Method for detecting content of poloxamer 188

Abstract

The invention provides a method for detecting the content of poloxamer 188. Specifically, a high performance liquid chromatography method is adopted, octadecylsilane chemically bonded silica is used as a filler, a formic acid aqueous solution is used as a mobile phase A, a mixed solution of formic acid and isopropanol or a mixed solution of acetic acid and isopropanol is used as a mobile phase B, and isocratic or gradient elution is carried out; and an evaporative light scattering detector is adopted for detection. The detection method is high in sensitivity, and the content of the poloxamer 188 can be rapidly and accurately detected with high sensitivity, so that the quality controllability of the product is ensured.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical analysis, and in particular relates to a method for detecting the content of poloxamer 188. Background Art

[0002] Poloxamer is a new type of polymer non-ionic surfactant, commonly used in pharmaceutical preparation excipients. Among them, poloxamer 188 (P188 for short) is an ideal solubilizer and emulsifier. Due to its stable properties and low toxicity, it is added to pharmaceutical preparations and has been used in multiple marketed intravenous injections. This excipient has been included in the 2020 edition of the Chinese Pharmacopoeia, but the pharmacopoeia only uses titration to determine its average molecular weight, and there is no analytical method for detecting its content. Therefore, P188 is added as a solubilizer or emulsifier during the preparation of antibody drugs, and the content of poloxamer 188 in the antibody drug needs to be determined.

[0003] CN111289652A discloses an HPLC-ELSD detection method for poloxamer 188 in recombinant human interleukin-12 injection, using a Cypermethrin Poly RP-100 column as the chromatographic column, with a quantitative limit of 0.4 μg and low sensitivity. Existing literature reports the use of HPLC-ELSD to detect the content of poloxamer 188, using a spherical hydrophilic silica gel TSKgel G3000SW column as the chromatographic column, with a quantitative limit of 12.5 μg / mL and low sensitivity (Teng Baoxia et al. ELSD-HPLC determination of poloxamer 188 in recombinant human interferon α2b injection, Chinese Journal of Pharmaceutical Industry. 2022, 53(05)). The existing HPLC-ELSD detection method has low sensitivity and low detection accuracy for preparations with low P188 content. Therefore, it is necessary to establish an analytical method for detecting the content of poloxamer 188 with high accuracy and sensitivity. Summary of the Invention

[0004] The present invention aims to provide a method for detecting the content of poloxamer 188. The method is rapid, highly accurate and highly sensitive.

[0005] On one hand, the present invention provides a method for detecting the content of poloxamer 188. The method is a high performance liquid chromatography method, using octadecylsilane bonded silica gel as a filler, an aqueous formic acid solution as a mobile phase A, a mixed solution of formic acid and isopropanol or a mixed solution of acetic acid and isopropanol as a mobile phase B, and isocratic or gradient elution; and detecting using an evaporative light scattering detector.

[0006] In some embodiments, the concentration of the formic acid aqueous solution is 0.5% to 5%, preferably 1% to 3%, and more preferably 2%.

[0007] In some embodiments, the concentration of formic acid or acetic acid in the mixed solution of formic acid and isopropyl alcohol or the mixed solution of acetic acid and isopropyl alcohol is 0.5% to 5%, preferably 1% to 3%, and more preferably 2%.

[0008] In some embodiments, aqueous formic acid solution is used as mobile phase A, and a mixed solution of formic acid and isopropanol is used as mobile phase B.

[0009] In some embodiments, the volume ratio of mobile phase A to mobile phase B is 0-80:20-100, and isocratic or gradient elution is performed, preferably gradient elution.

[0010] In some embodiments, the mobile phase A and mobile phase B are eluted according to the following gradient:

[0011]

[0012] In some embodiments, the flow rate of the mobile phase is 0.2 to 2.0 mL / min; preferably 0.4 to 1.2 mL / min; more preferably 0.4 to 0.6 mL / min; further preferably 0.5 mL / min.

[0013] In some embodiments, the chromatographic column model is Agilent Poroshell EC-C18 or an equivalent chromatographic column, with a specification of 4.6 mm×100 mm and a diameter of 2.7 μm.

[0014] In some embodiments, the drift tube temperature of the evaporative light scattering detector is 70° C., the nebulizer temperature is 40° C., and the gas flow rate is 1.2 SLM.

[0015] In some embodiments, the column temperature of the chromatographic column is 20-50°C, preferably 25-45°C, more preferably 35-45°C, and even more preferably 40°C.

[0016] In some embodiments, the injection volume is 10 to 40 μL, preferably 30 μL.

[0017] Another aspect of the present invention provides a method for detecting the content of poloxamer 188. The analysis method is performed on a high performance liquid chromatograph; the detector is an evaporative light scattering detector; a test solution and a reference solution of poloxamer 188 are injected separately; and the content of poloxamer 188 in the test sample is calculated using an external standard method.

[0018] In some embodiments, the detection method is used to detect the content of poloxamer 188 in antibodies.

[0019] In some embodiments, the test solution is precipitated by adding acetonitrile to the antibody test sample.

[0020] In some embodiments, a method for detecting the content of poloxamer 188 comprises the following steps:

[0021] (1) Preparation of reference solution: Dissolve poloxamer 188 in water and dilute to different concentrations. Add acetonitrile and mix well to obtain a series of reference solutions of different concentrations.

[0022] (2) Preparation of test solution: Take the antibody test sample, mix it with water, and centrifuge to obtain the antibody solution; add acetonitrile, mix it to precipitate the protein in the antibody, and centrifuge to obtain the supernatant, which is the test solution;

[0023] (3) Detection: The test solution and the reference solution were detected by high performance liquid chromatography, and the chromatograms were recorded. According to the external standard method, the linear regression equation was calculated using the logarithm of the concentration of poloxamer 188 in the reference solution and the logarithm of the corresponding peak area, and then the content of poloxamer 188 in the test solution was calculated.

[0024] In some embodiments, the concentrations of the series of control solutions are 0.003125 mg / mL, 0.00625 mg / mL, 0.0125 mg / mL, 0.025 mg / mL, 0.05 mg / mL, and 0.1 mg / mL.

[0025] In some embodiments, the external standard method is to calculate a linear regression equation using the logarithm of the poloxamer 188 concentration in the reference solution and the logarithm of the corresponding peak area, and substitute the logarithm of the poloxamer 188 peak area of ​​the test solution into the linear equation to calculate the logarithm of the poloxamer 188 concentration of the diluted test solution, and then calculate the poloxamer 188 concentration of the diluted test solution according to the antilogarithm, and then multiply it by the dilution factor to obtain the content of poloxamer 188 in the test sample.

[0026] In some embodiments, the specific calculation formula of step (3) is: the concentration of the test substance poloxamer 188 C = antilgC_ 供试品 × dilution factor, where C_ 供试品 is the poloxamer 188 concentration of the diluted test solution.

[0027] In the present invention, the chromatographic column Agilent Poroshell EC-C18 can be replaced by other chromatographic columns with equivalent performance.

[0028] Technical Effects

[0029] The detection method of the present invention has high sensitivity and can quickly, accurately and highly sensitively detect the content of poloxamer 188, thereby ensuring the controllability of product quality.

[0030] In the present invention, the "concentration of the formic acid aqueous solution" refers to the volume percentage of formic acid in the formic acid aqueous solution.

[0031] In the present invention, the “concentration of formic acid or acetic acid in a mixed solution of formic acid and isopropyl alcohol or a mixed solution of acetic acid and isopropyl alcohol” refers to the volume percentage of formic acid or acetic acid in the mixed solution of formic acid and isopropyl alcohol or the mixed solution of acetic acid and isopropyl alcohol. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The chromatograms are for the test solution, reference solution, and blank sample in Example 2. DETAILED DESCRIPTION

[0033] The present invention is described in more detail below through examples, but these specific descriptions are only used to illustrate the technical solutions of the present invention and do not constitute any limitation to the present invention.

[0034] The reagents and raw materials used in the present invention are all commercially available. Experimental methods without specific conditions are generally carried out under conventional conditions.

[0035] Example 1 Detection of Poloxamer 188 Content in Antibody Samples

[0036] Chromatographic conditions: Agilent Poroshell EC-C18 column, 4.6 mm × 100 mm, 2.7 μm; column temperature: 40°C; flow rate: 0.5 mL / min; injection volume: 30 μL, nebulizer temperature: 40°C; drift tube temperature: 70°C; gas flow rate: 1.20 SLM.

[0037] Mobile phase A: 2% formic acid in water, mobile phase B: 2% formic acid in isopropanol, gradient elution according to the table below.

[0038]

[0039]

[0040] Prepare a series of Poloxamer 188 reference solutions at different concentrations: Weigh an appropriate amount of poloxamer 188 and dilute it with water to create a 0.8 mg / mL stock solution. Pipette an appropriate amount to prepare a series of solutions with concentrations of 0.0125 mg / mL, 0.025 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, and 0.4 mg / mL. Pipette 200 μL of each solution into 600 μL of acetonitrile and vortex to mix.

[0041] Test solution: Pipette 180 μL of water, add 20 μL of antibody sample, vortex mix, centrifuge momentarily, then add 600 μL of acetonitrile, vortex mix, refrigerate at 2-8°C for 15 min, centrifuge (12000 rpm, 10 min), and collect the supernatant.

[0042] 30 μL of each solution was taken and measured according to the above chromatographic conditions, and the chromatogram was recorded. The linear regression equation was calculated by the logarithmic value of the concentration of poloxamer 188 in the reference solution and the logarithmic value of the corresponding peak area. The logarithmic value of the peak area of ​​poloxamer 188 in the test solution (logA 供试品 ) into the linear equation to calculate the common logarithm of the concentration of poloxamer 188 in the diluted test solution (logC 供试品 ), calculate the concentration of the test substance poloxamer 188 according to the calculation formula C (C = antilgC_ 供试品 × dilution factor, where C is the concentration of the test substance poloxamer 188; C_ 供试品 is the concentration of poloxamer 188 in the diluted test solution; dilution factor: 40). The test results are shown in Table 1.

[0043] Table 1 Sample test results

[0044]

[0045] Example 2 Specificity Experiment

[0046] Reference solution: Pipette 750 μL of water, add 50 μL of 0.8 mg / mL poloxamer 188 stock solution, and vortex to mix to obtain a poloxamer 188 solution with a concentration of 0.05 mg / mL. Pipette 200 μL of this solution, add 600 μL of acetonitrile, and vortex to mix.

[0047] The test solution was prepared according to the method described in Example 1.

[0048] Take the test solution, reference solution and blank solution, and the specific results are shown in Table 2 and Figure 1 .

[0049] Table 2

[0050] Sample name Retention time (min) blank solution / Poloxamer 188 reference solution 6.061 Test solution 6.061

[0051] The results showed that the blank solution was in the peak area of ​​Poloxamer 188, with no interference and good specificity.

[0052] Example 3 Linear Relationship Investigation

[0053] Prepare linear solutions according to the preparation method of a series of reference solutions of different concentrations of Poloxamer 188 in Example 1. Accurately measure the above solutions and measure them according to the chromatographic conditions in Example 1. Take the common logarithm (log A) of the average value of the peak area of ​​Poloxamer 188 obtained by three parallel injections of each linear solution. P188 ) as the ordinate (Y), the common logarithm of the concentration of poloxamer 188 (mg / mL) (logC P188) as the horizontal axis (X), a standard curve was drawn and a linear equation was obtained. The regression equation was Y = 1.5520X + 5.3702 (r = 0.9998). The results showed that when the mass concentration of poloxamer 188 was in the range of 0.0031 to 0.1 mg / mL, the common logarithm of the poloxamer 188 concentration (mg / mL) (logC P188 ) and the common logarithm of the peak area of ​​poloxamer 188 (logA P188 ) showed a good linear relationship.

[0054] Example 4 Accuracy

[0055] The method for preparing the spiked test solution is as follows: take an appropriate amount of poloxamer 188 and add water to prepare stock solutions 1, 2, and 3 with poloxamer 188 concentrations of 0.25 mg / mL, 0.5 mg / mL, and 0.75 mg / mL, respectively.

[0056] 50% spiked test solution: Pipette 160 μL of water, add 20 μL of sample and 20 μL of stock solution 1, vortex mix, centrifuge briefly, add 600 μL of acetonitrile, vortex mix, refrigerate at 2-8°C for 15 min, centrifuge (12000 rpm, 10 min), and collect the supernatant.

[0057] 100% spiked test solution: Pipette 160 μL of water, add 20 μL of sample and 20 μL of stock solution 2, vortex mix, centrifuge briefly, add 600 μL of acetonitrile, vortex mix, refrigerate at 2-8°C for 15 min, centrifuge (12000 rpm, 10 min), and collect the supernatant.

[0058] 150% spiked test solution: Pipette 160 μL of water, add 20 μL of sample and 20 μL of stock solution 3, vortex mix, centrifuge briefly, add 600 μL of acetonitrile, vortex mix, refrigerate at 2-8°C for 15 min, centrifuge (12000 rpm, 10 min), and collect the supernatant.

[0059] Three spiked test sample solutions were prepared in parallel. The test sample solution and each spiked test sample solution were assayed using the chromatographic conditions described in Example 1. The results are shown in Table 3. The results show that the recovery of poloxamer 188 ranged from 80% to 120%, with an RSD of ≤10%, demonstrating good accuracy.

[0060] Table 3 Accuracy test results (n=9)

[0061]

[0062]

[0063] Example 5 Limit of Quantitation

[0064] An appropriate amount of poloxamer 188 was dissolved and diluted in water to prepare a limit of quantitation solution with a signal-to-noise ratio of ≥ 10. The experimental results showed that the limit of quantitation of poloxamer 188 was 2.6 μg / mL, meeting the detection sensitivity requirements.

[0065] Example 6 Precision

[0066] Example 6-1 Repeatability

[0067] Six test solutions were prepared in parallel according to the preparation method of the test solution in Example 1, and the content of poloxamer 188 was measured. The results showed that the RSD of the poloxamer 188 content of the six test results was ≤10%, with good repeatability.

[0068] Example 6-2 Intermediate Precision

[0069] Six parallel test solutions were prepared using the same method as in Example 1. Different researchers performed the assays on different days using different instruments. The results are shown in Table 4. The results show that the RSD for poloxamer 188 content was ≤10% across 12 assays performed by different researchers using different instruments, demonstrating good intermediate precision.

[0070] Table 4 Intermediate precision results

[0071]

[0072] Example 7 Solution Stability

[0073] The test solution was prepared according to the method for preparing the test solution in Example 1. The test solution was placed at 6°C and tested at 0, 3, 14, 20, 24, 30, and 36 hours. The results showed that the RSD of the poloxamer 188 concentration measured at different time points after the test solution was placed at 6°C for at least 36 hours was 2.89%, indicating good sample stability.

Claims

1. A method for detecting the content of poloxamer 188, comprising high performance liquid chromatography (HPLC), using octadecylsilane bonded silica gel as a filler, aqueous formic acid as mobile phase A, a mixed solution of formic acid and isopropanol or a mixed solution of acetic acid and isopropanol as mobile phase B, and isocratic or gradient elution; detection is performed using an evaporative light scattering detector.

2. The detection method according to claim 1, wherein The concentration of the formic acid aqueous solution is 0.5% to 5%, preferably 1% to 3%, more preferably 2%; And / or, the concentration of formic acid or acetic acid in the mixed solution of formic acid and isopropyl alcohol or the mixed solution of acetic acid and isopropyl alcohol is 0.5% to 5%, preferably 1% to 3%, more preferably 2%.

3. The detection method according to claim 1, wherein The volume ratio of the mobile phase A to the mobile phase B is 0-80:20-100, and the elution is performed in an isocratic or gradient manner; preferably, the elution is performed in a gradient manner; more preferably, the mobile phase A and the mobile phase B are eluted in a gradient manner according to the following table:

4. The detection method according to claim 1, wherein The flow rate of the mobile phase is 0.2 to 2.0 mL / min; preferably 0.4 to 1.2 mL / min; more preferably 0.4 to 0.6 mL / min; further preferably 0.5 mL / min; and / or, the chromatographic column model is Agilent Poroshell EC-C18 or its equivalent, with specifications of 4.6 mm × 100 mm, 2.7 μm; and / or, the column temperature of the chromatographic column is 20 to 50° C., preferably 25 to 45° C., more preferably 35 to 45° C., and further preferably 40° C.; and / or, the injection volume is 10 to 40 μL, preferably 30 μL; And / or, the drift tube temperature of the evaporative light scattering detector is 70° C., the nebulizer temperature is 40° C., and the gas flow rate is 1.2 SLM.

5. The detection method according to claim 1, wherein The method further includes injecting a test solution and a reference solution of Poloxamer 188 respectively; and calculating the content of Poloxamer 188 in the test solution by using an external standard method.

6. The detection method according to claim 1, characterized in that The detection method is used for detecting the content of poloxamer 188 in antibodies.

7. The detection method according to claim 6, characterized in that The test solution is precipitated by adding acetonitrile to the antibody test sample.

8. The detection method according to claim 6, characterized in that The method further comprises the steps of: (1) Preparation of reference solution: Dissolve poloxamer 188 in water and dilute to different concentrations. Add acetonitrile and mix well to obtain a series of reference solutions of different concentrations. (2) Preparation of test solution: Take the antibody test sample, mix it with water, and centrifuge to obtain the antibody solution; add acetonitrile, mix it to precipitate the protein in the antibody, and centrifuge to obtain the supernatant, which is the test solution; (3) Detection: The test solution and the reference solution were detected by high performance liquid chromatography, and the chromatograms were recorded. According to the external standard method, the linear regression equation was calculated using the logarithm of the concentration of poloxamer 188 in the reference solution and the logarithm of the corresponding peak area, and then the content of poloxamer 188 in the test solution was calculated.

9. The detection method according to claim 8, characterized in that The concentrations of the series of reference solutions are 0.003125 mg / mL, 0.00625 mg / mL, 0.0125 mg / mL, 0.025 mg / mL, 0.05 mg / mL and 0.1 mg / mL.

10. The detection method according to claim 8, characterized in that The specific calculation formula of step (3) is: the concentration of the test sample poloxamer 188 C = antilgC_ 供试品 × dilution factor, where C_ 供试品 is the poloxamer 188 concentration of the diluted test solution.

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