Method for simultaneously detecting contents of two medicinal components in sample

Abstract

The invention discloses a method for simultaneously detecting the contents of two medicinal components in a sample, the two medicinal components are tafluprost, timolol, or pharmaceutically acceptable salts or solvates thereof, and the contents are determined by high performance liquid chromatography. Chromatographic conditions of the high performance liquid chromatography include that gradient elution is carried out by taking a reversed-phase column as a chromatographic column, and mobile phases adopted by the gradient elution include a mobile phase A and a mobile phase B; the mobile phase A comprises a mixed aqueous solution of dihydric phosphate and alkyl sulfonate, and the mobile phase B comprises acetonitrile. The method has the beneficial effects that the method is high in specificity, all auxiliary materials in the eye drop product have no interference on sample detection, and known impurities in the product have no interference on sample detection; the method disclosed by the invention is simple to operate, good in stability, good in repeatability and high in accuracy, and the quality of a tafluprost timolol maleate eye drop product can be better controlled.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis, and in particular to a method for simultaneously detecting the contents of two drug components in a sample. The method can be used to simultaneously determine the contents of tafluprost and timolol in a sample. Background Art

[0002] Tafluprost and timolol maleate eye drops are an ophthalmic compound preparation that contains two active ingredients with different mechanisms of action on aqueous humor dynamics, namely 15 μg / mL of tafluprost and 5 mg / mL of timolol. It is indicated for reducing intraocular pressure in patients with open-angle glaucoma or ocular hypertension. It is used for patients who have poor efficacy with topical beta-blockers or prostaglandin derivatives alone and may benefit from combined treatment. One eye drop per day can achieve the same effectiveness and safety as combined medication. Compared with the two eye drops, its ease of use, compliance and quality of life have been greatly improved.

[0003] Currently, there are few studies on the simultaneous determination of the contents of two active ingredients in tafluprost timolol maleate eye drops. Upon inquiry, it was found that the article "Green HPLC method with time programming for the determination of the co-formulated eye drops of tafluprost and timolol in their challengeable ratio" published by Abd-Alghafar WN et al. disclosed the use of high performance liquid chromatography, dual wavelength data acquisition, and standard curve method to determine the contents of tafluprost and timolol. The operation was complicated, the concentration of timolol in the test sample was too high, and the amount of reference sample used was large, which increased the cost. Furthermore, the literature only examined whether the excipients glycerol, Tween, sodium dihydrogen phosphate, and EDTA in the eye drops had no interference with the detection of the samples. However, the multi-dose specifications of tafluprost timolol maleate eye drops also contained the excipient benzalkonium chloride. The inventors found through testing that the method in the literature could not effectively separate benzalkonium chloride and known impurities, and the above substances interfered with the determination of the samples. It can be seen that the prior art has poor specificity and cannot effectively detect the contents of the two active ingredients in the sample at the same time.

[0004] In order to better control the quality of tafluprost timolol maleate eye drops, it is an urgent problem to develop a high performance liquid chromatography method that is specific, efficient, economical, and can simultaneously determine the contents of tafluprost and timolol. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a method for simultaneously determining the contents of two active ingredients, tafluprost and timolol, in a sample by high performance liquid chromatography. The method has strong specificity, good stability and repeatability, and high accuracy, and is of great significance for detecting and controlling the quality of tafluprost and timolol maleate eye drops.

[0006] The first aspect of the present invention provides a method for simultaneously detecting the content of two drug components in a sample, wherein the two drug components are: tafluprost, timolol, or pharmaceutically acceptable salts or solvates thereof, and the content is determined by high performance liquid chromatography, wherein the chromatographic conditions of the high performance liquid chromatography include gradient elution using a reverse phase column as a chromatographic column, wherein the mobile phase used in the gradient elution includes mobile phase A and mobile phase B;

[0007] The mobile phase A comprises a mixed aqueous solution of dihydrogen phosphate and alkyl sulfonate, and the mobile phase B comprises acetonitrile.

[0008] The present invention provides a method for simultaneously detecting the contents of two drug components in a sample. The two drug components are: tafluprost, timolol, or pharmaceutically acceptable salts or solvates thereof, and the contents are determined by high performance liquid chromatography. The chromatographic conditions of the high performance liquid chromatography include gradient elution using a reverse phase column as a chromatographic column, wherein the mobile phase used in the gradient elution includes a mobile phase A and a mobile phase B;

[0009] The mobile phase A is a mixed aqueous solution of dihydrogen phosphate and alkyl sulfonate with a pH of 2.5-3.5, and the mobile phase B is acetonitrile.

[0010] In the method described above, as a preferred embodiment, the reverse phase column is a reverse phase C18 column.

[0011] In the method described above, as a preferred embodiment, the specification of the reverse phase C18 column is 4.6 mm×250 mm.

[0012] In the method described above, as a preferred embodiment, the filler particle size of the reverse phase C18 column is 5 μm.

[0013] In the method described above, as a preferred embodiment, the reverse phase column is a reverse phase C18 column, the specification of the reverse phase C18 column is 4.6 mm×250 mm, and the filler particle size is 5 μm.

[0014] As a preferred embodiment of the method described above, the detection wavelength of the HPLC method is 218-222 nm, preferably 220 nm; the column temperature is 30-40°C, preferably 35°C.

[0015] The method described above, as a preferred embodiment, in the mixed aqueous solution, the concentration of dihydrogen phosphate is 0.01-0.015 mol / L, for example, 0.01 mol / L, 0.011 mol / L, 0.012 mol / L, 0.013 mol / L, 0.014 mol / L, 0.015 mol / L or any value therebetween.

[0016] The method described above, as a preferred embodiment, in the mixed aqueous solution, the concentration of the alkyl sulfonate is 0.01-0.015 mol / L, for example, 0.01 mol / L, 0.011 mol / L, 0.012 mol / L, 0.013 mol / L, 0.014 mol / L, 0.015 mol / L or any value therebetween.

[0017] In the method described above, as a preferred embodiment, the pH of the mixed aqueous solution is 2.5-3.5, for example, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5 or any value therebetween.

[0018] The method described above, as a preferred embodiment, the pH of the mixed aqueous solution is 2.8-3.0.

[0019] The method described above, as a preferred embodiment, the mobile phase A is a mixed aqueous solution of 0.01-0.015 mol / L dihydrogen phosphate and 0.01-0.015 mol / L alkyl sulfonate, and the pH is adjusted to 2.5-3.5 with phosphoric acid.

[0020] The method described above, as a preferred embodiment, the gradient elution procedure is:

[0021] 0-20min, the volume percentage of mobile phase A is uniformly reduced from 70% to 30%, and the volume percentage of mobile phase B is uniformly increased from 30% to 70%, and the flow rate is 0.9-1.1mL / min, preferably 1.0mL / min;

[0022] 20-25min, the volume percentage of mobile phase A is increased from 30% to 70% at a uniform rate, and the volume percentage of mobile phase B is decreased from 70% to 30% at a uniform rate, and the flow rate is 0.9-1.1mL / min, preferably 1.0mL / min;

[0023] 25-30min, the volume percentage of mobile phase A is 70%, the volume percentage of mobile phase B is 30%, and the flow rate is 0.9-1.1mL / min, preferably 1.0mL / min.

[0024] In the above-described method, as a preferred embodiment, the dihydrogen phosphate is a mixture of one or more of potassium dihydrogen phosphate, sodium dihydrogen phosphate, and ammonium dihydrogen phosphate.

[0025] In the above-described method, as a preferred embodiment, the alkyl sulfonate is a mixture of one or more of sodium pentane sulfonate, sodium hexane sulfonate, sodium heptane sulfonate and sodium octane sulfonate.

[0026] In the method described above, as a preferred embodiment, the dihydrogen phosphate is a mixture of one or more of potassium dihydrogen phosphate, sodium dihydrogen phosphate, and ammonium dihydrogen phosphate; the alkyl sulfonate is a mixture of one or more of sodium pentane sulfonate, sodium hexane sulfonate, sodium heptane sulfonate, and sodium octane sulfonate.

[0027] Preferably, the dihydrogen phosphate is sodium dihydrogen phosphate.

[0028] Preferably, the alkyl sulfonate is sodium octane sulfonate.

[0029] Preferably, the mobile phase A is sodium dihydrogen phosphate and sodium octane sulfonate.

[0030] Preferably, the mobile phase A is a mixed aqueous solution of 0.01 mol / L sodium dihydrogen phosphate and 0.01 mol / L sodium octane sulfonate, and the pH is adjusted to 2.8 with phosphoric acid.

[0031] The method described above, as a preferred embodiment, further comprises the following steps:

[0032] Tafluprost reference substance stock solution: weigh the tafluprost reference substance, dissolve it in diluent and dilute it to make a solution containing 150 μg of tafluprost per 1 mL;

[0033] Timolol reference substance stock solution: weigh the timolol reference substance, dissolve it in the diluent and dilute it to make a solution containing 200 μg of timolol per 1 mL;

[0034] Reference solution: measure the tafluprost reference stock solution and the timolol reference stock solution respectively and place them in the same volumetric flask, add the diluent and dilute to make a solution containing 15 μg of tafluprost and 50 μg of timolol per 1 mL;

[0035] Tafluprost test solution: Measure the test sample, add diluent and dilute to a solution containing 15 μg of tafluprost per 1 mL;

[0036] Timolol test solution: Take a sample of the test product, add diluent and dilute to a solution containing 50 μg of timolol per 1 mL;

[0037] Determination method: 50 μL of reference solution and test solution were measured and injected into the liquid chromatograph respectively, and the chromatogram was recorded. The contents of tafluprost and timolol in the test sample were calculated by the peak area according to the external standard method;

[0038] As a preferred embodiment of the method described above, the diluent is a methanol aqueous solution.

[0039] The method described above, as a preferred embodiment, the volume concentration of methanol in the methanol aqueous solution is 40-80%, for example, 50%, 55%, 60%, 65%, 70%, 75%, 80% or any value therebetween.

[0040] As a preferred embodiment, the above method has a volume concentration of methanol in the methanol aqueous solution of 50-70%.

[0041] The method described above, as a preferred embodiment, further comprises the following steps:

[0042] Tafluprost reference substance stock solution: weigh the tafluprost reference substance, dissolve it in 70% methanol aqueous solution and dilute it to make a solution containing 150 μg of tafluprost per 1 mL;

[0043] Timolol reference substance stock solution: weigh the timolol reference substance, add 50% methanol aqueous solution by volume to dissolve and dilute to make a solution containing 200 μg of timolol per 1 mL;

[0044] Reference solution: measure the tafluprost reference stock solution and the timolol reference stock solution respectively and place them in the same volumetric flask, add 50% methanol aqueous solution by volume and dilute to make a solution containing 15 μg of tafluprost and 50 μg of timolol per 1 mL;

[0045] Tafluprost test solution: Take a sample of the test product, add a 50% volume concentration of methanol in water and dilute to a solution containing about 15 μg of tafluprost per 1 mL;

[0046] Timolol test solution: Take a sample of the test product, add a 50% methanol aqueous solution and dilute to a solution containing approximately 50 μg of timolol per 1 mL;

[0047] Determination method: 50 μL of reference solution and test solution were measured and injected into liquid chromatograph respectively. Chromatograms were recorded and the contents of tafluprost and timolol in the test samples were calculated by peak areas according to the external standard method.

[0048] Preferably, the sample is tafluprost timolol maleate eye drops.

[0049] Compared with the prior art, the beneficial effects of the present invention are: the method described in the present invention has strong specificity, all auxiliary materials in the eye drop product have no interference with sample detection, and the known impurities in the product have no interference with sample detection.

[0050] The method described in the invention has the advantages of simple operation, good stability, good repeatability and high accuracy, and can better control the quality of the tafluprost timolol maleate eye drops product. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 It is a specific test chromatogram of the detection method of the present invention;

[0052] Figure 2 It is a chromatogram under the conditions of the buffer salt concentration of 0.01 mol / L and 0.015 mol / L of the present invention;

[0053] Figure 3 It is the chromatogram under the conditions of buffer salt pH=2.8 and pH=3.5 of the present invention. DETAILED DESCRIPTION

[0054] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the embodiments and drawings. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation to the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0055] The tafluprost timolol maleate eye drops and blank excipient solution (the two active ingredients of tafluprost and timolol maleate are removed when preparing the solution, and only other excipients are included) used in this article are prepared in-house according to the conventional preparation method of eye drops, and the tafluprost and timolol maleate reference substances, reagents, and excipients can all be obtained commercially, wherein the content of the tafluprost reference substance is 100%, and the content of the timolol maleate reference substance is 99.8%.

[0056] 1. Chromatographic conditions

[0057] Instrument: Waters Arc HPLC / 2998PDA

[0058] Chromatographic column: Welch Ultimate XB-C18, 5μm, 4.6*250mm

[0059] Mobile phase A: 0.01 mol / L sodium dihydrogen phosphate and 0.01 mol / L sodium octane sulfonate aqueous solution, phosphoric acid adjusted to pH = 2.8

[0060] Mobile phase B: acetonitrile

[0061] Flow rate: 1.0 mL / min, column temperature: 35 °C, detection wavelength: 220 nm, injection volume: 50 μL.

[0062] The elution gradient program is as follows:

[0063] Time (min) Mobile phase A (%) Mobile phase B (%) 0 70 30 20 30 70 25 70 30 30 70 30

[0064] The above mobile phase A and mobile phase B are both expressed in volume percentage.

[0065] 2. Solution preparation

[0066] Diluent / blank solution: 50% methanol in water.

[0067] Tafluprost reference substance stock solution: accurately weigh 15 mg of tafluprost reference substance and place it in a 100 mL volumetric flask, add an appropriate amount of 70% methanol aqueous solution to dissolve and dilute to the scale, and shake well.

[0068] Timolol reference substance stock solution: Accurately weigh 34.2 mg of timolol maleate reference substance and place it in a 50 mL volumetric flask, add an appropriate amount of diluent to dissolve and dilute to the scale, and shake well.

[0069] Reference solution: Accurately measure 2 mL of tafluprost reference stock solution and 2 mL of timolol reference stock solution respectively, place them in the same 20 mL volumetric flask, add diluent to dilute to the scale, and shake well.

[0070] Blank excipient solution: Pour the blank excipient (polysorbate 80, glycerol, sodium dihydrogen phosphate, disodium edetate, benzalkonium chloride) solution lacking tafluprost and timolol maleate directly into the liquid phase vial.

[0071] Tafluprost test solution: Pour tafluprost timolol maleate eye drops directly into the liquid phase vial.

[0072] Timolol test solution: accurately measure 1 mL of eye drop sample, place in a 100 mL volumetric flask, add diluent to dilute to the mark, and shake well;

[0073] Timolol impurity B localization solution: Accurately weigh 7 mg of impurity B reference substance, place in a 100 mL volumetric flask, add 40% volume concentration of methanol aqueous solution to dissolve and dilute to the scale, and shake well.

[0074] Timolol impurity G localization solution: Accurately weigh 3.5 mg of impurity G reference substance, place in a 100 mL volumetric flask, add 40% volume concentration of methanol aqueous solution to dissolve and dilute to the scale, and shake well.

[0075] Timolol impurity D localization solution: Accurately weigh 3.5 mg of impurity D reference substance, place in a 100 mL volumetric flask, add 40% volume concentration of methanol aqueous solution to dissolve and dilute to the scale, and shake well.

[0076] Tafluprost carboxylate impurity localization solution: accurately weigh 1.5 mg of carboxylate impurity reference substance, place in a 100 mL volumetric flask, add 70% by volume acetonitrile aqueous solution to dissolve and dilute to the scale, and shake well.

[0077] Linear solution 1: Accurately measure 1 mL of tafluprost reference stock solution and 1 mL of timolol reference stock solution, place them in the same 20 mL volumetric flask, add diluent to dilute to the scale, and shake well.

[0078] Linear solution 2: Accurately measure 2 mL of tafluprost reference stock solution and timolol reference stock solution, place them in the same 25 mL volumetric flask, add diluent to dilute to the scale, and shake well.

[0079] Linear solution 3: Accurately measure 1 mL of tafluprost reference stock solution and 1 mL of timolol reference stock solution, place them in the same 10 mL volumetric flask, add diluent to dilute to the scale, and shake well.

[0080] Linear solution 4: Accurately measure 3 mL of tafluprost reference stock solution and timolol reference stock solution, place them in the same 25 mL volumetric flask, add diluent to dilute to the scale, and shake well.

[0081] Linear solution 5: Accurately measure 3 mL of tafluprost reference stock solution and timolol reference stock solution, place them in the same 20 mL volumetric flask, add diluent to dilute to the scale, and shake well.

[0082] Tafluprost negative control solution: dissolve 0.5% timolol in purified water, add appropriate amounts of polysorbate 80, glycerol, disodium edetate, sodium dihydrogen phosphate, and benzalkonium chloride to obtain the solution.

[0083] Timolol negative control solution: dissolve 0.0015% tafluprost in purified water, and add appropriate amounts of polysorbate 80, glycerol, disodium edetate, sodium dihydrogen phosphate, and benzalkonium chloride to obtain the solution.

[0084] Tafluprost 80% recovery solution: Accurately measure 2 mL of tafluprost reference stock solution and place it in a 25 mL volumetric flask, then dilute to the mark with tafluprost negative control solution, shake well, and prepare 3 copies in parallel.

[0085] Tafluprost 100% recovery solution: Accurately measure 1 mL of tafluprost reference stock solution and place it in a 10 mL volumetric flask, then dilute to the mark with tafluprost negative control solution, shake well, and prepare 3 copies in parallel.

[0086] Tafluprost 120% recovery solution: Accurately measure 3 mL of tafluprost reference stock solution and place it in a 25 mL volumetric flask. Dilute to the mark with tafluprost negative control solution, shake well, and prepare 3 copies in parallel.

[0087] Timolol 80% recovery solution: Accurately measure 2 mL of timolol reference stock solution and place it in a 25 mL volumetric flask. Add 0.1 mL of timolol negative control solution and dilute to the scale with diluent. Shake well and prepare 3 copies in parallel.

[0088] Timolol 100% recovery solution: Accurately measure 1 mL of timolol reference stock solution and place it in a 10 mL volumetric flask, add 0.1 mL of timolol negative control solution, dilute to the scale with diluent, shake well, and prepare 3 copies in parallel.

[0089] Timolol 120% recovery solution: Accurately measure 3 mL of timolol reference stock solution and place it in a 25 mL volumetric flask, add 0.1 mL of timolol negative control solution, dilute to the scale with diluent, shake well, and prepare 3 copies in parallel.

[0090] The structural formulas of the above impurities are listed below:

[0091]

[0092] 3. Specificity test

[0093] Accurately measure 50 μL of blank solution, blank excipient solution, reference solution, and each impurity location solution, and inject them into the liquid chromatograph to record the chromatogram. Figure 1 .

[0094] from Figure 1 It can be seen that the blank solution, blank excipient solution and impurities had no interference on the detection of tafluprost and timolol, indicating that the method has good specificity.

[0095] 4. System adaptability test

[0096] The reference solution was prepared in parallel in two portions, 50 μL of the reference solution was accurately measured and injected into the liquid chromatograph respectively, and the chromatogram was recorded. The test results are shown in Table 1.

[0097] Table 1 System adaptability test results

[0098]

[0099] It can be seen from Table 1 that the system adaptability of the method of the present invention is good.

[0100] 5. Linearity test

[0101] Accurately measure 50 μL of the linear solutions of different concentrations prepared above, inject them into the liquid chromatograph, and record the chromatogram. Draw the standard curve with the concentration of each component as the horizontal axis and the peak area as the vertical axis. The test results are shown in Table 2 and Table 3.

[0102] Table 2 Tafluprost linearity test results

[0103]

[0104]

[0105] Table 3 Timolol linearity test results

[0106]

[0107] It can be seen from Table 2 and Table 3 that tafluprost showed a good linear relationship in the concentration range of 7.8250-23.4750 μg / mL; timolol showed a good linear relationship in the concentration range of 25.1978-75.5935 μg / mL.

[0108] 6. Accuracy test

[0109] Accurately measure 50 μL of the above-mentioned 80%-120% recovery rate solution of tafluprost (three portions of the recovery rate solution were prepared in parallel) and 80%-120% recovery rate solution of timolol, respectively, inject them into the liquid chromatograph, and record the chromatogram. Calculate the sample recovery rate, and the test results are shown in Tables 4 and 5.

[0110] Table 4 Tafluprost accuracy test results

[0111]

[0112]

[0113] Table 5 Timolol accuracy test results

[0114]

[0115] It can be seen from Table 4 and Table 5 that the average recoveries of tafluprost and timolol are both between 98% and 102%, which meet the requirements for content detection recovery, and the RSDs are all less than 2%, indicating that the detection method has good accuracy.

[0116] 7. Stability test

[0117] Accurately measure 50 μL of the above-mentioned reference solution, tafluprost test solution, and timolol test solution, respectively, and inject them into the liquid chromatograph to record the chromatogram. The test results are shown in Tables 6, 7, and 8.

[0118] Table 6 Stability of reference solution

[0119] time Tafluprost Peak area ratio 0h 768512 N / A 25h 775940 101.0％ time Timolol Peak area ratio 0h 1165999 N / A 25h 1163558 99.8％

[0120] Table 7 Tafluprost test solution stability

[0121] time Tafluprost Peak area ratio 0h 712768 N / A 11h 712367 99.9％ 22h 714217 100.2％

[0122] Table 8 Stability of timolol test solution

[0123] time Timolol Peak area ratio 0h 1154545 N / A 11h 1155236 100.1％ 22h 1153609 99.9％

[0124] It can be seen from Tables 6 to 8 that the reference solution was very stable when placed at room temperature for 25 hours, and the tafluprost test solution and timolol test solution were very stable when placed at room temperature for 22 hours.

[0125] 8. Repeatability test

[0126] The tafluprost test solution and the timolol test solution were prepared in six portions in parallel, and 50 μL of each test solution was accurately measured and injected into the liquid chromatograph, and the chromatogram was recorded. The test results are shown in Table 9.

[0127] Table 9 Repeatability test results

[0128]

[0129]

[0130] Note: RSD is the relative standard deviation. The smaller the value, the higher the precision and the better the repeatability.

[0131] As can be seen from Table 9, the average content of 6 samples of tafluprost was 96.6%, and the average content of 6 samples of timolol was 100.7%, and the RSDs were all less than 2%, indicating that the detection method had good repeatability.

[0132] 9. Durability test

[0133] By changing the detection wavelength, column temperature and flow rate in the chromatographic conditions, the sample is re-injected for testing. When changing one condition, other chromatographic conditions remain unchanged.

[0134] Accurately measure 50 μL of each of the blank solution, reference solution, tafluprost test solution, and timolol test solution, and inject them into the liquid chromatograph to record the chromatogram. The test results are shown in Tables 10 and 11.

[0135] Table 10 Tafluprost durability test results

[0136]

[0137] Table 11 Timolol durability test results

[0138]

[0139] It can be seen from Tables 10 and 11 that under each durability test condition, the difference between the tafluprost and timolol contents measured in the test solution and those measured under normal conditions was less than 2%, indicating that the method has good durability.

[0140] 10. Changes in buffer salt concentration

[0141] In the chromatographic conditions, mobile phase A was 0.015 mol / L sodium dihydrogen phosphate and 0.01 mol / L sodium octane sulfonate aqueous solution, phosphoric acid was used to adjust pH=2.8, and other conditions remained unchanged.

[0142] Accurately measure 50 μL of blank solution, tafluprost test solution, and timolol test solution, respectively, and inject them into the liquid chromatograph to record the chromatogram. Figure 2 .

[0143] from Figure 2 It can be seen that when using 0.015 mol / L buffer salt as the mobile phase, the elution time of tafluprost and timolol is basically the same as that when using 0.01 mol / L buffer salt, and the peak shape is better and the separation is good.

[0144] 11. Changes in pH of buffer salts

[0145] In the chromatographic conditions, mobile phase A was 0.015 mol / L sodium dihydrogen phosphate and 0.01 mol / L sodium octane sulfonate aqueous solution, phosphoric acid was used to adjust pH=3.5, and other conditions remained unchanged.

[0146] Accurately measure 50 μL of blank solution and reference solution, inject them into the liquid chromatograph, and record the chromatogram. Figure 3 .

[0147] Conclusion: When the buffer pH is 3.5, the peak time and peak shape of tafluprost and timolol meet the requirements.

[0148] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A method for simultaneously detecting the contents of two drug components in a sample, wherein the two drug components are: tafluprost, timolol, or pharmaceutically acceptable salts or solvates thereof, characterized in that: Performing content determination by high performance liquid chromatography, wherein the chromatographic conditions of the high performance liquid chromatography include gradient elution using a reverse phase column as a chromatographic column, wherein the mobile phase used in the gradient elution includes mobile phase A and mobile phase B; The mobile phase A comprises a mixed aqueous solution of dihydrogen phosphate and alkyl sulfonate, and the mobile phase B comprises acetonitrile.

2. The method according to claim 1, characterized in that The reverse phase column is a reverse phase C18 column. Preferably, the specification of the reverse phase C18 column is 4.6 mm×250 mm. Preferably, the filler particle size of the reverse phase C18 column is 5 μm.

3. The method according to claim 2, characterized in that The detection wavelength of the high performance liquid chromatography is 218-222 nm, preferably 220 nm; the column temperature is 30-40°C, preferably 35°C.

4. The method according to claim 1, characterized in that In the mixed aqueous solution, the concentration of dihydrogen phosphate is 0.01-0.015 mol / L, and / or In the mixed aqueous solution, the concentration of the alkyl sulfonate is 0.01-0.015 mol / L; and / or The pH of the mixed aqueous solution is 2.5-3.5, preferably 2.8-3.0; Preferably, phosphoric acid is used to adjust the pH of the mixed aqueous solution; Preferably, the mobile phase A is a mixed aqueous solution of 0.01-0.015 mol / L dihydrogen phosphate and 0.01-0.015 mol / L alkyl sulfonate, and phosphoric acid is used to adjust the pH to 2.5-3.

5.

5. The method according to claim 4, characterized in that The gradient elution procedure is: 0-20min, the volume percentage of mobile phase A is uniformly reduced from 70% to 30%, and the volume percentage of mobile phase B is uniformly increased from 30% to 70%, and the flow rate is 0.9-1.1mL / min, preferably 1.0mL / min; 20-25min, the volume percentage of mobile phase A is increased from 30% to 70% at a uniform rate, and the volume percentage of mobile phase B is decreased from 70% to 30% at a uniform rate, and the flow rate is 0.9-1.1mL / min, preferably 1.0mL / min; 25-30min, the volume percentage of mobile phase A is 70%, the volume percentage of mobile phase B is 30%, and the flow rate is 0.9-1.1mL / min, preferably 1.0mL / min.

6. The method according to claim 1, characterized in that The dihydrogen phosphate is a mixture of one or more of potassium dihydrogen phosphate, sodium dihydrogen phosphate, and ammonium dihydrogen phosphate; and / or The alkyl sulfonate is a mixture of one or more of sodium pentane sulfonate, sodium hexane sulfonate, sodium heptane sulfonate and sodium octane sulfonate.

7. The method according to claim 1, characterized in that The dihydrogen phosphate is sodium dihydrogen phosphate; and / or The alkyl sulfonate is sodium octane sulfonate; Preferably, the mobile phase A is sodium dihydrogen phosphate and sodium octane sulfonate.

8. The method according to claim 1, characterized in that The mobile phase A is a mixed aqueous solution of 0.01 mol / L sodium dihydrogen phosphate and 0.01 mol / L sodium octane sulfonate, and the pH is adjusted to 2.8 with phosphoric acid.

9. The method according to any one of claims 1 to 8, characterized in that: The following steps are also included: Tafluprost reference substance stock solution: weigh the tafluprost reference substance, add diluent to dissolve and dilute to make a solution containing 150 μg of tafluprost per 1 mL; Timolol reference substance stock solution: weigh the timolol reference substance, dissolve it in the diluent and dilute it to make a solution containing 200 μg of timolol per 1 mL; Reference solution: measure the tafluprost reference stock solution and the timolol reference stock solution respectively and place them in the same volumetric flask, add the diluent and dilute to make a solution containing 15 μg of tafluprost and 50 μg of timolol per 1 mL; Tafluprost test solution: Measure the test sample, add diluent and dilute to a solution containing 15 μg of tafluprost per 1 mL; Timolol test solution: Take a sample of the test product, add diluent and dilute to a solution containing 50 μg of timolol per 1 mL; Determination method: 50 μL of reference solution and test solution were measured and injected into the liquid chromatograph respectively, and the chromatogram was recorded. The contents of tafluprost and timolol in the test sample were calculated by the peak area according to the external standard method; Preferably, the diluent is a methanol aqueous solution, and preferably, the volume concentration of methanol in the methanol aqueous solution is 40-80%, preferably 50-70%; Preferably, the method further comprises the following steps: tafluprost reference substance stock solution: weigh the tafluprost reference substance, add a methanol aqueous solution with a volume concentration of 70% to dissolve and dilute to prepare a solution containing 150 μg of tafluprost per 1 mL; Timolol reference substance stock solution: weigh the timolol reference substance, add 50% methanol aqueous solution by volume to dissolve and dilute to make a solution containing 200 μg of timolol per 1 mL; Reference solution: measure the tafluprost reference stock solution and the timolol reference stock solution respectively and place them in the same volumetric flask, add 50% methanol aqueous solution by volume and dilute to make a solution containing 15 μg of tafluprost and 50 μg of timolol per 1 mL; Tafluprost test solution: Take a sample of the test product, add a 50% methanol aqueous solution and dilute to a solution containing 15 μg of tafluprost per 1 mL; Timolol test solution: Take a sample of the test product, add a 50% methanol aqueous solution and dilute to a solution containing 50 μg of timolol per 1 mL; Determination method: 50 μL of reference solution and test solution were measured and injected into liquid chromatograph respectively. Chromatograms were recorded and the contents of tafluprost and timolol in the test samples were calculated by peak areas according to the external standard method.

10. The method according to claim 9, characterized in that The sample is tafluprost timolol maleate eye drops.

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