WO2021135596A1 - Method for measuring carbohydrate content in ferric carboxymaltose - Google Patents

Abstract

A method for measuring a carbohydrate content in ferric carboxymaltose. The specific steps are: performing high-temperature acidification degradation treatment on a ferric carboxymaltose sample to be measured; then adjusting a pH value of a solution to produce a precipitate, so as to remove a ferric ligand; centrifugally filtering, and taking the filtrate; measuring the solution by means of an evaporative light scattering detector in high performance liquid chromatography; and quantitatively measuring a glucose standard substance by means of an external standard method, and calculating to obtain a standard curve, so as to obtain the content of carbohydrate in a ferric carboxymaltose sample solution. The method can rapidly and accurately obtain the carbohydrate content of ferric carboxymaltose, and can be used for the quality control of ferric carboxymaltose active pharmaceutical ingredient samples and injection products.

Description

Method for detecting carbohydrate content in carboxymaltose iron

METHOD FOR DETECTING CARBOHYDRATE CONTENT IN FERRIC CARBOXYMALTOSE

This application claims the priority of a Chinese patent application filed with the Chinese Patent Office on December 30, 2019, the application number is 201911395250.9, and the invention title is "Method for Detecting Carbohydrate Content in Ferric Carboxymaltose", the entire content of which is incorporated herein by reference. Applying.

Technical field

This application relates to the field of drug analysis, and in particular to a method for detecting the content of carbohydrates in iron carboxymaltose.

Background technique

Carboxymaltose iron belongs to the third generation of iron supplement products, the original research manufacturer Vifor, Switzerland, is a typical type I iron complex. It has the characteristics of stable product properties and strong binding capacity of iron and ligand. Compared with the current mainstream iron supplements, iron carboxymaltose has an iron utilization efficiency similar to that of iron sucrose, but it does not have the disadvantage of iron dextran which can cause allergic reactions. Due to the stable iron ligand binding capacity of carboxymaltose iron, iron can be released at a slow controlled rate, thereby greatly reducing the risk of iron poisoning caused by the generation of free radicals.

Carboxylic iron maltose is a nanoparticle formed by the combination of iron hydroxide and maltodextrin. The center of the particles is the iron hydroxide core, and the carbohydrate ligands are wrapped to form a shell. The main function of the carbohydrate ligand shell is to stabilize the iron core, control the release of iron, and maintain the particles in a suspended colloidal state. After intravenous injection into the human body, carboxymaltose iron gradually releases iron in the blood system, which is then absorbed in the bone marrow and liver to synthesize hemoglobin.

As an important component of carboxymaltose iron, carbohydrates determine the release rate and availability of iron in the human body. The detection of iron content in iron agents is more common, but the carbohydrates as ligands have not been reported in the literature because of the many impurities after the sample processing process and the low sensitivity of the instrument detection.

Summary of the invention

In view of this, this application provides a method for detecting carbohydrates in iron carboxymaltose, which includes the following steps:

Step 1. Weigh the carboxyl maltose iron, mix it with acid and then heat it to cool down, adjust the pH value, make the pure water constant volume, take the solution, centrifuge and filter, collect the filtrate to obtain the solution to be tested;

Step 2. Take the glucose standard to prepare a standard solution with a concentration gradient;

Step 3. Inject the standard solution, use acetonitrile-water-triethylamine as the mobile phase, and detect by high performance liquid chromatography-evaporative light scattering detection. The peak area obtained is the ordinate and the concentration of the standard solution is the horizontal Coordinates to get the standard curve;

Step 4. Inject the test solution, use acetonitrile-water-triethylamine as the mobile phase, detect by high performance liquid chromatography-evaporative light scattering detection method, obtain the peak area, and obtain the peak area according to the standard curve obtained in step 3. State the content of carbohydrates.

In some specific embodiments of the present application, the volume ratio of acetonitrile, water, and triethylamine in the mobile phase is (700～750):(200～250):(1～2); in some embodiments In the mobile phase, the volume ratio of acetonitrile, water, and triethylamine is 750:250:2.

In some specific embodiments of the present application, in step 3 or step 4, the high performance liquid chromatography adopts a 3.5 μm aperture amino chromatographic column, column temperature: 30-40°C, flow rate: 1.0-1.5 ml/min; in some embodiments Column temperature: 35°C, flow rate: 1ml/min.

In some specific embodiments of the present application, in step 3 or step 4, the detection temperature of evaporative light scattering detection is 45-55°C, and the flow rate is 1.0-2.0 L/min; in some embodiments, the detection temperature is 50°C, The flow rate is 1.5L/min.

In some specific embodiments of the present application, in step 1, in g/mL, the molar ratio of the carboxymaltose iron to the acid is a mass-volume ratio of (0.1～5.0):(6～12) g/ ml.

In some specific embodiments of the present application, in step 1, the acid is one or a mixture of two or more of hydrochloric acid, sulfuric acid or nitric acid; the concentration of the acid is 4-8 mol/L, preferably 6 mol/L The heating temperature is 100°C, the heating time is 0.5 to 1h; the cooling is reduced to 25°C ± 5°C.

In some specific embodiments of the present application, in step 1, the pH value is 6-8; the pH value is adjusted using lye, and the lye includes one or both of sodium hydroxide solution and potassium hydroxide solution. Kind.

In some specific embodiments of the present application, in step 1, the pH value is adjusted to 6.5-7.5 by using a 6 mol/L sodium hydroxide solution.

In some specific embodiments of the present application, in step 3 or 4, the HPLC column adopts XBridge™ Amide.

In some specific embodiments of the application, the carbohydrate is carboxymaltodextrin.

This application provides a method for measuring carboxymaltose iron carbohydrate suitable for industrial production. The HPLC-ELSD combination technology has high detection accuracy, and the linear correlation coefficient R ^{2 of the} standard curve can reach 0.99. It can efficiently and accurately detect the carbohydrate content of multiple batches of carboxymaltose iron at one time. There is no carbohydrate detection item for iron products. It is clearly reported in the literature that this is of great significance to the research of carbohydrate iron supplement drugs. The method can be used in the quality control of carboxymaltose iron bulk drug samples and injection products.

Description of the drawings

In order to more clearly describe the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art.

Figure 1 shows the standard curve spectrum;

Figure 2 is a superimposed spectrum of the samples of Example 1 and Example 2;

Figure 3 shows the spectrum of a standard glucose sample;

Figure 4 shows the glucose detection spectrum in the effect example.

Detailed ways

This application discloses a method for detecting the content of carbohydrates in iron carboxymaltose. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all deemed to be included in this application. The methods and applications of this application have been described through preferred embodiments. It is obvious that relevant personnel can make changes or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit and scope of this application to achieve and Apply the technology of this application.

The embodiment of the application provides a method for detecting the content of carbohydrates in iron carboxymaltose, which includes the following steps:

(1) Weigh the carboxyl maltose iron, mix it with acid and then heat it to cool down, adjust the pH value, make the volume of pure water constant, take the solution, centrifuge and filter, collect the filtrate to obtain the solution to be tested;

(2) Take glucose standard product to prepare a standard solution with concentration gradient;

(3) Injecting the standard solution, using acetonitrile-water-triethylamine as the mobile phase, and detecting by HPLC-ELSD, the peak area obtained is the ordinate and the concentration of the standard solution is the abscissa to obtain the standard curve;

(4) Inject the solution to be tested, use acetonitrile-water-triethylamine as the mobile phase, and detect by HPLC-ELSD method to obtain the peak area. According to the standard curve obtained in step (3), the ratio of the carbohydrate is obtained. content.

The examples of this application successfully provide a method for determining carbohydrates in carboxymaltose iron, which is degraded by acidification and adjusted the pH value to produce precipitation to remove the iron ligands to obtain the solution to be tested, using evaporative light scattering in high performance liquid chromatography The detector performs detection, and at the same time, the glucose standard is injected according to the external standard method, and the concentration and peak area of the standard solution are used for regression to calculate the standard curve, thereby obtaining the carbohydrate content in the carboxymaltose iron sample solution. The method can efficiently and accurately detect the carbohydrate content of multiple batches of carboxymaltose iron at one time, fills the gap in the carbohydrate detection method of iron products in the field, and is of great significance for the research of carbohydrate iron supplement drugs.

Optionally, the volume ratio of acetonitrile, water, and triethylamine in the mobile phase is (700-750): (200-250): (1-2). For the determination of carbohydrates in carboxymaltose iron, adjusting the components of the mobile phase to this range will help improve the peak shape and increase the accuracy of the detection results. Preferably, the volume ratio of the acetonitrile, water and triethylamine is 750:250:2.

Optionally, in step (3) or step (4), in order to further improve the accuracy of the detection result, HPLC adopts a 3.5 μm aperture amino chromatographic column, column temperature: 30-40° C., flow rate: 1.0-1.5 ml/min. It is understandable that those skilled in the art can adjust within the above range as required. For example, the column temperature can be 32°C, 35°C, 38°C, etc., and the flow rate can be 1.2ml/min, 1.3ml/min, 1.4ml/min. min etc.

Further, the HPLC column preferably adopts XBridge™ Amide.

Optionally, in step (3) or step (4), in order to further improve the accuracy of the detection result, the detection temperature of the ELSD is 45-55° C., and the flow rate is 1.0-2.0 L/min. It is understandable that those skilled in the art can adjust within the above range as required. For example, the detection temperature can be 48°C, 50°C, 52°C, etc., and the flow rate can be 1.2L/min, 1.5L/min, 1.8L/min. min etc.

Optionally, in step (1), in g/mL, the mass-volume ratio of the carboxymaltose iron to the acid is (0.1-5.0): (6-12) g/ml. It is understandable that those skilled in the art can adjust within the above range as required. For example, the mass of the iron carboxymaltose can be 0.2g, 0.5g, 1g, 2g, 3g, 4g, etc. and integer multiples of these values. The volume of the acid can be 8ml, 10ml, 11ml, etc. and integer multiples of these values.

Optionally, in step (1), the acid is one or a mixture of more than one of hydrochloric acid, sulfuric acid or nitric acid; the concentration of the acid is 4-8 mol/L; the heating temperature is 100°C , The heating time is 0.5-1h; the cooling is reduced to 25±5°C. It is understandable that those skilled in the art can adjust within the above range as required. For example, the concentration of the acid can be 5 mol/L, 6 mol/L, 7 mol/L, etc., and the heating time is 0.6 h, 0.7 h , 0.8h, 0.9h, etc.

Optionally, in step (1), in order to better control the dissociation of the sample in the analysis process and improve the detection accuracy, the pH value is 6-8; the pH value is adjusted using lye, and the lye includes One or both of sodium hydroxide solution or potassium hydroxide solution.

Further, as a preference, the pH value is adjusted to 6.5-7.5 by using a 6 mol/L sodium hydroxide solution.

Above, the carbohydrate in the iron carboxymaltose is carboxymaltodextrin.

The embodiment of the application provides a method for detecting carbohydrates in carboxymaltose iron. The specific steps include: accurately weighing carboxymaltose iron, adding an equivalent concentration of hydrochloric acid solution, heating at a high temperature and then reducing to room temperature, and adjusting the pH of the sodium hydroxide solution to a certain level Range, purified water to a constant volume; take the solution to centrifuge and filter to obtain the test sample; select the glucose standard, use acetonitrile-triethylamine mobile phase, use HPLC to separate and detect by evaporative light scattering detector, from the peak position to the peak end Position, integrate the signal peak along the baseline, and calculate the carbohydrate content based on the standard injection concentration.

In some embodiments, 0.1~5.0g of carboxymaltose iron is accurately weighed, 10ml 6mol/L hydrochloric acid solution is added to completely dissolve the sample, the reaction is heated at 100°C for 0.5~1h, and the pH is adjusted to with 6mol/L sodium hydroxide solution 6～8, dilute to a 200ml volumetric flask with purified water. Centrifuge and filter 10ml of the treated solution.

In some embodiments, when the sample is weighed, 0.1 to 1.2 g of carboxymaltose iron is accurately weighed.

In some embodiments, when the sample is processed, the pH is adjusted to 6.5-7.5 with a 6 mol/L sodium hydroxide solution.

In some embodiments, when performing separation and detection, the mobile phase used is 750 ml of acetonitrile and 250 ml of purified water, mix well, add 2 ml of triethylamine, and ultrasonically degas it.

In some embodiments, when performing separation and detection, it is characterized in that: the glucose standard is formulated into 0.2mg/ml, 0.4mg/ml, 0.6mg/ml, 0.8mg/ml, 1.0mg/ml, 1.2mg/ml standard solution.

In some embodiments, the HPLC separation system selects a 3.5 μm aperture amino chromatographic column, column temperature: 30-40° C., flow rate: 1.0-1.5 ml/min.

In some embodiments, when performing separation and detection, the HPLC separation system selects a 3.5 μm aperture amino chromatographic column, column temperature: 35° C., flow rate: 1 ml/min.

In some embodiments, ELSD detector temperature: 45-55°C, flow rate 1.0-2.0 L/min.

In some embodiments, when performing separation and detection, the detector is set at a temperature of 50° C. and a flow rate of 1.5 L/min.

Compared with the prior art, the beneficial effects of this application are: this application uses high performance liquid chromatography connected with an evaporative light scattering detector (HPLC-ELSD method) to determine the content of carboxymaltose iron carbohydrates, and uses glucose standard products to make a standard curve , The method of calculating the carbohydrate content of the sample by integrating the standard product and the sample. This application can quickly and accurately obtain the carbohydrate content of carboxymaltose iron, which has positive significance for the research on iron products and the improvement of the intrinsic quality of the medicine.

The raw materials and reagents used in the method for detecting the content of carbohydrates in iron carboxymaltose provided in this application are all commercially available.

The application will be further elaborated below in conjunction with the examples:

Example 1

(1) Instrument: High Performance Liquid Chromatography: Thermo Fisher Scientific (Model UltiMate3000), Evaporative Light Scattering Detector: Alltech (Model 3300). Other high-performance liquid chromatography instruments with the above-mentioned accessories and functions are also available.

(2) Chromatographic conditions:

Chromatographic column: Chromatographic column: XBridge ^TM Amide, 3.5μm, 4.6*250mm;

Mobile phase: accurately measure 750ml of acetonitrile and 250ml of purified water, mix well, and add 2ml of triethylamine.

Flow rate: 1.0ml/min.

Injection volume: 20μl.

Weigh accurately 0.4003g carboxymaltose iron, add 6ml 6mol/L hydrochloric acid solution, heat at 100℃ for 1h, add 30ml purified water, add 6mol/L sodium hydroxide solution to adjust the pH to 6.5, and transfer to a 200ml volumetric flask , Make up to the mark with purified water. Centrifuge 10ml of the solution and filter it through a 0.22μm filter membrane to obtain a sample.

Preparation of the reference solution: a solution containing 0.2mg, 0.4mg, 0.6mg, 0.8mg, 1.0mg, 1.2mg of anhydrous glucose per milliliter.

A 20μl standard solution was injected, and the concentration and peak area of the standard solution were used for regression to obtain a standard linear unary equation y = 335.37x-22.201, and the correlation coefficient was r ² = 0.999. Inject 20μl of the sample solution and bring the peak area into the standard curve to obtain the concentration of glucose in the sample solution.

Calculation formula:

Where:

T: The concentration of glucose in the sample solution (mg/ml)

V: constant volume of sample solution (ml)

m: the mass of the sample (mg)

The peak area of the FCM180420-AK01 batch was 198.1422, and the sample concentration was calculated to be 0.6570 (mg/ml). According to the results, the carbohydrate content of the sample data was calculated to be 29.54%.

The standard curve spectrum is shown in Figure 1, the sample spectrum is shown in Figure 2, and the glucose standard sample spectrum is shown in Figure 3.

The peak areas corresponding to the standards of different concentrations are shown in Table 1 below.

Table 1 Peak area of standard products

Standard mg/ml	Peak area
0.2011	50.5028
0.3965	109.2465
0.6031	175.3608
0.7987	242.1413
0.9969	311.4723
1.2074	387.2528

Example 2

(1) Instrument: High Performance Liquid Chromatography: Thermo Fisher Scientific (Model UltiMate3000), Evaporative Light Scattering Detector: Alltech (Model 3300). Other high-performance liquid chromatography instruments with the above-mentioned accessories and functions are also available.

(2) Chromatographic conditions:

Chromatographic column: Chromatographic column: XBridge ^TM Amide, 3.5μm, 4.6*250mm;

Mobile phase: accurately measure 750ml of acetonitrile and 250ml of purified water, mix well, and add 2ml of triethylamine.

Flow rate: 1.0ml/min.

Injection volume: 20μl.

Accurately weigh 0.2874g carboxymaltose iron, add 6ml 6mol/L hydrochloric acid solution, heat at 100℃ for 1h, add 30ml purified water, add 6mol/L sodium hydroxide solution to adjust the pH to 6.5, and transfer to a 200ml volumetric flask , Make up to the mark with purified water. Centrifuge 10ml of the solution and filter it through a 0.22μm filter membrane to obtain a sample.

Preparation of the reference solution: a solution containing 0.2mg, 0.4mg, 0.6mg, 0.8mg, 1.0mg, 1.2mg of anhydrous glucose per milliliter.

A 20μl standard solution was injected, and the concentration and peak area of the standard solution were used for regression to obtain a standard linear unary equation y = 335.37x-22.201, and the correlation coefficient was r ² = 0.999. Inject 20μl of the sample solution and bring the peak area into the standard curve to obtain the concentration of glucose in the sample solution.

Calculation formula:

Where:

T: The concentration of glucose in the sample solution (mg/ml)

V: constant volume of sample solution (ml)

m: the mass of the sample (mg)

The peak area of the FCM180608-AK01 batch is 122.1473, and the sample concentration is calculated to be 0.4374 (mg/ml). According to the results, the carbohydrate content of the sample data is calculated as: 27.39%.

The standard curve spectrum is shown in Figure 1, the sample spectrum is shown in Figure 2, and the glucose standard sample spectrum is shown in Figure 3.

The sample spectrum information of Example 1 and Example 2 is shown in Table 2 below.

Table 2 Spectrogram information of Example 1 and Example 2

The spectrum information of the glucose standard sample is shown in Table 3 below.

Table 3 Reference substance spectrum information

Effect example 1 Accuracy detection

In order to verify the accuracy of this method, we selected glucose standards for testing under the same sequence, and the accuracy of the method has been verified.

(1) Instrument: High Performance Liquid Chromatography: Thermo Fisher Scientific (Model UltiMate3000), Evaporative Light Scattering Detector: Alltech (Model 3300). Other high-performance liquid chromatography instruments with the above-mentioned accessories and functions are also available.

(2) Chromatographic conditions:

Chromatographic column: Chromatographic column: XBridge ^TM Amide, 3.5μm, 4.6*250mm;

Mobile phase: accurately measure 750ml of acetonitrile and 250ml of purified water, mix well, and add 2ml of triethylamine.

Flow rate: 1.0ml/min.

Injection volume: 20μl.

Accurately weigh 0.1202g glucose standard, transfer it to a 200ml volumetric flask, and dilute to the mark with purified water to prepare a 0.6mg/ml concentration solution. Centrifuge 10ml of the solution and filter it through a 0.22μm filter membrane to obtain a sample.

Preparation of the reference solution: a solution containing 0.2mg, 0.4mg, 0.6mg, 0.8mg, 1.0mg, 1.2mg of anhydrous glucose per milliliter.

A 20μl standard solution was injected, and the concentration and peak area of the standard solution were used for regression to obtain a standard linear unary equation y = 335.37x-22.201, and the correlation coefficient was r ² = 0.999. Inject 20μl of the sample solution and bring the peak area into the standard curve to obtain the concentration of glucose in the sample solution. The peak area of the glucose standard was measured to be 178.9323, the sample concentration was calculated to be 0.5997 (mg/ml), and the recovery rate was 99.95%. The glucose detection spectrum is shown in Figure 4, and the spectrum information is shown in Table 4 below.

Table 4 Glucose detection spectrum information

The above are only the preferred embodiments of this application. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of this application, several improvements and modifications can be made, and these improvements and modifications are also Should be regarded as the scope of protection of this application.

Claims (10)

1. The method for detecting the content of carbohydrates in iron carboxymaltose is characterized in that it comprises the following steps:

   Step 1. Weigh carboxyl maltose iron, mix it with acid and then heat it to cool down, adjust the pH value, make the pure water volume constant, take the solution, centrifuge and filter, collect the filtrate, and obtain the solution to be tested;

   Step 2. Take the glucose standard to prepare a standard solution with a concentration gradient;

   Step 3. Inject the standard solution, use acetonitrile-water-triethylamine as the mobile phase, and detect by high performance liquid chromatography-evaporative light scattering detection. The peak area obtained is the ordinate and the concentration of the standard solution is the horizontal Coordinates to get the standard curve;

   Step 4. Inject the test solution, use acetonitrile-water-triethylamine as the mobile phase, detect by high performance liquid chromatography-evaporative light scattering detection method, obtain the peak area, and obtain the peak area according to the standard curve obtained in step 3. State the content of carbohydrates.
2. The method of claim 1, wherein the volume ratio of acetonitrile, water, and triethylamine in the mobile phase is (700-750): (200-250): (1-2).
3. The method according to claim 1, wherein in step 3 or step 4, the high performance liquid chromatography adopts a 3.5 μm aperture amino chromatography column, the column temperature: 30-40°C, and the flow rate: 1.0-1.5 ml/min.
4. The method according to claim 1, wherein in step 3 or step 4, the detection temperature of the evaporative light scattering detection is 45-55°C, and the flow rate is 1.0-2.0 L/min.
5. The method of claim 1, wherein in step 1, the mass-volume ratio of the carboxymaltose iron to the acid is (0.1～5.0): (6～12) g/mL in g/mL ml.
6. The method of claim 1, wherein in step 1, the acid is one or a mixture of two or more of hydrochloric acid, sulfuric acid or nitric acid; the concentration of the acid is 4-8 mol/L; The heating temperature is 100°C, the heating time is 0.5-1 h; and the temperature drop is reduced to 25±5°C.
7. The method of claim 1, wherein in step 1, the pH value is 6-8; the pH value is adjusted using lye, and the lye includes one of sodium hydroxide solution or potassium hydroxide solution. Kind or two kinds.
8. The method according to claim 6 or 7, wherein in step 1, the pH value is adjusted to 6.5-7.5 by using a 6 mol/L sodium hydroxide solution.
9. The method of claim 1, wherein in step 3 or 4, the chromatographic column of the high performance liquid chromatography adopts XBridge™ Amide.
10. The method of claim 1, wherein the carbohydrate is carboxymaltodextrin.

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