A HPLC determination method for tyloxapol content

Abstract

The invention discloses a HPLC determination method of tyloxapol content, comprising: preparing a tyloxapol inerting column solution, a tyloxapol reference solution, and a sample solution to be tested, respectively, wherein the concentration of the tyloxapol inerting column solution is greater than the concentration of the tyloxapol reference solution; firstly injecting the tyloxapol inerting column solution into a high performance liquid chromatograph for specific adsorption on the chromatographic column; then respectively performing high performance liquid analysis on the tyloxapol reference solution and the sample solution to be tested, and using an external standard method to determine the content of tyloxapol in the sample to be tested. The HPLC determination method of tyloxapol content of the invention is simple, fast, accurate, and highly sensitive, greatly shortening the time for testing, and is particularly suitable for determining the content of tyloxapol in a super-strong moisturizing lubricating eye drop, helping to improve the quality control and medication safety of tyloxapol liquid preparations, and can be used as an effective method for evaluating the quality of tyloxapol-containing preparations.

Description

Technical Field

[0001] The present invention relates to a high performance liquid chromatography (HPLC) method for determining the content of tyloxapol, in particular to an HPLC method for determining the content of tyloxapol in a tyloxapol liquid preparation, and in particular to an HPLC method for determining the content of tyloxapol in a moisturizing lubricating eye drop. Background Art

[0002] Tyloxapol, also known as Anliville, tyloxapol, tyloxapol, TRITON WR-1339 (hyperlipidemia modeling agent), CAS number: 25301-02-4. Tyloxapol is an alkyl aryl polyether alcohol non-ionic liquid polymer. As a pharmaceutical excipient, it is widely used in various dosage forms and drugs for treating different diseases, such as being used as a surfactant during liquefaction, removing mucopurulent and bronchopulmonary secretions, and inhibiting plasma lipolysis activity, thereby decomposing lipoproteins rich in triglycerides.

[0003]

[0004] After searching, the inventor found that tyloxapol is not included in the pharmacopoeias of China, Britain, Europe, etc., and the United States Pharmacopoeia includes tyloxapol but does not provide a method for content detection; there is no related tyloxapol detection method in the literature. With the increasing application of tyloxapol in pharmaceutical preparations, it is necessary to establish a convenient and fast detection method to quickly detect the content of tyloxapol in drugs.

[0005] High performance liquid chromatography is an important branch of chromatography. It uses liquid as the mobile phase and a high-pressure infusion system to pump single solvents with different polarities or mixed solvents with different proportions, buffers and other mobile phases into a chromatographic column equipped with a stationary phase. After the components are separated in the column, they enter the detector for detection, thereby realizing the analysis of the sample. High performance liquid chromatography has become an important and mature technology in the fields of chemical engineering and medicine due to its advantages such as high pressure, high speed, high efficiency, high sensitivity, wide range of applications, repeated use of chromatographic columns, small sample volume requirements and easy recovery. Due to its long-term development and use, it is superior to other detection methods such as gas chromatography in terms of safety, compliance and cost.

[0006] Size exclusion chromatography is also called steric exclusion chromatography (SEC) and gel chromatography. Size exclusion chromatography uses the characteristics of porous gel stationary phase and the relative relationship between the pore size of the gel pores and the size of the molecular coils of the polymer sample to separate the solutes. Size exclusion chromatography is suitable for the exploratory separation of unknown samples and can quickly provide a comprehensive picture of the sample's molecular size composition. Summary of the invention

[0007] The object of the present invention is to provide a method for determining the content of tyloxapol by high performance liquid chromatography, which utilizes two exclusion chromatography columns in combination to determine the content of tyloxapol under a high performance liquid chromatography system.

[0008] Screening process of the technical solution of the present invention:

[0009] (1) Preliminary conception

[0010] Bausch + Lomb, Soothe, Super Moisturizing Lubricating Eye Drops: This super moisturizing lubricating eye drop is produced by Bausch + Lomb. It is a sterile eye drop for dry eye care, moisturizing, soothing, long-lasting and effective. It is mostly used to soothe dry eyes and prevent further irritation. The ingredients include povidone, tyloxapol, benzalkonium chloride, boric acid, disodium edetate dihydrate, water, sodium borate, sodium chloride, etc. This product is widely used for eye care and relieving eye fatigue.

[0011] In the formulation of the extra-strong moisturizing lubricating eye drops, povidone and tyloxapol are macromolecular compounds compared to other components in the formulation and can be separated using an exclusion chromatography column.

[0012] (2) Selection of absorption wavelength

[0013] After preliminary exploration and research on the prescription, the inventor determined that the maximum absorption wavelengths of tyloxapol and povidone are similar. The absorption wavelengths of the two excipients were scanned by ultraviolet spectrophotometer to obtain the maximum absorption wavelengths of the two: povidone has a maximum absorption wavelength at 202.118nm, which gradually decreases to 240nm; tyloxapol has a maximum absorption wavelength at 197.997nm, 193.392nm, 203.755nm, and 278.629nm, which gradually decreases to 240nm. The results show that tyloxapol has strong absorption at the terminal wavelength (190-200nm), but the system is greatly disturbed when using the terminal wavelength, and tyloxapol is also greatly affected by povidone, so the initial use is 220nm, which still has strong absorption. .

[0014] (3) Preliminary screening of mobile phase

[0015] 25% H3PO4: Measure 250mL of H3PO4, add pure water to dilute to 1000mL, and shake well.

[0016] Mobile phase A (pH2.5 50mmol Buffer: acetonitrile = 90:10 v / v): weigh about 7.80g of NaH2PO4, measure 1000mL of water, stir by ultrasonic, adjust the pH to 2.50 with 25% H3PO4, take 900mL, add 100mL of acetonitrile, and stir by ultrasonic.

[0017] Mobile phase B (pH2.5 80mmol Buffer): weigh about 12.48g of NaH2PO4, measure 1000mL of water, stir by ultrasonic, adjust the pH to 2.50 with 25% H3PO4, and stir by ultrasonic.

[0018] Solvent (pH2.5 80mmol Buffer): weigh about 12.48g of NaH2PO4, measure 1000mL of water, stir by ultrasonic, adjust the pH to 2.50 with 25% H3PO4, and stir by ultrasonic.

[0019] Tyloxapol solution (0.6 mg / mL): Accurately weigh about 60 mg of tyloxapol, place it in a 20 mL volumetric flask, add an appropriate amount of solvent to dissolve, add solvent to the scale, shake well, as the tyloxapol stock solution; accurately measure 2 mL of tyloxapol stock solution, place it in a 10 mL volumetric flask, add an appropriate amount of solvent to dissolve, add solvent to the scale, shake well.

[0020] Eye drop test solution: Measure about 3 mL of eye drops, filter through a 0.22 μm PTFE filter membrane, take 2 mL of the filtrate, put it into a 10 mL volumetric bottle, add an appropriate amount of solvent to dissolve, add solvent to the scale, and shake well.

[0021] The parameters of HPLC are as follows:

[0022] Table 1. Liquid chromatography conditions -1

[0023]

[0024] Method 1: Use mobile phase A (pH 2.5 50mmol Buffer: acetonitrile = 90:10) as the mobile phase, accurately measure 10μL of 0.6mg / mL tyloxapol solution, inject it into the liquid chromatograph, inject it twice continuously, and record the spectrum ( Figure 1 ), there are multiple peaks in the chromatogram and the target peak cannot be determined.

[0025] Method 2: Using mobile phase B (pH 2.5 80mmol Buffer) as the mobile phase, accurately measure 10μL of 0.6mg / mL tyloxapol solution, inject it into the liquid chromatograph, and record the spectrum ( Figure 2 ), tyloxapol peak, retention time is 6.057min. Accurately measure 10μL eye drop test solution, inject into liquid chromatograph, record the spectrum ( Figure 3 ), under this condition, tyloxapol (Rt = 6.038min) and the adjacent peaks did not achieve baseline separation, and the specificity did not meet the requirements. The method could be further optimized based on mobile phase B. Therefore, it was considered to add a chromatographic column of the same model to improve the separation.

[0026] (4) Two chromatographic columns of the same model connected in series

[0027] Solvent (pH 2.5 80 mmol Buffer), mobile phase B (pH 2.5 80 mmol Buffer).

[0028] Tyloxapol stock solution (3 mg / mL): Accurately weigh about 60 mg of tyloxapol and place it in a 20 mL volumetric flask. Add an appropriate amount of solvent to dissolve and dilute to the scale, and shake well.

[0029] Povidone solution: Accurately weigh about 25 mg of povidone, place it in a 50 mL volumetric bottle, add an appropriate amount of solvent to dissolve and dilute to the scale, and shake well.

[0030] Eye drop test solution: Measure about 2 mL of eye drops, filter through a 0.22 μm PTFE filter membrane, take about 2 mL of the filtrate, place it in a 10 mL volumetric flask, add an appropriate amount of solvent to dilute to the scale, and shake well.

[0031] Tyloxapol solution (0.6 mg / mL): Accurately measure 2 mL of tyloxapol stock solution and place it in a 10 mL volumetric flask. Add appropriate amount of solvent to dilute to the scale and shake well.

[0032] The parameters of HPLC are as follows:

[0033] Table 2. LC conditions for method 3

[0034]

[0035] Accurately measure 10 μL of 3 mg / mL tyloxapol stock solution, 0.6 mg / mL tyloxapol test solution, 0.5 mg / mL povidone solution, and eye drop test solution, respectively, inject into the liquid chromatograph, record the spectrum, and correspond to Figure 4 , Figure 5 , Figure 6 , Figure 7 .Depend on Figure 4 It can be seen that at high concentration, no peak was produced after two consecutive injections of the tyloxapol stock solution (3 mg / mL). Figure 5 It can be seen that at low concentration, tyloxapol solution (0.6 mg / mL) was injected twice continuously, and no peak appeared either; Figure 6 and Figure 7 It can be seen that povidone solution and eye drop test sample solvent were injected twice in a row, and povidone was detected in both, but the target peak of tyloxapol was not detected. Therefore, the target peak was not detected after adding a chromatographic column, and the method needs to be further adjusted.

[0036] (5) Selection of test solution concentration

[0037] Method 3 Under the conditions of liquid chromatography, the tyloxapol test sample did not produce a peak. The inventors found through analysis that the internal filler of the Welch XtimateSEC-120 chromatographic column had nonspecific adsorption sites. For different targets, a sample with a concentration higher than the target concentration must be prepared first, and the injection must be repeated multiple times to adsorb the nonspecific adsorption sites, and then normal injection must be performed to ensure that the subsequent normal concentration samples do not lose their own concentration due to adsorption of nonspecific adsorption sites during sample loss. According to the analysis results, a high-concentration tyloxapol solution higher than the target concentration (3 mg / mL) was first prepared to saturate the chromatographic column.

[0038] Mobile phase B (pH2.5 80mmol Buffer): weigh about 24.96g of NaH2PO4, measure 2000mL of water, stir by ultrasonic, adjust the pH to 2.50 with 25% H3PO4, and stir by ultrasonic.

[0039] Solvent (pH2.5 80mmol Buffer): Same as mobile phase.

[0040] High concentration tyloxapol solution (30 mg / mL): Accurately weigh about 600 mg of tyloxapol, place in a 20 mL volumetric flask, add appropriate amount of solvent, ultrasonically dissolve and dilute to the scale, and shake well.

[0041] Tyloxapol stock solution (3 mg / mL): Accurately weigh about 60 mg of tyloxapol and place it in a 20 mL volumetric flask. Add an appropriate amount of solvent to dissolve and dilute to the scale, and shake well.

[0042] Povidone solution: Accurately weigh about 25 mg of povidone, place in a 50 mL volumetric bottle, add appropriate amount of solvent to dissolve and dilute to the scale, and shake well.

[0043] Eye drop test solution: Measure about 2 mL of eye drops, filter through a 0.22 μm PTFE filter membrane, take about 2 mL of the filtrate, place it in a 10 mL volumetric flask, add an appropriate amount of solvent to dilute to the scale, and shake well.

[0044] Tyloxapol solution (0.6 mg / mL): Accurately measure 2 mL of tyloxapol stock solution and place it in a 10 mL volumetric flask. Add appropriate amount of solvent to dilute to the scale and shake well.

[0045] The liquid chromatography parameters are as follows:

[0046] Table 3. LC conditions for method 4

[0047]

[0048] Accurately measure 10 μL of 30 mg / mL tyloxapol test solution and inject it into the liquid chromatograph. Repeat the injection 5 times in a row to conduct subsequent investigations after specific adsorption on the chromatographic column.

[0049] The detection wavelength was set at 220 nm. 10 μL of each of the povidone solution, 3 mg / mL tyloxapol stock solution, eye drop test solution, and 0.6 mg / mL tyloxapol solution were accurately measured and injected into the liquid chromatograph in sequence. Each sample was injected twice. The results were as follows: Figure 8 , Fig. 9 , Fig.10 , Fig.11 As shown. Figure 8 It can be seen that the retention time of povidone is 10.677min. Fig. 9 It can be seen that the peak areas of the two injections of 3 mg / mL tyloxapol stock solution were 289.1171 mAU*min and 285.2989 mAU*min, the retention times were 11.918 min and 11.915 min, and the peak heights were 536.04 mAU and 529.37 mAU. Fig.10 It can be seen that the target substance was detected in the eye drop test sample, and the peak areas of the two injections were 4.6930mAU*min and 3.0341mAU*min, respectively, the retention times were 12.378min and 12.403min, the peak heights were 5.34mAU and 3.51mAU, respectively, and the separation between tyloxapol and povidone (Rt=10.707min) was good. However, the peak shape of the 0.6mg / mL tyloxapol solution was poor, while the chromatographic peak shape of the 3mg / mL tyloxapol solution was good.

[0050] It was determined that when two chromatographic columns of the same model were used in combination, after the HPLC system was balanced and before formal sampling, the chromatographic column was first saturated with a high concentration of tyloxapol sample, and the concentration of the tyloxapol test solution was selected to be 3 mg / mL.

[0051] After determining the above conditions, further adjustments and optimizations were made to the repeatability of the injection results and the preparation method of the test solution.

[0052] (6) Mobile phase re-optimization:

[0053] Methods 5 to 7 use the same chromatographic conditions, as shown in Table 4, but with different mobile phases.

[0054] Method 5: Mobile phase C (pH 6.80 150mmol Buffer): Weigh about 11.69g of sodium dihydrogen phosphate dihydrate and about 10.65g of anhydrous disodium hydrogen phosphate, measure 1L of pure water, stir by ultrasonic until completely dissolved, adjust the pH to 6.80, filter, and take the filtrate.

[0055] Method 6: Mobile phase D (pH6.80 50mmol Buffer:acetonitrile = 90:10v / v): Weigh about 3.90g of sodium dihydrogen phosphate dihydrate, about 3.55g of anhydrous disodium hydrogen phosphate, and 17.54g of sodium chloride, measure 1L of pure water, and stir by ultrasound until completely dissolved, adjust the pH to 6.80, filter, take 900mL of the filtrate, measure 100mL of acetonitrile, and stir by ultrasound.

[0056] Method 7: Mobile phase E (pH2.50 80mmol Buffer): Weigh about 12.48g of sodium dihydrogen phosphate, add 1000mL of water, stir by ultrasonic, and adjust the pH to 2.50 with phosphoric acid.

[0057] Tyloxapol reference solution (3 mg / mL): Accurately weigh about 30 mg of tyloxapol, place in a 10 mL volumetric flask, dissolve in mobile phase C, add mobile phase C to the scale, and shake well.

[0058] In the same way, use mobile phase D and mobile phase E as solvents to prepare one portion each.

[0059] Eye drop test solution: Measure about 2 mL of eye drops, filter through a 0.22 μm PTFE filter membrane, take the filtrate, and place it in a sample vial.

[0060] Blank eye drops without tyloxapol: Place povidone, benzalkonium chloride, boric acid, disodium edetate dihydrate, water, sodium borate, and sodium chloride in a 100 mL volumetric flask according to the commercial prescription amount, add water to dissolve and dilute to the scale, and shake well.

[0061] Blank solution without tyloxapol: Measure about 2 mL of blank eye drops without tyloxapol, filter it through a 0.22 μm PTFE filter membrane, take the filtrate, and place it in an injection vial.

[0062] The parameters of HPLC are as follows:

[0063] Table 4. Liquid chromatography conditions for methods 5 to 7

[0064] Column temperature 40℃ Injection volume 10μL Injection time 40min Flow rate 1 mL / min wavelength 220nm Solvents Mobile phase Elution method Isocratic elution Chromatographic columns Welch Xtimate SEC-120; 7.8×300 mm; 5μm; two in series

[0065] Before executing methods 5 to 7, the chromatographic column was inerted with a high concentration of tyloxapol solution, that is, 10 μL of 30 mg / mL tyloxapol test solution was accurately measured and injected into the liquid chromatograph, and the injection was repeated 5 times in succession.

[0066] Method 5: Using mobile phase C as the mobile phase, accurately measure 10 μL of tyloxapol reference solution (prepared with mobile phase C), eye drop test solution, and blank solution without tyloxapol, and inject them into the liquid chromatograph in sequence to record the spectrum ( Fig.12). It can be seen that the peak area of ​​tyloxapol in the tyloxapol reference solution is 6.2547mAU*min, the peak area of ​​the eye drop test solution is 189mAU*min, the peak height is above 200mAU, the sensitivity meets the requirements, and the target peak is not detected in the blank solution without tyloxapol. However, the concentration of the buffer is too high, and it is easy to cause salting out in the chromatographic system and chromatographic column of the university, which is very harmful to the system. It is not the preferred mobile phase system.

[0067] Method 6: Use mobile phase D as the mobile phase, accurately measure 10 μL of the eye drop test solution, repeat the injection 5 times, and record the spectrum ( Fig.13 ). The target peak area of ​​the first injection of the eye drop test solution was 114.8902mAU*min, and the target peak area of ​​the eye drop test solution from the second injection onwards was above 200mAU*min, which may be due to nonspecific adsorption of the chromatographic column. Remove the first injection of the eye drop test solution ( Fig.14 ), four injections of eye drop test solution and five injections of homemade sample test solution, the RSD% of the peak area was 3.79%. This shows that the system precision meets the requirements. The separation degree of the tyloxapol peak and the povidone peak is above 4, achieving baseline separation.

[0068] Method 7: Using mobile phase E as the mobile phase, accurately measure 10 μL of each eye drop test solution, repeat the injection 5 times, and record the spectrum ( Fig.15 ). The target peak area of ​​the first injection of the eye drop test solution was 38.2848 mAU*min, indicating that the chromatographic column needed to be further balanced at this concentration. From the second injection, the target peak areas of the eye drop and homemade sample test solutions were all above 200 mAU*min, and the software could not directly calculate the separation degree of povidone and tyloxapol.

[0069] In summary, comparing the separation results of mobile phase C, mobile phase D and mobile phase E, the separation degree of povidone and tyloxapol in mobile phase D is the largest, the theoretical plate number is high, and when the chromatographic column is saturated, 4 consecutive injections are repeated, and the RSD is 2.14%, which is less than 5%, and the repeatability is good. Therefore, mobile phase D is selected as the mobile phase for tyloxapol content determination.

[0070] (7) Optimization of the preparation method of tyloxapol solution

[0071] Since tyloxapol is a non-ionic liquid polymer of the alkyl aromatic polyether alcohol class, which is soluble in water, benzene, toluene, chloroform, carbon tetrachloride, carbon disulfide and acetic acid, tyloxapol has viscous, oily physical properties and is surface active, which can easily lead to large errors in the weighing process and produce bubbles that are difficult to remove during the dissolution process. Therefore, the preparation method of tyloxapol reference solution was further investigated.

[0072] Solvent (pH6.80 50mmol Buffer:acetonitrile = 90:10v / v): Weigh about 3.90g of sodium dihydrogen phosphate dihydrate, about 3.55g of anhydrous disodium hydrogen phosphate, and 17.54g of sodium chloride, measure 1L of pure water, and stir by ultrasonic until completely dissolved, adjust the pH to 6.80, filter, take 900mL of the filtrate, measure 100mL of acetonitrile, and stir by ultrasonic.

[0073] Solution 1: Accurately weigh about 600 mg of tyloxapol, place it in a 50 mL volumetric flask, add an appropriate amount of solvent to dissolve and dilute to the scale; accurately measure 5 mL of the solution, place it in a 20 mL volumetric flask, add solvent to dilute to the scale, and shake well.

[0074] In this scheme, a large number of tyloxapol clumps at the bottom of the volumetric flask and is dissolved by vortexing, shaking and ultrasound. The first two dissolution methods will produce a large number of bubbles that are difficult to remove. After standing overnight, bubbles are still present when observed the next day, making it impossible to accurately determine the volume. Ultrasound requires more than 4 hours.

[0075] Option 2:

[0076] Accurately weigh about 150 mg of tyloxapol, place it in a 50 mL volumetric flask, add an appropriate amount of solvent to dissolve and dilute to the scale.

[0077] In this scheme, the aggregation phenomenon of tyloxapol is lighter than that in scheme 1 (600 mg). Under an ultrasonic power of 200 W, it can be completely dissolved in 2 to 3 hours without generating bubbles.

[0078] In summary, the purpose of the present invention is achieved through the following technical solutions:

[0079] A HPLC determination method for tyloxapol content comprises: preparing a tyloxapol inerting column solution, a tyloxapol reference solution, and a test sample solution, respectively, wherein the concentration of the tyloxapol inerting column solution is greater than that of the tyloxapol reference solution; firstly injecting the tyloxapol inerting column solution into a high performance liquid chromatograph to perform specific adsorption on the chromatographic column; then performing high performance liquid phase analysis on the tyloxapol reference solution and the test sample solution, respectively, and determining the tyloxapol content in the test sample by an external standard method.

[0080] Preparation of the tyloxapol inerting column solution: accurately weigh about 600 mg of tyloxapol, place it in a 20 mL volumetric flask, add an appropriate amount of buffer solution-acetonitrile and ultrasonically dissolve it, add buffer solution-acetonitrile to the scale, shake well, and obtain a tyloxapol inerting column solution with a concentration of 30 mg / mL.

[0081] Preferably, first accurately measure 10 μL of the tyloxapol inerting column solution, inject it into the high performance liquid chromatograph, repeat the injection continuously, and perform specific adsorption on the chromatographic column. If the peak area ratio of the last two injections is within 100±1%, the inerting is completed. Generally, the tyloxapol inerting column solution is repeatedly injected 5 times.

[0082] Preparation of the tyloxapol reference solution: accurately weigh about 150 mg of tyloxapol, place in a 50 mL volumetric flask, add an appropriate amount of buffer solution-acetonitrile, sonicate until completely dissolved, let stand to room temperature, add buffer solution-acetonitrile to dilute to the scale, shake well, and obtain a 3 mg / mL tyloxapol reference solution.

[0083] Preferably, when preparing the tyloxapol inerted column solution and the tyloxapol reference solution, the ultrasonic power can be selected to be 200W.

[0084] The preparation of the test sample solution, wherein the test sample is eye drops, tyloxapol has been dissolved in an aqueous solution, and a commercially available sample is directly taken: the test sample is measured, filtered through a 0.22 μm PTFE filter membrane, about 2 mL of the filtrate is taken, placed in a 10 mL volumetric bottle, an appropriate amount of buffer solution-acetonitrile is added to dissolve, buffer solution-acetonitrile is added to the scale, and shaken to obtain the test sample solution.

[0085] The sample to be tested is a tyloxapol liquid preparation, specifically Bausch+Lomb, Soothe, extra-strong moisturizing lubricating eye drops.

[0086] The HPLC conditions are as follows: chromatographic column: two Welch Xtimate SEC-120 columns or other exclusion chromatography columns with the same effect are used in combination; Welch Xtimate SEC-120 column: inner diameter 7.8 mm, column length 300 mm, filler particle size 5 μm; column temperature 25-45°C; injection volume 10 μL, buffer-acetonitrile as mobile phase, flow rate 0.5-2.0 mL / min, ultraviolet detection wavelength 220 nm, tyloxapol is not interfered by solvent peaks and other components in the sample.

[0087] Preferably, the HPLC conditions are as follows: chromatographic column: two Welch Xtimate SEC-120 chromatographic columns are used in combination; Welch Xtimate SEC-120 chromatographic column: inner diameter 7.8 mm, column length 300 mm, filler particle size 5 μm; column temperature 40°C; injection volume 10 μL, buffer-acetonitrile as mobile phase, flow rate 1.0 mL / min, UV detection wavelength 220 nm

[0088] The volume ratio of the buffer solution to acetonitrile in the buffer solution-acetonitrile is 9:1.

[0089] The preparation method of the buffer solution-acetonitrile is as follows: weigh about 3.90 g of sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O), about 3.55 g of anhydrous disodium hydrogen phosphate (Na2HPO4), and 17.54 g of sodium chloride (NaCl), measure 1 L of pure water, stir them by ultrasonic until they are completely dissolved, adjust the pH to 6.80 with 25% phosphoric acid (H3PO4) aqueous solution and 0.1 M sodium hydroxide (NaOH) aqueous solution, filter, take 900 mL of the filtrate, measure 100 mL of acetonitrile, and stir them by ultrasonic.

[0090] The calculation method of tyloxapol content in the sample to be tested is:

[0091]

[0092] Wherein, Wr: mass of reference substance, mg; Ws: mass of test substance, mg; Ar: peak area of ​​reference substance; As: peak area of ​​test substance; Sr: dilution multiple of reference substance; Ss: dilution multiple of test substance; P: content of reference substance.

[0093] Tyloxapol has a peak within the range of 12min±0.5min, with a peak height of approximately 360-370mAu.

[0094] Beneficial effects of the present invention:

[0095] The HPLC determination method of the tyloxapol content of the present invention is simple, rapid, accurate and sensitive, has undergone complete development and method verification, and is particularly suitable for determining the content of tyloxapol in super-strong moisturizing and lubricating eye drops.

[0096] The method of the present invention greatly shortens the test time, is simple and feasible, fast and accurate, reduces the test pressure, has good precision, linear relationship, stability, repeatability, high recovery rate and good durability.

[0097] The presaturation of the chromatographic column in the present invention is innovative in practical application.

[0098] The present invention solves the problem that there is currently no reliable liquid phase detection method for detecting the tyloxapol content in the extra-strong moisturizing lubricating eye drops, helps to improve the quality control and medication safety of tyloxapol liquid preparations, and can be used as an effective method for evaluating the quality of tyloxapol-containing preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0099] Figure 1 This is the elution result of tyloxapol when mobile phase A is used as the mobile phase in method 1.

[0100] Figure 2 This is the elution result of tyloxapol when mobile phase B is used as the mobile phase in method 2 (peak 4 is tyloxapol).

[0101] Figure 3 This is the peak elution result of eye drops using method 2 with mobile phase B as the mobile phase (peak No. 2 is tyloxapol).

[0102] Figure 4 The peak results are obtained from two consecutive injections of tyloxapol stock solution (3 mg / mL) under the HPLC conditions of Method 3.

[0103] Figure 5 The peak results are obtained from two consecutive injections of tyloxapol solution (0.6 mg / mL) under the HPLC conditions of Method 3.

[0104] Figure 6 This is the peak result of two consecutive injections of the povidone test solution (0.5 mg / mL) under the liquid chromatography conditions of Method 3.

[0105] Figure 7 This is the peak result of two consecutive injections of the eye drop test solution under the HPLC conditions of Method 3.

[0106] Figure 8 Position the chromatogram for povidone.

[0107] Fig. 9 Peak results of two consecutive injections of tyloxapol stock solution (3 mg / mL).

[0108] Fig.10 This is the peak result of two consecutive injections of the eye drop test solution.

[0109] Fig.11 This is the peak result of two consecutive injections of tyloxapol solution (0.6 mg / mL).

[0110] Fig.12 This is the peak result of injection when Method 5 uses Mobile C as the mobile phase.

[0111] Fig.13 This is the peak result of injection when mobile phase D is used as the mobile phase in method 6.

[0112] Fig.14 for Fig.13 Results of removing the first injection of eye drop test solution.

[0113] Fig.15 This is the peak result of injection when mobile phase E is used as the mobile phase in method 7.

[0114] Fig.16 This is the peak situation of the solvent in the specificity experiment.

[0115] Fig.17 This is the peak of the tyloxapol reference solution (3 mg / mL) in the specificity experiment.

[0116] Fig.18 This is the linear range experimental result - the linear range standard curve of tyloxapol. DETAILED DESCRIPTION

[0117] Example 1

[0118] 1. Instruments and test drugs

[0119] 1.1 Instrument

[0120] ThermoUltimate 3000 high performance liquid chromatograph; Thermo U3000-Chromeleon7 chromatography data workstation; Ruisijie Unique-LC R10 multifunctional ultrapure water system.

[0121] 1.2 Drug testing

[0122]

[0123] 2. Methods

[0124] 2.1 Solution preparation

[0125] Solvent (pH6.80 50mmol Buffer:acetonitrile = 90:10v / v): Weigh about 3.90g of sodium dihydrogen phosphate dihydrate, about 3.55g of disodium hydrogen phosphate anhydrous, and 17.54g of sodium chloride, measure 1L of pure water, and stir by ultrasound until completely dissolved, adjust the pH to 6.80 with 25% phosphoric acid (H3PO4) aqueous solution and 0.1mol / L sodium hydroxide (NaOH) aqueous solution, filter, take 900mL of the filtrate, measure 100mL of acetonitrile, put them in the same beaker, and stir by ultrasound.

[0126] 25% phosphoric acid aqueous solution: Measure 25mL of phosphoric acid, dilute to 100ml with water, and shake well.

[0127] 0.1mol / L NaOH solution: weigh about 2g of NaOH into a 500mL volumetric flask, add pure water to dissolve, add pure water to dilute to the scale, shake well, and you have it.

[0128] Preparation of tyloxapol inerting column solution: accurately weigh about 600 mg of tyloxapol, place it in a 20 mL volumetric flask, add an appropriate amount of buffer solution-acetonitrile and sonicate to dissolve it, add buffer solution-acetonitrile to the scale, shake well, and obtain a tyloxapol inerting column test solution with a concentration of 30 mg / mL.

[0129] Eye drop test solution: measure commercially available eye drops, filter through a 0.22 μm PTFE filter membrane, take about 2 mL of the filtrate, put it into a 10 mL volumetric bottle, add an appropriate amount of solvent to dissolve, add solvent to the scale, shake well, and obtain the eye drop test solution.

[0130] 2.2 Chromatographic conditions

[0131] Chromatographic column: Welch Xtimate SEC-120 exclusion chromatography column, column inner diameter 7.8 mm, column length 300 mm, filler particle size 5 μm, Part No. 00237-31052, filler lot No. SEC05012.14; the column was saturated with 30 mg / mL tyloxapol solution before each formal injection;

[0132] Mobile phase (pH6.80 50mmol Buffer: acetonitrile = 90:10v / v): weigh about 3.90g of sodium dihydrogen phosphate dihydrate, about 3.55g of disodium hydrogen phosphate anhydrous, and 17.54g of sodium chloride, measure 1L of pure water, and stir by ultrasound until completely dissolved, adjust the pH to 6.80 with 25% phosphoric acid (H3PO4) aqueous solution and 0.1mol / L sodium hydroxide (NaOH) aqueous solution, filter, take 900mL of the filtrate, measure 100mL of acetonitrile, and stir by ultrasound;

[0133] Flow rate: 1.0 mL / min;

[0134] UV detection wavelength: 220nm;

[0135] Column temperature: 40°C;

[0136] Injection volume: 10 μL.

[0137] System suitability requirements: First, accurately measure 10 μL of tyloxapol inerting column solution, inject it into the HPLC, record the chromatogram, repeat the injection 5 times continuously, perform specific adsorption on the chromatographic column, and the peak area ratio of the last two injections is within 100±1%, which can be considered as inerting. Then accurately measure 10 μL of solvent and tyloxapol reference solution respectively, inject them into the HPLC, and record the chromatogram. The solvent chromatogram should not show a peak at the tyloxapol peak. The tyloxapol reference solution is continuously injected for 5 injections, and the theoretical plate number is calculated based on the tyloxapol peak and is not less than 2500. Inject 5 times continuously, and the RSD of the tyloxapol peak area in the chromatogram should be less than 5%. Determination: Accurately measure 10 μL of the reference solution and the test solution, inject them into the liquid chromatograph, record the chromatogram, and calculate by peak area according to the external standard method.

[0138] 2.3 Methodological investigation

[0139] 2.3.1 Chromatographic system suitability:

[0140] Accurately weigh about 150 mg of tyloxapol, place it in a 50 mL volumetric flask, add an appropriate amount of solvent, sonicate until completely dissolved, let stand to room temperature, add solvent to dilute to the scale, shake well, and obtain a tyloxapol reference solution; accurately measure 10 μL and inject it into the high performance liquid chromatograph, the theoretical plate number is calculated based on the tyloxapol peak to be 4400, which is greater than 2500; inject the sample continuously for 5 times, the RSD of the tyloxapol peak area in the chromatogram is 0.3%, which is less than 5%, and the system suitability meets the requirements.

[0141] 2.3.2 Method Specificity: Prepare the tyloxapol reference solution according to "2.3.1 Chromatographic System Suitability". Determine the tyloxapol reference solution and eye drop test solution according to this method. The corresponding retention time of tyloxapol is t = 12.3 min. Fig.16 and Fig.17 It can be seen that the tyloxapol peak is not interfered by the solvent peak and other components in the sample, and the peak shape is good.

[0142] 2.3.3 Linear range: Prepare reference solutions with concentrations of 2.7, 2.85, 3.00, 3.15, and 3.30 mg / mL. The preparation method is as follows:

[0143] L-1 Test solution (2.70 mg / mL): Accurately weigh about 135 mg of tyloxapol, place in a 50 mL volumetric flask, add an appropriate amount of solvent, sonicate until completely dissolved, let stand to room temperature, add solvent to dilute to the scale, and shake well.

[0144] L-2 Test solution (2.85 mg / mL): Accurately weigh about 143 mg of tyloxapol, place in a 50 mL volumetric flask, add an appropriate amount of solvent, sonicate until completely dissolved, let stand to room temperature, add solvent to dilute to the scale, and shake well.

[0145] L-3 Test solution (3.0000 mg / mL): Accurately weigh about 150 mg of tyloxapol, place in a 50 mL volumetric flask, add an appropriate amount of solvent, sonicate until completely dissolved, let stand to room temperature, add solvent to dilute to the scale, and shake well.

[0146] L-4 test solution (3.15 mg / mL): Accurately weigh about 158 ​​mg of tyloxapol, place in a 50 mL volumetric flask, add an appropriate amount of solvent, sonicate until completely dissolved, let stand to room temperature, add solvent to dilute to the scale, and shake well.

[0147] L-5 Test solution (3.30 mg / mL): Accurately weigh about 165 mg of tyloxapol, place in a 50 mL volumetric flask, add appropriate amount of T-20211130B1, sonicate until completely dissolved, let stand to room temperature, add T-20211130B1 to dilute to the scale, and shake well.

[0148] Table 5. Linear sample preparation table

[0149] Linear Name Relative concentration% Theoretical sample weight (mg) Absolute concentration (mg / mL) Volume of measuring flask (mL) L-1 90 135 2.70 50 L-2 95 143 2.85 50 L-3 100 150 3.00 50 L-4 105 158 3.15 50 L-5 110 165 3.30 50

[0150] Take 10 μL of the test solution and inject it into the high performance liquid chromatograph, analyze it according to the above chromatographic conditions, and plot the peak area A (n=6) of tyloxapol against the weight concentration C (mg / mL) ( Fig.18 ), and the linear regression equation is: y = 106.0342x-122.0437, R 2 =0.9994. The results showed that in the range of 2.7-3.3 mg / mL, the peak area A of tyloxapol and the concentration C showed a good linear relationship.

[0151] 2.3.4 Method accuracy (expressed as recovery rate): Prepare reference solutions with concentrations of 2.7, 3.0 and 3.3 mg / mL respectively, and determine the recovery rate according to the law. The preparation method is as follows:

[0152] Solvent: Weigh about 3.90 g of sodium dihydrogen phosphate dihydrate, about 3.55 g of disodium hydrogen phosphate anhydrous, and 17.54 g of sodium chloride, measure 1 L of pure water, stir by ultrasonic until completely dissolved, adjust the pH to 6.80, filter, take 900 mL of the filtrate, measure 100 mL of acetonitrile, and stir by ultrasonic.

[0153] Blank eye drops without tyloxapol: Place povidone, benzalkonium chloride, boric acid, disodium edetate dihydrate, purified water, sodium borate, and sodium chloride in a 100-mL volumetric flask according to the commercial prescription amount, add water to dissolve and dilute to the scale, and shake well.

[0154] Tyloxapol reference solution: accurately weigh about 150 mg of tyloxapol, place in a 50 mL volumetric bottle, add an appropriate amount of solvent, sonicate until completely dissolved, let stand at room temperature, add solvent to dilute to scale, shake well. Prepare two portions in the same way.

[0155] 90% recovery rate test solution: accurately weigh about 135 mg of tyloxapol, place in a 50 mL volumetric bottle, add an appropriate amount of blank eye drops without tyloxapol, sonicate until completely dissolved, let stand at room temperature, add extra moisturizing and lubricating eye drops without tyloxapol to dilute to scale, shake well, filter through a 0.22 μm PTFE filter membrane, and take the filtrate. Prepare 3 copies in parallel.

[0156] 100% recovery rate test solution: accurately weigh about 150 mg of tyloxapol, place in a 50 mL volumetric bottle, add an appropriate amount of blank eye drops without tyloxapol, sonicate until completely dissolved, let stand at room temperature, add extra moisturizing lubricating eye drops without tyloxapol to dilute to scale, shake well, filter through a 0.22 μm PTFE filter membrane, and take the filtrate. Prepare 3 copies in parallel.

[0157] 110% recovery rate test solution: accurately weigh about 165 mg of tyloxapol, place in a 50 mL volumetric bottle, add an appropriate amount of blank eye drops without tyloxapol, sonicate until completely dissolved, let stand at room temperature, add extra moisturizing lubricating eye drops without tyloxapol to dilute to scale, shake well, filter through a 0.22 μm PTFE filter membrane, and take the filtrate. Prepare 3 copies in parallel

[0158] The method recovery rates of the reference solution are shown in Table 6. It shows that the average recovery rates at three concentrations are between 92% and 105%, the relative standard deviation within the group (n=3) and the relative standard deviation between the groups (n=9) are both less than 5.0%, and the accuracy of this method is high.

[0159] Table 6. Accuracy sample preparation table

[0160]

[0161] 2.3.5 Method precision: A 3.0 mg / mL tyloxapol sample solution was prepared according to “2.3.1 Chromatographic system suitability” and analyzed under the above chromatographic conditions for six consecutive measurements. The results are shown in Table 7.

[0162] The content was calculated using the standard curve obtained from the linear experiment. The results showed that the RSD% of tyloxapol in the six samples was 0.5%, less than 5.0%; this method has good precision.

[0163] Table 7. Results of method precision determination

[0164]

[0165] 2.3.6 Method stability: A 3.0 mg / mL tyloxapol sample solution was prepared in accordance with “2.3.1 Chromatographic System Suitability”, and 10 μL of sample was injected at 0, 5.5, 8, 12, 15.5, 18, 22, 27, and 31.5 h, respectively. The solution was analyzed according to the above chromatographic conditions to investigate the stability of the solution. The results are shown in Table 8, which show that the ratio of the main peak area of ​​tyloxapol measured at each time point to the main peak area of ​​tyloxapol at 0 h was in the range of 90% to 110%, and the tyloxapol peak area did not change significantly within 31.5 h, and the sample measurement was stable within 31.5 h.

[0166] Table 8. Stability test results

[0167]

Claims

1. A HPLC determination method for the content of tyloxapol, characterized in that: Used for separating tyloxapol and povidone, comprising: preparing a tyloxapol inerting column solution, a tyloxapol reference solution, and a test sample solution, respectively, wherein the concentration of the tyloxapol inerting column solution is greater than the concentration of the tyloxapol reference solution; firstly injecting the tyloxapol inerting column solution into a high performance liquid chromatograph to perform specific adsorption on the chromatographic column; then performing high performance liquid analysis on the tyloxapol reference solution and the test sample solution, respectively, and determining the content of tyloxapol in the test sample by an external standard method; The high performance liquid chromatography conditions are as follows: two Welch Xtimate SEC-120 columns are connected in series; the Welch Xtimate SEC-120 column has an inner diameter of 7.8 mm, a column length of 300 mm, and a filler particle size of 5 μm; a mobile phase containing buffer-acetonitrile, wherein the volume ratio of the buffer to acetonitrile in the buffer-acetonitrile is 9:1, and the preparation method is as follows: 3.90 g of sodium dihydrogen phosphate dihydrate, 3.55 g of anhydrous disodium hydrogen phosphate, and 17.54 g of sodium chloride are weighed, 1 L of pure water is measured, ultrasonically stirred until completely dissolved, the pH is adjusted to 6.80, filtered, 900 mL of the filtrate is taken, 100 mL of acetonitrile is measured, and ultrasonically stirred; the ultraviolet detection wavelength is 220 nm.

2. The HPLC determination method of tyloxapol content according to claim 1, characterized in that: Preparation of the tyloxapol inerting column solution: accurately weigh 600 mg of tyloxapol, place it in a 20 mL volumetric flask, add an appropriate amount of buffer solution-acetonitrile and ultrasonically dissolve it, add buffer solution-acetonitrile to the scale, shake well, and obtain a tyloxapol inerting column solution with a concentration of 30 mg / mL.

3. The HPLC determination method of tyloxapol content according to claim 1, characterized in that: Preparation of the tyloxapol reference solution: accurately weigh 150 mg of tyloxapol, place in a 50 mL volumetric flask, add an appropriate amount of buffer solution-acetonitrile, sonicate until completely dissolved, let stand to room temperature, add buffer solution-acetonitrile to dilute to the scale, shake well, and obtain a 3 mg / mL tyloxapol reference solution.

4. The HPLC determination method of tyloxapol content according to claim 2 or 3, characterized in that: When preparing the tyloxapol inerting column solution and the tyloxapol reference solution, the ultrasonic power was 200W.

5. The HPLC determination method of tyloxapol content according to claim 1, characterized in that: Preparation of the test sample solution: measure the test sample, filter it through a 0.22 μm PTFE filter membrane, take 2 mL of the filtrate, put it into a 10 mL volumetric flask, add an appropriate amount of buffer solution-acetonitrile to dissolve it, add buffer solution-acetonitrile to the scale, shake well, and obtain the test sample solution.

6. The HPLC determination method of tyloxapol content according to claim 1 or 5, characterized in that: The sample to be tested is a tyloxapol liquid preparation.

7. The HPLC determination method of tyloxapol content according to claim 1, characterized in that: The HPLC conditions were as follows: chromatographic column: two Welch Xtimate SEC-120 columns were used in combination; Welch Xtimate SEC-120 column: inner diameter 7.8 mm, column length 300 mm, filler particle size 5 μm; column temperature 25-45°C; injection volume 10 μL, buffer-acetonitrile as mobile phase, flow rate 0.5-2.0 mL / min, UV detection wavelength 220 nm.

8. The HPLC determination method of tyloxapol content according to claim 7, characterized in that: The HPLC conditions were as follows: chromatographic column: two Welch Xtimate SEC-120 columns were used in combination; Welch Xtimate SEC-120 column: inner diameter 7.8 mm, column length 300 mm, filler particle size 5 μm; column temperature was 40°C; injection volume was 10 μL, buffer-acetonitrile was used as the mobile phase, the flow rate was 1.0 mL / min, and the UV detection wavelength was 220 nm.

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