A separation method and detection method for components in a bilastine sample

Abstract

The present invention provides a separation method and a detection method for components in a bilastine sample, and relates to the technical field of drug quality analysis. The present invention adopts HPLC chromatography, uses phosphate buffer solution and methanol-acetonitrile as a mobile phase system, adopts a specific mobile phase system and a gradient elution program, and adds ion pair reagents-triethylamine and / or n-octylamine in the phosphate buffer, which can highly separate bilastine, process impurities (impurity A, impurity B, impurity C and impurity D), degradation impurities (impurity E, impurity F), and antibacterial agents (methyl parahydroxybenzoate and propyl parahydroxybenzoate) at one time, with short time consumption, high separation efficiency, simple operation, and low separation cost. The present invention uses a detector or a mass spectrometer to detect the separation results, and the detection results of the components in the bilastine sample can be obtained. The detection method provided by the present invention is efficient and convenient, has good reproducibility, strong specificity, high accuracy, and high sensitivity.

Description

Technical Field

[0001] The invention relates to the technical field of drug quality analysis, and in particular to a method for separating and detecting components in a bilastine sample. Background Art

[0002] Bilastine, with the structural formula shown in 1, has a chemical name of 2-[4-(2-(4-(1-(2-ethoxyethyl)benzimidazol-2-yl)piperidin-1-yl)ethyl)phenyl]-2-methylpropionic acid, and a molecular formula of C 28 H 37 N3O3 is a second-generation histamine H1 receptor antagonist developed by the Spanish FAES Pharmaceutical Company. It does not have the side effects of sedation and cardiotoxicity found in commonly used antihistamines. It is highly safe and can be used to treat allergic rhinitis and chronic idiopathic urticaria.

[0003] As 1.

[0004] Bilastine oral solution may contain process impurities and oxidative degradation impurities, among which process impurities include impurities A, B, C and D, and oxidative degradation impurities include impurities E and F, which are cis-trans isomers. The above impurities are all introduced into the finished product by the API or produced by the degradation of the API, and no new impurities or new degradation impurities are produced in the preparation process.

[0005]

[0006] .

[0007] Chinese patent CN116165307A discloses a method for simultaneously separating bilastine and its impurities: using a chromatographic column filled with octadecylsilane bonded silica gel, using mobile phase A and mobile phase B as mobile phases for gradient elution, mobile phase A is phosphate buffer, mobile phase B is methanol and / or acetonitrile, this method can simultaneously separate and detect up to 12 process impurities within 50 minutes, and has good separation and obvious peak shape. However, when using the above method for separation, its impurity J (oxidative degradation impurity) does not distinguish between cis and trans isomers, and the above-mentioned oxidative degradation impurities E and F cannot be effectively separated. Summary of the invention

[0008] In view of this, the object of the present invention is to provide a separation method and a detection method for components in a bilastine sample. The method provided by the present invention can achieve effective separation of oxidative degradation (impurity E and impurity F) in a bilastine sample.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0010] The present invention provides a method for separating components in a bilastine sample, comprising the following steps:

[0011] The bilastine sample to be tested is subjected to high performance liquid chromatography separation to obtain separation results of components in the bilastine sample;

[0012] The conditions for the HPLC separation include: the mobile phase system includes mobile phase A and mobile phase B, the mobile phase A is a phosphate buffer, the phosphate buffer contains an ion pair reagent, and the ion pair reagent includes triethylamine and / or n-octylamine; the mobile phase B is a mixed solvent of methanol and acetonitrile; the elution method is gradient elution, and the procedure of the gradient elution includes:

[0013] 0-5min, the volume fraction of the mobile phase A is 50-70%;

[0014] 5-35 min, the volume fraction of the mobile phase A is reduced from 50-70% to 15-25%;

[0015] 35-40 min, the volume fraction of the mobile phase A is 15-25%;

[0016] 40-41 min, the volume fraction of the mobile phase A increased from 15-25% to 50-70%;

[0017] 41-50 min, the volume fraction of the mobile phase A is 50-70%;

[0018] The ingredients include one or more of bilastine, related impurities and excipients; the related impurities include one or more of impurity A, impurity B, impurity C, impurity D, impurity E and impurity F:

[0019] .

[0020] Preferably, the volume concentration of the ion pair reagent in the phosphate buffer is 0.07-0.27%;

[0021] The pH value of the phosphate buffer is 6.5-7.5.

[0022] Preferably, the volume fraction of methanol in the mobile phase B is 30-75%.

[0023] Preferably, the chromatographic column used for the HPLC separation includes a C18 chromatographic column, the column temperature is 15-40°C, the flow rate of the mobile phase is 0.5-1.5 mL / min, the separation wavelength is 210-290 nm, and the injection volume is 5-100 μL.

[0024] Preferably, the auxiliary materials include one or more of an antibacterial agent, cyclodextrin, hydroxyethyl cellulose, sucralose, flavor, hydrochloric acid and sodium hydroxide; the antibacterial agent includes methyl parahydroxybenzoate and / or propyl parahydroxybenzoate.

[0025] Preferably, the bilastine sample to be tested includes a liquid sample containing bilastine and / or a solid sample containing bilastine.

[0026] Preferably, the bilastine-containing liquid sample is diluted with a first diluent before use; the first diluent is a mixed solution of the mobile phase A and the mobile phase B;

[0027] The bilastine-containing solid sample is dissolved in methanol before use, and a second diluent is added for dilution; the second diluent includes a mixed solution of the mobile phase A and the mobile phase B.

[0028] The present invention also provides a method for detecting components in a bilastine sample, comprising the following steps: detecting the separation results of the components in the bilastine sample obtained by the separation method described in the above technical solution to obtain the detection results of the components in the bilastine sample; the detection includes detecting by using a detector or a mass spectrometer; and the detection results include qualitative detection results and / or quantitative detection results.

[0029] Preferably, the detection wavelength of the detector is 210~290nm.

[0030] Preferably, the method for obtaining the quantitative detection result includes area normalization method, internal standard method or external standard method.

[0031] The present invention uses phosphate buffer solution and methanol-acetonitrile as the mobile phase system, adopts a specific mobile phase system and a gradient elution program, and adds an ion pair reagent-triethylamine and / or n-octylamine in the phosphate buffer, which can completely separate the cis-trans isomers (impurity E, impurity F) of the degradation impurities, and can also highly separate bilastine, process impurities (impurity A, impurity B, impurity C and impurity D), and degradation impurities (impurity E, impurity F) at one time. Methylparaben and propylparaben have ultraviolet absorption and will produce peaks in liquid chromatography separation, and there is a risk of interfering with impurity detection. The separation method provided by the present invention can ensure that the antibacterial agent (methylparaben and propylparaben) does not interfere with the detection of other components, and the separation degree of each substance is high. The separation method provided by the present invention can completely separate bilastine, four process impurities, two degradation impurities, methylparaben and propylparaben at one time, with short time consumption, high separation efficiency, simple operation and low separation cost.

[0032] The present invention uses a detector or a mass spectrometer to detect the obtained separation results, and can obtain qualitative and / or quantitative detection results of the components in the bilastine sample. The detection method provided by the present invention is not affected by auxiliary materials in the detection of bilastine and related impurities, is efficient and convenient, has good reproducibility, strong specificity, high accuracy, high sensitivity, and can achieve quality control of bilastine samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is the blank solvent HPLC spectrum;

[0034] Figure 2 It is the HPLC spectrum of blank excipient;

[0035] Figure 3 It is the HPLC spectrum of system suitability solution;

[0036] Figure 4 The HPLC spectrum of Bilastine oral solution was not destroyed;

[0037] Figure 5 This is the HPLC spectrum of Bilas oral solution after high temperature destruction;

[0038] Figure 6 This is the HPLC spectrum of Bilas oral solution after 24 hours of light destruction;

[0039] Figure 7 This is the HPLC spectrum of Bilas oral solution after 10 days of light destruction;

[0040] Figure 8 This is the HPLC spectrum of Bilas oral solution after oxidation damage;

[0041] Fig. 9 This is the HPLC spectrum of Bilas oral solution after acid destruction;

[0042] Fig.10 This is the HPLC spectrum of Bilas oral solution after alkali destruction;

[0043] Fig.11 HPLC spectra of the detection limits of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F and bilastine;

[0044] Fig.12 HPLC spectra of the limits of quantitation of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F and bilastine;

[0045] Fig.13 is the linear curve of methyl paraben;

[0046] Fig.14 is the linear curve of impurity A;

[0047] Fig.15 is the linear curve of impurity B;

[0048] Fig.16 is the linear curve of impurity E;

[0049] Fig.17 is the linear curve of impurity F;

[0050] Fig.18 is the linear curve of propylparaben;

[0051] Fig.19 is the linear curve of impurity C;

[0052] Fig. 20 is the linear curve of Bilastine;

[0053] Fig.21 is the linear curve of impurity D;

[0054] Fig. 22 It is a spectrum of the solution suitable for separating the system using the HPLC chromatographic conditions in CN116165307A. DETAILED DESCRIPTION

[0055] The present invention provides a method for separating components in a bilastine sample, comprising the following steps: subjecting the bilastine sample to be tested to high performance liquid chromatography detection to obtain a separation result of the components in the bilastine sample;

[0056] The conditions for the HPLC separation include: the mobile phase system includes mobile phase A and mobile phase B, the mobile phase A is a phosphate buffer, the phosphate buffer contains an ion pair reagent, and the ion pair reagent includes triethylamine and / or n-octylamine; the mobile phase B is a mixed solvent of methanol and acetonitrile; the elution method is gradient elution, and the procedure of the gradient elution includes:

[0057] 0-5min, the volume fraction of the mobile phase A is 50-70%;

[0058] 5-35 min, the volume fraction of the mobile phase A is reduced from 50-70% to 15-25%;

[0059] 35-40 min, the volume fraction of the mobile phase A is 15-25%;

[0060] 40-41 min, the volume fraction of the mobile phase A increased from 15-25% to 50-70%;

[0061] 41-50 min, the volume fraction of the mobile phase A is 50-70%;

[0062] The ingredients include one or more of bilastine, related impurities and excipients; the related impurities include one or more of impurity A, impurity B, impurity C, impurity D, impurity E and impurity F:

[0063] .

[0064] Unless otherwise specified, the materials and equipment used in the present invention are all commercially available products in the art.

[0065] In the present invention, the bilastine sample to be tested preferably includes a bilastine-containing liquid sample and / or a bilastine-containing solid sample; the present invention has no special limitation on the bilastine-containing liquid sample and the bilastine-containing solid sample, as long as they contain bilastine. Specifically, the bilastine liquid preferably includes one or more of bilastine oral liquid, bilastine injection and bilastine suspension; the bilastine-containing solid sample preferably includes one or more of bilastine bulk drug, bilastine tablets, bilastine granules and bilastine orodisintegrating tablets.

[0066] In the present invention, the bilastine-containing liquid sample is preferably diluted with a first diluent before use to obtain a liquid sample test solution. In the present invention, the first diluent is preferably a mixed solution of the mobile phase A and the mobile phase B, and the volume fraction of the mobile phase A in the first diluent is preferably 50-70%, and in specific embodiments can be 50%, 55%, 60%, 65% or 70%. In the present invention, the concentration of bilastine in the liquid sample test solution is preferably 0.01-2 mg / mL, and in specific embodiments can be 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.7 mg / mL, 1.8 mg / mL, 1.9 mg / mL or 2 mg / mL.

[0067] In the present invention, the solid sample containing bilastine is preferably dissolved in methanol before use, and a second diluent is added for dilution to obtain a solid sample test solution. In the present invention, the mass volume ratio of the solid sample containing bilastine to methanol is preferably 1 mg: 2~20 mL, more preferably 1 mg: 5~10 mL. In the present invention, the second diluent is preferably a mixed solution of the mobile phase A and the mobile phase B, and the volume fraction of the mobile phase A in the second diluent is preferably 50~70%, and in specific embodiments it can be 50%, 55%, 60%, 65% or 70%. In the present invention, the concentration of the bilastine solid sample in the solid sample test solution is preferably 0.01-2 mg / mL, and in specific embodiments can be 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.7 mg / mL, 1.8 mg / mL, 1.9 mg / mL or 2 mg / mL.

[0068] In the present invention, the mobile phase A is a phosphate buffer, and the phosphate buffer contains an ion pair reagent. The preparation method of the phosphate buffer preferably includes the following steps: mixing an aqueous phosphoric acid solution and an ion pair reagent, adding water to make up the volume, and obtaining a phosphate buffer. In the present invention, the ion pair reagent includes triethylamine and / or n-octylamine, preferably triethylamine or n-octylamine; the volume concentration of the ion pair reagent in the phosphate buffer is preferably 0.07-0.27%, and in a specific embodiment, the volume concentration of the ion pair reagent in the phosphate buffer can be 0.07%, 0.1%, 0.15%, 0.2%, 0.25% or 0.27%. In the present invention, the volume concentration of phosphoric acid in the aqueous phosphoric acid solution is preferably 83%-98%, more preferably 85-90%; the volume fraction of the aqueous phosphoric acid solution in the phosphate buffer is preferably 0.02-0.1%, more preferably 0.05%. In the present invention, the pH value of the phosphate buffer is preferably 6.5-7.5. In a specific embodiment, the pH value of the phosphate buffer may be 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4 or 7.5; the pH value is preferably adjusted by phosphoric acid or n-octylamine.

[0069] In the present invention, the mobile phase B is a mixed solvent of methanol and acetonitrile, and the volume fraction of methanol in the mobile phase B is 30-75%. In a specific embodiment, the volume fraction of methanol in the mobile phase B can be 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%.

[0070] In the present invention, the elution mode of the high performance liquid chromatography separation is gradient elution, and the procedure of the gradient elution includes:

[0071] 0-5min, the volume fraction of the mobile phase A is 50-70%. In a specific embodiment, the volume fraction of the mobile phase A can be 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or 70%;

[0072] 5-35min, the volume fraction of the mobile phase A is reduced from 50-70% to 15-25%. In a specific embodiment, the volume fraction of the mobile phase A can be reduced from 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% and 70% to any one of 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24% and 25%;

[0073] 35-40 min, the volume fraction of the mobile phase A is 15-25%. In a specific embodiment, the volume fraction of the mobile phase A can be 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24% or 25%;

[0074] 40-41 min, the volume fraction of the mobile phase A is increased from 15-25% to 50-70%. In a specific embodiment, the volume fraction of the mobile phase A can be increased from 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24% and 25% to any one of 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% and 70%;

[0075] 41~50min, the volume fraction of the mobile phase A is 50~70%. In a specific embodiment, the volume fraction of the mobile phase A can be 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or 70%.

[0076] In the present invention, the chromatographic column used for the high performance liquid chromatography separation preferably includes a C18 chromatographic column; the column temperature is preferably 15-40°C. In a specific embodiment, the column temperature can be 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C.

[0077] In the present invention, the flow rate of the mobile phase for HPLC separation is preferably 0.5-1.5 mL / min. In a specific embodiment, the flow rate of the mobile phase can be 0.5 mL / min, 0.6 mL / min, 0.7 mL / min, 0.8 mL / min, 0.9 mL / min, 1 mL / min, 1.1 mL / min, 1.2 mL / min, 1.3 mL / min, 1.4 mL / min or 1.5 mL / min.

[0078] In the present invention, the injection volume of the HPLC detection is 5~100μL. In a specific embodiment, the injection volume can be 5μL, 10μL, 15μL, 20μL, 25μL, 30μL, 35μL, 40μL, 45μL, 50μL, 55μL, 60μL, 65μL, 70μL, 75μL, 80μL, 85μL, 90μL, 95μL or 100μL.

[0079] In the present invention, the excipients preferably include one or more of an antibacterial agent, cyclodextrin, hydroxyethyl cellulose, sucralose, flavor, hydrochloric acid and sodium hydroxide; the antibacterial agent preferably includes methyl parahydroxybenzoate (abbreviated as methylparaben) and / or propyl parahydroxybenzoate (abbreviated as propylparaben); the cyclodextrin preferably includes β-cyclodextrin.

[0080] The present invention also provides a method for detecting components in a bilastine sample, comprising the following steps: detecting the separation results of the components in the bilastine sample obtained by the separation method described in the above technical solution to obtain the detection results of the components in the bilastine sample; the detection includes detecting by using a detector or a mass spectrometer; and the detection results include qualitative detection results and / or quantitative detection results.

[0081] In the present invention, the detector preferably includes an ultraviolet detector; the detection wavelength of the detector is preferably 210~290nm. In a specific embodiment, the detection wavelength can be 210nm, 215nm, 220nm, 225nm, 230nm, 235nm, 240nm, 245nm, 250nm, 255nm, 260nm, 265nm, 270nm, 275nm, 280nm, 285nm or 290nm.

[0082] In the present invention, the method for obtaining the quantitative detection result includes area normalization method, internal standard method or external standard method. The present invention has no special limitation on the area normalization method, internal standard method or external standard method, and the area normalization method, internal standard method or external standard method well known to those skilled in the art can be used for quantitative determination.

[0083] In order to further illustrate the present invention, the detection method of the components in the bilastine sample provided by the present invention is described in detail below in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0084] In the embodiments of the present invention, the conditions for HPLC chromatographic separation and detection (optimal conditions) are as follows: the high performance liquid chromatograph is Agilent 1260; the chromatographic column is phenomenex Luna C18 100A 4.6mm×250mm, 5μm; the column temperature is 25°C; the mobile phase: the mobile phase A is a phosphate buffer (mix 0.5mL of 85v / v% phosphoric acid aqueous solution and 1.7mL of n-octylamine, add water to make the volume 1000mL, and adjust the pH value to 6.9±0.1 with phosphoric acid or n-octylamine); the mobile phase B is methanol-acetonitrile with a volume ratio of 1:1; the detection is performed by an ultraviolet detector with a detection wavelength of 275nm; the mobile phase flow rate is 0.9mL / min; the injection volume is 25μL; a gradient elution method is adopted, and the gradient elution program is shown in Table 1.

[0085] Table 1 Gradient elution program

[0086]

[0087] Diluent (solvent for dissolving reference substance and sample to be tested): mobile phase A: mobile phase B volume ratio = 1:1.

[0088] Example 1

[0089] Methodology Validation - System Suitability

[0090] 1 Solution preparation method

[0091] 1.1 Preparation of test solution

[0092] Test solution for oral liquid preparation (0.5 mg / mL): Dilute the bilastine oral solution with diluent and shake well to obtain a solution with a bilastine oral solution concentration of 0.5 mg / mL.

[0093] Bilastine API test solution: dissolve the bilastine API in methanol, dilute with a diluent, and shake well to obtain a bilastine API test solution with a bilastine API concentration of 0.5 mg / mL, wherein the mass volume ratio of bilastine API to methanol is 1 mg:5 mL.

[0094] Bilastine tablet test solution: dissolve the bilastine tablet in methanol, dilute with diluent, and shake well to obtain a bilastine tablet test solution with a bilastine tablet concentration of 0.5 mg / mL. The mass volume ratio of bilastine tablet to methanol is 1 mg:5 mL.

[0095] 1.2 Preparation of impurity stock solutions

[0096] Impurity A stock solution: Accurately weigh 1.02 mg of impurity A reference substance and place it in a 5 mL volumetric flask, add 0.6 mL of methanol, dissolve it by ultrasonication, dilute it to the scale with a diluent, and shake well.

[0097] Impurity B stock solution: Accurately weigh 1.14 mg of impurity B reference substance and place it in a 5 mL volumetric flask, add 0.6 mL of methanol, dissolve it by ultrasonication, dilute it to the scale with diluent, and shake well.

[0098] Impurity C stock solution: Accurately weigh 1.33 mg of impurity C reference substance and place it in a 5 mL volumetric flask, add 0.6 mL of methanol, dissolve it by ultrasonication, dilute it to the scale with diluent, and shake well.

[0099] Impurity D stock solution: Accurately weigh 0.96 mg of impurity D reference substance and place it in a 5 mL volumetric flask, add 0.6 mL of methanol, dissolve it by ultrasonication, dilute it to the scale with diluent, and shake well.

[0100] Impurity E stock solution: Accurately weigh 1.42 mg of impurity E reference substance and place it in a 5 mL volumetric flask, add 0.6 mL of methanol, dissolve it by ultrasonication, dilute it to the scale with diluent, and shake well.

[0101] Impurity F stock solution: Accurately weigh 1.15 mg of impurity F reference substance and place it in a 5 mL volumetric flask, add 0.6 mL of methanol, dissolve it by ultrasonication, dilute it to the scale with diluent, and shake well.

[0102] 1.3 Preparation of impurity location solutions

[0103] Impurity A localization solution: Accurately measure 1 mL of impurity A stock solution and place it in a 100 mL volumetric flask, add 12.5 mL of methanol, dilute to the scale with diluent, and shake well.

[0104] Impurity B localization solution: Accurately measure 1 mL of impurity B stock solution and place it in a 100 mL volumetric flask, add 12.5 mL of methanol, dilute to the scale with diluent, and shake well.

[0105] Impurity C localization solution: Accurately measure 1 mL of impurity C stock solution and place it in a 100 mL volumetric flask, add 12.5 mL of methanol, dilute to the scale with diluent, and shake well.

[0106] Impurity D localization solution: Accurately measure 1 mL of impurity D stock solution and place it in a 100 mL volumetric flask, add 12.5 mL of methanol, dilute to the scale with diluent, and shake well.

[0107] Impurity E localization solution: Accurately measure 1 mL of the impurity E stock solution and place it in a 100 mL volumetric flask, add 12.5 mL of methanol, dilute to the scale with a diluent, and shake well.

[0108] Impurity F localization solution: Accurately measure 1 mL of impurity F stock solution and place it in a 100 mL volumetric flask, add 12.5 mL of methanol, dilute to the scale with diluent, and shake well.

[0109] 1.4 Preparation of excipient stock solution

[0110] Cyclodextrin stock solution: Accurately weigh 17.83 mg of cyclodextrin and place it in a 20 mL volumetric flask, add 10 mL of water, dissolve it by ultrasonic, dilute it to the scale with a diluent, and shake well.

[0111] Hydroxyethyl cellulose stock solution: Accurately weigh 13.21 mg of hydroxyethyl cellulose and place it in a 20 mL volumetric flask, add 10 mL of water, dissolve it by ultrasonic, dilute it to the scale with a diluent, and shake well.

[0112] Methyl p-hydroxybenzoate stock solution: Accurately weigh 16.05 mg of methyl p-hydroxybenzoate and place it in a 25 mL volumetric flask, add 5 mL of methanol, dissolve it by ultrasonication, dilute it to the scale with a diluent, and shake well.

[0113] Propyl p-hydroxybenzoate stock solution: Accurately weigh 8.67 mg of propyl p-hydroxybenzoate and place it in a 25 mL volumetric flask, add 5 mL of methanol, dissolve it by ultrasonication, dilute it to the scale with a diluent, and shake well.

[0114] Sucralose stock solution: Accurately weigh 8.61 mg of sucralose and place it in a 100 mL volumetric flask. Add 20 mL of methanol and dissolve it by ultrasonic. Then dilute it to the scale with a diluent and shake well.

[0115] Essence stock solution: Accurately weigh 20.87 mg of essence and place it in a 50 mL volumetric flask, add 10 mL of water, dissolve it by ultrasonic, dilute it to the scale with a diluent, and shake well.

[0116] 1.5 Preparation of auxiliary material mixed solution: Accurately measure 1 mL each of cyclodextrin stock solution, hydroxyethyl cellulose stock solution, methyl parahydroxybenzoate stock solution, propyl parahydroxybenzoate stock solution, sucralose stock solution, and essence stock solution and place them in the same 10 mL volumetric flask, dilute to the scale with diluent, and shake well.

[0117] 1.6 Preparation of the main component solution: Accurately weigh 12.53 mg of bilastine and place it in a 25 mL volumetric flask, add 12.5 mL of methanol, dissolve it by ultrasonication, dilute it to the scale with a diluent, and shake well.

[0118] 1.7 Preparation of system suitability solution: Accurately measure 2 mL of impurity mixed solution and place it in a 10 mL volumetric flask. Then accurately measure 5 mL of auxiliary material mixed solution and place it in the same volumetric flask. Dilute to the scale with the main component solution and shake well.

[0119] 2 HPLC separation

[0120] The blank solvent (diluent), excipient mixed solution (blank excipient) and system adaptability solution were injected and separated by HPLC under the optimal HPLC detection conditions. Figure 1 is the blank solvent HPLC spectrum, Figure 2 is the HPLC spectrum of blank excipients, Figure 3 is the HPLC spectrum of the system suitability solution, Figure 1~Figure 3 It can be seen that the excipients in the bilastine oral solution can be completely separated from the impurities of the raw material, the excipients do not interfere with the impurity detection, and each impurity can be completely separated from the main peak of bilastine.

[0121] The results of HPLC test on the system suitability solution injection under different initial mobile phase A and mobile phase B volume ratios, column temperatures, pH values ​​of mobile phase A, column temperatures, and mobile phase flow rates are shown in Tables 2 and 3.

[0122] Table 2 Results of the durability test under changing chromatographic conditions

[0123]

[0124] Table 3 Results of the durability test under changing chromatographic conditions

[0125]

[0126] Example 2

[0127] Methodology Validation - Stability

[0128] Unspoiled solution: Accurately measure 5 mL of bilastine oral solution and place it in a 25 mL volumetric bottle. Dilute to the mark with diluent and shake well.

[0129] High temperature destruction: Take 2 mL of the undestroyed solution into a sampling vial and dry it in a drying oven at 105°C for 4 h.

[0130] Light damage: Take 2 mL of undamaged solution into a sampling vial and place it in a light box at 5000 lx ± 500 lx to dry for 24 hours or 10 days.

[0131] Oxidative damage: Take 2 mL of bilastine oral solution in a 10 mL volumetric flask, add 5 mL of 3 v / v% hydrogen peroxide solution, leave for 24 hours, dilute to the scale with a diluent, shake well, and the solution is ready.

[0132] Acid destruction: Take 2 mL of bilastine oral solution in a 10 mL volumetric flask, add 1.5 mL of 0.1 mol / L hydrochloric acid aqueous solution, leave for 24 hours, add 1.5 mL of 0.1 mol / L sodium hydroxide solution to neutralize, dilute to the scale with diluent, shake well, and obtain.

[0133] Alkali destruction: Take 2 mL of bilastine oral solution in a 10 mL volumetric flask, add 1.5 mL of 0.1 mol / L sodium hydroxide solution, let stand for 24 h, add 1.5 mL of 0.1 mol / L hydrochloric acid aqueous solution to neutralize, then dilute to the scale with a diluent, shake well, and the product is ready.

[0134] The undamaged bilastine oral solution and each damaged bilastine oral solution were sampled and separated by HPLC. The results are shown in Figure 4~Figure 9 .

[0135] Figure 4 Bilastine oral solution does not destroy the HPLC spectrum. Figure 4 It can be seen that methylparaben and propylparaben, as well as two process impurities: impurity A and impurity C, can be detected in bilastine oral solution.

[0136] Figure 5 This is the HPLC spectrum of Bilastine oral solution after high temperature destruction. Figure 5 It can be seen that the oral solution of bilastine is unstable under high temperature of 105°C, and degrades into oxidized impurities E, as well as process impurities B and impurities D; methylparaben and propylparaben degrade into p-hydroxybenzoic acid (i.e. Figure 5 Unknown impurity in the solution: retention time is 5.137min).

[0137] Figure 6 This is the HPLC spectrum of Bilastine oral solution after 24 hours of light destruction. Figure 6 It can be seen that the bilastine oral solution is relatively stable under 24 hours of light exposure and no new impurities are degraded.

[0138] Figure 7 This is the HPLC spectrum of Bilastine oral solution after 10 days of light destruction. Figure 7 It can be seen that under long-term light conditions, the oral solution of bilastine is unstable and degrades into oxidized impurities F and impurity D; methylparaben and propylparaben degrade into p-hydroxybenzoic acid (i.e. Figure 7 Unknown impurities in the solution: retention time is 5.110min), and brown bottles should be used to protect from light when packaging.

[0139] Figure 8 This is the HPLC spectrum of Bilastine oral solution after oxidation destruction. Figure 8 It can be seen that the bilastine oral solution can be degraded into impurities E and F under oxidative conditions and is unstable under oxidative conditions.

[0140] Fig. 9 is the HPLC spectrum of Bilastine oral solution after acid destruction, Fig. 9 It can be seen that the raw material of bilastine oral solution is relatively stable under acidic conditions, while the preservatives methyl paraben and propyl paraben are unstable and will hydrolyze to generate parahydroxybenzoic acid (i.e. Fig. 9 Unknown impurity in the solution: retention time is 5.147min).

[0141] Fig.10 The HPLC spectrum of Bilastine oral solution is destroyed by alkali. Fig.10 It can be seen that the raw material of bilastine oral solution is relatively stable under alkaline conditions, while the preservatives methyl paraben and propyl paraben are unstable and will hydrolyze to generate parahydroxybenzoic acid (i.e. Fig.10 Unknown impurity in the solution: retention time is 5.147min).

[0142] Example 3

[0143] Methodological Validation - Limit of Quantitation and Limit of Detection

[0144] Preparation of quantitation limit solution:

[0145] Reference substance stock solution (equivalent to 10wt% test substance solution): Accurately weigh 9.98mg of methylparaben, 10.02mg of propylparaben, 10.03mg of bilastine reference substance, 10.70mg of impurity A, 9.50mg of impurity B, 10.25mg of impurity C, 9.60mg of impurity D, 10.80mg of impurity E, and 10.70mg of impurity F, place them in the same 200mL volumetric flask, add 10ml of methanol, dissolve by ultrasonication, dilute to the scale with diluent, and shake well.

[0146] Reference substance mixed solution (equivalent to 1wt% test substance solution): Accurately measure 10mL of reference substance stock solution and place it in a 100mL volumetric flask, dilute to the scale with diluent, shake well, and obtain.

[0147] Preparation of quantitative limit solution: Accurately pipette 1 mL of the reference mixed solution (equivalent to 1 wt% test solution) into a 20 mL volumetric flask, add diluent to dilute to the scale, shake well, and obtain.

[0148] Preparation of detection limit solution: Accurately pipette 2.0 mL of quantitation limit solution into a 10 mL volumetric flask, add diluent to dilute to the scale, shake well, and the solution is ready.

[0149] The quantitative limit solution was continuously injected into HPLC for separation and detection 6 times, and the detection limit solution was continuously injected into HPLC for detection 3 times. The ratio of the main peak (Bilas) peak height to the noise (signal-to-noise ratio) was calculated, and the chromatogram was recorded. The results are shown in Tables 4 to 5 and Figure 11~Figure 12 , Fig.11 The HPLC spectra of the quantitative limits of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F and bilastine are shown in FIG. Fig.12 HPLC spectra of the detection limits of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F and bilastine. The quantitation limit is expressed as the concentration present in bilastine.

[0150] Limit of quantitation = limit of quantitation concentration / sample concentration × 100%.

[0151] Table 4 Results of quantitation limit determination

[0152]

[0153] From Table 4 and Fig.11 It can be seen that under the HPLC chromatographic conditions of the present invention, the quantitative limits of bilastine and its four process impurities (impurity A, impurity B, impurity C and impurity D), two oxidative degradation impurities (impurity E and impurity F), and excipients (methyl parahydroxybenzoate and propyl parahydroxybenzoate) meet the requirement that S / N is greater than 10, and the quantitative limit of each component is equivalent to 0.05wt% of the bilastine concentration in the test solution.

[0154] Table 5 Detection limit determination results

[0155]

[0156] From Table 5 and Fig.12 It can be seen that under the HPLC chromatographic conditions of the present invention, the detection limits of bilastine and its four process impurities (impurity A, impurity B, impurity C and impurity D), two oxidative degradation impurities (impurity E and impurity F), and excipients (methyl paraben and propyl paraben) meet the requirement that S / N is greater than 3, and the detection limit of each component is equivalent to 0.01wt% of the bilastine concentration in the test solution.

[0157] Example 4

[0158] Methodological Validation - Linear Relationship

[0159] Preparation of linear solution

[0160] Reference substance stock solution (equivalent to 10wt% test substance solution): Accurately weigh 9.98mg of methylparaben, 10.02mg of propylparaben, 10.03mg of bilastine reference substance, 10.70mg of impurity A, 9.50mg of impurity B, 10.25mg of impurity C, 9.60mg of impurity D, 10.80mg of impurity E, and 10.70mg of impurity F, place them in the same 200mL volumetric flask, add 10ml of methanol, dissolve by ultrasonication, dilute to the scale with diluent, and shake well.

[0161] Reference substance mixed solution (equivalent to 1wt% test substance solution): Accurately measure 10mL of reference substance stock solution and place it in a 100mL volumetric flask, dilute to the scale with diluent, shake well, and obtain.

[0162] Linear solution L1 (equivalent to 0.05wt% test solution): Accurately measure 1mL of the reference mixed solution and place it in a 20mL volumetric flask, dilute to the scale with diluent, shake well, and obtain.

[0163] Linear solution L2 (equivalent to 0.1wt% test solution): Accurately measure 1mL of the reference solution into a 10mL volumetric flask, dilute to the mark with diluent, and shake well.

[0164] Linear solution L3 (equivalent to 0.2wt% test solution): Accurately measure 1mL of the reference mixed solution and place it in a 5mL volumetric flask, dilute to the scale with diluent, shake well, and obtain.

[0165] Linear solution L4 (equivalent to 0.5wt% test solution): Accurately measure 5mL of the reference solution into a 10mL volumetric flask, dilute to the mark with diluent, and shake well.

[0166] Linear solution L5 (equivalent to 0.8wt% test solution): Accurately measure 8mL of the reference solution into a 10mL volumetric flask, dilute to the mark with diluent, and shake well.

[0167] Linear solution L6 (equivalent to 1wt% test solution): that is, the reference sample mixed solution.

[0168] Take the above linear solutions L1~L6 and inject them once, and then separate and detect them by HPLC. The experimental results are shown in Figure 13~Figure 21 and Tables 6 to 14, among which, Figure 13~Figure 21 The linear curves are methylparaben, impurity A, impurity B, impurity E, impurity F, propylparaben, impurity C, bilastine and impurity D, respectively.

[0169] According to the slope of the linear equation of the main component and each impurity, calculate the correction factor f (f=k 主成分 / k 斜率 ), the results are shown in Tables 6 to 14, where k 主成分 The slope of the linear regression equation of the principal component concentration (X)-response value (Y), k 杂质 It is the slope of the linear regression equation of impurity concentration (X)-response value (Y), and the impurity concentration is measured in bilastine. The correction factor is in the range of 0.9~1.1 and is calculated as 1.0.

[0170] Table 6 Linear determination results of methyl paraben

[0171]

[0172] Table 7 Linear determination results of impurity A

[0173]

[0174] Table 8 Linear determination results of impurity B

[0175]

[0176] Table 9 Impurity E linear determination results

[0177]

[0178] Table 10 Impurity F linearity determination results

[0179]

[0180] Table 11 Linear determination results of propyl paraben

[0181]

[0182] Table 12 Impurity C linear determination results

[0183]

[0184] Table 13 Bilastine linearity determination results

[0185]

[0186] Table 14 Impurity D linearity determination results

[0187]

[0188] Depend on Figure 13~Figure 21As shown in Tables 6 to 14, under the HPLC chromatographic conditions of the present invention, the concentrations of bilastine and its four process impurities (impurity A, impurity B, impurity C and impurity D), two oxidative degradation impurities (impurity E and impurity F), and excipients (methyl parahydroxybenzoate and propyl parahydroxybenzoate) have a good linear relationship with the peak area.

[0189] Example 5

[0190] Methodological Validation - Accuracy

[0191] Preparation of accuracy solution:

[0192] Accuracy solution A1 (equivalent to 0.05wt% of the test sample concentration): accurately measure 4mL of bilastine oral solution and place it in a 20mL volumetric bottle, add 1mL of the reference sample mixed solution prepared in Example 4, dilute to the mark with a diluent, shake well, and obtain. Prepare 3 portions in parallel.

[0193] Accuracy solution A2 (equivalent to 0.1wt% of the test sample concentration): accurately measure 4mL of bilastine oral solution and place it in a 20mL volumetric bottle, add 2mL of the reference sample mixed solution prepared in Example 4, dilute to the mark with a diluent, shake well, and obtain. Prepare 3 portions in parallel.

[0194] Accuracy solution A3 (equivalent to 0.5wt% of the test sample concentration): accurately measure 4mL of bilastine oral solution and place it in a 20mL volumetric bottle, add 10mL of the reference sample mixed solution prepared in Example 4, dilute to the mark with a diluent, shake well, and obtain. Prepare 3 portions in parallel.

[0195] Accuracy solution A4 (equivalent to 1% of the test sample concentration): Accurately measure 4 mL of bilastine oral solution and place it in a 20 mL volumetric bottle, dilute to the mark with the reference sample mixed solution prepared in Example 4, and shake well. Prepare 3 portions in parallel.

[0196] Each accuracy solution was injected, separated and detected by HPLC, and the recovery rate was calculated by the external standard method. The results are shown in Table 15.

[0197] Table 15 Spiked recovery results

[0198]

[0199] It can be seen from Table 15 that under the HPLC chromatographic conditions of the present invention, the accuracy of bilastine and its four process impurities (impurity A, impurity B, impurity C and impurity D), two oxidative degradation impurities (impurity E and impurity F), and excipients (methyl parahydroxybenzoate and propyl parahydroxybenzoate) is good.

[0200] Example 6

[0201] Methodology Validation - Precision

[0202] Spiked test solution (equivalent to 0.5wt% of the test solution concentration): Accurately measure 4mL of bilastine oral solution and place it in a 20mL volumetric flask, add 10mL of the reference solution prepared in Example 4 (equivalent to 1wt% of the test solution), dilute to the mark with a diluent, shake well, and prepare 6 portions in parallel.

[0203] Each solution was injected into HPLC for separation and detection, and the recovery rate was calculated by the external standard method. The results are shown in Table 16.

[0204] Table 16 Precision results (wt%)

[0205]

[0206] It can be seen from Table 16 that the precision of the spiked test solution of the related substance detection method of bilastine oral solution is good.

[0207] Example 7

[0208] Methodology Validation - Solution Stability

[0209] Reference substance mixed solution (equivalent to 1 wt % test substance solution): the reference substance mixed solution prepared in Example 4.

[0210] Spiked test solution (equivalent to 0.5 wt % of the test sample concentration): the spiked test solution prepared in Example 6.

[0211] The reference mixed solution and the spiked test solution were placed at room temperature for 0 h, 24 h, 48 h, and 72 h, respectively, and then injected and separated and detected by HPLC. The results are shown in Tables 17 and 18.

[0212] Table 17 Stability of reference substance mixed solution

[0213]

[0214] Table 18 Stability of spiked test solution

[0215]

[0216] It can be seen from Tables 17 and 18 that under the HPLC chromatographic conditions provided by the present invention, the reference solution and the test solution of bilastine and its four process impurities (impurity A, impurity B, impurity C and impurity D), two oxidative degradation impurities (impurity E and impurity F), and excipients (methyl parahydroxybenzoate and propyl parahydroxybenzoate) are stable within 72 hours.

[0217] Comparative Example 1

[0218] The system suitability solution prepared in Example 1 of the present invention was separated using the HPLC chromatographic conditions in paragraphs

[0038] to

[0042] of the CN116165307A specification. The chromatogram is shown in FIG. Fig. 22 It can be seen that bilastine and impurity D merge into a peak, indicating that impurity D and bilastine are not completely separated; propylparaben merges with the blank peak, which will affect the quantitative analysis results of propylparaben. Impurity E and impurity F merge into a peak, indicating that impurity E and impurity F are not completely separated.

[0219] 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 separating components in a bilastine sample, comprising the following steps: The bilastine sample to be tested is subjected to high performance liquid chromatography separation to obtain separation results of components in the bilastine sample; The conditions for the high performance liquid chromatography separation include: the chromatographic column is a C18 chromatographic column, the mobile phase system includes mobile phase A and mobile phase B, the mobile phase A is a phosphate buffer, the phosphate buffer contains an ion pair reagent, and the ion pair reagent includes triethylamine and / or n-octylamine; the mobile phase B is a mixed solvent of methanol and acetonitrile; the elution method is gradient elution, and the procedure of the gradient elution includes: 0-5min, the volume fraction of the mobile phase A is 50-70%; 5-35 min, the volume fraction of the mobile phase A is reduced from 50-70% to 15-25%; 35-40 min, the volume fraction of the mobile phase A is 15-25%; 40-41 min, the volume fraction of the mobile phase A increased from 15-25% to 50-70%; 41-50 min, the volume fraction of the mobile phase A is 50-70%; The ingredients include bilastine, related impurities and excipients; the related impurities include impurities E and F, and also include one or more of impurities A, B, C and D: ； The auxiliary materials include one or more of an antibacterial agent, cyclodextrin, hydroxyethyl cellulose, sucralose, flavor, hydrochloric acid and sodium hydroxide; the antibacterial agent includes methyl parahydroxybenzoate and / or propyl parahydroxybenzoate.

2. The separation method according to claim 1, characterized in that The volume concentration of the ion pair reagent in the phosphate buffer is 0.07-0.27%; The pH value of the phosphate buffer is 6.5-7.

5.

3. The separation method according to claim 1, characterized in that The volume fraction of methanol in the mobile phase B is 30-75%.

4. The separation method according to any one of claims 1 to 3, characterized in that The column temperature of the high performance liquid chromatography separation is 15-40° C., the flow rate of the mobile phase is 0.5-1.5 mL / min, and the injection volume is 5-100 μL.

5. The separation method according to claim 1, characterized in that The bilastine sample to be tested includes a liquid sample containing bilastine and / or a solid sample containing bilastine.

6. The separation method according to claim 5, characterized in that The bilastine-containing liquid sample is diluted with a first diluent before use; the first diluent is a mixed solution of the mobile phase A and the mobile phase B; The bilastine-containing solid sample is dissolved in methanol before use, and a second diluent is added for dilution; the second diluent includes a mixed solution of the mobile phase A and the mobile phase B.

7. A method for detecting components in a bilastine sample, comprising the following steps: The separation result of the components in the bilastine sample obtained by the separation method according to any one of claims 1 to 6 is detected to obtain the detection result of the components in the bilastine sample; the detection includes detection using an ultraviolet detector or a mass spectrometer; the detection result includes a qualitative detection result and / or a quantitative detection result; the detection wavelength of the ultraviolet detector is 210 to 290 nm.

8. The detection method according to claim 7, characterized in that: The method for obtaining the quantitative detection result includes area normalization method, internal standard method or external standard method.

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