorus content.

According to the method of the present invention, the lanthanum-containing phosphorus binder is lanthanum carbonate and/or lanthanum polystyrene sulfonate, or a composition containing lanthanum carbonate and/or lanthanum polystyrene sulfonate.

According to the method of the present invention, wherein in step a), the organic acid buffer is prepared using a metal or ammonium salt of an organic acid, wherein the acid group in the metal or ammonium salt of the organic acid is selected from formate , acetate, propionate, alkyl sulfate, alkyl sulfonate and citrate. Wherein, the metal ion in the metal salt of the organic acid is selected from sodium ion and potassium ion.

According to the method of the present invention, the following formula is specifically used to calculate:

in,

A: The absorbance of the test solution;

A ₀ : the absorbance of the control solution;

C ₀ : the phosphorus concentration in the phosphate standard solution, in g/ml;

V: the amount of phosphate standard solution, the unit is ml;

W: The weighing sample amount of the lanthanum-containing reagent, the unit is g;

F: The percentage of lanthanum in the lanthanum-containing reagent.

According to the method of the present invention, in step a), the mass fraction of the metal salt or ammonium salt of the organic acid is 0.5-10%.

According to the method of the present invention, in step a), the pH is 3-8.

According to the method of the present invention, wherein in step a), the pH is adjusted using hydrochloric acid, sulfuric acid, formic acid, acetic acid, propionic acid, alkylsulfuric acid, alkylsulfonic acid or citric acid.

According to the method of the present invention, wherein in step b), the phosphate is selected from sodium phosphate, sodium monohydrogen phosphate, sodium dihydrogen phosphate, potassium phosphate, potassium monohydrogen phosphate, potassium dihydrogen phosphate and ammonium phosphate and one or more of their hydrates.

According to the method of the present invention, wherein in step c) and step d), the described molybdenum-antimony antibody method for detecting phosphorus content comprises the following steps:

① Preparation of ascorbic acid solution: Weigh ascorbic acid and dilute with water;

②Preparation of molybdate solution: dissolve ammonium molybdate and antimony potassium tartrate in water, add ammonium molybdate solution to sulfuric acid solution under stirring, then add antimony potassium tartrate solution and mix well;

3. Take the test solution of step c) and the reference substance solution of step d) in equal amounts, add ascorbic acid solution respectively, add molybdate after mixing and stand, and use ultraviolet spectrophotometer to measure at the selected wavelength respectively. absorbance.

Compared with the prior art, the invention has the following advantages: through the screening of the release system of lanthanum polystyrene sulfonate, a detection method for the binding ability of lanthanum sulfonate to phosphorus is established, and the experiment proves that the operation is simple and the cost is low, which is similar to Digestive tract body fluid environment can accurately evaluate the binding capacity of lanthanum-phosphorus polystyrene sulfonate.

Detailed ways

The present invention will be further described in detail below with reference to the specific embodiments, and the given examples are only for illustrating the present invention, rather than for limiting the scope of the present invention.

Example 1

a) Prepared pH3.0 sodium formate buffer: weigh 41 g of sodium formate, add 1000 ml of water to dissolve it, and adjust pH3.0 with formic acid;

b) Preparation of phosphate standard solution: Weigh 0.62g of ammonium phosphate and dilute to 100ml with pH3.0 sodium formate buffer;

c) Preparation of sample solution and detection: Preparation of molybdate solution: Dissolve 13 g of ammonium molybdate and 0.35 g of antimony potassium tartrate in 100 ml of water, respectively. Under constant stirring, slowly add the ammonium molybdate solution to 300 ml of 50% concentrated sulfuric acid, then add the antimony potassium tartrate solution and mix well.

Preparation of ascorbic acid solution: Weigh 5 g of ascorbic acid and dilute to 50 ml with water.

Preparation of sample solution: Weigh about 178 mg of the sample, accurately weigh it, put it in a 250 ml conical flask, add 90 ml of pH 6.8 sodium acetate solution, and then accurately add 20.0 ml of phosphate standard solution, shake it in a water bath at 37 °C for 4 hours, Take 1.0ml of the reaction solution, centrifuge at 12000rpm for 10min, take 0.5ml of the supernatant after centrifugation, put it in a 25ml volumetric flask, dilute it with water to the mark, take 5.0ml and put it in a 25ml volumetric flask, add 1ml of ascorbic acid solution, mix well and let stand for 30s , add 2ml of molybdate solution, dilute to the mark with an appropriate amount of water, mix well, and use it as the test solution.

Take 0.05ml of phosphate standard solution, put it in a 25ml measuring bottle, dilute it with water to the mark, take 5ml and put it in a 25ml volumetric flask, add 1ml of ascorbic acid solution, mix well and let stand for 30s, add 2ml of molybdate solution, and make up with an appropriate amount of water. Volume to the mark, mix well, as a control solution. After the test solution and the control solution were allowed to stand for 15 minutes, the absorbance was measured at a wavelength of 600 nm using an ultraviolet spectrophotometer. The test results are shown in Table 1.

Table 1 The measurement results of phosphorus binding capacity obtained by the method of the present invention

It can be seen from the data in Table 1 that the method of the present invention can measure the phosphorus binding capacity of lanthanum carbonate and lanthanum polystyrene sulfonate with good repeatability and stable numerical value.

Comparative Example 1

This comparative example uses the existing method (the method described in CN96193918.4) to determine the phosphorus binding capacity of lanthanum carbonate and lanthanum polystyrene sulfonate, and the results are shown in Table 2.

Table 2 The measurement results of phosphorus binding capacity obtained by the existing method

It can be seen from the data in Table 2 that the phosphorus binding capacity of lanthanum carbonate can be determined by using the existing method, and the three determination results are relatively consistent and have good repeatability. However, when this method is used to measure the phosphorus binding capacity of lanthanum polystyrene sulfonate, the result fluctuates greatly and the repeatability is relatively poor, so the existing method is not suitable for the detection of lanthanum polystyrene sulfonate.

By analyzing the characteristics of lanthanum carbonate and lanthanum polystyrene sulfonate, the applicant believes that the reason for this result may be that the detection methods in the prior art are all established based on lanthanum salts of inorganic acids, which can dissolve lanthanum salts. In concentrated hydrochloric acid, it is ionic in pH3 solution, and lanthanum ions and phosphate radicals immediately generate lanthanum phosphate precipitation. Lanthanum polystyrene sulfonate is an organic acid lanthanum salt formed by combining lanthanum ions with strongly acidic cationic resins. It is insoluble in water and general acid, alkali solutions and organic solvents. It is a polymer polymer with good chemical stability. thing. The ion exchange of ion exchange resin is selective and has nothing to do with the pH value of the solution. Lanthanum ions will not be completely released in the pH3 solution, and the method cannot accurately detect the phosphorus binding capacity of lanthanum polystyrene sulfonate. Lanthanum styrene sulfonate also exchanges with other cations, and lanthanum ions are released from the stomach (acid environment) to the intestinal tract (neutral environment), and it has a certain ability to remove phosphorus. Phosphorus-binding capacity of lanthanum styrene sulfonate at different pH environments. Therefore, further development of new detection methods is required.

Comparing the data in Table 1 and Table 2, it can be seen that the method of the present invention determines the lanthanum carbonate with the same measurement results as the prior art, with high accuracy, and the method of the present invention can also accurately measure the phosphorus binding capacity of lanthanum polystyrene sulfonate.

Example 2

Different batches of lanthanum polystyrene sulfonate samples were detected using the method of Example 1, and the measurement results were as follows:

Table 3 Determination results of phosphorus binding capacity of lanthanum polystyrene sulfonate

batch number 1014 1018 1109 1114 1115 1g lanthanum to remove phosphorus (g) 0.259 0.256 0.239 0.234 0.244

It can be seen from the data in Table 3 that the difference in the binding capacity of polystyrene sulfonate lanthanum phosphorus is small between different batches, which proves that this method can be used for the determination of the binding capacity of polystyrene lanthanum sulfonate lanthanum phosphorus.

Example 3

In this example, the method of the present invention is used to detect the phosphorus binding capacity of lanthanum polystyrene sulfonate at different pH.

The characteristic of lanthanum polystyrene sulfonate is that it can show phosphorus binding capacity at different pH, which is not available in lanthanum carbonate, and the detection method of the prior art cannot realize the determination of phosphorus binding capacity at different pH. , the measurement results are shown in Table 4.

The measurement method under neutral conditions is as follows:

a) Prepared pH6.8 sodium acetate buffer: Weigh 100 g of potassium citrate, add 1000 ml of water to dissolve it, and adjust the pH to 6.8 with hydrochloric acid.

b) Preparation of phosphate standard solution: Weigh 0.72 g of sodium dihydrogen phosphate, and dilute to 100 ml with pH6.8 potassium citrate buffer.

c) Preparation of sample solution and detection: Preparation of molybdate solution: Dissolve 13 g of ammonium molybdate and 0.35 g of antimony potassium tartrate in 100 ml of water, respectively. Under constant stirring, slowly add the ammonium molybdate solution to 300 ml of 50% concentrated sulfuric acid, then add the antimony potassium tartrate solution and mix well.

Preparation of ascorbic acid solution: Weigh 5 g of ascorbic acid and dilute to 50 ml with water.

Weigh about 178 mg of lanthanum polystyrene sulfonate, accurately weigh it, put it in a 250 ml conical flask, add 90 ml of sodium acetate solution with pH 6.8, and then accurately add 15.0 ml of phosphate standard solution, shake it in a water bath at 37 ° C for 4 hours, Take 1.0ml of the reaction solution, centrifuge at 12000rpm for 10min, take 0.5ml of the supernatant after centrifugation, put it in a 25ml volumetric flask, dilute it with water to the mark, take 5.0ml and put it in a 25ml volumetric flask, add 1ml of ascorbic acid solution, mix well and let stand for 30s , add 2ml of molybdate solution, dilute to the mark with an appropriate amount of water, mix well, and use it as the test solution.

Take 0.05ml of phosphate standard solution, put it in a 25ml measuring bottle, dilute it with water to the mark, take 5ml and put it in a 25ml volumetric flask, add 1ml of ascorbic acid solution, mix well and let stand for 30s, add 2ml of molybdate solution, and make up with an appropriate amount of water. Volume to the mark, mix well, as a control solution. After the test solution and the control solution were allowed to stand for 15 minutes, the absorbance was measured at a wavelength of 700 nm using an ultraviolet spectrophotometer.

Table 4 Determination results of lanthanum phosphorus binding capacity of polystyrene sulfonate

batch number 1014 1018 1109 1114 1115 1g lanthanum to remove phosphorus (g) 0.244 0.266 0.234 0.234 0.251

It can be seen that the method of the present invention can realize the detection of the phosphorus binding ability of lanthanum polystyrene sulfonate under neutral conditions.

Example 4

In this example, the method of the present invention is used to detect the phosphorus binding capacity under alkaline conditions.

a) Potassium acetate buffer solution of pH 8.0 prepared: Weigh 5 g of potassium acetate, add 1000 ml of water to dissolve it, and adjust pH 8.0 with acetic acid.

b) Preparation of phosphate standard solution: Weigh 0.85 g of anhydrous dipotassium hydrogen phosphate, and dilute to 100 ml with pH 8.0 potassium acetate buffer.

c) Preparation of sample solution and detection: Preparation of molybdate solution: Dissolve 13 g of ammonium molybdate and 0.35 g of antimony potassium tartrate in 100 ml of water, respectively. Under constant stirring, slowly add the ammonium molybdate solution to 300 ml of 50% concentrated sulfuric acid, then add the antimony potassium tartrate solution and mix well.

Preparation of ascorbic acid solution: Weigh 5 g of ascorbic acid and dilute to 50 ml with water.

Preparation of sample solution: Weigh about 178 mg of lanthanum polystyrene sulfonate, accurately weigh it, put it in a 250 ml conical flask, add 90 ml of sodium acetate solution with pH 6.8, and then accurately add 5.0 ml of phosphate standard solution, at 37 ℃ Shake in a water bath for 4h, take 1.0ml of the reaction solution, centrifuge at 12000rpm for 10min, take 0.5ml of the supernatant after centrifugation, put it in a 25ml volumetric flask, dilute with water to the mark, take 5.0ml and put it in a 25ml volumetric flask, add 1ml of ascorbic acid solution, Mix well and let stand for 30s, add 2ml of molybdate solution, dilute to the mark with an appropriate amount of water, mix well, and use it as the test solution.

Take 0.05ml of phosphate standard solution, put it in a 25ml measuring bottle, dilute it with water to the mark, take 5ml and put it in a 25ml volumetric flask, add 1ml of ascorbic acid solution, mix well and let stand for 30s, add 2ml of molybdate solution, and make up with an appropriate amount of water. Volume to the mark, mix well, as a control solution. After the test solution and the control solution were left standing for 15 minutes, the absorbance was measured at a wavelength of 800 nm using an ultraviolet spectrophotometer. The test results are shown in Table 5.

Table 5 Determination results of phosphorus binding capacity of lanthanum polystyrene sulfonate

batch number 1014 1018 1109 1114 1115 1g lanthanum to remove phosphorus (g) 0.239 0.246 0.246 0.237 0.239

It can be seen that the method of the present invention can realize the detection of the phosphorus binding ability of lanthanum polystyrene sulfonate under alkaline conditions.

To sum up, the present invention provides a method for accurately determining the phosphorus-binding capacity of lanthanum polystyrene sulfonate, which is also applicable to other lanthanum-containing reagents, and can measure the phosphorus-binding capacity under different pH conditions, which is the largest of the phosphorus-binding reagents. Quality control of large-scale industrial production and clinical application testing provide methodological guarantee.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone who is familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, The protection scope of the present invention should be defined by the claims.

Claims (9)

1. A method for detecting the phosphorus binding capacity of a lanthanum containing reagent, the method comprising the steps of:

a) preparing an organic acid buffer:

b) preparing a phosphate standard solution: preparing a phosphate standard solution by using the organic acid buffer solution obtained in the step a);

c) preparing a lanthanum-containing sample solution by using the organic acid buffer solution obtained in the step a), adding a phosphate standard solution, counting as a test sample solution, and detecting the phosphorus content by using a molybdenum-antimony resistance method;

d) taking a phosphate standard solution with the same volume as that in the step c), adding water to fix the volume to the same volume as that in the step c), counting as a reference solution, and detecting the phosphorus content by using a molybdenum-antimony resistance method;

e) the ability of lanthanum to bind phosphorus per unit amount was calculated by the decrease in phosphorus content.

2. The method of claim 1, wherein the phosphorus-containing reagent is lanthanum carbonate and/or lanthanum polystyrenesulfonate, or a composition comprising lanthanum carbonate and/or lanthanum polystyrenesulfonate.

3. The process according to claim 1 or 2, wherein in step a) the organic acid buffer is prepared using a metal or ammonium salt of an organic acid, wherein the acid group in the metal or ammonium salt of an organic acid is selected from the group consisting of formate, acetate, propionate, alkylsulfate, alkylsulfonate and citrate; the metal ion in the metal salt of the organic acid is selected from sodium ion and potassium ion.

4. The method according to any one of claims 1 to 3, in particular using the following formula:

wherein,

a: absorbance of the test solution;

A₀: absorbance of the control solution;

C₀: the phosphorus concentration in the phosphate standard solution is g/ml;

v: the dosage of the phosphate standard solution is ml;

w: weighing a lanthanum-containing reagent by weight, wherein the unit is g;

f: the percentage of lanthanum in the lanthanum containing reagent.

5. The method according to any one of claims 1 to 4, wherein in step a), the mass fraction of the metal or ammonium salt of the organic acid is 0.5 to 10%.

6. The process according to any one of claims 1 to 5, wherein in step a) the pH is between 3 and 8.

7. The process according to any one of claims 1 to 6, wherein in step a) the pH is adjusted using hydrochloric acid, sulfuric acid, formic acid, acetic acid, propionic acid, alkyl sulfuric acid, alkyl sulfonic acid and/or citric acid.

8. The process according to any one of claims 1-6, wherein in step b) the phosphate is selected from one or more of sodium phosphate, sodium monohydrogen phosphate, sodium dihydrogen phosphate, potassium monohydrogen phosphate, potassium dihydrogen phosphate and ammonium phosphate, and hydrates thereof.

9. The method according to any one of claims 1-8, wherein in step c) and step d), the molybdenum-antimony resistance method for detecting the phosphorus content comprises the following steps:

preparing an ascorbic acid solution: weighing ascorbic acid, and diluting with water;

preparing molybdate solution: dissolving ammonium molybdate and potassium antimony tartrate in water, adding the ammonium molybdate solution into a sulfuric acid solution under stirring, adding the potassium antimony tartrate solution, and uniformly mixing;

and thirdly, respectively adding an ascorbic acid solution into the equal amount of the test solution in the step c) and the equal amount of the reference solution in the step d), uniformly mixing and standing, adding molybdate, and respectively measuring the absorbance at the selected wavelength by using an ultraviolet spectrophotometer.