Method for detecting phosphate, pyrophosphate and tripolyphosphate in diquafosol sodium

Abstract

The invention relates to a method for detecting phosphate, pyrophosphate and tripolyphosphate in diquafosol sodium, which has the advantages of high sensitivity, strong specificity, good accuracy and simple and rapid operation, can effectively analyze the drug quality, and has practical significance for ensuring the safety and reliability of diquafosol sodium.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of analytical chemistry, and particularly relates to a method for detecting phosphate, pyrophosphate and tripolyphosphate in diquafosol sodium. BACKGROUND

[0002] Diquafosol sodium is a dinucleotide derivative, which acts on P2Y2 receptors on the conjunctival tissue and the membrane of goblet cells to increase the intracellular calcium ion concentration, promote the secretion of tear fluid containing water and mucin, and improve the damage of the corneal epithelium in terms of quality and quantity.

[0003] The impurity level is one of the important factors for the safety and effectiveness of a drug, and therefore, how to accurately determine the content of phosphate, pyrophosphate and tripolyphosphate in diquafosol sodium becomes a problem to be solved.

[0004] At present, there is no method for detecting phosphate, pyrophosphate and tripolyphosphate in diquafosol sodium in the pharmacopoeias, patents and literatures of various countries.

[0005] In order to effectively analyze the quality of a drug and ensure the safety of medication, a method for detecting phosphate, pyrophosphate and tripolyphosphate with high sensitivity, good specificity, strong durability, convenience and effectiveness needs to be developed. SUMMARY

[0006] The technical problem to be solved by the present application is to provide a method for detecting phosphate, pyrophosphate and tripolyphosphate in diquafosol sodium, which has high sensitivity, good specificity and high accuracy, can strictly control the quality of phosphate, pyrophosphate and tripolyphosphate in diquafosol sodium, ensures the safety and reliability of diquafosol sodium, and has practical significance.

[0007] To solve the above technical problem, the technical scheme adopted by the present application is as follows:

[0008] The method for detecting phosphate, pyrophosphate and tripolyphosphate in diquafosol sodium adopts ion chromatography, a Dionex IonPac TM AS11 column is used as the chromatographic column, a Dionex IonPac TM AG11 column is used as the guard column, a potassium hydroxide aqueous solution is used as the eluent, the flow rate is 1.0 ml / min, the column temperature is 30℃, a conductivity detector is used, the detection method is suppressive conductivity detection, the current value is 248mA, the conductivity cell temperature is 35℃, the injection volume is 25μl, and gradient elution is performed.

[0009] Preferably, the gradient elution procedure is as follows:

[0010]

[0011] Preferably, the chromatographic column is an anion exchange column; in this embodiment of the invention, the Dionex IonPac anion exchange column is used. TM The AS11 column, measuring 4mm x 250mm, is supplied by Thermo Fisher Scientific; the guard column is an anion exchange guard column, preferably Dionex IonPac. TM The AG11 column, measuring 4mm x 50mm, is supplied by Thermo Fisher Scientific.

[0012] Preferably, the flow rate is 0.5 ml / min to 1.5 ml / min, and more preferably 1.0 ml / ml.

[0013] Preferably, the column temperature is 25℃~35℃, and more preferably 30℃.

[0014] Preferably, the temperature of the conductivity cell is 30°C to 40°C, and more preferably 35°C.

[0015] A conductivity detector was used, and the detection method was suppressed conductivity detection with a current value of 248mA.

[0016] Preferably, the injection volume is 25 μl.

[0017] Preferably, the method includes the following steps:

[0018] (1) Test solution: Take an appropriate amount of this product, weigh it accurately, add 50 mmol / L potassium hydroxide solution to dissolve and quantitatively dilute to prepare a solution containing about 5 mg per 1 ml;

[0019] (2) Reference solution: Accurately weigh appropriate amounts of potassium pyrophosphate, sodium tripolyphosphate, and potassium dihydrogen phosphate dried to constant weight at 105℃, dissolve and quantitatively dilute with 50 mmol / L potassium hydroxide solution to prepare a solution containing approximately phosphate ions (in PO42-) per ml. 3- (calculated as P2O7), pyrophosphate (as P2O7) 4- (calculated) and tripolyphosphate (in P3O) 10 5- A mixed solution of approximately 25 μg each;

[0020] (3) The reference solution and the test solution were determined by ion chromatography, respectively;

[0021] The detection conditions for the ion chromatography method are as follows: chromatographic column: Dionex IonPac TM AS11 column; Dionex IonPac protective column TMAG11 column; potassium hydroxide aqueous solution as eluent, flow rate of 1.0 ml / min; column temperature of 30℃; conductivity detector, detection method of suppressed conductivity detection, current value of 248 mA, conductivity cell temperature of 35℃; injection volume of 25 μl; gradient elution.

[0022] Result calculation: Calculated using the external standard method.

[0023] Due to the adoption of the above technical solutions, the present invention has the following advantages compared with the prior art:

[0024] 1. The determination method of the present invention provides a comprehensive analysis of phosphate, pyrophosphate and tripolyphosphate in diquaphosphophosphate sodium, and can achieve effective separation of phosphate, pyrophosphate and tripolyphosphate in diquaphosphophosphate sodium.

[0025] 2. The method of this invention has high sensitivity, strong specificity and good accuracy, providing a simple and convenient detection method for the examination of phosphate, pyrophosphate and tripolyphosphate in diquaphosphodium. It can strictly control the quality of diquaphosphodium, ensuring the safety and reliability of diquaphosphodium, and has practical significance. Attached Figure Description

[0026] Figure 1 This is the chromatogram of the blank solution in this invention;

[0027] Figure 2 These are the chromatograms of the various positioning solutions in this invention;

[0028] Figure 3 This is the chromatogram of the reference solution in this invention;

[0029] Figure 4 This is the chromatogram of the test solution in this invention; Figure 5 This is a chromatogram of the spiked solution of the test sample in this invention. Detailed Implementation

[0030] To make the technical solution and beneficial effects of the present invention more apparent and understandable, a detailed description is provided below in conjunction with the accompanying drawings and specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any product identical or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0031] Experimental methods not specified in the following examples are generally performed under standard experimental conditions.

[0032] In the specific embodiments of the present invention, sodium diquafosol, potassium dihydrogen phosphate, potassium pyrophosphate, sodium tripolyphosphate, and equipment used are all known products. Sodium diquafosol, potassium dihydrogen phosphate, potassium pyrophosphate, and sodium tripolyphosphate are obtained by purchasing commercially available products.

[0033] Table 1 Equipment Information

[0034] Equipment name Model Manufacturer Ion chromatograph ICS2000 Thermo Fisher Scientific Electronic balance 125SM-FR Precisa Weighing Instruments Ltd. Electronic balance PX623ZH / E Ohaus Instruments (Changzhou) Co., Ltd. Pure water instrument EUE-10UV Haisi Company Vacuum drying oven DZ-1BCIV Tianjin Tester Instrument Co., Ltd.

[0035] Table 2 Material Information

[0036] Name Source Batch number Potassium hydroxide National Pharmaceutical Group Chemical Reagent Co., Ltd. 20230511 Potassium hydroxide eluent Thermo Fisher Scientific 074532 Diquafos sodium Guangdong Xianqiang Pharmaceutical Co., Ltd. 230901R

[0037] Table 3. Reference Standard Information

[0038] Name Manufacturer Batch number Content (%) Monopotassium phosphate National Pharmaceutical Group Chemical Reagent Co., Ltd. 20230927 100.0 Potassium pyrophosphate Mai Rui 67907009 99.6 Sodium tripolyphosphate Mcclin C16279246 98.112

[0039] Example 1

[0040] Methodological study of the detection and analysis method of the present invention

[0041] The following conditions were used for all experiments in this embodiment:

[0042] Column: Dionex IonPac TM AS11 column (4mm*250mm);

[0043] Protective pillar: Dionex IonPac TM AG11 column (4mm*50mm);

[0044] Flow rate: 1.0 ml / min;

[0045] Column temperature: 30℃;

[0046] Detector: Conductivity detector;

[0047] Detection method: Suppressed conductivity detection;

[0048] Current value: 248mA;

[0049] Injection volume: 25 μl

[0050] The gradient elution procedure is as follows:

[0051]

[0052] Testing steps:

[0053] Blank solution: 50 mmol / L potassium hydroxide solution.

[0054] Test solution: Take an appropriate amount of this product, accurately weigh it, dissolve it in 50 mmol / L potassium hydroxide solution and dilute it quantitatively to prepare a solution containing about 5 mg per ml.

[0055] Reference solution: Accurately weigh appropriate amounts of potassium pyrophosphate, sodium tripolyphosphate, and potassium dihydrogen phosphate dried to constant weight at 105℃. Dissolve and quantitatively dilute with 50 mmol / L potassium hydroxide solution to prepare a solution containing approximately phosphate ions per ml (in PO42-). 3- (calculated as P2O7), pyrophosphate (as P2O7) 4- (calculated) and tripolyphosphate (in P3O) 10 5- A mixed solution of approximately 25 μg each.

[0056] 1. Specificity test

[0057] Solution preparation:

[0058] Blank solution: 50 mmol / L potassium hydroxide solution.

[0059] Phosphate stock solution: Accurately weigh 89.6 mg of potassium dihydrogen phosphate dried to constant weight at 105 °C, place it in a 50 mL volumetric flask, dissolve and dilute to the mark with 50 mmol / L potassium hydroxide solution, and shake well to obtain the solution. [Phosphate (as PO4)] 3- The concentration (calculated) is approximately 1250 μg / ml.

[0060] Pyrophosphate stock solution: Accurately weigh 119.2 mg of potassium pyrophosphate, place it in a 50 ml volumetric flask, dissolve and dilute to the mark with 50 mmol / L potassium hydroxide solution, and shake well. [Pyrophosphate (as P2O7)] 4- The concentration (calculated) is approximately 1250 μg / ml.

[0061] Tripolyphosphate stock solution: Accurately weigh 92.7 mg of sodium tripolyphosphate, place it in a 50 ml volumetric flask, dissolve and dilute to the mark with 50 mmol / L potassium hydroxide solution, and shake well to obtain the solution. [Tripolyphosphate (as P3O4)] 10 5- The concentration (calculated) is approximately 1250 μg / ml.

[0062] Positioning solutions: Accurately measure 1 ml each of the stock solutions of phosphate, pyrophosphate, and tripolyphosphate, and place them separately in 50 ml volumetric flasks. Dilute to the mark with 50 mmol / L potassium hydroxide solution, and shake well. [Phosphate (as PO4)] 3- (calculated as P2O7), pyrophosphate (as P2O7) 4- (calculated), tripolyphosphate (as P3O) 10 5- The concentration (calculated) is approximately 25 μg / ml.

[0063] Reference solutions: Accurately measure 1 ml each of the stock solutions of phosphate, pyrophosphate, and tripolyphosphate, place them in the same 50 ml volumetric flask, dilute to the mark with 50 mmol / L potassium hydroxide solution, and shake well. [Phosphate (as PO4)] 3- (calculated as P2O7), pyrophosphate (as P2O7) 4- (calculated), tripolyphosphate (as P3O) 10 5- The concentration (calculated) is approximately 25 μg / ml.

[0064] Test solution: Accurately weigh 138.2 mg of sample from batch 230901R, place it in a 25 ml volumetric flask, dissolve and dilute to the mark with 50 mmol / L potassium hydroxide solution, and shake well. (The concentration of diquafosol sodium is approximately 5 mg / ml.)

[0065] Spiked solution for the test sample: Accurately weigh 138.2 mg of sample from batch 230901R and place it in a 25 ml volumetric flask. Accurately measure 0.5 ml each of the stock solutions of phosphate, pyrophosphate, and tripolyphosphate and place them in the same 25 ml volumetric flask. Dissolve and dilute to the mark with 50 mmol / L potassium hydroxide solution, and shake well. [Phosphate (as PO4)] 3 The concentrations of pyrophosphate (calculated as P2O74-), tripolyphosphate (calculated as P3O105-) were all approximately 25 μg / ml, and the concentration of diquafosol sodium was approximately 5 mg / ml.

[0066] Accurately measure 25 μl each of the blank solution, each positioning solution, the reference solution, the test solution, and the spiked test solution, and inject them into the ion chromatograph. Record the chromatograms. The detection results are shown in the appendix. Figure 1 ~Attached Figure 5 And Table 4 below.

[0067] Table 4 Results of specificity test

[0068]

[0069] The results above show that the blank solution does not interfere with the detection; in the reference solution, phosphate, pyrophosphate and tripolyphosphate peaks appear sequentially, and the resolutions between the impurity peaks are 16.54 and 9.48, respectively, both not less than 1.5; no unknown impurities were detected in the test solution, and it does not interfere with the detection of known impurities, indicating that this method has good specificity.

[0070] 2. Precision test

[0071] Solution preparation:

[0072] Blank solution: 50 mmol / L potassium hydroxide solution.

[0073] Reference stock solution: Accurately weigh 119.2 mg potassium pyrophosphate, 92.7 mg sodium tripolyphosphate, and 89.6 mg potassium dihydrogen phosphate dried to constant weight at 105 °C, place them in a 50 ml volumetric flask, dissolve and dilute to the mark with 50 mmol / L potassium hydroxide solution, and shake well to obtain the solution.

[0074] Reference solution: Accurately measure 1 ml of the reference stock solution, place it in a 50 ml volumetric flask, dilute to the mark with 50 mmol / L potassium hydroxide solution, and shake well to obtain the solution.

[0075] Five consecutive injections of the reference solution were performed. The peak area and retention time were recorded, and the RSD values ​​of the peak area and retention time were calculated. The detection results are shown in Tables 5 to 7 below.

[0076] Table 5 Results of Phosphate Injection Precision Test

[0077]

[0078] Table 6 Results of Pyrophosphate Injection Precision Test

[0079]

[0080] Table 7 Results of Tripolyphosphate Injection Precision Test

[0081]

[0082] The results above show that: after five consecutive injections of the reference solution, the RSD of the phosphate peak area was 0.38%, and the RSD of the retention time was 0.53%; the RSD of the pyrophosphate peak area was 0.26%, and the RSD of the retention time was 0.62%; the RSD of the tripolyphosphate peak area was 0.69%, and the RSD of the retention time was 0.85%; the RSD of the peak area and the RSD of the retention time of each impurity peak were all no greater than 2.0%, indicating that the injection precision of this method is good.

[0083] 3. Linearity and Range Tests

[0084] Solution preparation:

[0085] Blank solution: 50 mmol / L potassium hydroxide solution.

[0086] Linear stock solution: Accurately weigh 119.2 mg potassium pyrophosphate, 92.7 mg sodium tripolyphosphate, and 89.6 mg potassium dihydrogen phosphate dried to constant weight at 105 °C, place them in a 50 ml volumetric flask, dissolve and dilute to the mark with 50 mmol / L potassium hydroxide solution, and shake well to obtain the solution.

[0087] Take an appropriate amount of linear stock solution and prepare linear solutions of various concentrations according to the table below.

[0088] Linear concentration Volume of linear stock solution (ml) Make up to (ml) 25％ 0.5 100 50％ 0.5 50 100％ 1.0 50 150％ 1.5 50 200％ 2.0 50

[0089] Accurately measure 25 μl of each linear solution, inject it into the ion chromatograph, record the chromatogram, and perform linear regression analysis on the concentration using peak area. The detection results are shown in Tables 8 to 10 below.

[0090] Table 8 Results of Phosphate Linearity Test

[0091]

[0092] Table 9 Results of linearity test for pyrophosphate

[0093]

[0094] Table 10 Results of the linearity test for tripolyphosphate

[0095]

[0096] The results above show that within the concentration range of phosphate from 6.301 μg / ml to 50.41 μg / ml, the correlation coefficient R is [value missing]. 2 The correlation coefficient R is 1.0000, greater than 0.998; for pyrophosphate in the concentration range of 6.294 μg / ml to 50.35 μg / ml, the correlation coefficient R is... 2 The correlation coefficient R is 1.0000, which is greater than 0.998; for tripolyphosphate in the concentration range of 6.263 μg / ml to 50.11 μg / ml, the correlation coefficient R is... 2 The value is 1.0000, which is greater than 0.998, indicating that the linear relationship of this method is relatively good.

[0097] 4. Limit of detection test

[0098] Solution preparation:

[0099] Blank solution: 50 mmol / L potassium hydroxide solution.

[0100] Take the linear point solutions prepared under item 3. Linearity test and dilute them stepwise. Use a signal-to-noise ratio of not less than 10:1 as the limit of quantitation solution and not less than 3:1 as the limit of detection solution.

[0101] Each of the above diluted solutions was injected into the ion chromatograph, and the chromatograms were recorded. The detection results are shown in Tables 11 to 13 below.

[0102] Table 11 Results of Phosphate Detection Limits

[0103]

[0104] Table 12 Results of Limits for Pyrophosphate Detection

[0105]

[0106]

[0107] Table 13 Results of Tripolyphosphate Detection Limits

[0108]

[0109] The results above show that: the detection limit for phosphate is 0.01260 μg / ml, with a ratio of 0.05% to the reference concentration (not exceeding 20%), and the quantitation limit is 0.02520 μg / ml, with a ratio of 0.1% to the reference concentration (not exceeding 50%); the detection limit for pyrophosphate is 0.02518 μg / ml, with a ratio of 0.1% to the reference concentration (not exceeding 20%), and the quantitation limit is 0.05035 μg / ml, with a ratio of 0.2% to the reference concentration (not exceeding 50%); the detection limit for tripolyphosphate is 0.02505 μg / ml, with a ratio of 0.1% to the reference concentration (not exceeding 20%), and the quantitation limit is 0.05011 μg / ml, with a ratio of 0.2% to the reference concentration (not exceeding 50%). This method exhibits high detection sensitivity.

[0110] 5. Repeatability test

[0111] Solution preparation:

[0112] Blank solution: 50 mmol / L potassium hydroxide solution.

[0113] Reference stock solution: Accurately weigh 119.2 mg potassium pyrophosphate, 92.7 mg sodium tripolyphosphate, and 89.6 mg potassium dihydrogen phosphate dried to constant weight at 105 °C, place them in a 50 ml volumetric flask, dissolve and dilute to the mark with 50 mmol / L potassium hydroxide solution, and shake well to obtain the solution.

[0114] Reference solution: Accurately measure 1 ml of the reference stock solution, place it in a 50 ml volumetric flask, dilute to the mark with 50 mmol / L potassium hydroxide solution, and shake well to obtain the solution.

[0115] Test solution (background solution): Accurately weigh 138.2 mg of sample from batch 230901R, place it in a 25 ml volumetric flask, dissolve and dilute to the mark with 50 mmol / L potassium hydroxide solution, and shake well. Prepare two parallel solutions.

[0116] 100% recovery solution: Accurately weigh 138.2 mg of sample from batch 230901R and place it in a 25 ml volumetric flask. Accurately measure 0.5 ml of the reference stock solution and place it in the same 25 ml volumetric flask. Dissolve and dilute to the mark with 50 mmol / L potassium hydroxide solution, and shake well. Prepare 6 parallel aliquots using the same method.

[0117] Accurately measure 25 μl of each of the blank solution, reference solution, test solution, and 100% recovery solution and inject them into the ion chromatograph. Record the chromatograms, calculate the measured amount using the external standard method, subtract the background amount and compare it with the added amount to calculate the recovery rate. The detection results are shown in Tables 14 to 16 below.

[0118] Table 14 Results of Phosphate Repeatability Tests

[0119]

[0120] Table 15 Results of repeatability test for pyrophosphate

[0121]

[0122] Table 16 Results of Tripolyphosphate Repeatability Tests

[0123]

[0124]

[0125] The results show that the average recoveries of phosphate, pyrophosphate and tripolyphosphate in the six 100% recovery solutions were 99.9%, 100.7% and 101.2%, respectively, all within the range of 90% to 110%. The RSD values ​​were 0.62%, 0.69% and 0.64%, respectively, all not exceeding 10%, indicating that the method has good repeatability.

[0126] 6. Accuracy Test

[0127] Solution preparation:

[0128] Blank solution: 50 mmol / L potassium hydroxide solution.

[0129] Reference stock solution: Accurately weigh 119.2 mg potassium pyrophosphate, 92.7 mg sodium tripolyphosphate, and 89.6 mg potassium dihydrogen phosphate dried to constant weight at 105 °C, place them in a 50 ml volumetric flask, dissolve and dilute to the mark with 50 mmol / L potassium hydroxide solution, and shake well to obtain the solution.

[0130] Reference solution: Accurately measure 1 ml of the reference stock solution, place it in a 50 ml volumetric flask, dilute to the mark with 50 mmol / L potassium hydroxide solution, and shake well to obtain the solution.

[0131] Test solution (background solution): Accurately weigh 138.2 mg of sample from batch 230901R, place it in a 25 ml volumetric flask, dissolve and dilute to the mark with 50 mmol / L potassium hydroxide solution, and shake well. Prepare two parallel solutions.

[0132] 50% recovery solution: Accurately weigh 138.2 mg of sample from batch 230901R and place it in a 25 ml volumetric flask. Accurately measure 0.25 ml of the reference stock solution and place it in the same 25 ml volumetric flask. Dissolve and dilute to the mark with 50 mmol / L potassium hydroxide solution, and shake well. Prepare three parallel aliquots using the same method.

[0133] 100% recovery solution: Accurately weigh 138.2 mg of sample from batch 230901R and place it in a 25 ml volumetric flask. Accurately measure 0.5 ml of the reference stock solution and place it in the same 25 ml volumetric flask. Dissolve and dilute to the mark with 50 mmol / L potassium hydroxide solution, and shake well. Prepare 6 parallel aliquots using the same method.

[0134] 150% recovery solution: Accurately weigh 138.2 mg of sample from batch 230901R and place it in a 25 ml volumetric flask. Accurately measure 0.75 ml of the reference stock solution and place it in the same 25 ml volumetric flask. Dissolve and dilute to the mark with 50 mmol / L potassium hydroxide solution, and shake well. Prepare three parallel aliquots using the same method.

[0135] Accurately measure 25 μl of each of the blank solution, reference solution, test solution, and recovery solution and inject them into the sample. Record the chromatograms, calculate the measured amount using the external standard method, subtract the background amount and compare it with the added amount to calculate the recovery rate. The detection results are shown in Tables 17 to 19 below.

[0136] Table 17 Results of Phosphate Accuracy Test

[0137]

[0138] Table 18 Results of Pyrophosphate Accuracy Test

[0139]

[0140]

[0141] Table 19 Results of Tripolyphosphate Accuracy Test

[0142]

[0143] The results show that: the average recovery rate of 12 samples at various phosphate concentrations was 99.9%, all within the range of 90% to 110%, with an RSD of 0.78%, not exceeding 10%; the average recovery rate of 12 samples at various pyrophosphate concentrations was 100.6%, all within the range of 90% to 110%, with an RSD of 1.29%, not exceeding 10%; and the average recovery rate of 12 samples at various tripolyphosphate concentrations was 101.4%, all within the range of 90% to 110%, with an RSD of 1.06%, not exceeding 10%. The method demonstrates good accuracy.

[0144] 7. Solution stability test

[0145] Solution preparation:

[0146] Blank solution: 50 mmol / L potassium hydroxide solution.

[0147] Take the reference solution (prepared as described in section 1. Specificity test) and place it at room temperature.

[0148] Inject 25 μl of the above solution into the ion chromatograph, record the chromatogram, and the detection results are shown in Table 20 below.

[0149] Table 20 Results of the stability test of the reference solution.

[0150]

[0151] The results above show that the ratio of phosphate concentration to 0-day concentration in the reference solution after 4 days at room temperature is 99.6, the ratio of pyrophosphate concentration to 0-day concentration is 100.8%, and the ratio of tripolyphosphate concentration to 0-day concentration is 101.1%, all of which meet the requirement of 98.0% to 102.0%. Therefore, the reference solution is stable after at least 4 days at room temperature.

[0152] 8. Durability test

[0153] Solution preparation:

[0154] Blank solution: 50 mmol / L potassium hydroxide solution.

[0155] The reference solution and the test solution shall be prepared in the same manner as described in section 1, Specificity Test.

[0156] Inject 25 μl of the above solutions into the ion chromatograph, record the chromatograms, and the detection results are shown in Tables 21 and 22 below.

[0157] Table 21 Durability-Separation Test Results

[0158]

[0159] Table 22 Durability - Impurity Detection Test Results

[0160]

[0161] The results above show that, under various robustness conditions, taking the reference solution as an example, phosphate, pyrophosphate, and tripolyphosphate peaks appear sequentially, and the resolution between each impurity peak is not less than 1.5, which meets the system suitability requirements. Under various robustness conditions, the maximum deviation of the total amount of phosphate, pyrophosphate, and tripolyphosphate in the test solution from the standard conditions is 0.001%, which is not greater than 10% (0.05%) specified in the standard. This method has good robustness.

[0162] In summary, this invention provides a method for detecting phosphate, pyrophosphate, and tripolyphosphate in diquaphosphodium. This method exhibits good specificity, achieving baseline separation between the phosphate, pyrophosphate, and tripolyphosphate peaks. It also demonstrates good system applicability, extremely high sensitivity (with detection limits as low as 0.01260 μg / ml for phosphate, pyrophosphate, and tripolyphosphate), and good repeatability and accuracy. This invention provides a convenient detection method for detecting phosphate, pyrophosphate, and tripolyphosphate in diquaphosphodium, thereby controlling the impurity levels of these components in diquaphosphodium.

[0163] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations included in the claims. Various modifications and changes can be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments can be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only illustrate several implementations of the present invention and do not limit the scope of protection of this patent.

Claims

1. A method for testing phosphate, pyrophosphate, and tripolyphosphate in diquafosol sodium, characterized in that, Ion chromatography was used, with an anion exchange column; gradient elution was performed using potassium hydroxide aqueous solution as the eluent.

2. The method for testing phosphate, pyrophosphate, and tripolyphosphate in diquafosol sodium according to claim 1, characterized in that, The chromatographic column is an anion exchange column, preferably a Dionex IonPac column. TM The AS11 column, measuring 4mm x 250mm, is supplied by Thermo Fisher Scientific; the guard column is an anion exchange guard column, preferably Dionex IonPac. TM The AG11 column, measuring 4mm x 50mm, is supplied by Thermo Fisher Scientific.

3. The determination method according to claim 1, characterized in that, The flow rate is 0.5 ml / min to 1.5 ml / min, and more preferably 1.0 ml / min.

4. The determination method according to claim 1, characterized in that, The column temperature is 25℃~35℃, and more preferably 30℃.

5. The determination method according to claim 1, characterized in that, The temperature of the conductivity cell is 30℃~40℃, and more preferably 35℃.

6. The determination method according to claim 1, characterized in that, A conductivity detector was used, and the detection method was suppressed conductivity detection with a current value of 248mA.

7. The determination method according to claim 1, characterized in that, The injection volume was 25 μl.

8. The method for testing phosphate, pyrophosphate and tripolyphosphate in diquafosol sodium according to any one of claims 1 to 7, characterized in that, Includes the following steps: (1) Test solution: Take an appropriate amount of this product, weigh it accurately, add 50 mmol / L potassium hydroxide solution to dissolve and quantitatively dilute to prepare a solution containing about 5 mg per 1 ml; (2) Reference solution: Accurately weigh appropriate amounts of potassium pyrophosphate, sodium tripolyphosphate, and potassium dihydrogen phosphate dried to constant weight at 105℃, dissolve and quantitatively dilute with 50 mmol / L potassium hydroxide solution to prepare a solution containing approximately phosphate ions (in PO42-) per ml. 3- (calculated as P2O7), pyrophosphate (as P2O7) 4- (calculated) and tripolyphosphate (in P3O) 10 5- A mixed solution of approximately 25 μg each; (3) The reference solution and the test solution were determined by ion chromatography, respectively; The detection conditions for the ion chromatography method are as follows: chromatographic column: Dionex IonPac TM AS11 column; protective column Dionex IonPac TM AG11 column; using potassium hydroxide aqueous solution as eluent, flow rate 1.0 ml / min; The column temperature was 30℃; a conductivity detector was used, the detection method was suppressed conductivity detection, the current value was 248mA, the conductivity cell temperature was 35℃; the injection volume was 25μl; gradient elution was performed.

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