Method for measuring content of iron in sucrose iron oxyhydroxide by high performance liquid chromatography

Abstract

The invention relates to a method for determining the iron content in sucrose iron oxyhydroxide by high performance liquid chromatography. The chromatographic conditions of the liquid chromatograph are as follows: octadecylsilane chemically bonded silica is selected as a filler, the column length is 150mm, the column temperature is 30 DEG C, and the flow velocity is 1.0 ml / min; the liquid chromatograph adopts reversed-phase high-performance liquid chromatography for isocratic elution, a mobile phase is a buffer solution and a polar organic solvent, the buffer solution is a phase A, the polar organic solvent is a phase B, and the polar organic solvent of the phase B in the mobile phase is acetonitrile; pre-column derivatization is carried out by adopting a complex reagent, an iron element in sucrose hydroxyl iron oxide is converted into bivalent iron, and then derivatization is carried out by using the complex reagent. The method has the advantages that the stable ferrous complex is generated by using the complex, and the derivatization-high performance liquid chromatography is adopted for determination, so that the detection specificity and accuracy are improved, background interference is effectively removed, and the iron content in a test sample is accurately determined.

Description

Technical Field

[0001] This invention relates to the field of quality control of sucrose ferric hydroxide raw materials, specifically to a method for determining the iron content in sucrose ferric hydroxide by high performance liquid chromatography. Background Technology

[0002] Sucrose ferric hydroxide is a novel oral iron-based, non-calcium-containing phosphate binder. Approved by the FDA in 2013, it is currently approved in the United States, Canada, Europe, Japan, and other regions for controlling serum phosphorus levels in dialysis patients. In 2025, sucrose ferric hydroxide chewable tablets became the first iron-based, non-calcium phosphate binder to be approved for marketing in my country by the NMPA (National Medical Products Administration) for controlling serum phosphorus levels in CKD patients undergoing hemodialysis or peritoneal dialysis. It consists of polynuclear iron(III)-hydroxyhydroxide, sucrose, and starch. The added sucrose prevents the aging of iron(III)-hydroxyhydroxide, thus maintaining its phosphate-binding capacity. The polynuclear iron(III)-hydroxyhydroxide is an insoluble component that is not absorbed or metabolized. It binds to dietary phosphate in the digestive tract through ligand exchange of hydroxyl groups and / or related water molecules, and is then excreted in feces, reducing intestinal phosphorus absorption and lowering serum phosphorus. Studies have found that ferric hydroxide has a strong phosphate-binding capacity within the physiologically relevant pH range of the gastrointestinal tract and does not increase iron overload.

[0003] Sucrose hydroxyl oxide has an effective phosphorus-lowering effect and is non-inferior to sevelamer. In a phase III clinical trial, the mean (SD) change in serum phosphorus levels from baseline to week 12 was comparable between the sucrose hydroxyl oxide group and the sevelamer group: −0.71 (0.60) mmol / L and −0.63 (0.52) mmol / L, with a difference of 0.08 mmol / L (0.05), 95% CI of −0.02 and 0.18, respectively. The lower limit of the difference is above the non-inferiority margin, thus demonstrating the non-inferiority of sucrose hydroxyl oxide to sevelamer. The results of this clinical trial indicate that sucrose ferric hydroxide was superior to sevelamer in reducing phosphorus levels in the early period (week 1): the mean (SD) change from baseline compared to sevelamer was −0.69 (0.55) mmol / L at week 4 and −0.37 (0.47) mmol / L at week 8, and −0.74 (0.56) and −0.59 (0.50) mmol / L at week 8. These clinical trial results are consistent with those of the pivotal phase III study PA-CL-05A and the Japanese population. This study provides evidence for the application of sucrose ferric hydroxide in reducing phosphorus in dialysis patients with hyperphosphatemia in China. Phosphate is a risk factor affecting vascular calcification, and a level of ≤55 mg / dL is recommended. In one study, phosphate levels decreased to the target range at week 8 and thereafter, suggesting that treatment of hyperphosphatemia with sucrose ferric hydroxide can inhibit vascular calcification. This study observed an increase in hemoglobin and a decrease in the accumulation of intravenous iron (IV-iron) after the application of sucrose hydroxyl oxide, suggesting that the drug can improve renal anemia. Some studies have reported that hemoglobin affects life outcomes, and excessive IV-iron may also increase cardiovascular and infection-related risks. Therefore, the use of sucrose hydroxyl oxide holds promise for improving survival by improving renal anemia and avoiding the risks associated with IV-iron. Furthermore, studies have shown that sucrose hydroxyl oxide pills are less burdensome than sevelamer, which will be associated with reducing patient medication burden and improving patient adherence.

[0004] Iron in this active pharmaceutical ingredient is an inorganic element, and derivatization methods are frequently used for its determination in analytical chemistry. For the determination of metallic elements, the traditional derivatization-colloid method involves treating the sample with derivatization reagents and colloidal flocculation, followed by spectrophotometric determination in the visible light region. This method has inherent errors, and the sucrose groups in this product also provide background interference, resulting in drawbacks such as a narrow measurement range and low accuracy. Therefore, through research, we have developed a new detection method and rigorously validated it to ensure its scientific rigor and to meet the needs of research and development and production. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for determining the iron content in sucrose ferric hydroxide by high performance liquid chromatography, which can solve the problems of existing technologies that use derivatization reagents and colloidal flocculation to treat samples and then use spectrophotometry in the visible light region for determination, which have certain errors and are also subject to interference from sucrose aggregates.

[0006] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: the iron content in sucrose ferric hydroxide is determined by high performance liquid chromatography, wherein the chromatographic conditions of the liquid chromatograph are as follows: octadecylsilane-bonded silica gel is used as the packing material, the column length is 150 mm, the column temperature is 30 °C, and the flow rate is 1.0 ml / min. The detector used is an ultraviolet-visible detector; The liquid chromatograph uses reversed-phase high-performance liquid chromatography with isocratic elution. The mobile phase is a buffer solution and a polar organic solvent. The buffer solution is phase A, and the polar organic solvent is phase B. The polar organic solvent in phase B of the mobile phase is acetonitrile. The mixing volume ratio of phase A to phase B is 50:50.

[0007] Furthermore, the buffer solution of phase A is a phosphate buffer solution with a pH of 4.0, and its components include one or more of phosphoric acid, sodium dihydrogen phosphate, disodium hydrogen phosphate, and potassium dihydrogen phosphate, with a buffer concentration of 30-60 mmol / L.

[0008] Furthermore, pre-column derivatization was performed using a complex reagent. First, the ferric element in sucrose hydroxyl iron oxide was converted to divalent iron using a reducing agent, and then derivatization was performed using a complex reagent.

[0009] Furthermore, the complex reagent contains a triazine group, which can form a stable complex compound with ferrous iron. The molar ratio of the complex reagent to iron in the test solution is 5 to 10:1. The reducing agent is hydroxylamine hydrochloride, and the amount used is 1 ml.

[0010] A method for determining the iron content in sucrose ferric hydroxide by high performance liquid chromatography, characterized by comprising the following steps: (1) Take the sucrose hydroxy ferric oxide test sample, weigh it accurately, place it in a volumetric flask, add the mobile phase to dissolve and dilute it to a solution containing 25 μg of iron per 1 mL, and use it as the test sample solution; (2) Take an appropriate amount of ferrous ammonium sulfate reference standard, accurately weigh it, dissolve it in water and quantitatively dilute it to prepare a solution containing 25 μg of divalent iron per 1 ml, as the reference solution; (3) Accurately measure 1 ml of the test solution and the reference solution, place them in 20 ml volumetric flasks, add 1 ml of 1.0 mg / ml 5,6-diphenyl-3-(2-pyridyl)-1,2,4-triazine (PDT) methanol solution and 1 ml of 0.1 g / ml hydroxylamine hydrochloride solution, and dilute to the mark with the mobile phase; (4) Take 20 μl each of the derivatized test sample and reference solution, inject them into the chromatograph, record the chromatogram, and calculate the iron content by summing the peak areas of the complexes formed by iron ions according to the following formula: ; In the formula: ---The sum of the peak areas of the main peak of the test sample solution; ---The sum of the peak areas of the main peak of the reference solution.

[0011] The advantages of this invention are as follows: This invention uses high performance liquid chromatography to determine the iron content of sucrose hydroxyl iron oxide after pre-column derivatization, thereby enabling the invention to effectively control the content of the active pharmaceutical ingredient, which is of indispensable significance for the quality control of sucrose hydroxyl iron oxide. By using a complexation method to generate a stable divalent iron complex, and then using liquid chromatography for determination, the specificity and accuracy of the detection are greatly improved by employing derivatization-high performance liquid chromatography. This method can effectively remove background interference and accurately determine the iron content in the test sample. From the perspectives of improving product quality, enhancing the safety of clinical drug use, and optimizing the process flow, this invention has significant commercial value and can form a technical barrier in enterprise standards, thereby increasing the added value of enterprise products. Attached Figure Description

[0012] Figure 1 This is the chromatogram of the test sample of the present invention; Figure 2 This is the blank interference chromatogram of the present invention; Figure 3 This is a comparison chart showing the pH value screening of the buffer solution according to the present invention. Detailed Implementation

[0013] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. The following embodiments will enable those skilled in the art to more fully understand this invention, but do not limit the invention to the scope of the described embodiments.

[0014] Sucrose ferric hydroxide, a mixture of ferric hydroxide and sucrose, can be written as: pn-FeOOH+x C 12 H 22 O 11+y (C6H 10 O5) n This invention employs a complexation approach to generate stable divalent iron complexes, which are then analyzed using a liquid chromatography method, significantly improving the specificity and accuracy of the detection.

[0015] This specific implementation method adopts the following technical solution, including the following steps: (1) Take the sucrose hydroxy ferric oxide test sample, weigh it accurately, place it in a volumetric flask, add the mobile phase to dissolve and dilute it to a solution containing 25 μg of iron per 1 mL, and use it as the test sample solution; (2) Take an appropriate amount of ferrous ammonium sulfate reference standard, accurately weigh it, dissolve it in water and quantitatively dilute it to prepare a solution containing 25 μg of divalent iron per 1 ml, as the reference solution; (3) Accurately measure 1 ml of the test solution and 1 ml of the reference solution, and place them in 20 ml volumetric flasks respectively. Add the complex reagent and the reducing agent. The complex reagent contains a triazine group, which can form a stable complex compound with ferrous iron. The molar ratio of the complex reagent to iron in the test solution is 5 to 10:1. The reducing agent is hydroxylamine hydrochloride, and the amount used is 1 ml.

[0016] In this embodiment, 1 ml of 1.0 mg / ml 5,6-diphenyl-3-(2-pyridyl)-1,2,4-triazine (PDT) methanol solution and 1 ml of 0.1 g / ml hydroxylamine hydrochloride solution were added, and the solution was diluted to the mark with the mobile phase. (4) Take 20 μl each of the derivatized test sample and reference solution, inject them into the chromatograph, record the chromatogram, and calculate the iron content by summing the peak areas of the complexes formed by iron ions according to the following formula: ; In the formula: ---The sum of the peak areas of the main peak of the test sample solution; ---The sum of the peak areas of the main peak of the reference solution.

[0017] The iron content in sucrose ferric hydroxide was determined by high performance liquid chromatography (HPLC). The chromatographic conditions of the HPLC instrument were as follows: octadecylsilane-bonded silica gel was used as the packing material, the column length was 150 mm, the column temperature was 30 ℃, and the flow rate was 1.0 ml / min. The detector used is an ultraviolet-visible detector; The liquid chromatograph uses reversed-phase high-performance liquid chromatography with isocratic elution. The mobile phase consists of a buffer solution and a polar organic solvent. The buffer solution is phase A, and the polar organic solvent is phase B. The polar organic solvent in phase B of the mobile phase is acetonitrile. The mixing volume ratio of phase A to phase B is 50:50.

[0018] Phase A buffer is a pH 4.0 phosphate buffer, which contains one or more of the following: phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, and potassium dihydrogen phosphate, with a concentration of 30-60 mmol / L.

[0019] This invention creatively explored a derivatization reagent system, using hydroxylamine hydrochloride to reduce iron in the analyte to divalent, and then using 5,6-diphenyl-3-(2-pyridyl)-1,2,4-triazine (PDT) as a complexing reagent to form a stable derivative. By adjusting the mobile phase ratio, the high-performance liquid chromatography system of this invention was finally established and the methodology was validated.

[0020] (1) Exploration of Derivatives 5,6-Diphenyl-3-(2-pyridyl)-1,2,4-triazine (PDT) can form stable compounds with metal ions under weakly acidic conditions. In this example, using a 40 mmol / L potassium dihydrogen phosphate buffer, and adjusting the pH with phosphoric acid, the derivative remained stable for 24 hours when the pH of the buffer in the mobile phase was 4.0. The concentrations of the derivative at different pH values ​​are shown in the table below, and the comparison graph of the buffer pH screening is shown in the figure below. Figure 3 As shown.

[0021] Time (h) pH 3.0 (μg / ml) pH 4.0 (μg / ml) pH 5.0 (μg / ml) 1 25.11 25.32 24.89 2 24.11 25.15 23.33 4 23.21 25.08 22.51 8 22.35 25.63 20.09 12 23.12 25.19 19.87 (2) Exploration of mobile phase Using buffer:acetonitrile ratios of 50:50 and 60:40, based on experimental results, the 50:50 ratio was determined to have the most suitable peak shape and retention time, allowing PDT to achieve baseline separation from the formed chelate. Results are shown in the appendix. Figure 1 and attached Figure 2 .

[0022] (3) Methodological verification Related matter methodological validation results project CP2020 Acceptable Standards Verification results Exclusivity The blank solvent should be free of interference. The solvent does not interfere with the determination of iron. Sample injection precision The RSD% of peak area and retention time after 6 repeated injections is no greater than 2%. Experimental results show that when the test sample and reference solution are injected continuously for 6 injections, the RSD of retention time and peak area is less than 2.0%, indicating that the injection precision of this method is good. Detection limit The concentration at a signal-to-noise ratio (S / N) of ≈3 is the limit of detection, and the concentration at a signal-to-noise ratio (S / N) of ≈10 is the limit of quantitation. The detection limit is approximately 0.1 μg / ml (1% of the sample). This method has good sensitivity and meets the detection requirements. Linearity and Range The correlation coefficient r of linear regression is not less than 0.995. Within the concentration range of 5–30 μg / ml, the concentration of this product exhibits a good linear relationship with the peak area. Solution stability The RSD% of the main peak area before and after sample placement is no greater than 2%. The experimental results show that the test solution, control solution, and system suitability solution are stable after being placed at room temperature for 24 hours. Repeatability The RSD% of the content and recovery results from the six determinations was no greater than 2%. Experimental results show that when the same batch of samples is measured in parallel six times, the RSD% of the content is no greater than 2%. This method has good repeatability. Durability After changing the conditions, the separation degree between the chelate main peak and the PDT peak under each condition met the requirements, and the change in the detected content was not significant. When the relative flow rate changes by ±10%, the column temperature changes by ±5℃, and the proportion of organic phase in the mobile phase changes by ±2%, the system applicability and test results show no significant changes. This method has good robustness. The validation results show that all indicators of this method meet the requirements of the 2020 edition of the Chinese Pharmacopoeia and are suitable for detection.

[0023] The foregoing has shown and described the basic principles and main features of the present invention, as well as its advantages. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A method for determining the iron content in sucrose ferric hydroxide by high performance liquid chromatography, characterized in that: The iron content in sucrose ferric hydroxide was determined by high performance liquid chromatography (HPLC). The chromatographic conditions of the HPLC instrument were as follows: octadecylsilane-bonded silica gel was used as the packing material, the column length was 150 mm, the column temperature was 30 ℃, and the flow rate was 1.0 ml / min. The detector used is an ultraviolet-visible detector; The liquid chromatograph uses reversed-phase high-performance liquid chromatography with isocratic elution. The mobile phase is a buffer solution and a polar organic solvent. The buffer solution is phase A, and the polar organic solvent is phase B. The polar organic solvent in phase B of the mobile phase is acetonitrile. The mixing volume ratio of phase A to phase B is 50:

50.

2. The method for determining the iron content in sucrose hydroxyl iron oxide by high performance liquid chromatography according to claim 1, characterized in that: The buffer solution of phase A is a phosphate buffer solution with a pH of 4.0, and its components include one or more of phosphoric acid, sodium dihydrogen phosphate, disodium hydrogen phosphate, and potassium dihydrogen phosphate, with a buffer concentration of 30-60 mmol / L.

3. The method for determining the iron content in sucrose hydroxyl iron oxide by high performance liquid chromatography according to claim 1, characterized in that: Pre-column derivatization was performed using a complex reagent. First, the ferric element in sucrose hydroxyl iron oxide was converted to divalent iron using a reducing agent, and then derivatization was performed using a complex reagent.

4. The method for determining the iron content in sucrose hydroxyl iron oxide by high performance liquid chromatography according to claim 3, characterized in that: The complex reagent contains a triazine group, which can form a stable complex compound with ferrous iron. The molar ratio of the complex reagent to iron in the test solution is 5 to 10:

1. The reducing agent is hydroxylamine hydrochloride, and the amount used is 1 ml.

5. The method for determining the iron content in sucrose hydroxyl iron oxide by high performance liquid chromatography according to claim 1, characterized in that: Includes the following steps: (1) Take the sucrose hydroxy ferric oxide test sample, weigh it accurately, place it in a volumetric flask, add the mobile phase to dissolve and dilute it to a solution containing 25 μg of iron per 1 mL, and use it as the test sample solution; (2) Take an appropriate amount of ferrous ammonium sulfate reference standard, accurately weigh it, dissolve it in water and quantitatively dilute it to prepare a solution containing 25 μg of divalent iron per 1 ml, as the reference solution; (3) Accurately measure 1 ml of the test solution and the reference solution, place them in 20 ml volumetric flasks, add 1 ml of 1.0 mg / ml 5,6-diphenyl-3-(2-pyridyl)-1,2,4-triazine methanol solution and 1 ml of 0.1 g / ml hydroxylamine hydrochloride solution, and dilute to the mark with the mobile phase; (4) Take 20 μl each of the derivatized test sample and reference solution, inject them into the chromatograph, record the chromatogram, and calculate the iron content by summing the peak areas of the complexes formed by iron ions according to the following formula: In the formula: ---The sum of the peak areas of the main peak of the test sample solution; ---The sum of the peak areas of the main peak of the reference solution.

Images