Related substance detection methods for mometasone furoate nasal spray

Abstract

This invention discloses a method for detecting related substances in mometasone furoate nasal spray. The method uses an octadecylsilane-bonded silica column, with 0.02 mol / L sodium perchlorate as mobile phase A and acetonitrile as mobile phase B, employing gradient elution at a flow rate of 0.8–1.2 mL / min. The detection wavelength is 240 nm–260 nm, the column temperature is 30–50 °C, and the injection volume is 10–25 μL. The sum of the volume fractions of mobile phase A and mobile phase B is 100, and at elution min 0, the volume fraction of mobile phase A is 57–59. This method can simultaneously detect unknown photodegradation impurities, the excipient peak position is flat and does not affect impurity quantification, preservatives are completely eluted, and the method exhibits good precision and robustness. It also improves detection sensitivity, shortens detection time, and is simple to operate, highly specific, and resistant to interference.

Description

Technical Field

[0001] This invention relates to the field of detection of mometasone furoate nasal spray. More specifically, this invention relates to a method for detecting related substances in mometasone furoate nasal spray. Background Technology

[0002] Currently, known related substances in mometasone furoate include impurities D, K, L, M, H, O, Q, G, P, F, C, A, E, and T. However, the current standards for mometasone furoate nasal sprays lack quality control for the detection of these related substances. Furthermore, there are numerous types of mometasone furoate products on the market, each using different excipients. Prolonged elution times for these excipients can negatively impact the quantification of weaker impurities. Additionally, some preservatives are difficult to elute, particularly benzalkonium chloride, which may elute intermittently during testing, interfering with subsequent impurity detection and hindering the testing of this type of product. Figure 1 As shown, Figure 1 The image shows a liquid chromatogram for the detection of mometasone furoate-related substances using the existing imported registration standard. The figure indicates that the imported registration method has low detection efficiency. When using the imported registration method, the main peak elution time is approximately 23 minutes. According to the imported drug registration standard for mometasone furoate, the run time must be 2.5 times the main peak elution time, therefore the run time should be at least approximately 60 minutes. These impurities that elute later exhibit some peak broadening, leading to reduced sensitivity. During development, detection of photodegradation solutions revealed that photodegradation produces some unknown impurities that cannot be eluted within the 2.5 times elution time window of the main peak using the imported registration standard method, and are typically eluted during the third sample run (see [link to relevant documentation]). Figure 2 Meanwhile, it was discovered that an unknown impurity in the active pharmaceutical ingredient co-eluted with impurity C and could not be separated. The impurity 5 minutes earlier appeared on the excipient peak, which may lead to inaccurate impurity quantification.

[0003] For the reasons mentioned above, there is an urgent need to develop a detection method for mometasone furoate nasal spray to solve the problem of interference from preservatives and excipients, thereby controlling the impurity level within a safe and reasonable range and ensuring the quality and safety of the drug. Summary of the Invention

[0004] One object of the present invention is to solve at least the above-mentioned problems and to provide a method for detecting related substances in mometasone furoate nasal spray. This method uses an octadecylsilane-bonded silica gel column, with 0.02 mol / L sodium perchlorate as mobile phase A and acetonitrile as mobile phase B, performing gradient elution. It can simultaneously detect unknown photodegradation impurities, and the excipient peak positions are flat, not affecting impurity quantification. Preservatives are completely eluted, avoiding the influence of intermittent elution on detection. It has good precision and durability, while also improving detection sensitivity, shortening detection time, and is simple to operate, highly specific, and strongly resistant to interference.

[0005] To achieve these objectives and other advantages according to the present invention, a method for detecting related substances in mometasone furoate nasal spray is provided. This method employs high-performance liquid chromatography (HPLC) with an octadecylsilane-bonded silica column. Gradient elution is performed using 0.02 mol / L sodium perchlorate as mobile phase A and acetonitrile as mobile phase B. The flow rate is 0.8–1.2 mL / min, the detection wavelength is 240 nm–260 nm, the column temperature is 30–50 °C, and the injection volume is 10–25 μL. The sum of the volume fractions of mobile phase A and mobile phase B is 100, and at the 0-minute elution, the volume fraction of mobile phase A is 57–59.

[0006] Preferably, the flow rate is 0.8–1.0 mL / min, the detection wavelength is 252 nm–256 nm, the column temperature is 38–42 °C, and the injection volume is 10–20 μL.

[0007] Preferably, the flow rate is 0.9 mL / min, the detection wavelength is 254 nm, the column temperature is 40 °C, and the injection volume is 10 μL.

[0008] Preferably, the chromatographic column has a size of 4.6 × 150 mm and the packing material has a particle size of 2.7 μm.

[0009] Preferably, the gradient elution parameters are as follows: at 0 min, mobile phase A: mobile phase B = 58%: 42%; at 10 min, mobile phase A: mobile phase B = 48%: 52%; at 25 min, mobile phase A: mobile phase B = 48%: 52%; at 40 min, mobile phase A: mobile phase B = 20%: 80%; at 41 min, mobile phase A: mobile phase B = 58%: 42%; and at 45 min, mobile phase A: mobile phase B = 58%: 42%.

[0010] Preferably, the preparation of the mobile phase A is as follows: weigh 2.8 g of sodium perchlorate monohydrate, add water to 1000 mL, shake well, and adjust the pH to 3.0 with perchloric acid.

[0011] Preferably, the test solution is used to detect the photodegradation of mometasone furoate nasal spray.

[0012] The present invention has at least the following beneficial effects:

[0013] This invention also provides a method for detecting related substances, their content, and benzalkonium chloride in mometasone furoate nasal spray. This method is simple to operate, highly specific (capable of simultaneously detecting unknown photodegradation impurities), highly resistant to interference (the baseline of excipient peak positions is flat and does not affect impurity quantification; preservatives are completely eluted, avoiding the impact of intermittent elution on detection), has good linearity, and good precision and durability. It also improves detection sensitivity, shortens detection time, and can simultaneously detect the content of the preservative benzalkonium chloride, thus improving detection efficiency.

[0014] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0015] Figure 1 The high-performance liquid chromatogram is shown for the detection of related substances in mometasone furoate nasal spray using existing imported registration standard methods.

[0016] Figure 2 The high-performance liquid chromatogram of the third injection of mometasone furoate nasal spray when the test solution is exposed to light is obtained using the imported registered standard method.

[0017] Figure 3 This is a high-performance liquid chromatogram of the test solution in Example 1 of the present invention;

[0018] Figure 4 This is a high-performance liquid chromatogram of the test solution in Example 2 of the present invention;

[0019] Figure 5 This is a high-performance liquid chromatogram of the test solution in Example 3 of the present invention;

[0020] Figure 6 This is a high-performance liquid chromatogram of the test solution in Example 4 of the present invention;

[0021] Figure 7 This is a high-performance liquid chromatogram of the test solution in Example 5 of the present invention;

[0022] Figure 8 This is a high-performance liquid chromatogram of the test solution in Example 6 of the present invention;

[0023] Figure 9 This is a high-performance liquid chromatogram of the test solution in Example 7 of the present invention;

[0024] Figure 10 This is a high-performance liquid chromatogram of the test solution in Example 8 of the present invention;

[0025] Figure 11 This is a high-performance liquid chromatogram of the test solution in Example 9 of the present invention;

[0026] Figure 12 This is the high-performance liquid chromatogram of the blank excipient solution in Comparative Example 2 of this invention;

[0027] Figure 13 This is a high-performance liquid chromatogram of the 0.1% mixed impurity sample solution in Comparative Example 2 of this invention;

[0028] Figure 14 This is a high-performance liquid chromatogram of the blank excipient solution of Comparative Example 3 of the present invention;

[0029] Figure 15 This is a high-performance liquid chromatogram of the single impurity T localization solution of Comparative Example 3 of the present invention;

[0030] Figure 16 This is a high-performance liquid chromatogram of the blank excipient solution of Comparative Example 4 of the present invention;

[0031] Figure 17 This is a high-performance liquid chromatogram of the 0.1% mixed impurity sample solution of Comparative Example 4 of the present invention;

[0032] Figure 18 This is a high-performance liquid chromatogram of the blank excipient solution of Comparative Example 5 of the present invention;

[0033] Figure 19 This is a high-performance liquid chromatogram of the single impurity T localization solution of Comparative Example 5 of the present invention;

[0034] Figure 20 The image shows a high-performance liquid chromatogram of a 0.1% impurity sample solution detected using the method of this invention.

[0035] Figure 21 This is a high-performance liquid chromatogram of the test sample solution for photodegradation of mometasone furoate using the method of the present invention.

[0036] Note: The horizontal axis of the above high performance liquid chromatograms is in minutes (min), and the vertical axis is the response value (AU). Detailed Implementation

[0037] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0038] <List of Reagents and Instruments>

[0039] This invention provides a method for simultaneously determining the content of mometasone furoate nasal spray, related substances, and benzalkonium chloride, using high-performance liquid chromatography (HPLC). The list of reagents and instruments used in the experiment is shown in Table 1. Other reagents or instruments not listed in Table 1 are readily known and available to those skilled in the art.

[0040] Table 1. List of reagents and instruments used in the experiment

[0041] Reagents and Instruments factory model Acetonitrile Ningbo Gaoteng Chemical Technology Co., Ltd. HPLC Sodium perchlorate monohydrate Sinopharm Chemical Reagent Co., Ltd. AR perchloric acid Sinopharm Chemical Reagent Co., Ltd. AR Mometasone furoate reference standard China National Institutes for Food and Drug Control / Mometasone furoate nasal spray Zhejiang Xianju Pharmaceutical Co., Ltd. / Impurity D Zhejiang Xianju Pharmaceutical Co., Ltd. / impurity K Zhejiang Xianju Pharmaceutical Co., Ltd. / impurity L Zhejiang Xianju Pharmaceutical Co., Ltd. / impurity M Zhejiang Xianju Pharmaceutical Co., Ltd. / impurity H TRC / impurity O Zhejiang Xianju Pharmaceutical Co., Ltd. / impurity Q Zhejiang Xianju Pharmaceutical Co., Ltd. / Impurity G TRC / impurity P Zhejiang Xianju Pharmaceutical Co., Ltd. / impurity F TRC / Impurity C TRC / Impurity A TRC / Impurity E TRC / impurity T TLC / High performance liquid chromatography Agilent 1260VWD

[0042] Note: The above-mentioned impurities and mometasone furoate are reference standards.

[0043] <Testing Methods>

[0044] 1. Solution preparation

[0045] Acidic acetonitrile: Measure 10 mL of glacial acetic acid, add acetonitrile to 2 L, and mix well to obtain acidic acetonitrile;

[0046] Diluent solution: Take 500 mL of acidic acetonitrile and 500 mL of water, mix well to prepare the diluent;

[0047] Reference solution: Dissolve mometasone furoate reference standard in acidic acetonitrile and dilute to volume. Dilute with diluent to a 70 μg / mL solution as the reference stock solution. Accurately measure 5 mL of mometasone furoate reference stock solution into a 50 mL volumetric flask, add 20 mL of acidic acetonitrile, then add water to dilute to volume and shake well to obtain the reference solution.

[0048] Limit of Quantification Solution: Dilute the reference solution 2000 times with diluent to a concentration of 0.035 μg / mL.

[0049] System suitability solution: Accurately weigh an appropriate amount of mometasone furoate impurity D reference standard, dilute it with diluent to 60 μg / mL, measure 1 mL, add 10 mL of the mometasone furoate reference standard solution, and shake well. This is the system suitability solution. The peak resolution between mometasone furoate and mometasone furoate impurity D should not be less than 2.0.

[0050] Blank solution: diluent solution.

[0051] Mobile phase A (0.02 mol / mL sodium perchlorate solution): Weigh 2.8 g of sodium perchlorate monohydrate, add water to 1000 mL, shake well, and adjust the pH to 3.0 with perchloric acid.

[0052] Impurity mixture (1%): Accurately measure an appropriate amount of each impurity stock solution and dilute with acidic acetonitrile to a concentration of 0.7 μg / mL for each impurity reference standard;

[0053] 0.1% Mixed Impurities Spray Solution: Accurately weigh an appropriate amount of mometasone furoate nasal spray, equivalent to 3.5 mg mometasone furoate, place it in a 50 mL volumetric flask, add 5 mL of mixed impurities solution (1%), add 20 mL of acidic acetonitrile, shake for 15 minutes, sonicate for 5 minutes, add 10 mL of water, let it cool to room temperature, dilute with water to the mark, shake well and centrifuge, take the supernatant and filter it through a 0.45 μm PTFE filter, discard 2 mL of the initial filtrate, and take the subsequent filtrate.

[0054] 2. Detection method:

[0055] The determination was performed using high-performance liquid chromatography (HPLC). Specifically, 10 μL each of the blank solution, limit of quantitation solution, system suitability solution, and test solution were accurately pipetted and injected sequentially into the HPLC instrument, and the chromatograms were recorded. The chromatographic conditions were as follows:

[0056] Chromatographic column: CAPCELL CORE C18 4.6×150mm 2.7μm;

[0057] Detection wavelength: 254nm; Detector (ultraviolet detector, VWD)

[0058] Column temperature: 40℃;

[0059] Flow rate: 0.9 mL / min;

[0060] Injection volume: 10 μL;

[0061] Mobile phase: Mobile phase A: 0.02 mol / mL sodium perchlorate solution, Mobile phase B: acetonitrile; The ratio of mobile phase A to mobile phase B at 0 min is 58:42;

[0062] The gradient elution parameters are shown in Table 2:

[0063] Table 2 Gradient elution parameters

[0064] Time (min) Mobile phase A (%) Mobile phase B (%) 0 58 42 10 48 52 25 48 52 40 20 80 41 58 42 45 58 42

[0065] In the following experimental examples, unless otherwise specified, the corresponding solutions were prepared according to the above method and the tests were performed according to the detection method. The test solution is the solution to be tested.

[0066] <Example 1>

[0067] 1. Preparation of test solution

[0068] Accurately weigh an appropriate amount of mometasone furoate nasal spray, equivalent to 3.5 mg of mometasone furoate, place it in a 50 mL volumetric flask, add 25 mL of acidic acetonitrile, shake for 15 minutes, sonicate for 5 minutes, add 10 mL of water, let it cool to room temperature, dilute with water to the mark, shake well, centrifuge, and filter the supernatant as the test solution of this example.

[0069] 2. Testing

[0070] Following the above detection method, accurately pipette 10 μl each of the blank solution, limit of quantitation solution, system suitability solution, and test solution, and inject them into the liquid chromatograph, recording the chromatograms; the high-performance liquid chromatogram of the test solution is shown below. Figure 3 Results analysis: From Figure 3 As can be seen, using the perchloric acid system as the mobile phase allows for the timely elution of benzalkonium chloride (the benzalkonium chloride excipient peaks are at approximately 31 min and 38 min), avoiding the impact of irregular elution of benzalkonium chloride on the detection. Furthermore, the main peak and adjacent impurity peaks are baseline separated, and impurities are also well separated from each other. Unknown impurities can be separated from impurity C. Figure 3 The peak to the left of impurity C, close to the peak where impurity C is located, is the peak of the unknown impurity. At the same time, the baselines of the solvent and excipient peaks are relatively flat, reducing the interference of excipients on detection. Moreover, compared with the imported registration standard method, the detection time is shortened (the imported registration standard method takes about 60 minutes to run, while the method of this invention takes about 45 minutes to run).

[0071] <Example 2>

[0072] 1. Preparation of test solution

[0073] Accurately weigh an appropriate amount of mometasone furoate nasal spray, equivalent to 3.5 mg of mometasone furoate, place it in a 50 mL volumetric flask, add 25 mL of acidic acetonitrile, shake for 15 minutes, sonicate for 5 minutes, add 10 mL of water, let it cool to room temperature, dilute with water to the mark, shake well, centrifuge, and filter the supernatant as the test solution of this example.

[0074] 2. Testing

[0075] Following the above detection method, accurately pipette 10 μl each of the blank solution, limit of quantitation solution, system suitability solution, and test solution, and inject them sequentially into the liquid chromatograph, recording the chromatograms. The chromatographic conditions in this embodiment differ from those in Example 1 in the elution parameters. The gradient elution parameters for this embodiment are shown in Table 3; all other parameters are the same as in Example 1. The high-performance liquid chromatogram of the test solution is shown in [Table 1]. Figure 4 .

[0076] Table 3 Gradient elution parameters for Example 2

[0077] Time (min) Mobile phase A (%) Mobile phase B (%) 0 57 43 10 47 53 25 47 53 40 20 80 41 57 43 45 57 43

[0078] Results analysis: From Figure 4 As can be seen, compared with Example 1, by changing the proportion of the elution mobile phase and increasing the proportion of the organic phase, the detection method still has good durability and the various impurities are still well separated.

[0079] <Example 3>

[0080] 1. Preparation of test solution

[0081] Accurately weigh an appropriate amount of mometasone furoate nasal spray, equivalent to 3.5 mg of mometasone furoate, place it in a 50 mL volumetric flask, add 25 mL of acidic acetonitrile, shake for 15 minutes, sonicate for 5 minutes, add 10 mL of water, let it cool to room temperature, dilute with water to the mark, shake well, centrifuge, and filter the supernatant as the test solution of this example.

[0082] 2. Testing

[0083] Following the above detection method, accurately pipette 10 μl each of the blank solution, limit of quantitation solution, system suitability solution, and test solution, and inject them sequentially into the liquid chromatograph, recording the chromatograms. The chromatographic conditions in this embodiment differ from those in Example 1 in the gradient elution parameters, which are shown in Table 4. All other parameters are the same as in Example 1. The high-performance liquid chromatogram of the test solution is shown in [Table 4]. Figure 5 ;

[0084] Table 4 Gradient elution parameters for Example 3

[0085] Time (min) Mobile phase A (%) Mobile phase B (%) 0 59 41 10 49 51 25 49 51 40 20 80 41 59 41 45 59 41

[0086] Results analysis: From Figure 5 As can be seen, compared with Example 1, by changing the proportion of the elution mobile phase to reduce the proportion of the organic phase, the detection method still has good durability and the various impurities are still well separated.

[0087] <Example 4>

[0088] 1. Preparation of test solution

[0089] Accurately weigh an appropriate amount of mometasone furoate nasal spray, equivalent to 3.5 mg of mometasone furoate, place it in a 50 mL volumetric flask, add 25 mL of acidic acetonitrile, shake for 15 minutes, sonicate for 5 minutes, add 10 mL of water, let it cool to room temperature, dilute with water to the mark, shake well, centrifuge, and filter the supernatant as the test solution of this example.

[0090] 2. Testing

[0091] Following the above detection method, accurately pipette 10 μl each of the blank solution, limit of quantitation solution, system suitability solution, and test solution, and inject them sequentially into the liquid chromatograph, recording the chromatograms. The chromatographic conditions in this embodiment differ from those in Example 1 except for the column temperature; in this embodiment, the column temperature is 38°C. All other conditions are the same as in Example 1. The high-performance liquid chromatogram of the test solution is shown below. Figure 6 .

[0092] Results analysis: From Figure 6 As can be seen, when the column temperature is 38℃, the detection method still has good durability and the various impurities are still well separated.

[0093] <Example 5>

[0094] 1. Preparation of test solution

[0095] Accurately weigh an appropriate amount of mometasone furoate nasal spray, equivalent to 3.5 mg of mometasone furoate, place it in a 50 mL volumetric flask, add 25 mL of acidic acetonitrile, shake for 15 minutes, sonicate for 5 minutes, add 10 mL of water, let it cool to room temperature, dilute with water to the mark, shake well, centrifuge, and filter the supernatant as the test solution of this example.

[0096] 2. Testing

[0097] Following the above detection method, accurately pipette 10 μl each of the blank solution, limit of quantitation solution, system suitability solution, and test solution, and inject them sequentially into the liquid chromatograph, recording the chromatograms. The chromatographic conditions in this embodiment differ from those in Example 1 in that the column temperature is 42°C. The high-performance liquid chromatogram of the test solution is shown below. Figure 7 .

[0098] Results analysis: From Figure 7 As can be seen, when the column temperature is 42℃, the detection method still has good durability and the various impurities are still well separated.

[0099] <Example 6>

[0100] 1. Preparation of test solution

[0101] Accurately weigh an appropriate amount of mometasone furoate nasal spray, equivalent to 3.5 mg of mometasone furoate, place it in a 50 mL volumetric flask, add 25 mL of acidic acetonitrile, shake for 15 minutes, sonicate for 5 minutes, add 10 mL of water, let it cool to room temperature, dilute with water to the mark, shake well, centrifuge, and filter the supernatant as the test solution of this example.

[0102] 2. Testing

[0103] Following the above detection method, precisely pipette 10 μl each of the blank solution, limit of quantitation solution, system suitability solution, and test solution, and inject them sequentially into the liquid chromatograph, recording the chromatograms. The difference between the chromatographic conditions in this embodiment and Example 1 lies in the flow rate; in this embodiment, the flow rate is 0.8 mL / min. The high-performance liquid chromatogram of the test solution is shown below. Figure 8 .

[0104] Results analysis: From Figure 8As can be seen, when the flow rate is 0.8 mL / min, the detection method still has good durability, and the various impurities are still well separated.

[0105] <Example 7>

[0106] 1. Preparation of test solution

[0107] Accurately weigh an appropriate amount of mometasone furoate nasal spray, equivalent to 3.5 mg of mometasone furoate, place it in a 50 mL volumetric flask, add 25 mL of acidic acetonitrile, shake for 15 minutes, sonicate for 5 minutes, add 10 mL of water, let it cool to room temperature, dilute with water to the mark, shake well, centrifuge, and filter the supernatant as the test solution of this example.

[0108] 2. Testing

[0109] Following the above detection method, accurately pipette 10 μl each of the blank solution, limit of quantitation solution, system suitability solution, and test solution, and inject them sequentially into the liquid chromatograph, recording the chromatograms. The difference between the chromatographic conditions in this embodiment and Example 1 lies in the flow rate; in this embodiment, the flow rate is 1.0 mL / min. The high-performance liquid chromatogram of the test solution is shown below. Figure 9 .

[0110] Results analysis: From Figure 8 As can be seen, when the flow rate is 1.0 mL / min, the detection method still has good durability, and the various impurities are still well separated.

[0111] <Example 8>

[0112] 1. Preparation of test solution

[0113] Accurately weigh an appropriate amount of mometasone furoate nasal spray, equivalent to 3.5 mg of mometasone furoate, place it in a 50 mL volumetric flask, add 25 mL of acidic acetonitrile, shake for 15 minutes, sonicate for 5 minutes, add 10 mL of water, let it cool to room temperature, dilute with water to the mark, shake well, centrifuge, and filter the supernatant as the test solution of this example.

[0114] 2. Testing

[0115] Following the above detection method, precisely pipette 10 μl each of the blank solution, limit of quantitation solution, system suitability solution, and test solution, and inject them sequentially into the liquid chromatograph, recording the chromatograms. The chromatographic conditions in this embodiment differ from those in Example 1 in that the detection wavelength is 252 nm in this embodiment. The high-performance liquid chromatogram of the test solution is shown below. Figure 10 .

[0116] Results analysis: From Figure 10As can be seen, the detection method still exhibits good durability at a detection wavelength of 252 nm. The various impurities remain well separated.

[0117] <Example 9>

[0118] 1. Preparation of test solution

[0119] Accurately weigh an appropriate amount of mometasone furoate nasal spray, equivalent to 3.5 mg of mometasone furoate, place it in a 50 mL volumetric flask, add 25 mL of acidic acetonitrile, shake for 15 minutes, sonicate for 5 minutes, add 10 mL of water, let it cool to room temperature, dilute with water to the mark, shake well, centrifuge, and filter the supernatant as the test solution of this example.

[0120] 2. Testing

[0121] Following the above detection method, precisely pipette 10 μl each of the blank solution, limit of quantitation solution, system suitability solution, and test solution, and inject them sequentially into the liquid chromatograph, recording the chromatograms. The chromatographic conditions in this embodiment differ from those in Example 1 in that the detection wavelength is 256 nm in this embodiment. The high-performance liquid chromatogram of the test solution is shown below. Figure 11 .

[0122] Results analysis: From Figure 11 As can be seen, the detection method still has good durability when the detection wavelength is 256nm.

[0123] <Comparative Example 1>

[0124] 1. Preparation of test solution

[0125] Accurately weigh an appropriate amount of mometasone furoate nasal spray, equivalent to 3.5 mg of mometasone furoate, place it in a 50 mL volumetric flask, add 25 mL of acidic acetonitrile, shake for 15 minutes, sonicate for 5 minutes, add 10 mL of water, let it cool to room temperature, dilute with water to the mark, shake well, centrifuge, and filter the supernatant as the test solution for this comparative example.

[0126] 2. Testing

[0127] In this comparative example, the imported registration standard method was used to detect related substances in mobisol furoate nasal spray. The results are shown in [Figure Number]. Figure 1The results showed that the imported registration method had low detection efficiency. The main peak elution time was approximately 23 minutes. According to the imported drug registration standard JX20140277 for mometasone furoate, the running time should be 2.5 times the main peak elution time, therefore the running time should be at least approximately 60 minutes. These impurities that eluted later exhibited some peak broadening, leading to reduced sensitivity. During development, detection of the photodegradation solution revealed that photodegradation produces some unknown impurities that could not be eluted within a 2.5 times the main peak elution time using the imported registration standard method, and were typically eluted during the third sample run (see [link to relevant documentation]). Figure 2 At 51 minutes (approximately 171 minutes in total), an unknown impurity in the active pharmaceutical ingredient was co-eluted with impurity C, making separation impossible. The imported registration standard method has many problems.

[0128] <Comparative Example 2>

[0129] 1. Test solution

[0130] A. Blank excipient solution: Weigh 7.0 g of blank excipient (excluding mometasone furoate, including other blank excipients) into a 50 mL volumetric flask, add 35 mL of acidic acetonitrile, sonicate for 5 min, dilute to volume with water and mix well, centrifuge and filter, and take the filtrate as the blank excipient solution.

[0131] B. 0.1% mixed impurity spiking solution.

[0132] 2. Testing

[0133] In this comparative example, mobile phase A was 0.1% aqueous acetic acid solution, mobile phase B was acetonitrile, and all other parameters were the same as in Example 1. The blank excipient solution and the 0.1% mixed impurity loading solution were tested, and the results are shown in [Figure 1]. Figures 12-13 , Figure 12 This is the high-performance liquid chromatogram of a blank excipient solution. Figure 13 The high-performance liquid chromatography (HPLC) chromatogram of a 0.1% mixed impurity spiked solution is shown. The results indicate that when acetonitrile and 0.1% acetic acid solution are used as the mobile phase, the excipient peak in the blank excipient (…) Figure 12 At 16.975 min, at the main peak of mometasone furoate ( Figure 13 The peak appears at 16.700 min, which will interfere with the main peak.

[0134] <Comparative Example 3>

[0135] 1. Test solution

[0136] A. Blank excipient solution: Weigh 7.0 g of blank excipient (excluding mometasone furoate, including other blank excipients) into a 50 mL volumetric flask, add 35 mL of acidic acetonitrile, sonicate for 5 min, dilute to volume with water and mix well, centrifuge and filter, and take the filtrate as the blank excipient solution.

[0137] B. Single impurity T localization solution (0.7 μg / mL): Accurately measure impurity T reference standard, dissolve and dilute with acidic acetonitrile to 0.7 μg / mL.

[0138] 2. Testing

[0139] In this comparative example, mobile phase A was 0.1% trifluoroacetic acid (TFA) aqueous solution, mobile phase B was acetonitrile, and all other parameters were the same as in Example 1. The blank excipient solution and the single impurity T localization solution were tested, and the results are shown in [Figure 1]. Figures 14-15 , Figure 14 This is the high-performance liquid chromatogram of a blank excipient solution. Figure 15 The image shows a high-performance liquid chromatography (HPLC) chromatogram of a single impurity T-positioning solution. The results indicate that when acetonitrile and 0.1% trifluoroacetic acid aqueous solution (TFA) are used as the mobile phase, the excipient peak in the blank excipient (…) Figure 14 At 26.647 min, at impurity T ( Figure 15 The peak appears near 26 min (26.694 min), which will interfere with the detection of impurities T.

[0140] <Comparative Example 4>

[0141] 1. Test solution

[0142] A. Blank excipient solution: Weigh 7.0 g of blank excipient (excluding mometasone furoate, including other blank excipients) into a 50 mL volumetric flask, add 35 mL of acidic acetonitrile, sonicate for 5 min, dilute to volume with water and mix well, centrifuge and filter, and take the filtrate as the blank excipient solution.

[0143] B. 0.1% mixed impurity spiking solution.

[0144] 2. Testing

[0145] In this comparative example, mobile phase A was 0.05% TFA aqueous solution, and mobile phase B was 0.05% TFA acetonitrile solution. All other parameters were the same as in Example 1. The blank excipient solution and the 0.1% mixed impurity spiked solution were tested, and the results are shown below. Figures 16-17 , Figure 16 This is the high-performance liquid chromatogram of a blank excipient solution. Figure 17 The high-performance liquid chromatography (HPLC) chromatogram of a 0.1% mixed impurity spiking solution is shown. The results indicate that when 0.05% TFA acetonitrile and 0.05% trifluoroacetic acid solution (TFA) are used as the mobile phase, the excipient peak in the blank excipient (…) Figure 16 At 26.462 min, at impurity T ( Figure 17 The peak appears around 26 min (26.507 min), which will interfere with the detection of impurities T and the baseline is not stable.

[0146] <Comparative Example 5>

[0147] 1. Test solution

[0148] A. Blank excipient solution: Weigh 7.0 g of blank excipient (excluding mometasone furoate, including other blank excipients) into a 50 mL volumetric flask, add 35 mL of acidic acetonitrile, sonicate for 5 min, dilute to volume with water and mix well, centrifuge and filter, and take the filtrate as the blank excipient solution.

[0149] B. Single impurity T localization solution (0.7 μg / mL): Accurately measure impurity T reference standard, dissolve and dilute with acidic acetonitrile to 0.7 μg / mL.

[0150] 2. Testing

[0151] In this embodiment, mobile phase A was 0.2% TFA aqueous solution, mobile phase B was acetonitrile, and all other parameters were the same as in Example 1. The blank excipient solution and the single impurity T localization solution were tested, and the results are shown in [Figure 1]. Figures 18-19 , Figure 18 This is the high-performance liquid chromatogram of the blank excipient. Figure 19 The image shows the high-performance liquid chromatogram of a single impurity T-targeted solution. The results indicate that when acetonitrile and 0.2% trifluoroacetic acid solution (TFA) are used as the mobile phase, the excipient peak in the blank excipient (…) Figure 18 (27.036 min) in impurity T ( Figure 19 The peak appears near 26 min (26.732 min), which will interfere with the detection of impurity T.

[0152] In summary, the method of the present invention provides faster and more accurate detection of mometasone furoate nasal spray content, related substances, and benzalkonium chloride, and has strong anti-interference capabilities.

[0153] The following is the verification process of the method of the present invention.

[0154] System Applicability

[0155] Detection method: Blank solution, limit of quantitation solution, and system suitability solution were injected into the liquid chromatograph under the same chromatographic conditions as in Example 1 to test system suitability. Results showed no interference from the blank solution; the signal-to-noise ratio of the limit of quantitation solution was 23, greater than 10; the resolution of mometasone furoate and mometasone furoate impurity D in the system suitability solution was 7.81, greater than 2.0. All system suitability results met the requirements.

[0156] <Method Specificity> (Study on known impurities)

[0157] Impurity mixture (1%): Accurately measure an appropriate amount of each impurity stock solution and dilute with acidic acetonitrile to a concentration of 0.7 μg / mL for each impurity reference standard;

[0158] 0.1% Impurity Addition Solution: Accurately weigh an appropriate amount of mometasone furoate nasal spray, equivalent to 3.5 mg mometasone furoate, place it in a 50 mL volumetric flask, add 5 mL of impurity mixed solution (1%), add 20 mL of acidic acetonitrile, shake for 15 minutes, sonicate for 5 minutes, add 10 mL of water, let it cool to room temperature, dilute with water to the mark, shake well and centrifuge, take the supernatant and filter it through a 0.45 μm PTFE filter, discard 2 mL of the initial filtrate, and take the subsequent filtrate.

[0159] Detection: Inject 0.1% impurity spiking solution under the same chromatographic conditions as in Example 1 (i.e., replace the test solution in Example 1 with 0.1% impurity spiking solution). Record the relative retention time, resolution of each impurity, purity angle, and purity threshold. The results are shown in Table 5 and... Figure 20 ;

[0160] Table 5. Test results of 0.1% impurity sample solution.

[0161]

[0162]

[0163] As shown in Table 5, the minimum resolution between each impurity and its adjacent peak in the 0.1% impurity solution was 1.32, which is greater than 1.2; the minimum resolution between the main peak of mometasone furoate and its adjacent peak was 3.22, which is greater than 1.5; the purity angles of each impurity were all less than the purity threshold, indicating spectral purity. The preservative benzalkonium chloride peaks (at approximately 31 min and 38 min) showed good resolution with no interference from other impurities.

[0164] <Destructive Testing>

[0165] 1. Solution preparation

[0166] Test solution: Accurately weigh an appropriate amount of mometasone furoate nasal spray, equivalent to 3.5 mg of mometasone furoate, place it in a 50 mL volumetric flask, add 25 mL of acidic acetonitrile, shake for 15 minutes, sonicate for 5 minutes, add 10 mL of water, let it cool to room temperature, dilute with water to the mark, shake well, centrifuge, and filter the supernatant as the test solution.

[0167] Undegraded test solution: Accurately weigh an appropriate amount of mometasone furoate nasal spray, equivalent to 3.5 mg mometasone furoate, place it in a 50 mL volumetric flask, add 25 mL of acidic acetonitrile, shake for 15 minutes, sonicate for 5 minutes, dilute with water to the mark, shake well, centrifuge, take the supernatant and filter it through a 0.45 μm PTFE filter membrane, discard 2 mL of the initial filtrate, and take the subsequent filtrate.

[0168] Acid degradation test solution: Accurately weigh an appropriate amount of mometasone furoate nasal spray, equivalent to 3.5 mg mometasone furoate, place it in a 50 mL volumetric flask, add 5 mL of 0.1 mol / L hydrochloric acid solution, shake well, let stand for 24 hours, add 5 mL of 0.1 mol / L sodium hydroxide solution for neutralization, shake well, let stand for 5 minutes, add 25 mL of acidic acetonitrile, shake for 15 minutes, sonicate for 5 minutes, dilute with water to the mark, shake well, centrifuge, take the supernatant and filter it through a 0.45 μm PTFE filter membrane, discard 2 mL of the initial filtrate, and take the subsequent filtrate.

[0169] Alkaline degradation of the test solution: Accurately weigh an appropriate amount of mometasone furoate nasal spray, equivalent to 3.5 mg mometasone furoate, place it in a 50 mL volumetric flask, add 3 mL of 0.1 mol / L sodium hydroxide solution, shake well, let stand for 3 hours, add 3 mL of 0.1 mol / L hydrochloric acid solution for neutralization, shake well, let stand for 5 minutes, add 25 mL of acidic acetonitrile, shake for 15 minutes, sonicate for 5 minutes, dilute with water to the mark, shake well, centrifuge, take the supernatant and filter it through a 0.45 μm PTFE filter membrane, discard 2 mL of the initial filtrate, and take the subsequent filtrate.

[0170] Oxidative degradation test solution: Accurately weigh an appropriate amount of mometasone furoate nasal spray, equivalent to 3.5 mg mometasone furoate, place it in a 50 mL volumetric flask, add 1 mL of 30% hydrogen peroxide solution, shake well, let stand for 24 hours, add 25 mL of acidic acetonitrile, shake for 15 minutes, sonicate for 5 minutes, add 10 mL of water, shake well, let stand at room temperature, dilute with water to the mark, shake well, centrifuge, take the supernatant and filter it through a 0.45 μm PTFE filter membrane, discard 2 mL of the initial filtrate, and take the subsequent filtrate.

[0171] Photodegradation of the test solution: Take an appropriate amount of the contents of mometasone furoate nasal spray, place it in a transparent glass bottle and expose it to light (4500±500 lux) for 5 days. After exposure to light, shake it thoroughly. Take an appropriate amount of the solution, equivalent to 3.5 mg of mometasone furoate, accurately weigh it, place it in a 50 mL volumetric flask, add 25 mL of acidic acetonitrile, shake for 15 minutes, sonicate for 5 minutes, add 10 mL of water, let it cool to room temperature, dilute with water to the mark, shake well, centrifuge, take the supernatant and filter it through a 0.45 μm PTFE filter membrane, discard 2 mL of the initial filtrate, and take the subsequent filtrate.

[0172] High-temperature degradation of the test solution: Take mometasone furoate nasal spray, place it at 60℃ for 5 days, shake thoroughly, take an appropriate amount of the solution, equivalent to 3.5 mg mometasone furoate, accurately weigh it, place it in a 50 mL volumetric flask, add 25 mL of acidic acetonitrile, shake for 15 minutes, sonicate for 5 minutes, add 10 mL of water, let it cool to room temperature, dilute with water to the mark, shake well, centrifuge, take the supernatant and filter it through a 0.45 μm PTFE filter membrane, discard 2 mL of the initial filtrate, and take the subsequent filtrate.

[0173] 2. Sample Determination: Determine according to the test method, replacing the test solution in the test method with the degradation test solutions mentioned above. Record the chromatographic data of each degradation solution. The results are shown in Tables 6 and 7. The sum of the total impurities and content of the degradation solutions under each condition should be between 90% and 110% of that of the undegraded test solution.

[0174] Table 6 Chromatographic data of undegraded, acid-degraded, and alkali-degraded test sample solutions

[0175]

[0176]

[0177] Table 7 Chromatographic data of test sample solutions for oxidative degradation, high-temperature degradation, and photodegradation.

[0178]

[0179] Note: "ND" means Not Detected

[0180] 3. Results Analysis

[0181] As shown in Tables 6 and 7, the minimum resolution between mometasone furoate and degradation impurities in the degradation solutions of the test samples under each condition was 3.89, which is greater than 1.5. The minimum resolution between known impurities H, G, F, C, and D (impurities that need to be controlled) and other degradation impurities was 2.80, which is greater than 1.2. The total impurities and their contents in the degradation solutions of the test samples under each condition were all between 90% and 110% of those in the undegraded test sample solutions, which meets the standard.

[0182] <Detection of Unknown Impurities in Test Sample Solution Due to Photodegradation>

[0183] 1. Sample: Take the aforementioned photodegradation test solution and perform testing according to the test method (simply replace the test solution in the test method with the photodegradation test solution) and the imported registration standard method, respectively. The chromatogram obtained according to the test method is shown in the figure. Figure 20 The chromatogram obtained according to the import registration standard method is shown below. Figure 2 .

[0184] 2. Results Analysis

[0185] according to Figure 20 as well as Figure 2 The impurity content (area%) was determined. Figure 20 Unknown impurities at a retention time of approximately 37 min and Figure 2 The unknown impurity at a retention time of approximately 51 min is the same substance, because Figure 2The detection method used in this invention is based on the imported registration standard, which requires elution on the third injection after sample injection. Therefore, when using the imported registration standard to degrade the test solution by light, it takes a total of 171 minutes (60 minutes + 60 minutes + 51 minutes) for the sample to be eluted. However, the detection method of this invention can elute the sample in about 37 minutes. Therefore, the detection method of this invention has better impurity detection capability and higher detection efficiency.

[0186] <Determination of Limit of Quantitation and Limit of Detection>

[0187] Method: Accurately weigh an appropriate amount of mometasone furoate reference standard, dissolve and dilute it with acidic acetonitrile to prepare a stock solution. Dilute stepwise with diluent to prepare solutions of different concentrations.

[0188] The limit of detection (S / N≥10) and limit of quantitation (S / N≥3) of mometasone furoate were determined. The limit of quantitation solution was injected continuously for 6 injections, the peak area was measured, and the relative standard deviation of the peak area was calculated. The results of the limit of quantitation detection are shown in Table 8, and the results of the limit of detection are shown in Table 9.

[0189] Table 8 Results of Limit of Quantitation Detection

[0190]

[0191]

[0192] Table 9 Detection Limit Results

[0193]

[0194] Results analysis: The limits of quantitation (LOQ) and detection (LOD) concentrations were 0.0142 μg / mL and 0.0071 μg / mL, respectively, equivalent to 0.02% and 0.01% of the test solution. The final test solution concentration of 0.05% was used as the routine LQ solution: 0.035 μg / mL. Compared to the imported registered standard method with a LQ of 0.035 μg / mL and a LOD of 0.014 μg / mL, this invention exhibits higher sensitivity and a lower LQ.

[0195] <Impurity linearity>

[0196] Prepare a linear solution with a concentration of 0.014 μg / mL to 105 μg / mL, equivalent to 0.02% to 150% of the test solution, using mometasone furoate reference standard.

[0197] The limits for known impurities H, G, F, C, and D are 0.1% (0.07 μg / mL), and the concentration range of the impurities to be prepared is 0.014 μg / mL to 0.28 μg / mL, which is equivalent to 20% to 400% of the known impurity limits.

[0198] According to the test method, the above linear solutions were tested respectively, the chromatographic data were recorded and analyzed, and the results are shown in Table 10; according to the test method, the above known impurity solutions were tested respectively, the chromatographic data were recorded and analyzed, and the results are shown in Table 11.

[0199] Table 10 Results of linear solution chromatographic data analysis

[0200]

[0201] Table 11. Chromatographic data analysis results for each known impurity.

[0202]

[0203]

[0204] <Precision>

[0205] 1. Repeatability test

[0206] Take mometasone furoate nasal spray, determine its content, impurities and benzalkonium chloride, repeat the determination 6 times, and calculate the results. The results are shown in Tables 12 to 14.

[0207] Table 12 Content repeatability results

[0208]

[0209] Table 13 Reproducibility Results of Related Substances

[0210]

[0211] Table 14 Repeatability Results of Benzalkonium Chloride

[0212]

[0213] 2. Intermediate precision test

[0214] Impurities were measured by different personnel at different times using different instruments. The results are shown in Tables 15 to 17.

[0215] Table 15 Results of intermediate precision content

[0216]

[0217] Table 16 Results related to intermediate precision

[0218]

[0219] Table 17 Results of intermediate precision benzalkonium chloride content

[0220]

[0221] Tables 15-17 show that the average content of mometasone furoate in the 6 test solutions was 97.1%, with an RSD of 0.3% and no greater than 2.0%. The average content of mometasone furoate in the 12 samples was 97.0%, with an RSD of 0.3% and no greater than 2.0%, which meets the acceptable standard.

[0222] Under the robustness test conditions, the absolute value of the change in the content of each impurity in the test solution, compared with the average value of the repeatability results, does not exceed 0.02%, and the absolute value of the change in the total impurity content does not exceed 0.02%, which meets the acceptable standard.

[0223] The benzalkonium chloride content in the six test solutions ranged from 0.0180% to 0.0220%; the average value of the six solutions was 0.02061%, with an RSD of 0.3%, which is less than 2.0%. The combined repeatability of the 12 test solutions showed an average content ranging from 0.0180% to 0.0220%, with an RSD (n=12) of 0.4%, which is less than 2.0%, meeting the acceptable standard.

[0224] <Durability>

[0225] Take an appropriate amount of mometasone furoate nasal spray and determine the impurities. The impurity peaks should be separated from the baselines of adjacent peaks. Under the robustness test conditions, compared with the average value of the repeatability results, the absolute value of the change in the content of each impurity in the test solution should not exceed 0.05%, and the absolute value of the change in the total impurity content should not exceed 0.05%. The robustness test conditions include different column temperatures (38℃ and 42℃), different flow rates (0.8 mL / min and 1.0 mL / min), and different detection wavelengths (252 nm and 256 nm).

[0226] The detection method was as follows: 10 μl each of blank solution, limit of quantitation solution, and resolution solution were injected into the liquid chromatograph and detected under the above-mentioned robustness test conditions. The chromatograms were recorded and analyzed. The results are shown in Table 18.

[0227] Table 18 Statistical Analysis of Durability Test Results

[0228]

[0229] The results showed that the absolute value of the change in the content of each impurity did not exceed 0.05%, and the absolute value of the change in the total impurity content did not exceed 0.05%.

[0230] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and embodiments shown and described herein.

Claims

1. A method for detecting related substances in mometasone furoate nasal spray, using high performance liquid chromatography, characterized in that, The chromatographic column filled with octadecylsilane bonded silica gel is selected, 0.02 mol / L sodium perchlorate is used as mobile phase A, acetonitrile is used as mobile phase B, gradient elution is carried out, the flow rate is 0.8-1.2 mL / min, the detection wavelength is 240 nm-260 nm, the column temperature is 30-50 DEG C, and the injection amount is 10-25 muL; The parameters of the gradient elution are as follows: at 0 min, mobile phase A: mobile phase B = 58%:42%, at 10 min, mobile phase A: mobile phase B = 48%:52%, at 25 min, mobile phase A: mobile phase B = 48%:52%, at 40 min, mobile phase A: mobile phase B = 20%:80%, at 41 min, mobile phase A: mobile phase B = 58%:42%, at 45 min, mobile phase A: mobile phase B = 58%:42%; The preparation of the mobile phase A is as follows: 2.8 g of sodium perchlorate monohydrate is weighed, water is added to 1000 mL, and then shaken, and the pH is adjusted to 3.0 with perchloric acid; The related substances are impurity D, impurity K, impurity L, impurity M, impurity H, impurity O, impurity Q, impurity G, impurity P, impurity F, impurity C, impurity A, impurity E and impurity T.

2. The method of detecting related substances of mometasone fruroate nasal spray according to claim 1, wherein, The flow rate is 0.8-1.0 mL / min, the detection wavelength is 252 nm-256 nm, the column temperature is 38-42 DEG C, and the injection amount is 10-20 muL.

3. The method of detecting related substances of mometasone fruroate nasal spray according to claim 2, wherein, The flow rate is 0.9 mL / min, the detection wavelength is 254 nm, the column temperature is 40 DEG C, and the injection amount is 10 muL.

4. The method of claim 1, wherein the method is for the determination of related substances of mometasone furoate nasal spray. The specification of the chromatographic column is 4.6*150 mm, and the particle size of the filler is 2.7 mu m.

5. The method for detecting related substances in mometasone furoate nasal spray as described in claim 1, characterized in that, The test is used for detecting the light degradation of mometasone furoate nasal spray test sample solution.

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