A method for simultaneously detecting the contents of poloxamer 188, tyloxapol and liquid paraffin in a test sample

Abstract

The present invention belongs to the technical field of component detection, and provides a method for simultaneously detecting the content of poloxamer 188, tyloxapol, and liquid paraffin in a test sample, the method comprising the following steps: mixing poloxamer 188, tyloxapol, liquid paraffin, and dichloromethane to obtain a mixed solution; mixing the mixed solution with isopropanol to obtain a reference solution; performing liquid phase detection on the reference solution to obtain a standard curve of poloxamer 188, tyloxapol, and liquid paraffin in the reference solution; mixing a test sample with isopropanol to obtain a test solution; performing liquid phase detection on the test solution, comparing and calculating the test result with the standard curve, and determining the content of poloxamer 188, tyloxapol, and liquid paraffin in the test sample. The method of the present invention is simple, accurate, has a short analysis time, and has good precision, reproducibility, and stability, and can provide a theoretical basis and scientific basis for comprehensively evaluating and controlling the quality of test samples.

Description

Technical Field

[0001] The present invention relates to the technical field of component detection, and in particular to a method for simultaneously detecting the contents of poloxamer 188, tyloxapol and liquid paraffin in a test sample. Background Art

[0002] Poloxamer 188, English name Poloxamer 188, trade name Pluronic, CAS number: 9003-11-6. It is a block copolymer of α-hydrogen-ω-hydroxy poly(ethylene oxide) a-poly(propylene oxide) b-poly(ethylene oxide) a. Propylene oxide and propylene glycol react to form polyoxypropylene glycol, and then ethylene oxide is added to form a block copolymer. Molecular formula: HO(C2H4O)a(C3H6O)b(C2H4O)aH. Poloxamer is a non-ionic surfactant. As a pharmaceutical excipient, it can be used as a dispersant, emulsifier, solubilizer, wetting agent, etc. It is widely used in various dosage forms and drugs for treating different diseases. It is a commonly used emulsifier for fat emulsion products.

[0003]

[0004] a=80,b=27

[0005] Poloxamer structure

[0006] Tyloxapol, English name; Tyloxapol, also known as tetrabutylphenol, tetrabutylphenol alcohol, CAS number: 25301-02-4. Tyloxapol is an alkyl aryl polyether alcohol non-ionic liquid polymer that can reduce tension and surface tension. The hydrophobic alkyl group can increase the solubility of oil in this molecule, while a large number of hydrophilic groups increase the solubility of water in this molecule. Therefore, it is used as an emulsifier for water / oil and oil / water emulsions. It has many uses in the medical field and is often used in ophthalmic preparations, such as solution-type eye drops, suspensions, emulsions, etc.

[0007]

[0008] Tyloxapol structure

[0009] Liquid paraffin is a mixture of refined liquid saturated aliphatic hydrocarbons (C14-C18) and higher alkanes such as cyclic hydrocarbons. CAS number: 8012-95-1. Liquid paraffin has the functions and effects of lubrication, defecation promotion, skin protection, anti-ulcer, and wound healing. In ophthalmic preparations, it can lubricate the eyes, reduce sourness, reduce tear evaporation, and increase eye wettability.

[0010] The ophthalmic emulsion product containing poloxamer 188, tyloxapol and liquid paraffin has good efficacy. The content of drug ingredients directly affects the effect of the drug, and accurate and reliable content detection methods are of great significance to product quality.

[0011] There are public documents such as "Determination of Poloxamer 188 in Recombinant Human Interferon α2b Injection by ELSD-HPLC", "Determination of Poloxamer 188 in Recombinant Human Growth Hormone Injection by Evaporative Light Scattering", "Determination of Poloxamer 188 in Recombinant Human IL-12 Injection by Evaporative Light Scattering Detection", and "Determination of Poloxamer 188 in Paclitaxel Fat Emulsion Injection by Gel Chromatography-Evaporative Light Scattering Detection". The evaporative light scattering detection method is used to determine the poloxamer content in drugs, but only the poloxamer 188 in the prescription is detected, and the matrix interference is relatively easy to control.

[0012] Chinese patents CN116609460A, CN111289652A, and CN113424057B disclose analytical methods for determining poloxamer 188 in drugs, using ELSD or CAD detectors, and the chromatographic analysis system uses the molecular exclusion principle or the reverse phase principle, but the disclosed methods are also unable to effectively determine multiple components.

[0013] Chinese patent CN117571642A discloses a tyloxapol spectrophotometric detection method, which is not suitable for complex matrix drug analysis and multi-component analysis.

[0014] Chinese patent CN116026953A discloses a method for detecting the content of tyloxapol. In the high performance liquid chromatography analysis process, a Welch Xtimate SEC-120 type exclusion chromatography column (two chromatography columns are connected in series) is used, with pH 6.80 50mmol Buffer (weighing about 3.90g of dihydrogen phosphate dihydrate, about 3.55g of disodium hydrogen phosphate anhydrous, 17.54g of sodium chloride, measuring 1L of pure water, ultrasonically stirring until completely dissolved, and adjusting the pH to 6.80 with 25% phosphoric acid aqueous solution and 0.1mol / L sodium hydroxide aqueous solution) : acetonitrile = 90:10 (v / v) as the mobile phase, the flow rate is 1.0ml / min, the ultraviolet detection wavelength is 220nm, the column temperature is 40°C, the injection volume is 10μL, and the detection time is 30 minutes. This method involves the combination of two chromatographic columns of the same model. After the HPLC system is balanced, the chromatographic column needs to be saturated with a 3 mg / ml tyloxapol test solution before formal sampling. This is rather cumbersome, and this method cannot detect poloxamer 188 and liquid paraffin in drugs.

[0015] Chinese patent CN118311158A discloses a liquid phase analysis method for simultaneously determining the contents of poloxamer 188 and tyloxapol in a drug. The two components are detected simultaneously using evaporative light scattering detection. In the disclosed typical separation spectrum, the tyloxapol and matrix peaks in the drug analysis spectrum cannot be baseline separated, and there is a certain interference. In addition, this method cannot detect the liquid paraffin content in the drug.

[0016] After searching, the inventor found no literature or patents disclosing a method for detecting the content of liquid paraffin in drugs.

[0017] In summary, there is currently no analytical method that can quickly and simultaneously detect the contents of poloxamer 188, tyloxapol, and liquid paraffin in drugs. Summary of the invention

[0018] The purpose of the present invention is to overcome the defects in the prior art and provide a method for simultaneously detecting the contents of poloxamer 188, tyloxapol and liquid paraffin in a test sample.

[0019] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0020] The present invention provides a method for simultaneously detecting the contents of poloxamer 188, tyloxapol and liquid paraffin in a test sample, comprising the following steps:

[0021] (1) mixing poloxamer 188, tyloxapol, liquid paraffin and dichloromethane to obtain a mixed solution; mixing the mixed solution with isopropanol to obtain a reference solution;

[0022] (2) performing liquid phase detection on the reference solution to obtain a standard curve of poloxamer 188, tyloxapol, and liquid paraffin in the reference solution;

[0023] (3) mixing the test sample and isopropanol to obtain a test sample solution;

[0024] (4) The test solution is subjected to liquid phase detection, and the test results are compared and calculated with the standard curve to determine the content of poloxamer 188, tyloxapol, and liquid paraffin in the test sample.

[0025] Preferably, in the mixed solution in step (1), the concentration of poloxamer 188 is 0.1-1 mg / mL, the concentration of tyloxapol is 1-2 mg / mL, and the concentration of liquid paraffin is 4-6 mg / mL.

[0026] Preferably, the volume ratio of the mixed solution to isopropanol in step (1) is 0.5-1.5:9.5-8.5.

[0027] Preferably, the column temperature of the liquid phase detection in step (2) is 40-45°C, the nebulizing gas flow rate is 1.5-2 L / min, the drift tube temperature is 50-60°C, and the injection volume is 10-15 μL.

[0028] Preferably, in step (2), the mobile phase A of the liquid phase detection is 1% acetic acid solution, and the mobile phase B is 1% acetic acid isopropanol solution;

[0029] The gradient elution program is as follows:

[0030] Time / minute Mobile phase A / % Mobile phase B% 0 80 20 3.9 80 20 4.0 59 41 5.4 59 41 5.5 0 100 9.5 0 100 9.6 80 20 15 80 20

[0031] The flow rate of the mobile phase is 1-2 mL / min.

[0032] Preferably, in step (3), the volume ratio of the test sample to isopropanol is 0.5-1:9.5-9.

[0033] Preferably, the column temperature of the liquid phase detection in step (4) is 40-45°C, the nebulizing gas flow rate is 1.5-2 L / min, the drift tube temperature is 50-60°C, and the injection volume is 10-15 μL.

[0034] Preferably, in step (4), the mobile phase A of the liquid phase detection is 1% acetic acid solution, and the mobile phase B is 1% acetic acid isopropanol solution;

[0035] The gradient elution program is as follows:

[0036]

[0037]

[0038] The flow rate of the mobile phase is 1-2 mL / min.

[0039] The invention provides a method for simultaneously detecting the contents of poloxamer 188, tyloxapol and liquid paraffin in a test sample, comprising the following steps: mixing poloxamer 188, tyloxapol, liquid paraffin and dichloromethane to obtain a mixed solution; mixing the mixed solution with isopropanol to obtain a reference solution; performing liquid phase detection on the reference solution to obtain a standard curve of poloxamer 188, tyloxapol and liquid paraffin in the reference solution; mixing a test sample with isopropanol to obtain a test sample solution; performing liquid phase detection on the test sample solution, comparing and calculating the detection result with the standard curve, and determining the contents of poloxamer 188, tyloxapol and liquid paraffin in the test sample.

[0040] The present invention adopts a high performance liquid chromatography method and a gradient elution detection method, and innovatively realizes the simultaneous determination of the contents of poloxamer 188, tyloxapol and liquid paraffin in a test sample. The chromatographic method established by the present invention can better separate and determine the contents of the effective ingredients of poloxamer 188, tyloxapol and liquid paraffin in the test sample, and the chromatographic peak has a good peak shape and a high degree of separation. Under the chromatographic conditions, the concentrations of poloxamer 188, tyloxapol and liquid paraffin show a good linear relationship with the peak area.

[0041] The method of the present invention is simple and accurate, has a short analysis time, and has good precision, reproducibility and stability, and can provide a theoretical basis and scientific basis for comprehensively evaluating and controlling the quality of test products. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a specificity test diagram of the detection method of Example 1;

[0043] Figure 2 is the standard curve diagram of Poloxamer 188 in Example 1;

[0044] Figure 3 is the standard curve of tyloxapol in Example 1;

[0045] Figure 4 This is the standard curve diagram of liquid paraffin in Example 1. DETAILED DESCRIPTION

[0046] The present invention provides a method for simultaneously detecting the contents of poloxamer 188, tyloxapol and liquid paraffin in a test sample, comprising the following steps:

[0047] (1) mixing poloxamer 188, tyloxapol, liquid paraffin and dichloromethane to obtain a mixed solution; mixing the mixed solution with isopropanol to obtain a reference solution;

[0048] (2) performing liquid phase detection on the reference solution to obtain a standard curve of poloxamer 188, tyloxapol, and liquid paraffin in the reference solution;

[0049] (3) mixing the test sample and isopropanol to obtain a test sample solution;

[0050] (4) The test solution is subjected to liquid phase detection, and the test results are compared and calculated with the standard curve to determine the content of poloxamer 188, tyloxapol, and liquid paraffin in the test sample.

[0051] In the present invention, the concentration of poloxamer 188 in the mixed solution in step (1) is preferably 0.1-1 mg / mL, more preferably 0.2-0.8 mg / mL, and more preferably 0.4-0.6 mg / mL; the concentration of tyloxapol is preferably 1-2 mg / mL, more preferably 1.2-1.8 mg / mL, and more preferably 1.4-1.6 mg / mL; the concentration of liquid paraffin is preferably 4-6 mg / mL, more preferably 4.5-5.5 mg / mL, and more preferably 4.8-5.2 mg / mL.

[0052] In the present invention, the volume ratio of the mixed solution to isopropanol in step (1) is preferably 0.5-1.5:9.5-8.5, more preferably 0.8-1.2:9.2-8.8, and more preferably 1:9.

[0053] In the present invention, the column temperature of the liquid phase detection in step (2) is preferably 40-45°C, more preferably 41-44°C, and more preferably 42-43°C; the nebulizing gas is nitrogen, and the nebulizing gas flow rate is preferably 1.5-2 L / min, more preferably 1.6-1.9 L / min, and more preferably 1.7-1.8 L / min; the drift tube temperature is preferably 50-60°C, more preferably 52-58°C, and more preferably 54-56°C; the injection volume is preferably 10-15 μL, more preferably 11-14 μL, and more preferably 12-13 μL.

[0054] In the present invention, the mobile phase A of the liquid phase detection in step (2) is a 1% acetic acid solution, and the mobile phase B is a 1% acetic acid isopropanol solution.

[0055] In the present invention, the gradient elution procedure is as follows:

[0056] Time / minute Mobile phase A / % Mobile phase B% 0 80 20 3.9 80 20 4.0 59 41 5.4 59 41 5.5 0 100 9.5 0 100 9.6 80 20 15 80 20

[0057] In the present invention, the flow rate of the mobile phase is preferably 1 to 2 mL / min, more preferably 1.2 to 1.8 mL / min, and even more preferably 1.4 to 1.6 mL / min.

[0058] In the present invention, the volume ratio of the test sample to isopropanol in step (3) is preferably 0.5-1:9.5-9, more preferably 0.6-0.9:9.4-9.1, and more preferably 0.7-0.8:9.3-9.2.

[0059] In the present invention, the column temperature of the liquid phase detection in step (4) is preferably 40-45°C, more preferably 41-44°C, and more preferably 42-43°C; the nebulizing gas is nitrogen, and the nebulizing gas flow rate is preferably 1.5-2 L / min, more preferably 1.6-1.9 L / min, and more preferably 1.7-1.8 L / min; the drift tube temperature is preferably 50-60°C, more preferably 52-58°C, and more preferably 54-56°C; the injection volume is preferably 10-15 μL, more preferably 11-14 μL, and more preferably 12-13 μL.

[0060] In the present invention, the mobile phase A of the liquid phase detection in step (4) is a 1% acetic acid solution, and the mobile phase B is a 1% acetic acid isopropanol solution.

[0061] In the present invention, the gradient elution procedure is as follows:

[0062] Time / minute Mobile phase A / % Mobile phase B% 0 80 20 3.9 80 20 4.0 59 41 5.4 59 41 5.5 0 100 9.5 0 100 9.6 80 20 15 80 20

[0063] In the present invention, the flow rate of the mobile phase is preferably 1 to 2 mL / min, more preferably 1.2 to 1.8 mL / min, and even more preferably 1.4 to 1.6 mL / min.

[0064] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0065] Example 1

[0066] Take 25 mg of poloxamer 188, 75 mg of tyloxapol and 250 mg of liquid paraffin respectively, mix them with dichloromethane to obtain a mixed solution (wherein the concentration of poloxamer 188 is 0.5 mg / mL, the concentration of tyloxapol is 1.5 mg / mL, and the concentration of liquid paraffin is 5 mg / mL), then take 0.5 mL, 0.8 mL, 1.0 mL, 1.2 mL, and 1.5 mL of the mixed solution respectively, place them in 10 mL volumetric flasks, and make up the volume with isopropanol to obtain reference solution (1), (2), (3), (4), and (5) in turn.

[0067] The test sample is a contact lens lubricant. Take 0.5 mL of commercially available contact lens lubricant, accurately weigh it, and dissolve it in 9.5 mL of isopropanol to obtain a test sample solution.

[0068] The reference solution and the test solution were tested according to the following parameters: high performance liquid chromatograph UltiMate-3000 (Thermo Fisher Scientific Inc.); evaporative light scattering detector (Alltech Inc.); ME204E 1 / 100000 electronic analytical balance (METTLERTOLEDO, USA); silica gel was used as filler (FLM Mercury Tween, 4.6mm×150mm, 5μm or chromatographic column of equivalent performance), column temperature was 40°C, nebulizing gas was nitrogen, flow rate was 1.5L per minute, drift tube temperature was 55°C, injection volume was 10μL, mobile phase A was 1% acetic acid solution, mobile phase B was 1% acetic acid isopropanol solution, and gradient elution program was shown in Table 1.

[0069] Table 1 Gradient elution program

[0070] Time / minute Mobile phase A / % Mobile phase B% 0 80 20 3.9 80 20 4.0 59 41 5.4 59 41 5.5 0 100 9.5 0 100 9.6 80 20 15 80 20

[0071] Specificity test chart Figure 1 As shown, Figure 1 1 is the reference solution (3), 2 is the test solution, 3 is isopropanol, and 4 is the blank emulsion solution. Figure 1 It can be seen that there is no interference from the blank emulsion solution and other components, indicating that the method has good specificity.

[0072] After testing the reference solutions (1) to (5) respectively, regression was performed with the logarithm of the reference solution concentration as the abscissa and the logarithm of the peak area as the ordinate. The results are shown in Table 2.

[0073] Table 2 Linearity results

[0074]

[0075]

[0076] The standard curve of Poloxamer 188 is shown in the figure below: Figure 2 The standard curve of tyloxapol is shown in Figure 3 The standard curve of liquid paraffin is shown in Figure 4 As shown in Table 2 and Figures 2 to 4 It can be seen that the results show a good linear relationship within the range.

[0077] 10 μL of the reference solution (3) was taken and tested according to the above parameters for a total of six times, the peak areas were recorded, and the RSDs of the peak areas of poloxamer 188, tyloxapol, and liquid paraffin were calculated. The results are shown in Table 3. As can be seen from Table 3, the RSDs of the peak areas of poloxamer 188, tyloxapol, and liquid paraffin were 0.6%, 0.3%, and 1.2%, respectively, and the injection precision was good.

[0078] Table 3 Precision test results

[0079] serial number Poloxamer 188 Peak Area The area of ​​Tyloxapol Liquid paraffin peak area 1 185819 1809772 3954017 2 185482 1821782 3829799 3 185160 1822682 3941141 4 186748 1810231 3901299 5 187725 1815436 3939429 6 184726 1813316 3948495 RSD(%) 0.6 0.3 1.2

[0080] A total of 6 contact lens lubricant test solutions were prepared using the same method, and the test was carried out according to the above conditions. The peak areas were recorded and the contents of poloxamer 188, tyloxapol and liquid paraffin in the contact lens lubricant were calculated. The results are shown in Table 4, indicating that the method has good repeatability.

[0081] Table 4 Repeatability test results

[0082]

[0083]

[0084] It can be seen from the above embodiments that the present invention adopts high performance liquid chromatography and gradient elution detection method to innovatively achieve the simultaneous determination of the contents of poloxamer 188, tyloxapol and liquid paraffin in the test sample. The chromatographic method established by the present invention can better separate and determine the contents of the effective ingredients of poloxamer 188, tyloxapol and liquid paraffin in the test sample. The chromatographic peak shape is good and the separation degree is high. Under this chromatographic condition, the concentration of poloxamer 188, tyloxapol and liquid paraffin shows a good linear relationship with the peak area.

[0085] The method of the present invention is simple and accurate, has a short analysis time, and has good precision, reproducibility and stability, and can provide a theoretical basis and scientific basis for comprehensively evaluating and controlling the quality of test products.

[0086] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for simultaneously detecting the contents of poloxamer 188, tyloxapol and liquid paraffin in a test sample, characterized in that: It includes the following steps: (1) mixing poloxamer 188, tyloxapol, liquid paraffin and dichloromethane to obtain a mixed solution; mixing the mixed solution with isopropanol to obtain a reference solution; (2) performing liquid phase detection on the reference solution to obtain a standard curve of poloxamer 188, tyloxapol, and liquid paraffin in the reference solution; (3) mixing the test sample and isopropanol to obtain a test sample solution; (4) The test solution is subjected to liquid phase detection, and the test results are compared and calculated with the standard curve to determine the content of poloxamer 188, tyloxapol, and liquid paraffin in the test sample.

2. The method for simultaneously detecting the contents of poloxamer 188, tyloxapol and liquid paraffin in a test sample according to claim 1, characterized in that: In the mixed solution in step (1), the concentration of poloxamer 188 is 0.1-1 mg / mL, the concentration of tyloxapol is 1-2 mg / mL, and the concentration of liquid paraffin is 4-6 mg / mL.

3. The method for simultaneously detecting the contents of poloxamer 188, tyloxapol and liquid paraffin in a test sample according to claim 1 or 2, characterized in that: The volume ratio of the mixed solution to isopropanol in step (1) is 0.5-1.5:9.5-8.

5.

4. The method for simultaneously detecting the contents of poloxamer 188, tyloxapol and liquid paraffin in a test sample as claimed in claim 3, characterized in that: In step (2), the column temperature of the liquid phase detection is 40-45° C., the nebulizing gas flow rate is 1.5-2 L / min, the drift tube temperature is 50-60° C., and the injection volume is 10-15 μL.

5. The method for simultaneously detecting the contents of poloxamer 188, tyloxapol and liquid paraffin in a test sample according to claim 1 or 4, characterized in that: In step (2), the mobile phase A of the liquid phase detection is 1% acetic acid solution, and the mobile phase B is 1% acetic acid isopropanol solution; The gradient elution program is as follows: The flow rate of the mobile phase is 1-2 mL / min.

6. The method for simultaneously detecting the contents of poloxamer 188, tyloxapol and liquid paraffin in a test sample as claimed in claim 5, characterized in that: In step (3), the volume ratio of the test sample to isopropanol is 0.5-1:9.5-9.

7. The method for simultaneously detecting the contents of poloxamer 188, tyloxapol and liquid paraffin in a test sample as claimed in claim 6, characterized in that: In step (4), the column temperature of the liquid phase detection is 40-45° C., the nebulizing gas flow rate is 1.5-2 L / min, the drift tube temperature is 50-60° C., and the injection volume is 10-15 μL.

8. The method for simultaneously detecting the contents of poloxamer 188, tyloxapol and liquid paraffin in a test sample according to claim 7, characterized in that: In step (4), the mobile phase A of the liquid phase detection is 1% acetic acid solution, and the mobile phase B is 1% acetic acid isopropanol solution; The gradient elution program is as follows: The flow rate of the mobile phase is 1-2 mL / min.

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