Method for detecting pharmaceutical adjuvant or sodium phosphate for preparing aluminum phosphate immunologic adjuvant and inorganic anions of sodium phosphate

Abstract

The invention relates to a method for detecting sodium phosphate for preparing an aluminum phosphate immunologic adjuvant and inorganic anions of the sodium phosphate, and the detection method is used for simultaneously detecting 9 inorganic anions or 8 inorganic impurity anions in a sodium phosphate raw material by using ion chromatography, and comprises the following steps: providing an ion chromatograph and an electric conductivity detector; providing a standard substance of each anion; preparing a reference substance solution, and mixing the reference substance solution and a test solution; precisely measuring each reference substance solution, the system applicability solution and the test solution, respectively injecting into an ion chromatograph, and recording chromatograms; and measuring and calculating the content of the eight inorganic anion impurities in the test sample according to the peak area of the chromatogram of the reference substance solution by an external standard method. The invention further relates to application of the sodium phosphate detected by the method to preparation of an aluminum phosphate vaccine immunologic adjuvant.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, relates to a sodium phosphate, also relates to an aluminum phosphate vaccine immune adjuvant prepared by using the sodium phosphate, and further relates to a method for detecting inorganic anions in the sodium phosphate, especially a method for simultaneously detecting the contents of 9 inorganic anions in the sodium phosphate, especially the contents of 8 inorganic anion impurities, by using the same method. Background Art

[0002] Sodium phosphate, also known as trisodium phosphate, exists in three forms: anhydrous sodium phosphate, sodium phosphate monohydrate, and sodium phosphate dodecahydrate [USP43-NF38[S]. 2022: Issue 3]. Sodium phosphate dodecahydrate has been included in Part IV of the 2020 edition of the Chinese Pharmacopoeia. Sodium phosphate is a commonly used pharmaceutical excipient, mainly used as a buffer and pH regulator, and is also used to produce aluminum phosphate adjuvants in vaccines.

[0003] Aluminum adjuvant is the most widely used immune adjuvant. As an adsorbent, aluminum adjuvant mainly includes aluminum hydroxide adjuvant and aluminum phosphate adjuvant. It can strongly adsorb protein antigens from the solution to form a precipitate. When it is inoculated into the body, it can form an "antigen library" and slowly release antigens, fully prolonging the action time of the antigen. At the same time, it can also promote the response of local macrophages.

[0004] Aluminum adjuvants have a high degree of biosafety. People have proved through isotope labeling that aluminum adjuvants injected intramuscularly can be effectively dissolved and absorbed in the body and eventually excreted through urine. Aluminum adjuvants can enhance the immunogenicity of antigens and improve immunoresponsiveness through various mechanisms. The mechanism of action includes: the granular form of soluble antigen combined with adjuvant is conducive to APCs phagocytosis; deposition at the injection site; inducing significant proliferation of T cells and enhancing Th2 immune response; inducing monocytes to differentiate into mature CD cells. Therefore, aluminum adjuvants are widely used in vaccine preparation processes, especially in vaccines containing protein or polysaccharide antigens. Aluminum adjuvant vaccines that have been marketed at home and abroad include: diphtheria-tetanus-acellular pertussis vaccine (DTaP), Haemophilus influenzae type b conjugate vaccine, group A and group C meningococcal polysaccharide conjugate vaccine, pneumonia conjugate vaccine, hepatitis B vaccine, human papillomavirus vaccine, etc.

[0005] Under physiological conditions, aluminum phosphate adjuvant is suitable for adsorbing cationic antigens and is used in multivalent pneumococcal conjugate vaccines. The general method for preparing aluminum phosphate adjuvant is to mix a soluble aluminum salt solution and a phosphate solution in a certain proportion. Depending on the mixing method, it usually includes a continuous reaction method and a batch reaction method. The preparation of aluminum phosphate adjuvant requires a soluble aluminum salt solution, a phosphate solution, and a suitable replacement buffer solution system.

[0006] The prior art has provided many methods for preparing aluminum phosphate adjuvants, and the raw materials used are usually water-soluble aluminum chloride and sodium phosphate. For example, CN106729702B (Chinese patent application No. 201611237397.1) relates to an aluminum phosphate adjuvant and its preparation method and application, the method comprising the preparation of an aluminum phosphate solution, a replacement treatment of the aluminum phosphate solution, and a resuspension treatment of the aluminum phosphate solution. The present method can achieve the surface charge and particle size of the aluminum phosphate adjuvant to be stable and the pH value is closer to the pH value of human body fluids, which is suitable for the adsorption of one or more antigens and meets the application requirements of multiple vaccines in preparations.

[0007] Pharmaceutical excipients are different from drugs. One excipient can be used in the production of multiple drugs. Safety problems of a certain excipient will inevitably lead to safety problems of multiple drugs. Impurities are an important factor causing adverse drug reactions [Zhu Lin, Chen Xinru, Shi Haiwei, et al. Ion chromatography-conductivity / ultraviolet detection of trace nitrite and nitrate in the pharmaceutical excipient lactose [J]. Pharmacy and Clinical Research, 2024, 32 (2): 115-120]. The impurities in pharmaceutical excipients are mainly divided into organic impurities, residual solvents, and inorganic impurities, among which inorganic impurities include reagents, ligands, inorganic salts, etc. Most of the pharmaceutical excipients sodium phosphate dodecahydrate included in the fourth volume of the 2020 edition of the Pharmacopoeia of the People's Republic of China use volumetric methods or chemical reaction methods to control inorganic anion impurities. These methods have poor specificity, sensitivity and accuracy, subjective visual errors, and are not convenient for simultaneous determination of multiple anion impurities.

[0008] Common detection techniques for anions include titration, colorimetry, ultraviolet spectrophotometry, electrochemical method, high performance liquid chromatography, ion chromatography, etc. Ion chromatography is widely used due to its strong specificity, high sensitivity and accurate results [Zhu Lin, Chen Xinru, Shi Haiwei, et al. Ion chromatography-conductivity / ultraviolet detection of trace nitrite and nitrate in pharmaceutical excipient lactose [J]. Pharmacy and Clinical Research, 2024, 32(2): 115-120; Wu Yuping, Li Tianfei, Wang Dongdan, et al. Determination of impurity content in food additive tripolyphosphate by ion chromatography [J]. Chromatography, 2003, 21(3): 291-293; Wu Jun, Cai Peng. Determination of phosphite and phosphate in risedronate sodium tablets by ion chromatography [J]. West China Journal of Pharmacy, 2018, 33(1): 092-094].

[0009] Simultaneously determining the contents of nine anions, including hypophosphite, chloride, phosphite, carbonate, nitrate, sulfate, phosphate, pyrophosphate, and trimetaphosphate, in the pharmaceutical excipient sodium phosphate, especially determining the contents of the other eight impurity anions except phosphate, in order to control the quality of sodium phosphate, is beneficial for preparing aluminum phosphate adjuvant using sodium phosphate. Therefore, those skilled in the art expect to provide a method, such as ion chromatography, that can simultaneously determine the contents of the above nine anions in the pharmaceutical excipient sodium phosphate, which has the characteristics of good separation, high sensitivity, high throughput, and wide application range, and also expect to provide an aluminum phosphate immune adjuvant prepared using the pharmaceutical excipient sodium phosphate controlled by the above method. Summary of the invention

[0010] The purpose of the present invention is to provide an aluminum phosphate immune adjuvant prepared using the pharmaceutical excipient sodium phosphate detected by the method of the present invention, or the purpose of the present invention is to provide a method such as ion chromatography that can simultaneously determine the content of nine inorganic anions in the pharmaceutical excipient sodium phosphate with the characteristics of good separation, high sensitivity, high throughput, and wide application range. It is expected that the aluminum phosphate immune adjuvant provided by the present invention, which can be used as a vaccine adjuvant, has excellent performance in one or more aspects.

[0011] To this end, the first aspect of the present invention provides a method for simultaneously detecting 9 inorganic anions or 8 inorganic impurity anions in the pharmaceutical excipient sodium phosphate using ion chromatography, the method comprising the following steps: (1) providing an ion chromatograph and a conductivity detector (for example, the ion chromatograph is a Thermo DioNEX AS-AP ion chromatograph, and the conductivity detector is a conductivity detector); (2) providing carbonate ion standard solution, chlorine standard substance in water, sulfate standard substance in water, nitrate standard substance in water, phosphate standard substance in water, providing standard substances of sodium pyrophosphate, hypophosphorous acid, phosphorous acid, and sodium trimetaphosphate; providing sodium phosphate test sample (for example, anhydrous sodium phosphate, sodium phosphate dodecahydrate, sodium phosphate monohydrate); (3) Preparation of reference solution and mixed reference solution: accurately weigh or measure appropriate amounts of 9 anion standard substances, namely hypophosphite, chloride, phosphite, carbonate, nitrate, sulfate, phosphate, pyrophosphate and trimetaphosphate, and add appropriate amount of ultrapure water to prepare reference stock solution with a mass concentration of 1 mg / ml; accurately measure appropriate amount of reference stock solution to prepare reference solutions for qualitative or quantitative determination of each ion or mixed reference solution for system suitability test at a concentration suitable for detection; the concentration of hypophosphite, phosphite, phosphate, pyrophosphate and trimetaphosphate in the system suitability solution is 1 μg / ml, the concentration of chloride is 0.1 μg / ml, the concentration of nitrate and sulfate is 0.2 μg / ml, and the concentration of carbonate is 10 μg / ml; (4) Preparation of test solution: Accurately weigh an appropriate amount of sample, place in a 10 ml volumetric flask, add water to dissolve and dilute to the mark, which is equivalent to a phosphate concentration of 2 mg / ml; (5) Chromatographic conditions: Column: Dionex IonPac TM AS11-HC Analytical (4mm×250mm, other chromatographic columns with equivalent performance can also be used), Guard column: Dionex IonPac TM AG11-HC Analytical (4mm×50mm, other guard columns with equivalent performance can also be used), Suppressor: Thermo Scientific Dionex ADRS 600 (other suppressors of equivalent performance may also be used), Detector: Conductivity detector, Eluent: A is 100mmol / L potassium hydroxide solution, B is water, gradient elution: 0~10min (A: 22%) → 10.1~30min (A: 22% → A: 80%) → 30.1~51min (A: 80%) → 51.1~60min (A: 22%), flow rate 1.0mL / min, Column temperature: 35°C, Injection volume: 20 μL; (6) Determination method: Accurately measure 20 μl of each reference solution, system suitability solution, and test solution, inject them into the ion chromatograph respectively, and record the chromatogram; use the chromatogram obtained from the system suitability solution to conduct a methodological investigation, calculate the contents of 8 inorganic anion impurities in the test sample by the peak area of ​​the chromatogram of the reference solution according to the external standard method, and calculate the phosphate content in the test sample; the separation between the phosphate peak and its adjacent peaks and other anion impurity peaks is greater than 2.0 (in line with the general requirements in this field); for unknown impurities, use phosphate as a reference and calculate the impurity content by the peak area according to the external standard method.

[0012] Furthermore, the second aspect of the present invention provides a method for preparing an aluminum phosphate adjuvant, comprising the following steps: (i) using ion chromatography to simultaneously detect the contents of the following eight anions in the sodium phosphate raw material: hypophosphite, chloride, phosphorous acid, carbonate, nitrate, sulfate, pyrophosphate, and trimetaphosphate, and selecting sodium phosphate with a low anion impurity content as the raw material (for example, selecting sodium phosphate that meets pharmaceutical standards and has eight anions less than 0.1%, other impurities less than 0.1%, and a total impurity content less than 0.2% as the raw material); the sodium phosphate is selected from: anhydrous sodium phosphate, sodium phosphate dodecahydrate, and sodium phosphate monohydrate; the method for detecting eight anions in the sodium phosphate raw material comprises the following steps: (1) providing an ion chromatograph and a conductivity detector (for example, the ion chromatograph is a Thermo DioNEX AS-AP ion chromatograph, and the conductivity detector is a conductivity detector); (2) providing carbonate ion standard solution, chlorine standard substance in water, sulfate standard substance in water, nitrate standard substance in water, phosphate standard substance in water, providing standard substances of sodium pyrophosphate, hypophosphorous acid, phosphorous acid, and sodium trimetaphosphate; providing sodium phosphate sample to be tested (for example, anhydrous sodium phosphate, sodium phosphate dodecahydrate, sodium phosphate monohydrate); (3) Preparation of reference solution and mixed reference solution: accurately weigh or measure appropriate amounts of 9 anion standard substances, namely hypophosphite, chloride, phosphite, carbonate, nitrate, sulfate, phosphate, pyrophosphate and trimetaphosphate, and add appropriate amount of ultrapure water to prepare reference stock solution with a mass concentration of 1 mg / ml; accurately measure appropriate amount of reference stock solution to prepare reference solutions for qualitative or quantitative determination of each ion or mixed reference solution for system suitability test at a concentration suitable for detection; the concentration of hypophosphite, phosphite, phosphate, pyrophosphate and trimetaphosphate in the system suitability solution is 1 μg / ml, the concentration of chloride is 0.1 μg / ml, the concentration of nitrate and sulfate is 0.2 μg / ml, and the concentration of carbonate is 10 μg / ml; (4) Preparation of test solution: Accurately weigh an appropriate amount of sample, place in a 10 ml volumetric flask, add water to dissolve and dilute to the mark to prepare a solution with a phosphate concentration of about 2 mg / ml. (5) Chromatographic conditions: Column: Dionex IonPac TM AS11-HC Analytical (4mm×250mm, other chromatographic columns with equivalent performance can also be used), Guard column: Dionex IonPac TM AG11-HC Analytical (4mm×50mm, other guard columns with equivalent performance can also be used), Suppressor: Thermo Scientific Dionex ADRS 600 (other suppressors of equivalent performance may also be used), Detector: Conductivity detector, Eluent: A is 100mmol / L potassium hydroxide solution, B is water, gradient elution: 0~10min (A: 22%) → 10.1~30min (A: 22% → A: 80%) → 30.1~51min (A: 80%) → 51.1~60min (A: 22%), flow rate 1.0mL / min, Column temperature: 35°C, Injection volume: 20 μL; (6) Determination method: Accurately measure 20 μl of each reference solution, system suitability solution, and test solution, respectively, and inject them into the ion chromatograph to record the chromatogram; use the chromatogram obtained from the system suitability solution to conduct a methodological investigation, and calculate the contents of 8 inorganic anion impurities in the test sample by the peak area of ​​the reference solution chromatogram according to the external standard method; the separation between the phosphate peak and its adjacent peaks and other anion impurity peaks is greater than 2.0 (in line with the general requirements in this field); for unknown impurities, use phosphate as a reference, and calculate the impurity content by the peak area according to the external standard method; (ii) weighing 10 mmol of aluminum chloride or its hydrate, such as AlCl3·6H2O, and 9-11 mmol, such as 9 mmol or 10 mmol or 11 mmol, of a sodium phosphate raw material (such as anhydrous sodium phosphate, sodium phosphate dodecahydrate, sodium phosphate monohydrate) meeting the detection requirements of step (1), and dissolving them in 30-50 ml, such as 30 ml or 40 ml or 50 ml, and 25-35 ml, such as 25 ml or 30 ml or 35 ml, of a solvent, respectively, wherein the solvent is a mixture of ethylene glycol and deionized water in a volume ratio of 10-15:100, such as 10:100, 12:100 or 15:100; (iii) placing the aluminum chloride solution on a magnetic stirrer, uniformly adding the sodium phosphate solution to the aluminum chloride solution at room temperature at a stirring speed of 700-800 rpm, for example, 700 rpm, 750 rpm or 800 rpm, over a period of 18-23 minutes, for example, 18 minutes, 20 minutes or 23 minutes, and continuing to stir for 40-50 minutes, for example, 40 minutes, 45 minutes or 50 minutes after the addition is completed, and then transferring the reaction solution to a 150 ml glass bottle and autoclaving at 121° C. for 30 minutes; (iv) The reaction solution was allowed to stand and naturally cool to room temperature, centrifuged at 10,000 rpm for 15 min, the supernatant was discarded, and the precipitate was washed three times with sterile water for injection. After each washing, the precipitate was centrifuged at 10,000 rpm for 15 min and the supernatant was discarded. The precipitate was dried under reduced pressure at 45° C. to a water content of less than 3%, thereby obtaining a micro-powdered aluminum phosphate adjuvant sample.

[0013] The method according to the second aspect of the present invention comprises the following steps: (i) using ion chromatography to simultaneously detect the contents of the following eight anions in the sodium phosphate raw material: hypophosphite, chloride, phosphorous acid, carbonate, nitrate, sulfate, pyrophosphate, and trimetaphosphate, and selecting sodium phosphate with a low anion impurity content as the raw material (for example, selecting sodium phosphate that meets pharmaceutical standards and has eight anions less than 0.1%, other impurities less than 0.1%, and a total impurity content less than 0.2% as the raw material); the sodium phosphate is selected from: anhydrous sodium phosphate, sodium phosphate dodecahydrate, and sodium phosphate monohydrate; (ii) 2.415 g (Mr 241.46, 10 mmol) of AlCl3·6H2O and 1.639 g (Mr 163.94, 10 mmol) of Na3PO4 were weighed and dissolved in 40 ml and 30 ml of a solvent, respectively, wherein the solvent was a mixture of ethylene glycol and deionized water in a volume ratio of 12:100; (iii) placing the aluminum chloride solution on a magnetic stirrer, uniformly adding the sodium phosphate solution to the aluminum chloride solution at room temperature at a stirring speed of 750 rpm over a period of 20 minutes, and continuing to stir for 45 minutes after the addition is completed. Then, the reaction solution is transferred to a 150 ml glass bottle and autoclaved at 121° C. for 30 minutes; (iv) The reaction solution was allowed to stand and naturally cool to room temperature, centrifuged at 10,000 rpm for 15 min, the supernatant was discarded, and the precipitate was washed three times with sterile water for injection. After each washing, the precipitate was centrifuged at 10,000 rpm for 15 min and the supernatant was discarded. The precipitate was dried under reduced pressure at 45° C. to a water content of less than 3%, thereby obtaining a micro-powdered aluminum phosphate adjuvant sample.

[0014] The method according to the second aspect of the present invention comprises the following steps: (i) using ion chromatography to simultaneously detect the contents of the following eight anions in the sodium phosphate raw material: hypophosphite, chloride, phosphorous acid, carbonate, nitrate, sulfate, pyrophosphate, and trimetaphosphate, and selecting sodium phosphate with a low anion impurity content as the raw material (for example, selecting sodium phosphate that meets pharmaceutical standards and has eight anions less than 0.1%, other impurities less than 0.1%, and a total impurity content less than 0.2% as the raw material); the sodium phosphate is selected from: anhydrous sodium phosphate, sodium phosphate dodecahydrate, and sodium phosphate monohydrate; (ii) 2.415 g (Mr 241.46, 10 mmol) of AlCl3·6H2O and 3.8013 g (Mr 380.13, 10 mmol) of Na3PO4·12H2O were weighed and dissolved in 40 ml and 30 ml of a solvent, respectively, wherein the solvent was a mixture of ethylene glycol and deionized water in a volume ratio of 12:100; (iii) placing the aluminum chloride solution on a magnetic stirrer, uniformly adding the sodium phosphate solution to the aluminum chloride solution at room temperature at a stirring speed of 750 rpm over a period of 20 minutes, and continuing to stir for 45 minutes after the addition is completed. Then, the reaction solution is transferred to a 150 ml glass bottle and autoclaved at 121° C. for 30 minutes; (iv) The reaction solution was allowed to stand and naturally cool to room temperature, centrifuged at 10,000 rpm for 15 min, the supernatant was discarded, and the precipitate was washed three times with sterile water for injection. After each washing, the precipitate was centrifuged at 10,000 rpm for 15 min and the supernatant was discarded. The precipitate was dried under reduced pressure at 45° C. to a water content of less than 3%, thereby obtaining a micro-powdered aluminum phosphate adjuvant sample.

[0015] The method according to the second aspect of the present invention comprises the following steps: (i) using ion chromatography to simultaneously detect the contents of the following eight anions in the sodium phosphate raw material: hypophosphite, chloride, phosphorous acid, carbonate, nitrate, sulfate, pyrophosphate, and trimetaphosphate, and selecting sodium phosphate with a low anion impurity content as the raw material (for example, selecting sodium phosphate that meets pharmaceutical standards and has eight anions less than 0.1%, other impurities less than 0.1%, and a total impurity content less than 0.2% as the raw material); the sodium phosphate is selected from: anhydrous sodium phosphate, sodium phosphate dodecahydrate, and sodium phosphate monohydrate; (ii) 2.415 g (Mr 241.46, 10 mmol) of AlCl3·6H2O and 3.421 g (Mr 380.13, 9 mmol) of Na3PO4·12H2O were weighed and dissolved in 50 ml and 25 ml of a solvent, respectively, wherein the solvent was a mixture of ethylene glycol and deionized water in a volume ratio of 10:100; (iii) placing the aluminum chloride solution on a magnetic stirrer, uniformly adding the sodium phosphate solution to the aluminum chloride solution at room temperature at a stirring speed of 800 rpm over a period of 23 minutes, and continuing to stir for 50 minutes after the addition is completed. Then, the reaction solution is transferred to a 150 ml glass bottle and autoclaved at 121° C. for 30 minutes; (iv) The reaction solution was allowed to stand and naturally cool to room temperature, centrifuged at 10,000 rpm for 15 min, the supernatant was discarded, and the precipitate was washed three times with sterile water for injection. After each washing, the precipitate was centrifuged at 10,000 rpm for 15 min and the supernatant was discarded. The precipitate was dried under reduced pressure at 45° C. to a water content of less than 3%, thereby obtaining a micro-powdered aluminum phosphate adjuvant sample.

[0016] The method according to the second aspect of the present invention comprises the following steps: (i) using ion chromatography to simultaneously detect the contents of the following eight anions in the sodium phosphate raw material: hypophosphite, chloride, phosphorous acid, carbonate, nitrate, sulfate, pyrophosphate, and trimetaphosphate, and selecting sodium phosphate with a low anion impurity content as the raw material (for example, selecting sodium phosphate that meets pharmaceutical standards and has eight anions less than 0.1%, other impurities less than 0.1%, and a total impurity content less than 0.2% as the raw material); the sodium phosphate is selected from: anhydrous sodium phosphate, sodium phosphate dodecahydrate, and sodium phosphate monohydrate; (ii) 2.415 g (Mr 241.46, 10 mmol) of AlCl3·6H2O and 4.181 g (Mr 380.13, 11 mmol) of Na3PO4·12H2O were weighed and dissolved in 30 ml and 35 ml of a solvent, respectively, wherein the solvent was a mixture of ethylene glycol and deionized water in a volume ratio of 15:100; (iii) placing the aluminum chloride solution on a magnetic stirrer, uniformly adding the sodium phosphate solution to the aluminum chloride solution at room temperature at a stirring speed of 700 rpm over a period of 18 minutes, and continuing to stir for 40 minutes after the addition is completed. Then, the reaction solution is transferred to a 150 ml glass bottle and autoclaved at 121° C. for 30 minutes; (iv) The reaction solution was allowed to stand and naturally cool to room temperature, centrifuged at 10,000 rpm for 15 min, the supernatant was discarded, and the precipitate was washed three times with sterile water for injection. After each washing, the precipitate was centrifuged at 10,000 rpm for 15 min and the supernatant was discarded. The precipitate was dried under reduced pressure at 45° C. to a water content of less than 3%, thereby obtaining a micro-powdered aluminum phosphate adjuvant sample.

[0017] Furthermore, the third aspect of the present invention provides an aluminum phosphate adjuvant, which is prepared by any method described in the second aspect of the present invention.

[0018] Any embodiment of any aspect of the present invention can be combined with other embodiments, as long as they do not conflict. In addition, in any embodiment of any aspect of the present invention, any technical feature can be applied to the technical features in other embodiments, as long as they do not conflict.

[0019] The aluminum chloride used in the present invention, unless otherwise specified, refers to aluminum chloride hexahydrate, also known as crystalline aluminum chloride, with a molecular formula of AlCl3·6H2O and a molecular weight of Mr = 241.43. Aluminum chloride hexahydrate is easily soluble in water, anhydrous ethanol, ether and glycerol, and its aqueous solution is acidic.

[0020] The sodium phosphate used in the present invention can be anhydrous sodium phosphate (molecular formula Na3PO4, molecular weight 163.94), sodium phosphate monohydrate (molecular formula Na3PO4·H2O, molecular weight 181.96), or sodium phosphate dodecahydrate (molecular formula Na3PO4·12H2O, Mr=380.124), all of which are soluble in water but insoluble in ethanol.

[0021] A theoretical product of the reaction of aluminum chloride and sodium phosphate is aluminum phosphate (molecular formula AlPO4, molecular weight 121.953), which can be used as a raw material for inorganic chemical products. It is a white crystalline powder, insoluble in water, ethanol and acetic acid, soluble in concentrated hydrochloric acid, concentrated nitric acid, alkali and alcohol. It is usually used as an antacid to neutralize and buffer gastric acid, increase the pH value in the stomach, and thus relieve the symptoms of excessive gastric acid.

[0022] In addition, in some aspects of the present invention, it was found that sulfate ions in the sodium phosphate raw material may be detrimental to the preparation of the adjuvant. In the aluminum chloride reaction system, sulfate ions can theoretically react with aluminum ions to form aluminum sulfate (molecular formula Al2(SO4)3, molecular weight 342.151) that is soluble in water but insoluble in ethanol.

[0023] The method of the invention can simultaneously detect nine kinds of anions in sodium phosphate. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 : Typical chromatogram of system suitability solution (in the figure, the peak numbers and names are: 1 hypophosphite, 2 chloride, 3 phosphite, 4 carbonate, 5 nitrate, 6 sulfate, 7 phosphate, 8 pyrophosphate, 9 trimetaphosphate).

[0025] Figure 2 : Chromatogram of the test conditions (in the figure, the peak numbers and names are: 1 phosphate, 2 chloride, 3 phosphite, carbonate, 4 nitrate, 5 sulfate, 6 phosphate, 7 pyrophosphate).

[0026] Figure 3 : Chromatogram of the test conditions (in the figure, the peak numbers and names are: 1 phosphate, 2 chloride, 3 phosphite, carbonate, 4 nitrate, 5 sulfate, 6 phosphate, 7 pyrophosphate).

[0027] Figure 4 : Chromatogram of test conditions investigation (in the figure, the peak numbers and names are: 1 hypophosphite, 2 chloride, 3 phosphite, 4 carbonate, 5 nitrate, 6 sulfate, 7 phosphate, 8 pyrophosphate, 9 trimetaphosphate).

[0028] Figure 5: Chromatogram of test conditions investigation (in the figure, the peak numbers and names are: 1 hypophosphite, 2 chloride, 3 phosphite, 4 carbonate, 5 nitrate, 6 sulfate, 7 phosphate, 8 pyrophosphate, 9 trimetaphosphate). DETAILED DESCRIPTION

[0029] The present invention is further illustrated below by specific various types of examples, however, it should be understood that these examples are only for the purpose of describing in more detail and should not be understood as limiting the present invention in any form. The present invention provides a general and / or specific description of the materials and test methods used in the experiment. Although many materials and operating methods used to achieve the purpose of the present invention are well known in the art, the present invention is still described as detailed as possible herein. It is clear to those skilled in the art that, hereinafter, unless otherwise specified, the materials and operating methods used in the present invention are well known in the art. The aluminum chloride and sodium phosphate used in the present invention are products purchased from the market, such as sodium phosphate dodecahydrate, which complies with the various provisions of the standard for the variety contained in the fourth part of the 2020 edition of the Chinese Pharmacopoeia, and the method of the present invention is also used to simultaneously detect 9 anions in sodium phosphate as a raw material.

[0030] Test Example 1: Simultaneous detection of 9 anions in sodium phosphate raw materials using ion chromatography This detection method is particularly used to detect 8 kinds of inorganic anion impurities in sodium phosphate raw materials.

[0031] 1. Instruments: Thermo DioNEX AS-AP ion chromatograph, Dionex ICS 5000+ conductivity detector (Thermo Fisher Scientific), XSR205DU electronic analytical balance (Mettler).

[0032] 2. Reagents: carbonate ion standard solution, chlorine standard substance in water, sulfate standard substance in water, nitrate standard substance in water, phosphate standard substance in water, all from Northern Weiye Metrology Technology Research Institute, with a concentration of 1000 μg / ml; sodium pyrophosphate (H2Na2O7P2, Sigma, ≥99.0%), hypophosphorous acid (KH2PO2, McLean, 99%), phosphorous acid (HNa2O3P·5H2O, Sigma, ≥98%) and sodium trimetaphosphate (Na3O9P3, Sigma, ≥95%) standard substances; 100 mmol / L potassium hydroxide solution; experimental water is deionized water treated by MilliporeMill-Q water treatment system. If not otherwise specified, the deionized water used in the present invention is deionized water obtained by this method.

[0033] 3. Test samples: 14 batches of sodium phosphate samples, numbered S1~S14, and the samples came from 6 manufacturers (A~F). Among them, the samples from manufacturer A were S1~S4, the samples from manufacturer B were S5, the samples from manufacturer C were S6~S8, the sample from manufacturer D was S10, the samples from manufacturer E were S9 and S11~S13, and the sample from manufacturer F was S14. S9 and S14 are anhydrous sodium phosphate, and the remaining samples are sodium phosphate dodecahydrate.

[0034] 4. Preparation of reference solution and mixed reference solution: accurately weigh or measure appropriate amount of 9 anion standard substances including hypophosphite, chloride, phosphite, carbonate, nitrate, sulfate, phosphate, pyrophosphate and trimetaphosphate, and add appropriate amount of ultrapure water to prepare reference stock solution with a mass concentration of 1 mg / ml; accurately measure appropriate amount of reference stock solution to prepare reference solutions for qualitative or quantitative determination of each ion at a concentration suitable for detection or mixed reference solution for system suitability test; the concentration of hypophosphite, phosphite, phosphate, pyrophosphate and trimetaphosphate in the system suitability solution is 1 μg / ml, the concentration of chloride is 0.1 μg / ml, the concentration of nitrate and sulfate is 0.2 μg / ml, and the concentration of carbonate is 10 μg / ml.

[0035] 5. Preparation of test solution: Accurately weigh an appropriate amount of sample, place in a 10 ml volumetric flask, add water to dissolve and dilute to the scale to prepare a solution with a phosphate concentration of about 2 mg / ml.

[0036] 6. Chromatographic conditions: Column: Dionex IonPac TM AS11-HC Analytical (4mm×250mm, other chromatographic columns with equivalent performance can also be used), Guard column: Dionex IonPac TM AG11-HC Analytical (4mm×50mm, other guard columns with equivalent performance can also be used), Suppressor: Thermo Scientific Dionex ADRS 600 (other suppressors of equivalent performance may also be used), Detector: Conductivity detector, Eluent: A is 100mmol / L potassium hydroxide solution, B is water, gradient elution: 0~10min (A: 22%) → 10.1~30min (A: 22% → A: 80%) → 30.1~51min (A: 80%) → 51.1~60min (A: 22%), flow rate: 1.0mL / min, Column temperature: 35°C, Injection volume: 20 μL.

[0037] 7. Determination method: Accurately measure 20 μl of each reference solution, system suitability solution, and test solution, respectively, and inject them into the ion chromatograph to record the chromatogram; use the chromatogram obtained from the system suitability solution to conduct a methodological investigation, and calculate the various inorganic anion contents in the test sample by the peak area of ​​the chromatogram of the reference solution according to the external standard method; the separation between the phosphate peak and its adjacent peaks and other anion impurity peaks is greater than 2.0 (in line with the general requirements in this field); for unknown impurities, use phosphate as a reference, and calculate the impurity content by peak area according to the external standard method.

[0038] 8. Methodological Validation 8.1. Specificity test: Take blank solvent (water) and system suitability solution, measure them according to "6. Chromatographic conditions" and record the chromatograms. Each anion peak is well separated. The blank solvent has no interference in the determination of the 9 anions. The typical chromatogram of the system suitability solution is as follows: Figure 1 As shown, the method has good specificity. To further investigate the specificity, the present invention further designs a forced degradation test (acid, alkali, oxidation, high temperature, light, high humidity), which mainly investigates the separation of degradation products and main peak phosphate under various severe conditions and the peak purity of the main peak, so as to comprehensively evaluate the specificity of the chromatographic conditions.

[0039] 8.2. Forced degradation test of sample solution: Take one portion of each of the following samples (described in Table 3 of the present invention): S1, S5, S6, S9, S10 (see Table 3 of the present invention for sample numbers), accurately weigh an appropriate amount of the sample according to the method of "5. Preparation of test solution", place it in a 10 ml volumetric bottle, add water to dissolve it, and then perform degradation tests under different conditions: (1) Add 1 ml of 1 mol / L hydrochloric acid solution, place at room temperature for 24 h, add 1 ml of 1 mol / L sodium hydroxide solution, dilute to scale with water, and shake well to obtain an acid-destroying solution; (2) Add 1 ml of 1 mol / L sodium hydroxide solution, place at room temperature for 24 h, add 1 ml of 1 mol / L hydrochloric acid solution, dilute to scale with water, and shake well to obtain an alkali destruction solution; (3) Add 1 ml of 6% hydrogen peroxide solution, place at room temperature for 24 hours, dilute to scale with water, and shake well to obtain an oxidative destruction solution; (4) Place in a 60°C water bath for 24 hours, then dilute to volume with water and shake well to obtain a high temperature destruction solution; (5) Place under 4000 lx light conditions for about 24 hours, then dilute to scale with water and shake well to obtain a light-damaged solution; (6) Place under ultraviolet light conditions for about 24 hours, then dilute to scale with water and shake well to obtain an ultraviolet light destruction solution; (7) Dilute to scale with water and shake well to obtain an undamaged solution, which serves as the sample control solution.

[0040] The above solution was measured according to "6. Chromatographic conditions" and "7. Measurement method" above.

[0041] 8.3. Sample powder forced degradation test: Take one portion of each of the following samples (described in Table 3 of the present invention): about 1 g of S1, S5, S6, S9, and S10, respectively, place them in a flat weighing bottle of constant weight, accurately weigh them, and place them under ultraviolet light, 4000 lx visible light, high temperature, and high humidity for 5 days and 10 days, respectively. Accurately weigh the total weight of the flat weighing bottle after placement, and calculate the coefficient of change of the total weight of the sample powder; then according to the method of "5. Preparation of test solution", accurately weigh an appropriate amount of the sample, place it in a 10 ml volumetric bottle, add water to dissolve it, and prepare the test solution under ultraviolet light, 4000 lx visible light, high temperature, and high humidity.

[0042] Take the above test sample solution and measure the above solution according to the above "6. Chromatographic conditions" and "7. Determination method", record the chromatogram, the samples are basically affected by the destruction conditions in the same way, large impurity peaks are detected under acid, alkali and oxidation destruction conditions, no degradation impurities are detected under other destruction conditions, and the impurity peaks can be well separated from the main peak, indicating that the method has good specificity.

[0043] 8.4 Linearity Test Accurately measure an appropriate amount of the reference substance stock solution in "4. Preparation of mixed reference substance solution", dilute with water to prepare a series of linear solutions with concentrations ranging from 0.0005μg / ml to 100μg / ml, and perform sample analysis. Perform linear regression with the concentration of each component as the abscissa (X) and the peak area as the ordinate (Y). The components have a good linear relationship within the corresponding concentration range, and the results are shown in Table 1.

[0044] Table 1: Linear relationship results

[0045] 8.5. Limit of Detection and Limit of Quantitation Through the determination of the above series of concentration linear solutions, the detection limit and quantification limit of each ion can be obtained. The results are shown in Table 1.

[0046] 8.6 Precision test Take 20µl of the system suitability solution and repeat the measurement 6 times according to the above “6. Chromatographic conditions” and “7. Determination method”, record the chromatogram, the RSD of the retention time of each peak is less than 0.3% (n=6), and the RSD of the peak area is less than 10.0 (n=6), indicating that the injection precision is good.

[0047] Take sample S4 and prepare a test solution containing 2 mg / ml phosphate according to the above method. Prepare 6 copies in parallel. The RSD of the main peak retention time is 0.46% (n=6), and the RSD of the peak area is 0.04% (n=6), indicating that the method has good precision and repeatability.

[0048] 8.7 Stability test Take sample S4, prepare a test solution containing 2 mg / ml phosphate according to the above method, place it at room temperature, and inject it according to the chromatographic conditions at different times (inject once every 2 hours from 0h to 24h), and record the chromatogram. The change in the inorganic anion content (peak area percentage) of the test solution is used as an indicator to examine the stability of the solution. The results show that within 24h, the RSD of the main component peak area is less than 0.1%, and the content of each impurity has no significant change, indicating that the sample solution is stable for at least 24h.

[0049] 8.8 Recovery test Take about 0.8g of the same batch of samples (S4) with known content, accurately weigh 6 portions, put them in 100mL volumetric flasks, accurately add eight anions except the main component phosphate according to the limit concentration, add water to dissolve and dilute to the scale to obtain the spiked sample solution. The average recovery rates of hypophosphorous acid, chloride ion, phosphorous acid, carbonate, nitrate, sulfate, pyrophosphate and trimetaphosphate ions are 97.4%, 109.3%, 114.9%, 115.4%, 96.1%, 86.0%, 111.2% and 88.3%, respectively.

[0050] 8.9 Durability test Take the system suitability solution, change the flow rate, column temperature, mobile phase buffer ratio, mobile phase buffer concentration in "6. Chromatographic conditions" and use different batches of chromatographic columns. Take the separation degree between the main peak of the test solution and the adjacent impurity peaks, the main peak retention time and the main peak theoretical plate number as indicators to investigate the durability of the method. The results are shown in Table 2. When the flow rate, column temperature, initial ratio of mobile phase buffer and different batches of columns are changed, the separation degree between the main peak and the adjacent impurity peaks in the durability test solution is greater than 1.5, which meets the requirements, indicating that when the flow rate, different batches of columns and other factors change slightly, the method is durable. When the column temperature, the initial ratio of mobile phase buffer, the column temperature is 25℃, 30℃ and the initial ratio of mobile phase buffer is 75%, the main peak appears on a steep slope, affecting the spectral analysis. It can be seen that the method has good durability when the flow rate, column temperature is 40℃, the initial ratio of mobile phase buffer is 80% and different batches of columns are changed.

[0051] Table 2: Durability test results

[0052] Note: * Normal conditions are flow rate 1.0 ml / min, column temperature 35°C, initial proportion of mobile phase B water 78%; # Chromatographic column 2 is Dionex IonPacTM AS11-HC Analytical (4 mm × 250 mm).

[0053] 9. Sample determination The main purpose of the present invention is to determine the contents of 8 anions other than the main component phosphate in sodium phosphate. Take 14 batches of samples, prepare the test solution according to the method of this test example, and use the chromatographic conditions of this test example for determination. Use the external standard method to calculate the contents of the other 8 anions except the main component phosphate by peak area. The results are shown in Table 3.

[0054] Table 3: Sample measurement results (%)

[0055] In the table, " / " indicates that it is below the detection limit and not detected, the term "other impurities" indicates the total amount of other unknown impurities, the term "quality" in the column Y indicates that it can be tried as a sodium phosphate raw material for preparing aluminum phosphate adjuvants due to its low impurity content, and the quality column No indicates that it is not suitable as a sodium phosphate raw material for preparing aluminum phosphate adjuvants due to its high impurity content. In addition, the samples numbered S1 to S14 come from 6 manufacturers (A to F), of which the samples of manufacturer A are S1 to S4, the samples of manufacturer B are S5, the samples of manufacturer C are S6 to S8, the samples of manufacturer D are S10, the samples of manufacturer E are S9 and S11 to S13, and the sample of manufacturer F is S14; S9 and S14 are anhydrous sodium phosphate (Na3PO4, Mr 163.94), and the remaining samples are sodium phosphate dodecahydrate (Na3PO4·12H2O, Mr 380.13).

[0056] Example 1: Preparation of aluminum phosphate adjuvant In this Example 1, aluminum phosphate adjuvant was prepared by referring to the method described in the literature with appropriate modifications.

[0057] (1) Using the ion chromatography method of Detection Example 1, the following eight anions in anhydrous or dodecahydrate sodium phosphate raw materials are simultaneously detected: hypophosphite, chloride, phosphorous acid, carbonate, nitrate, sulfate, pyrophosphate, and trimetaphosphate, and sodium phosphate with a low anion impurity content is selected as the raw material (in this example, sodium phosphate that meets the pharmaceutical standard and has eight anions less than 0.1%, other impurities less than 0.1%, and a total impurity content less than 0.2% is selected as the raw material); (2) Weigh 2.415 g (Mr 241.46, 10 mmol) of AlCl3·6H2O and 1.639 g (Mr 163.94, 10 mmol) of Na3PO4 and dissolve them in 40 ml and 30 ml of a solvent, respectively. The solvent is a mixture of ethylene glycol and deionized water in a volume ratio of 12:100. (3) placing the aluminum chloride solution on a magnetic stirrer, uniformly adding the sodium phosphate solution to the aluminum chloride solution at room temperature at a stirring speed of 750 rpm over a period of 20 minutes, and continuing to stir for 45 minutes after the addition is completed. Then, the reaction solution is transferred to a 150 ml glass bottle and autoclaved at 121° C. for 30 minutes; (4) The reaction solution was allowed to stand and naturally cool to room temperature, centrifuged at 10,000 rpm for 15 min, the supernatant was discarded, and the precipitate was washed three times with sterile water for injection. After each washing, the precipitate was centrifuged at 10,000 rpm for 15 min and the supernatant was discarded. The precipitate was dried under reduced pressure at 45° C. until the water content was less than 3%, thereby obtaining a micro-powdered aluminum phosphate adjuvant sample.

[0058] Example 2: Preparation of aluminum phosphate adjuvant (1) Using the ion chromatography method of Detection Example 1, the following eight anions in anhydrous or dodecahydrate sodium phosphate raw materials are simultaneously detected: hypophosphite, chloride, phosphorous acid, carbonate, nitrate, sulfate, pyrophosphate, and trimetaphosphate, and sodium phosphate with a low anion impurity content is selected as the raw material (in this example, sodium phosphate that meets the pharmaceutical standard and has eight anions less than 0.1%, other impurities less than 0.1%, and a total impurity content less than 0.2% is selected as the raw material); (2) Weigh 2.415 g (Mr 241.46, 10 mmol) of AlCl3·6H2O and 3.8013 g (Mr 380.13, 10 mmol) of Na3PO4·12H2O, and dissolve them in 40 ml and 30 ml of a solvent, respectively. The solvent is a mixture of ethylene glycol and deionized water in a volume ratio of 12:100. (3) placing the aluminum chloride solution on a magnetic stirrer, uniformly adding the sodium phosphate solution to the aluminum chloride solution at room temperature at a stirring speed of 750 rpm over a period of 20 minutes, and continuing to stir for 45 minutes after the addition is completed. Then, the reaction solution is transferred to a 150 ml glass bottle and autoclaved at 121° C. for 30 minutes; (4) The reaction solution was allowed to stand and naturally cool to room temperature, centrifuged at 10,000 rpm for 15 min, the supernatant was discarded, and the precipitate was washed three times with sterile water for injection. After each washing, the precipitate was centrifuged at 10,000 rpm for 15 min and the supernatant was discarded. The precipitate was dried under reduced pressure at 45° C. until the water content was less than 3%, thereby obtaining a micro-powdered aluminum phosphate adjuvant sample.

[0059] Example 3: Preparation of aluminum phosphate adjuvant (1) Using the ion chromatography method of Detection Example 1, the following eight anions in anhydrous or dodecahydrate sodium phosphate raw materials are simultaneously detected: hypophosphite, chloride, phosphorous acid, carbonate, nitrate, sulfate, pyrophosphate, and trimetaphosphate, and sodium phosphate with a low anion impurity content is selected as the raw material (in this example, sodium phosphate that meets the pharmaceutical standard and has eight anions less than 0.1%, other impurities less than 0.1%, and a total impurity content less than 0.2% is selected as the raw material); (2) Weigh 2.415 g (Mr 241.46, 10 mmol) of AlCl3·6H2O and 3.421 g (Mr 380.13, 9 mmol) of Na3PO4·12H2O, and dissolve them in 50 ml and 25 ml of a solvent, respectively. The solvent is a mixture of ethylene glycol and deionized water in a volume ratio of 10:100. (3) placing the aluminum chloride solution on a magnetic stirrer, and uniformly adding the sodium phosphate solution to the aluminum chloride solution at room temperature at a stirring speed of 800 rpm over a period of 23 minutes. After the addition is completed, stirring is continued for 50 minutes. Then, the reaction solution is transferred to a 150 ml glass bottle and autoclaved at 121° C. for 30 minutes. (4) The reaction solution was allowed to stand and naturally cool to room temperature, centrifuged at 10,000 rpm for 15 min, the supernatant was discarded, and the precipitate was washed three times with sterile water for injection. After each washing, the precipitate was centrifuged at 10,000 rpm for 15 min and the supernatant was discarded. The precipitate was dried under reduced pressure at 45° C. until the water content was less than 3%, thereby obtaining a micro-powdered aluminum phosphate adjuvant sample.

[0060] Example 4: Preparation of aluminum phosphate adjuvant (1) Using the ion chromatography method of Detection Example 1, the following eight anions in anhydrous or dodecahydrate sodium phosphate raw materials are simultaneously detected: hypophosphite, chloride, phosphorous acid, carbonate, nitrate, sulfate, pyrophosphate, and trimetaphosphate, and sodium phosphate with a low anion impurity content is selected as the raw material (in this example, sodium phosphate that meets the pharmaceutical standard and has eight anions less than 0.1%, other impurities less than 0.1%, and a total impurity content less than 0.2% is selected as the raw material); (2) Weigh 2.415 g (Mr 241.46, 10 mmol) of AlCl3·6H2O and 4.181 g (Mr 380.13, 11 mmol) of Na3PO4·12H2O, and dissolve them in 30 ml and 35 ml of a solvent, respectively. The solvent is a mixture of ethylene glycol and deionized water in a volume ratio of 15:100. (3) placing the aluminum chloride solution on a magnetic stirrer, uniformly adding the sodium phosphate solution to the aluminum chloride solution at room temperature at a stirring speed of 700 rpm over a period of 18 minutes, and continuing to stir for 40 minutes after the addition is completed. Then, the reaction solution is transferred to a 150 ml glass bottle and autoclaved at 121° C. for 30 minutes; (4) The reaction solution was allowed to stand and naturally cool to room temperature, centrifuged at 10,000 rpm for 15 min, the supernatant was discarded, and the precipitate was washed three times with sterile water for injection. After each washing, the precipitate was centrifuged at 10,000 rpm for 15 min and the supernatant was discarded. The precipitate was dried under reduced pressure at 45° C. until the water content was less than 3%, thereby obtaining a micro-powdered aluminum phosphate adjuvant sample.

[0061] Example 5: Preparation of aluminum phosphate adjuvant (1) Using the ion chromatography method of Detection Example 1, the following eight anions in anhydrous or dodecahydrate sodium phosphate raw materials are simultaneously detected: hypophosphite, chloride, phosphorous acid, carbonate, nitrate, sulfate, pyrophosphate, and trimetaphosphate, and sodium phosphate with a low anion impurity content is selected as the raw material (in this example, sodium phosphate that meets the pharmaceutical standard and has eight anions less than 0.1%, other impurities less than 0.1%, and a total impurity content less than 0.2% is selected as the raw material); (2) Weigh 2.415 g (Mr 241.46, 10 mmol) of AlCl3·6H2O and 1.639 g (Mr 163.94, 10 mmol) of Na3PO4 and dissolve them in 40 ml and 30 ml of deionized water, respectively; (3) placing the aluminum chloride solution on a magnetic stirrer, uniformly adding the sodium phosphate solution to the aluminum chloride solution at room temperature at a stirring speed of 750 rpm over a period of 20 minutes, and continuing to stir for 45 minutes after the addition is completed. Then, the reaction solution is transferred to a 150 ml glass bottle and autoclaved at 121° C. for 30 minutes; (4) The reaction solution was allowed to stand and naturally cool to room temperature, centrifuged at 10,000 rpm for 15 min, the supernatant was discarded, and the precipitate was washed three times with sterile water for injection. After each washing, the precipitate was centrifuged at 10,000 rpm for 15 min and the supernatant was discarded. The precipitate was dried under reduced pressure at 45° C. until the water content was less than 3%, thereby obtaining a micro-powdered aluminum phosphate adjuvant sample.

[0062] Example 6: Preparation of aluminum phosphate adjuvant (1) Using the ion chromatography method of Detection Example 1, the following eight anions in anhydrous or dodecahydrate sodium phosphate raw materials are simultaneously detected: hypophosphite, chloride, phosphorous acid, carbonate, nitrate, sulfate, pyrophosphate, and trimetaphosphate, and sodium phosphate with a low anion impurity content is selected as the raw material (in this example, sodium phosphate that meets the pharmaceutical standard and has eight anions less than 0.1%, other impurities less than 0.1%, and a total impurity content less than 0.2% is selected as the raw material); (2) Weigh 2.415 g (Mr 241.46, 10 mmol) of AlCl3·6H2O and 3.8013 g (Mr 380.13, 10 mmol) of Na3PO4·12H2O and dissolve them in 40 ml and 30 ml of deionized water, respectively; (3) placing the aluminum chloride solution on a magnetic stirrer, uniformly adding the sodium phosphate solution to the aluminum chloride solution at room temperature at a stirring speed of 750 rpm over a period of 20 minutes, and continuing to stir for 45 minutes after the addition is completed. Then, the reaction solution is transferred to a 150 ml glass bottle and autoclaved at 121° C. for 30 minutes; (4) The reaction solution was allowed to stand and naturally cool to room temperature, centrifuged at 10,000 rpm for 15 min, the supernatant was discarded, and the precipitate was washed three times with sterile water for injection. After each washing, the precipitate was centrifuged at 10,000 rpm for 15 min and the supernatant was discarded. The precipitate was dried under reduced pressure at 45° C. until the water content was less than 3%, thereby obtaining a micro-powdered aluminum phosphate adjuvant sample.

[0063] In a supplementary example which may be referred to as Example 7, the method of Example 2 is referred to, except that the ethylene glycol is replaced with equal amounts of methanol, ethanol, 1,2-propylene glycol, and glycerol to obtain four aluminum phosphate adjuvant samples which may be respectively referred to as Examples 71 to 74.

[0064] Test Example 2: Methodological Study on Simultaneous Detection of Nine Anions in Sodium Phosphate Raw Materials by Ion Chromatography In the detection example 1 of the present invention, an analytical method for detecting the content of nine kinds of inorganic anion impurities in sodium phosphate pharmaceutical excipients by ion chromatography was developed. Among them, an AS11 type anion chromatographic column was used, with 100mmol / L potassium hydroxide solution and water as eluents, gradient elution, and a flow rate of 1.0mL / min. The contents of impurity ions such as phosphate and hypophosphite, chloride, phosphite, carbonate, nitrate, sulfate, pyrophosphate, and trimetaphosphate in sodium phosphate pharmaceutical excipients were determined at the same time. Each inorganic anion impurity has a good linear relationship under the detection conditions, and the correlation coefficient R is greater than 0.99; the sample recovery rate is excellent; in the precision test, the RSD of the retention time of each peak is less than 0.3% (n=6), and the RSD of the peak area is less than 10.0 (n=6); nine kinds of inorganic anions in the 14 batches of samples collected were measured. The experimental results show that the method is simple to operate, accurate and sensitive, and the peak shape of the 9 measured components is good and the separation is good.

[0065] In the pharmaceutical industry, sodium phosphate is synthesized from sodium hydroxide and phosphoric acid through processes such as synthesis, filtration, concentration, crystallization, and dehydration. The synthetic raw materials in the production process may introduce chloride ions, carbonates, sulfates, and nitrates; degradation impurities may be mutually converted due to changes in conditions, and may include pyrophosphate, hypophosphorous acid, phosphorous acid, and hexametaphosphate. In addition, as one of the raw materials for the production of aluminum phosphate adjuvants, compounds of phosphorus with different valence states affect the synthesis of adjuvants. It is necessary to explore a method with high sensitivity and the ability to simultaneously determine hypophosphite, phosphite, phosphate, pyrophosphate, and trimetaphosphate. Therefore, accurate determination of the content of these impurities plays a key role and is of great significance in controlling the function of sodium phosphate and the quality of preparations.

[0066] The inventors have tried to select complex mobile phases. For example, the inventors have tried to use an alkaline mobile phase of 100 mM potassium hydroxide solution for isocratic elution. The chromatogram obtained is shown in FIG. Figure 2, indicating that the nine ions cannot be completely separated. For another example, the inventors have tried to use a neutral mobile phase of pure water for isocratic elution, and the chromatogram obtained is shown in Figure 3 , which also shows that the nine ions cannot be completely separated.

[0067] The use of ion chromatography to detect chloride, nitrate and sulfate has been reported in various industries, but the determination of carbonate is difficult to detect due to the influence of carbon dioxide in the sample, and because the main component of sodium phosphate, PO4 3- The concentration is extremely high, which will interfere with the detection of trace inorganic anions. In addition, the inventors also refer to the mobile phase in the literature [Song Ziyu, Ding Dazhong, Zhang Lei, et al. Determination of the content of three anions in sodium dihydrogen phosphate by ion chromatography [J]. Medical Herald, 2022, 41(9): 1351-1353] for elution. The typical elution chromatogram can be found in Figure 4 and Figure 5 , showing that the elution result is not ideal. After unremitting efforts and creative labor, the inventor finally found a method of gradient elution using the hydroxide eluent described in Detection Example 1, which makes PO4 3- The later elution peak avoids interference with other anionic components. At the same time, the system can minimize baseline shift during gradient elution, achieving better reproducibility, lower background noise and more sensitive detection limit.

[0068] In addition to being able to detect chloride ions, sulfate ions and carbonate ions, the method of the present invention can also determine other inorganic anions. The known anion impurities are calculated by the external standard method, and the other anion impurities are calculated by the phosphate reference method. According to the measurement results of 14 batches of samples, the chloride ions are all lower than 0.005%, the sulfate ions and nitrate ions are all lower than 0.01%, and the carbonate ions are all lower than 0.5%. Only one batch of samples was detected with a pyrophosphate content of 0.759%. Referring to the inspection method for chloride, sulfate and carbonate in sodium phosphate dodecahydrate included in the 2020 edition of the Chinese Pharmacopoeia and the newly established ion chromatography method; the limit of the newly established ion chromatography method can cover the pharmacopoeia method. The test results of 14 batches of samples by the method of detection example 1 of the present invention are compared with the test results of the ChP2020 version [Pharmacopoeia of the People's Republic of China 2020 edition. Part Four [S]. 2020: 838] method, and the results are shown in Table 4 for details.

[0069] Table 4: Comparison of inorganic anion detection methods

[0070] The method of the present invention can be used to simultaneously detect the contents of nine anions, including hypophosphite, chloride, phosphite, carbonate, nitrate, sulfate, phosphate, pyrophosphate and trimetaphosphate, in sodium phosphate pharmaceutical excipients, and is not affected by the high concentration of the main component phosphate ion. The method of the present invention is suitable for detecting sodium phosphate raw materials for preparing immune adjuvants, and can also provide a reference for the quality control of inorganic anions in samples in multiple fields such as other inorganic salt pharmaceutical excipients, water quality, beverages and food additives.

[0071] It is obvious to those skilled in the art that the present application is not limited to the details of the above exemplary embodiments, and that the present application can be implemented in other specific forms without departing from the spirit or essential features of the present application. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive, and the scope of the present application is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims are included in the present application.

Claims

1. A method for simultaneously detecting 9 inorganic anions or 8 inorganic impurity anions in a sodium phosphate raw material using ion chromatography, the method comprising the following steps: (1) providing an ion chromatograph and a conductivity detector (for example, the ion chromatograph is a Thermo DioNEX AS-AP ion chromatograph, and the conductivity detector is a conductivity detector); (2) providing carbonate ion standard solution, chlorine standard substance in water, sulfate standard substance in water, nitrate standard substance in water, phosphate standard substance in water, providing standard substances of sodium pyrophosphate, hypophosphorous acid, phosphorous acid, and sodium trimetaphosphate; providing sodium phosphate sample to be tested (for example, anhydrous sodium phosphate, sodium phosphate dodecahydrate, sodium phosphate monohydrate); (3) Preparation of reference solution and mixed reference solution: Accurately weigh or measure appropriate amounts of 9 anion standard substances, including hypophosphite, chloride, phosphite, carbonate, nitrate, sulfate, phosphate, pyrophosphate, and trimetaphosphate, and add appropriate amount of ultrapure water to prepare reference stock solution with a mass concentration of 1 mg / ml; Accurately measure an appropriate amount of reference stock solution to prepare reference solutions for qualitative or quantitative determination of each ion at a concentration suitable for detection or mixed reference solutions for system suitability testing; System suitability solution The concentration of hypophosphite, phosphite, phosphate, pyrophosphate and trimetaphosphate is 0.01 μg / ml~100 μg / ml (e.g. 1 μg / ml), the concentration of chloride ion is 0.005 μg / ml~100 μg / ml (e.g. 0.1 μg / ml), the concentration of nitrate and sulfate is 0.05 μg / ml~100 μg / ml (e.g. 0.2 μg / ml), and the concentration of carbonate is 0.5 μg / ml~100 μg / ml (e.g. 10 μg / ml); (4) Preparation of test solution: Accurately weigh an appropriate amount of sample, place in a 10 ml volumetric flask, add water to dissolve and dilute to scale to prepare a test solution (e.g., equivalent to a phosphate concentration of about 2 mg / ml); (5) Chromatographic conditions: Column: Dionex IonPac TM AS11-HC Analytical (4mm×250mm, other chromatographic columns with equivalent performance can also be used), Guard column: Dionex IonPac TM AG11-HC Analytical (4mm×50mm, other guard columns with equivalent performance can also be used), Suppressor: Thermo Scientific Dionex ADRS 600 (other suppressors of equivalent performance may also be used), Detector: Conductivity detector, Eluent: A is 100mmol / L potassium hydroxide solution, B is water, gradient elution: 0~10min (A: 20%~25%)→10.1~30min (A: 22%→A: 75%~85%)→30.1~51min (A: 75%~85%)→51.1~60min (A: 20%~25%), flow rate 0.8mL / min~1.2mL / min; for example: 0~10min (A: 22%)→10.1~30min (A: 22%→A: 80%)→30.1~51min (A: 80%)→51.1~60min (A: 22%), flow rate 1.0mL / min, Column temperature: 30℃~40℃, for example 35℃; Injection volume: 1μL~100μL, for example 20μL; (6) Determination method: Accurately measure 20 μl of each reference solution, system suitability solution, and test solution, respectively, and inject them into the ion chromatograph to record the chromatogram; use the chromatogram obtained from the system suitability solution to conduct a methodological investigation, calculate the contents of 8 inorganic anion impurities in the test sample by the peak area of ​​the reference solution chromatogram according to the external standard method, and optionally calculate the phosphate content in the test sample; the separation between the phosphate peak and its adjacent peaks and other anion impurity peaks is greater than 2.0 (in line with the general requirements in this field); for unknown impurities, use phosphate as a reference and calculate the impurity content by peak area according to the external standard method.

2. A method for preparing an aluminum phosphate adjuvant, comprising the steps of: (i) using ion chromatography to simultaneously detect the contents of the following eight anions in the sodium phosphate raw material: hypophosphite, chloride, phosphorous acid, carbonate, nitrate, sulfate, pyrophosphate, and trimetaphosphate, and selecting sodium phosphate with a low anion impurity content as the raw material (for example, selecting sodium phosphate that meets pharmaceutical standards and has eight anions less than 0.1%, other impurities less than 0.1%, and a total impurity content less than 0.2% as the raw material); the method for detecting eight anions in the sodium phosphate raw material comprises the following steps: (1) Providing an ion chromatograph and a conductivity detector; (2) providing carbonate ion standard solution, chlorine standard substance in water, sulfate standard substance in water, nitrate standard substance in water, phosphate standard substance in water, providing standard substances of sodium pyrophosphate, hypophosphorous acid, phosphorous acid, and sodium trimetaphosphate; providing sodium phosphate sample to be tested (for example, anhydrous sodium phosphate, sodium phosphate dodecahydrate, sodium phosphate monohydrate); (3) Preparation of reference solution and mixed reference solution: accurately weigh or measure appropriate amounts of 9 anion standard substances, namely hypophosphite, chloride, phosphite, carbonate, nitrate, sulfate, phosphate, pyrophosphate and trimetaphosphate, and add appropriate amount of ultrapure water to prepare reference stock solution with a mass concentration of 1 mg / ml; accurately measure appropriate amount of reference stock solution to prepare reference solutions for qualitative or quantitative determination of each ion or mixed reference solution for system suitability test at a concentration suitable for detection; the concentration of hypophosphite, phosphite, phosphate, pyrophosphate and trimetaphosphate in the system suitability solution is 1 μg / ml, the concentration of chloride is 0.1 μg / ml, the concentration of nitrate and sulfate is 0.2 μg / ml, and the concentration of carbonate is 10 μg / ml; (4) Preparation of test solution: Accurately weigh an appropriate amount of sample (about 0.21 mmol), place in a 10 ml volumetric flask, add water to dissolve and dilute to the mark, which is equivalent to a phosphate concentration of 2 mg / ml; (5) Chromatographic conditions: Column: Dionex IonPac TM AS11-HC Analytical (4mm×250mm, other chromatographic columns with equivalent performance can also be used), Guard column: Dionex IonPac TM AG11-HC Analytical (4mm×50mm, other guard columns with equivalent performance can also be used), Suppressor: Thermo Scientific Dionex ADRS 600 (other suppressors of equivalent performance may also be used), Detector: Conductivity detector, Eluent: A is 100mmol / L potassium hydroxide solution, B is water, gradient elution: 0~10min (A: 22%) → 10.1~30min (A: 22% → A: 80%) → 30.1~51min (A: 80%) → 51.1~60min (A: 22%), flow rate 1.0mL / min, Column temperature: 35°C, Injection volume: 20 μL; (6) Determination method: Accurately measure 20 μl of each reference solution, system suitability solution, and test solution, inject them into the ion chromatograph, and record the chromatogram; The chromatogram obtained from the system suitability solution was used for methodological investigation, and the contents of 8 inorganic anion impurities in the test sample were calculated using the peak area of ​​the chromatogram of the reference solution according to the external standard method; The separation between the phosphate peak and its adjacent peaks and other anionic impurity peaks is greater than 2.0 (in line with the general requirements in this field); for unknown impurities, phosphate is used as a reference, and the impurity content is calculated by the peak area according to the external standard method; (ii) weighing 10 mmol of aluminum chloride or its hydrate, such as AlCl3·6H2O, and 9-11 mmol, such as 9 mmol or 10 mmol or 11 mmol, of a sodium phosphate raw material (such as anhydrous sodium phosphate, sodium phosphate dodecahydrate, sodium phosphate monohydrate) meeting the detection requirements of step (1), and dissolving them in 30-50 ml, such as 30 ml or 40 ml or 50 ml, and 25-35 ml, such as 25 ml or 30 ml or 35 ml, of a solvent, respectively, wherein the solvent is a mixture of ethylene glycol and deionized water in a volume ratio of 10-15:100, such as 10:100, 12:100 or 15:100; (iii) placing the aluminum chloride solution on a magnetic stirrer, uniformly adding the sodium phosphate solution to the aluminum chloride solution at room temperature at a stirring speed of 700-800 rpm, for example, 700 rpm, 750 rpm or 800 rpm, over a period of 18-23 minutes, for example, 18 minutes, 20 minutes or 23 minutes, and continuing to stir for 40-50 minutes, for example, 40 minutes, 45 minutes or 50 minutes after the addition is completed, and then transferring the reaction solution to a 150 ml glass bottle and autoclaving at 121° C. for 30 minutes; (iv) The reaction solution was allowed to stand and naturally cool to room temperature, centrifuged at 10,000 rpm for 15 min, the supernatant was discarded, and the precipitate was washed three times with sterile water for injection. After each washing, the precipitate was centrifuged at 10,000 rpm for 15 min and the supernatant was discarded. The precipitate was dried under reduced pressure at 45° C. to a water content of less than 3%, thereby obtaining a micro-powdered aluminum phosphate adjuvant sample.

3. The method according to claim 2, wherein the sodium phosphate is selected from the group consisting of anhydrous sodium phosphate, sodium phosphate dodecahydrate, and sodium phosphate monohydrate.

4. The method according to claim 2, wherein the ion chromatograph is a Thermo DioNEX AS-AP ion chromatograph.

5. The method according to claim 2, wherein the conductivity detector is a conductivity detector.

6. The method according to claim 2, comprising the steps of: (i) using ion chromatography to simultaneously detect the contents of the following eight anions in the sodium phosphate raw material: hypophosphite, chloride, phosphorous acid, carbonate, nitrate, sulfate, pyrophosphate, and trimetaphosphate, and selecting sodium phosphate with a low anion impurity content as the raw material (for example, selecting sodium phosphate that meets pharmaceutical standards and has eight anions less than 0.1%, other impurities less than 0.1%, and a total impurity content less than 0.2% as the raw material); the sodium phosphate is selected from: anhydrous sodium phosphate, sodium phosphate dodecahydrate, and sodium phosphate monohydrate; (ii) 2.415 g (Mr 241.46, 10 mmol) of AlCl3·6H2O and 1.639 g (Mr 163.94, 10 mmol) of Na3PO4 were weighed and dissolved in 40 ml and 30 ml of a solvent, respectively, wherein the solvent was a mixture of ethylene glycol and deionized water in a volume ratio of 12:100; (iii) placing the aluminum chloride solution on a magnetic stirrer, uniformly adding the sodium phosphate solution to the aluminum chloride solution at room temperature at a stirring speed of 750 rpm over a period of 20 minutes, and continuing to stir for 45 minutes after the addition is completed. Then, the reaction solution is transferred to a 150 ml glass bottle and autoclaved at 121° C. for 30 minutes; (iv) The reaction solution was allowed to stand and naturally cool to room temperature, centrifuged at 10,000 rpm for 15 min, the supernatant was discarded, and the precipitate was washed three times with sterile water for injection. After each washing, the precipitate was centrifuged at 10,000 rpm for 15 min and the supernatant was discarded. The precipitate was dried under reduced pressure at 45° C. to a water content of less than 3%, thereby obtaining a micro-powdered aluminum phosphate adjuvant sample.

7. The method according to claim 2, comprising the steps of: (i) using ion chromatography to simultaneously detect the contents of the following eight anions in the sodium phosphate raw material: hypophosphite, chloride, phosphorous acid, carbonate, nitrate, sulfate, pyrophosphate, and trimetaphosphate, and selecting sodium phosphate with a low anion impurity content as the raw material (for example, selecting sodium phosphate that meets pharmaceutical standards and has eight anions less than 0.1%, other impurities less than 0.1%, and a total impurity content less than 0.2% as the raw material); the sodium phosphate is selected from: anhydrous sodium phosphate, sodium phosphate dodecahydrate, and sodium phosphate monohydrate; (ii) 2.415 g (Mr 241.46, 10 mmol) of AlCl3·6H2O and 3.8013 g (Mr 380.13, 10 mmol) of Na3PO4·12H2O were weighed and dissolved in 40 ml and 30 ml of a solvent, respectively, wherein the solvent was a mixture of ethylene glycol and deionized water in a volume ratio of 12:100; (iii) placing the aluminum chloride solution on a magnetic stirrer, uniformly adding the sodium phosphate solution to the aluminum chloride solution at room temperature at a stirring speed of 750 rpm over a period of 20 minutes, and continuing to stir for 45 minutes after the addition is completed. Then, the reaction solution is transferred to a 150 ml glass bottle and autoclaved at 121° C. for 30 minutes; (iv) The reaction solution was allowed to stand and naturally cool to room temperature, centrifuged at 10,000 rpm for 15 min, the supernatant was discarded, and the precipitate was washed three times with sterile water for injection. After each washing, the precipitate was centrifuged at 10,000 rpm for 15 min and the supernatant was discarded. The precipitate was dried under reduced pressure at 45° C. to a water content of less than 3%, thereby obtaining a micro-powdered aluminum phosphate adjuvant sample.

8. The method according to claim 2, comprising the steps of: (i) using ion chromatography to simultaneously detect the contents of the following eight anions in the sodium phosphate raw material: hypophosphite, chloride, phosphorous acid, carbonate, nitrate, sulfate, pyrophosphate, and trimetaphosphate, and selecting sodium phosphate with a low anion impurity content as the raw material (for example, selecting sodium phosphate that meets pharmaceutical standards and has eight anions less than 0.1%, other impurities less than 0.1%, and a total impurity content less than 0.2% as the raw material); the sodium phosphate is selected from: anhydrous sodium phosphate, sodium phosphate dodecahydrate, and sodium phosphate monohydrate; (ii) 2.415 g (Mr 241.46, 10 mmol) of AlCl3·6H2O and 3.421 g (Mr 380.13, 9 mmol) of Na3PO4·12H2O were weighed and dissolved in 50 ml and 25 ml of a solvent, respectively, wherein the solvent was a mixture of ethylene glycol and deionized water in a volume ratio of 10:100; (iii) placing the aluminum chloride solution on a magnetic stirrer, uniformly adding the sodium phosphate solution to the aluminum chloride solution at room temperature at a stirring speed of 800 rpm over a period of 23 minutes, and continuing to stir for 50 minutes after the addition is completed. Then, the reaction solution is transferred to a 150 ml glass bottle and autoclaved at 121° C. for 30 minutes; (iv) The reaction solution was allowed to stand and naturally cool to room temperature, centrifuged at 10,000 rpm for 15 min, the supernatant was discarded, and the precipitate was washed three times with sterile water for injection. After each washing, the precipitate was centrifuged at 10,000 rpm for 15 min and the supernatant was discarded. The precipitate was dried under reduced pressure at 45° C. to a water content of less than 3%, thereby obtaining a micro-powdered aluminum phosphate adjuvant sample.

9. The method according to claim 2, comprising the steps of: (i) using ion chromatography to simultaneously detect the contents of the following eight anions in the sodium phosphate raw material: hypophosphite, chloride, phosphorous acid, carbonate, nitrate, sulfate, pyrophosphate, and trimetaphosphate, and selecting sodium phosphate with a low anion impurity content as the raw material (for example, selecting sodium phosphate that meets pharmaceutical standards and has eight anions less than 0.1%, other impurities less than 0.1%, and a total impurity content less than 0.2% as the raw material); the sodium phosphate is selected from: anhydrous sodium phosphate, sodium phosphate dodecahydrate, and sodium phosphate monohydrate; (ii) 2.415 g (Mr 241.46, 10 mmol) of AlCl3·6H2O and 4.181 g (Mr 380.13, 11 mmol) of Na3PO4·12H2O were weighed and dissolved in 30 ml and 35 ml of a solvent, respectively, wherein the solvent was a mixture of ethylene glycol and deionized water in a volume ratio of 15:100; (iii) placing the aluminum chloride solution on a magnetic stirrer, uniformly adding the sodium phosphate solution to the aluminum chloride solution at room temperature at a stirring speed of 700 rpm over a period of 18 minutes, and continuing to stir for 40 minutes after the addition is completed. Then, the reaction solution is transferred to a 150 ml glass bottle and autoclaved at 121° C. for 30 minutes; (iv) The reaction solution was allowed to stand and naturally cool to room temperature, centrifuged at 10,000 rpm for 15 min, the supernatant was discarded, and the precipitate was washed three times with sterile water for injection. After each washing, the precipitate was centrifuged at 10,000 rpm for 15 min and the supernatant was discarded. The precipitate was dried under reduced pressure at 45° C. to a water content of less than 3%, thereby obtaining a micro-powdered aluminum phosphate adjuvant sample.

10. An aluminum phosphate adjuvant prepared by the method according to any one of claims 2 to 9.

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