Determination and analysis method for related substances of diquafosol sodium eye drops

Abstract

The invention belongs to the technical field of medicine analysis, and particularly relates to a diquafosol sodium eye drop related substance determination and analysis method which comprises the following specific steps: preparing a test solution; preparing a reference substance solution; preparing a sensitivity solution; a system applicability solution; the test solution, the reference solution, the sensitivity solution and the system applicability solution are precisely measured and respectively injected into a liquid chromatograph, and chromatograms are recorded until the retention time of the main components is 2 times that of the main components. According to the method for determining the related substances of the diquafosol sodium eye drops, the preparation process of a system applicable solution is optimized, the purposes of convenient preparation, low cost and high specificity are achieved, the method is high in sensitivity and good in separation degree and reproducibility, and quality control of preparations of the diquafosol sodium eye drops can be better achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of medical analysis, and particularly relates to a method for determining and analyzing related substances of diquafosol sodium eye drops. Background Art

[0002] Diquafosol sodium eye drops are a receptor agonist of the purine receptor P2Y2, mainly used to treat dry eye disease and improve dry eye symptoms by stabilizing the tear film; its mechanism of action is to stimulate the P2Y2 receptors of the ocular surface tissue to achieve the purpose of treating dry eyes.

[0003] The chemical name of diquafosol sodium is: p1, p4-di(5′-uridine)tetrasodium tetraphosphate, and the chemical structural formula is:

[0004]

[0005] At present, the analytical methods for related substances of diquafosol sodium eye drops are not included in the pharmacopoeias of various countries. The original drug has an imported drug registration standard (standard number: JX20130043), which contains the detection methods for related substances of diquafosol sodium eye drops. In addition, the raw material standards are not included in the pharmacopoeias of various countries.

[0006] However, the raw material quality standards of the raw material manufacturer (Suzhou Fourth Pharmaceutical Factory Co., Ltd.) include the relevant substance detection methods for diquafosol sodium raw materials. The relevant substance analysis methods were established with reference to the imported drug registration standards (standard number: JX20130043) and the relevant substance detection methods of the raw material manufacturer (Suzhou Fourth Pharmaceutical Factory Co., Ltd.) and combined with the quality characteristics of this product.

[0007] There is no system suitability solution in the import registration standard. The system suitability solution of the raw material manufacturer's quality standard is complicated to prepare and requires impurity reference substances, which is costly. This paper studies a detection method for related substances in diquafosol sodium eye drops that is easy to prepare, low in cost, and has strong specificity, good separation, high sensitivity, high precision and accuracy, and good durability. Summary of the invention

[0008] A method for determining and analyzing related substances of diquafosol sodium eye drops, wherein the method adopts high performance liquid chromatography to complete the detection, and the specific steps are as follows:

[0009] Preparation of test solution: Take 5 bottles of this product, mix well, accurately measure an appropriate amount of this product, and dilute with mobile phase to make a solution containing about 0.6 mg of diquafosol sodium per 1 ml;

[0010] Preparation of reference solution: Accurately weigh an appropriate amount of diquafosol sodium reference substance and prepare a solution containing about 6 μg of diquafosol sodium per 1 ml with mobile phase;

[0011] Preparation of sensitivity solution: accurately measure 1 ml of reference solution, place in a 20 ml volumetric flask, dilute to scale with mobile phase, and shake well;

[0012] System suitability solution: Take about 6 mg of diquafosol sodium reference substance, place it in a 10 ml volumetric flask, add 1 ml of 1 mol / L hydrochloric acid solution, heat in a 40°C water bath for 30 minutes, take it out, cool it, add 1 ml of 1 mol / L sodium hydroxide solution, then add the mobile phase to dilute to the scale, and shake well;

[0013] Assay The assay was completed.

[0014] In any of the above schemes, preferably, in the process of high performance liquid chromatography for detection, octylsilane bonded silica gel is used as a filler (Thermo AcclaimTM120 C8 column, 4.6mm×250mm, 5μm or a chromatography column with equivalent performance).

[0015] In any of the above schemes, it is preferred to use phosphate buffer (take 10.9g of potassium dihydrogen phosphate and 3.4g of tetrabutylammonium hydrogen sulfate, add 1000ml of water to dissolve, and adjust the pH value to 6.7 with 10% potassium hydroxide solution or phosphoric acid)-methanol (84:16) as the mobile phase.

[0016] In any of the above schemes, it is preferred that the detection wavelength is 250-270 nm; the column temperature is 25-35° C.; the flow rate is 0.8-1.2 ml / min; and the injection volume is 10-20 μl.

[0017] In any of the above schemes, it is preferred that the system suitability requirement is: in the system suitability solution chromatogram, UMP, UDP, UTP and diquafosol sodium appear in sequence, and the separation between the main peak and the chromatographic peaks of each known impurity should meet the requirements.

[0018] In any of the above schemes, preferably, the steps of completing the determination by the determination method include: accurately measuring the obtained test solution, reference solution, sensitivity solution, and system suitability solution, injecting them into the liquid chromatograph respectively, and recording the chromatogram to twice the retention time of the main component.

[0019] In any of the above schemes, it is preferred that in the chromatogram of the sensitivity solution, the signal-to-noise ratio of the peak height of the main component should be no less than 10.

[0020] Beneficial Effects

[0021] The method for determining related substances of diquafosol sodium eye drops designed in the invention optimizes the preparation process of the system suitability solution, achieving convenient preparation, low cost and high specificity. The method has high sensitivity, good separation and reproducibility, and can better achieve quality control of its preparations. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 It is a typical diagram of the present invention. DETAILED DESCRIPTION

[0022] The method for analyzing the related substances of diquafosol sodium eye drops by high performance liquid chromatography described in this patent uses octylsilane bonded silica gel as a filler (ThermoAcclaim TM 120 C8 column, 4.6mm×250mm, 5μm or chromatographic column with equivalent performance); phosphate buffer (take 10.9g of potassium dihydrogen phosphate and 3.4g of tetrabutylammonium hydrogen sulfate, add 1000ml of water to dissolve, and adjust the pH value to 6.7 with 10% potassium hydroxide solution or phosphoric acid)-methanol (84:16) as the mobile phase; the detection wavelength is 250-270nm; the column temperature is 25-35℃; the flow rate is 0.8-1.2ml / min; the injection volume is 10-20μl.

[0023] Detection requirements: solvent, mobile phase.

[0024] Test solution Take 5 bottles of this product, mix well, accurately measure an appropriate amount of this product, and dilute with mobile phase to make a solution containing about 0.6 mg of diquafosol sodium per 1 ml.

[0025] Reference solution: Accurately weigh an appropriate amount of diquafosol sodium reference substance and prepare a solution containing approximately 6 μg of diquafosol sodium per 1 ml using mobile phase.

[0026] Sensitivity solution: Accurately measure 1 ml of the reference solution, place it in a 20 ml volumetric flask, dilute to the mark with the mobile phase, and shake well.

[0027] System suitability solution: Take about 6 mg of diquafosol sodium reference substance, place it in a 10 ml volumetric flask, add 1 ml of 1 mol / L hydrochloric acid solution, heat in a 40°C water bath for 30 minutes, take it out, cool it, add 1 ml of 1 mol / L sodium hydroxide solution, then add the mobile phase to dilute to the scale and shake well.

[0028] System suitability requirements In the system suitability solution chromatogram, UMP, UDP, UTP and diquafosol sodium should appear in sequence, and the separation between the main peak and the chromatographic peaks of each known impurity should meet the requirements. In the sensitivity solution chromatogram, the signal-to-noise ratio of the main component peak height should be no less than 10.

[0029] Determination method: Accurately measure the test solution, reference solution, sensitivity solution, and system suitability solution, and inject them into the liquid chromatograph respectively, and record the chromatogram until twice the retention time of the main component.

[0030] (1) Specificity test results:

[0031]

[0032] Conclusion: Blank solvent and blank excipient solution have no interference to the detection of related substances of this product.

[0033] (2) Destruction test:

[0034] Solution preparation:

[0035]

[0036] result

[0037]

[0038] Conclusion: There are no impurities after the main peak, and the minimum separation degree of impurities before the main peak is 3.64, all greater than 1.5. The minimum separation degree between other impurities is 2.22, all not less than 1.5. The minimum purity factor of the main peak is 998, all greater than 980. All conditions destroy the conservation of mass (90%~110%).

[0039] (3) System precision

[0040] The reference solution was measured 6 times continuously, and the results are as follows:

[0041] Serial number Retention time min Peak area 1 17.580 86302 2 17.588 85558 3 17.570 85667 4 17.511 85464 5 17.464 86110 6 17.446 85773 RSD% 0.35 0.38

[0042] Conclusion: The method has good system precision.

[0043] (4) Limit of quantification and detection limit

[0044] Taking the signal-to-noise ratio (S / N) ≥ 10 as the quantitative limit solution, the quantitative limit results of each component are as follows:

[0045]

[0046] Taking the signal-to-noise ratio (S / N) ≥ 3 as the quantitative limit solution, the quantitative limit results of each component are as follows:

[0047]

[0048] Conclusion: This method has high sensitivity.

[0049] (5) Linearity and range

[0050] Linear stock solution preparation process

[0051]

[0052] The preparation process of linear solution is shown in the table below.

[0053] Linear solution preparation process

[0054]

[0055]

[0056] result:

[0057] Impurity Name Concentration range Linear equations Correlation coefficient r Correction Factor URD 0.3099μg / ml~9.2983μg / ml y=24099.2199x-296.6032 1.0000 0.53 UMP 0.2960μg / ml~8.8796μg / ml y=17528.7610x-451.8525 1.0000 0.73 UDP 0.3115μg / ml~9.3451μg / ml y=10320.0795x+57.8722 1.0000 1.23 Diquafosol Sodium 0.3117μg / ml~1.8702μg / ml y=12713.6849x-192.3667 0.9997 / UTP 0.3114μg / ml~9.3419μg / ml y=10630.8430x-244.3738 0.9999 1.21 Diquafosol Sodium 0.3117μg / ml~1.8702μg / ml y=12737.4257x-226.9333 0.9996 /

[0058] (6) Repeatability and intermediate precision

[0059] Reference solution, sensitivity solution, and system suitability solution: Prepare according to the concentration specified in the method.

[0060] Test solution: Take 5 bottles of this product, mix well, accurately measure 1 ml of this product, put it in a 50 ml volumetric flask, add diluent to dilute to the scale, shake well, as the test solution, prepare 6 portions in the same way.

[0061] The second analyst prepared 6 solutions in parallel at different times and tested them using different instruments.

[0062] result:

[0063] Repeatability: The number of impurities in the 6 test solutions was consistent, among which the single impurities were known, and the contents of other single impurities and total impurities were far below the limit of quantification. The RSD% of UMP, UDP, UTP, RRT=0.79 impurities, RRT=0.90 impurities and total impurities were 15.49%, 4.02%, 8.21%, 7.03%, 7.00% and 2.72%, respectively, which were less than 20.0%, indicating that this method had good repeatability.

[0064] Intermediate precision: The number of impurities in the 12 test solutions was consistent, among which the single impurities were known, and the contents of other single impurities and total impurities were far below the limit of quantification. The RSD% of UMP, UDP, UTP, RRT=0.79 impurity, RRT=0.90 impurity and total impurities were 13.43%, 4.68%, 15.34%, 9.51%, 14.07% and 3.67%, respectively, which were less than 20.0%, indicating that the intermediate precision of this method was good.

[0065] (7) Accuracy

[0066] Preparation of mixed reference stock solution

[0067] Concentration of impurity reference stock solution Dilution steps URD (300 μg / ml) 15mg (15.100mg) 50ml UMP (300 μg / ml) 17mg (17.230mg) 50ml UDP (300 μg / ml) 15mg(15.416mg)50ml UTP (300 μg / ml) 18mg (18.790mg) 50ml

[0068] URD, UMP and UDP reference solution preparation table

[0069]

[0070] Preparation of URD, UMP, UDP impurity recovery test solution: Take 5 bottles of this product, mix well, accurately measure 1 ml of diquafosol sodium eye drops, put it in a 50 ml volumetric bottle, accurately add specific solution 1, add diluent to dilute to the scale, shake well, see the table below for details.

[0071] URD, UMP and UDP impurity accuracy solution preparation table

[0072] name Specific solution 1 Sampling volume (ml) Unspecified Thinner Dilute to volume 5% (LOQ) recovery URD, UMP and UDP reference solution 1 10 80% recovery rate URD, UMP and UDP reference solution 2 10 100% recovery rate URD, UMP and UDP reference solution 3 10 120% recovery rate URD, UMP and UDP reference solution 4 10

[0073] UTP reference solution preparation table

[0074]

[0075] Prepare 3 solutions of each recovery rate in parallel according to the above table.

[0076] Preparation of UTP impurity recovery test solution: Take 5 bottles of this product, mix well, accurately measure 1 ml of diquafosol sodium eye drops, put it in a 50 ml volumetric bottle, accurately add specific solution 2, add diluent to dilute to the scale, shake well, see the table below for details.

[0077] UTP Impurity Accuracy Solution Preparation Table

[0078]

[0079]

[0080] Each recovery rate solution was prepared in triplicate according to the above table.

[0081] Sensitivity solution and system suitability solution: prepare according to the concentration specified in the method.

[0082] Reference solution: Prepare according to the concentration specified in the method.

[0083] result:

[0084] Impurity Name Average recovery rate% RSD% URD 99.97 1.18 UMP 98.46 1.47 UDP 101.25 3.32 UTP 100.69 3.53

[0085] Conclusion: The average recovery rate of each impurity (n=12) is in the range of 80.0-120.0%; the maximum RSD is 3.53%, which is less than 10.0%, and the accuracy of this method is good.

[0086] (8) Durability

[0087] Summary of changes in conditions

[0088] Summary of robust chromatography conditions

[0089]

[0090] result

[0091] Durability test results 1 (system suitability)

[0092]

[0093]

[0094] Durability Test Results II

[0095]

[0096] Conclusion: When there are slight changes in the chromatographic column (different batches), pH value of the mobile phase, flow rate, column temperature, mobile phase ratio, and wavelength, the separation of the system suitability solution meets the requirements, and the signal-to-noise ratio of the sensitivity solution is greater than 10; compared with the standard conditions, the difference in impurity content is within 0.05%, and the method has good durability.

[0097] Figure 1 The order of the peaks is URD, UMP, U2P2, UDP, UTP, U2P3, diquafosol sodium, U2P5, and U2P6.

[0100] In summary, it can be seen that the preparation process of the system suitability solution in the determination method of the present invention is optimized, which achieves convenient preparation, low cost and high specificity, and the method has high sensitivity, good separation and reproducibility, and can better achieve the quality control of its preparation.

[0101] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments can still be modified, or some or all of the technical features can be replaced by equivalents. These modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention. For those skilled in the art, any replacement improvements or changes made to the implementation methods of the present invention fall within the protection scope of the present invention.

[0102] The matters not described in detail in the present invention are all known technologies to those skilled in the art.

Claims

1. A method for determining and analyzing related substances of diquafosol sodium eye drops, characterized in that: The determination method adopts high performance liquid chromatography to complete the detection, and the specific steps are as follows: Preparation of test solution: Take 5 bottles of this product, mix well, accurately measure an appropriate amount of this product, and dilute with mobile phase to make a solution containing about 0.6 mg of diquafosol sodium per 1 ml; Preparation of reference solution: Accurately weigh an appropriate amount of diquafosol sodium reference substance and prepare a solution containing about 6 μg of diquafosol sodium per 1 ml with mobile phase; Preparation of sensitivity solution: accurately measure 1 ml of reference solution, place in a 20 ml volumetric flask, dilute to scale with mobile phase, and shake well; System suitability solution: take about 6 mg of diquafosol sodium reference substance, place in a 10 ml volumetric flask, add 1 ml of 1 mol / L hydrochloric acid solution, heat in a 40°C water bath for 30 minutes, take out, cool, add 1 ml of 1 mol / L sodium hydroxide solution, and then dilute to scale with mobile phase, and shake well; Assay The assay was completed.

2. A method for determining and analyzing related substances of diquafosol sodium eye drops according to claim 1, characterized in that: In the process of detection by high performance liquid chromatography, octylsilane bonded silica gel is used as a filler (ThermoAcclaimTM120 C8 column, 4.6mm×250mm, 5μm or a chromatographic column with equivalent performance).

3. A method for determining and analyzing related substances of diquafosol sodium eye drops according to claim 2, characterized in that: Phosphate buffer (10.9 g of potassium dihydrogen phosphate and 3.4 g of tetrabutylammonium hydrogen sulfate are dissolved in 1000 ml of water and the pH value is adjusted to 6.7 with 10% potassium hydroxide solution or phosphoric acid)-methanol (84:16) is used as the mobile phase.

4. A method for determining and analyzing related substances of diquafosol sodium eye drops according to claim 3, characterized in that: wherein, The detection wavelength is 250-270nm; the column temperature is 25-35°C; the flow rate is 0.8-1.2ml / min; and the injection volume is 10-20μl.

5. A method for determining and analyzing related substances of diquafosol sodium eye drops according to claim 4, characterized in that: System suitability requirements: In the system suitability solution chromatogram, UMP, UDP, UTP and diquafosol sodium appear in sequence, and the separation between the main peak and the chromatographic peaks of each known impurity should meet the requirements.

6. A method for determining and analyzing related substances of diquafosol sodium eye drops according to claim 5, characterized in that: The steps of completing the determination by the determination method include: accurately measuring the obtained test solution, reference solution, sensitivity solution, and system suitability solution, injecting them into the liquid chromatograph respectively, and recording the chromatogram to twice the retention time of the main component.

7. A method for determining and analyzing related substances of diquafosol sodium eye drops according to claim 6, characterized in that: In the chromatogram of the sensitivity solution, the signal-to-noise ratio of the main component peak height should be no less than 10.

Images