A method for detecting the content of active ingredients in nemonoxacin malate capsules

Abstract

The present invention discloses a method for detecting the content of an active ingredient in nemonofloxacin malate capsules, relating to the technical field of drug detection and analysis. The method comprises the following steps: detecting the content of the active ingredient in the nemonofloxacin malate capsules using high performance liquid chromatography. The present invention provides, for the first time, a method for detecting the content of an active ingredient in nemonofloxacin malate capsules. The inventors have discovered that selecting a specific chromatographic column and a specific mobile phase for isocratic elution can effectively avoid interference from non-active ingredients such as impurities and excipients in the nemonofloxacin malate capsules, thereby improving the accuracy of detecting the content of the active ingredient in the nemonofloxacin malate capsules and achieving accurate quantification of the content of the nemonofloxacin malate capsules, thus filling a gap in the prior art.

Description

Technical Field

[0001] The present invention relates to the technical field of drug detection and analysis, and in particular to a method for detecting the content of active ingredients in nemonoxacin malate capsules. Background Art

[0002] Nemonofloxacin malate capsules, whose active ingredient, nemonofloxacin, has the chemical structure shown in Formula (I), are a broad-spectrum antibacterial drug primarily used to treat respiratory tract infections (such as pneumonia and bronchitis), urinary tract infections (such as cystitis and pyelonephritis), and skin and soft tissue infections caused by sensitive bacteria. They have a wide range of uses.

[0003] The formula (I): .

[0004] As a core link in drug quality control, drug content control plays a decisive role in ensuring the safety and effectiveness of drugs. In the entire process of drug research and development, production, and supervision, accurate measurement of drug content can ensure that the dosage of the active ingredient of the drug meets the prescribed standards, thereby ensuring that it can achieve the expected therapeutic effect in clinical applications, while reducing the safety risks caused by dosage deviations. However, in public technical literature and industry reports, no effective method for content detection of nemonoxacin malate capsules has been found. This technical gap not only poses a challenge to the quality control of nemonoxacin malate capsules, but also limits the accuracy and reliability of this drug in clinical applications.

[0005] Given the shortcomings of existing technologies, it is imperative to develop a new method for determining the content of nemonofloxacin malate capsules. This new method must be efficient, convenient, and accurate, enabling rapid and precise determination of nemonofloxacin malate content in capsules. This method would provide scientific and reliable technical support for drug quality control, thereby ensuring the stable quality of nemonofloxacin malate capsules and safeguarding patient safety and efficacy. Summary of the Invention

[0006] In order to solve the above problems, the present invention provides a method for detecting the content of active ingredients in Nemonofloxacin Malate Capsules.

[0007] The present invention provides a method for detecting the content of an active ingredient in nemofloxacin malate capsules, the method comprising the following steps:

[0008] High performance liquid chromatography was used to detect the content of the active ingredient in the nemonoxacin malate capsules;

[0009] The conditions of the high performance liquid chromatography method include:

[0010] Column: Inersustain C18, 4.6 × 150 mm, 5 μm or Inertsustain C18, 4.6 × 125 mm, 5 μm;

[0011] Mobile phase: The mobile phase is composed of triethylamine phosphoric acid aqueous solution and acetonitrile in a volume ratio of (74-78): (22-26); the volume fractions of triethylamine and phosphoric acid in the triethylamine phosphoric acid aqueous solution are both 0.3-0.6 vol%.

[0012] Elution mode: isocratic elution.

[0013] Furthermore, the mobile phase is composed of triethylamine phosphoric acid aqueous solution and acetonitrile in a volume ratio of 76:24.

[0014] Furthermore, the volume fractions of triethylamine and phosphoric acid in the triethylamine phosphoric acid aqueous solution are both 0.5 vol%.

[0015] Furthermore, the conditions of the high performance liquid chromatography method also include:

[0016] Detection wavelength: 252~256 nm;

[0017] Column temperature: 28~32℃;

[0018] Flow rate: 0.9~1.1 ml / min;

[0019] Injection volume: 3~8μl;

[0020] Diluent: composed of the triethylamine phosphoric acid aqueous solution and acetonitrile in a volume ratio of (74~78): (22~26).

[0021] Furthermore, the detection wavelength is 254 nm;

[0022] and / or, the column temperature: 30°C;

[0023] and / or, the flow rate: 1.0 ml / min;

[0024] And / or, the injection volume: 5 μl;

[0025] And / or, the diluent is composed of the triethylamine phosphoric acid aqueous solution and acetonitrile in a volume ratio of 76:24.

[0026] Furthermore, when preparing the solution, the solvent used is consistent with the mobile phase.

[0027] Furthermore, the solution includes a test solution and a reference solution.

[0028] Furthermore, the test results meet the following requirements: the separation between the main peak of the active ingredient and the adjacent impurity peaks is greater than 1.5, each impurity peak does not interfere with the main peak of the active ingredient, and the tailing factor of the main peak of the active ingredient is ≤1.2;

[0029] The impurities include impurity A, impurity B, impurity C, impurity D and impurity E;

[0030] The chemical structural formula of the impurity A is shown below:

[0031] ;

[0032] The chemical structural formula of the impurity B is as follows:

[0033] ;

[0034] The chemical structural formula of the impurity C is shown below:

[0035] ;

[0036] The chemical structural formula of the impurity D is as follows:

[0037] ;

[0038] The chemical structural formula of the impurity E is shown below:

[0039] .

[0040] Furthermore, the theoretical plate number of the main peak of the active ingredient in the chromatogram of the test solution is not less than 2000.

[0041] The above technical solution provided by the embodiment of the present invention has at least the following advantages compared with the prior art:

[0042] The embodiments of the present invention provide a method for detecting the content of an active ingredient in nemonofloxacin malate capsules. The present invention provides a method for detecting the content of an active ingredient in nemonofloxacin malate capsules for the first time. The inventors have found that selecting a specific chromatographic column and a specific mobile phase for isocratic elution can effectively avoid interference from excipients and impurities, excipients, and other non-active ingredients in the nemonofloxacin malate capsules, thereby improving the accuracy of detecting the content of the active ingredient in the nemonofloxacin malate capsules and achieving accurate quantification of the content of the nemonofloxacin malate capsules, thus filling a gap in the prior art.

[0043] Specifically, the beneficial effects of the present invention are embodied in the following aspects:

[0044] 1. High innovation: The present invention innovatively provides a method for detecting the content of active ingredients in Nemonofloxacin Malate Capsules for the first time, filling the gap in the prior art.

[0045] 2. Strong specificity: The detection method of the present invention can accurately detect the content of nemonoxacin malate capsules. Neither the diluent nor the blank excipient interferes with the detection of the main component. In the spiked test solution, the separation between the active ingredient and the adjacent peak is greater than 1.5.

[0046] 3. Short analysis time and low cost: The present invention optimizes the mobile phase composition and chromatographic conditions to achieve effective separation of the main peak and impurities in a relatively short time, greatly reducing chromatographic column consumption and labor costs, and saving social resources.

[0047] 4. Good precision and high accuracy: The present invention has good precision and accuracy through multiple batches of data and verification results, which provides reliable technical support for the detection of the content of nemonoxacin malate capsules and the quality control of nemonoxacin malate capsules.

[0048] 5. Suitable for commercial production: The detection method of the present invention is not only accurate and reliable, but also easy to operate and low in cost, making it very suitable for quality control needs in commercial production.

[0049] In summary, the present invention innovatively provides a method for detecting the content of the active ingredient in nemonofloxacin malate capsules for the first time, which avoids the interference of blank excipients and nemonofloxacin malate impurities in the accurate quantification of nemonofloxacin malate, solves the problem of no method for determining the content of nemonofloxacin malate capsules, and has strong specificity, good precision, and high accuracy. It can be applied to the quality control of nemonofloxacin malate capsules. The conditions have the characteristics of good stability, simplicity, high efficiency, and low cost during the application process, and are more suitable for product content control in commercial production. Therefore, the present invention has significant beneficial effects and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0051] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0052] Figure 1 This is a chromatogram of the method for detecting the content of the active ingredient in Nemonoxacin Malate Capsules in Example 1 of the present invention;

[0053] Figure 2 This is a chromatogram of the method for detecting the content of the active ingredient in Nemonoxacin Malate Capsules in Example 2 of the present invention;

[0054] Figure 3 This is a chromatogram of the method for detecting the content of the active ingredient in Nemonoxacin Malate Capsules in Example 3 of the present invention;

[0055] Figure 4 This is a chromatogram of the method for detecting the content of the active ingredient in Nemofloxacin Malate Capsules in Comparative Example 1 of the present invention;

[0056] Figure 5 This is a chromatogram of the method for detecting the content of the active ingredient in Nemonoxacin Malate Capsules in Comparative Example 2 of the present invention;

[0057] Figure 6 This is a chromatogram of the method for detecting the content of the active ingredient in Nemonoxacin Malate Capsules in Comparative Example 3 of the present invention;

[0058] Figure 7 The chromatogram is a method for detecting the content of the active ingredient in Nemonoxacin Malate Capsules in Comparative Example 4 of the present invention. DETAILED DESCRIPTION

[0059] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0060] Unless otherwise specified, all raw materials, reagents, instruments, and equipment used in the present invention can be purchased commercially or prepared by existing methods. Furthermore, unless otherwise specified or specified, the steps and parameters involved can be performed according to existing high-performance liquid chromatography methods or directly using existing equipment, and are not described in detail in this document.

[0061] The technical solutions provided by the present invention are as follows:

[0062] The present invention provides a method for detecting the content of an active ingredient in nemofloxacin malate capsules, the method comprising the following steps:

[0063] High performance liquid chromatography was used to detect the content of the active ingredient in the nemonoxacin malate capsules;

[0064] The conditions of the high performance liquid chromatography method include:

[0065] Column: Inersustain C18, 4.6 × 150 mm, 5 μm or Inertsustain C18, 4.6 × 125 mm, 5 μm;

[0066] Mobile phase: The mobile phase is composed of triethylamine phosphoric acid aqueous solution and acetonitrile in a volume ratio of (74-78): (22-26); the volume fractions of triethylamine and phosphoric acid in the triethylamine phosphoric acid aqueous solution are both 0.3-0.6 vol%.

[0067] Elution mode: isocratic elution.

[0068] Compared with the existing technologies such as CN119985822A - A method for detecting diastereomers in nemonoxacin malate sodium chloride injection, the technical solution provided by the present invention has at least the following significant differences:

[0069] 1) The detection objects of the technical solutions provided by the present invention and the prior art are different; a comparative analysis is provided using CN119985822A as an example.

[0070] The detection object of CN119985822A is the diastereomers in nemonofloxacin malate sodium chloride injection, aiming to focus on the separation and detection of chiral impurities (such as (3S, 5R) and (3R, 5S) type isomers); while the detection object of the present invention is nemonofloxacin, the active ingredient in nemonofloxacin malate capsules, aiming to focus on the content accuracy of nemonofloxacin, the active ingredient in nemonofloxacin malate capsules, and it is necessary to eliminate the interference of excipients (such as starch, magnesium stearate) and degradation impurities, filling the gap in the existing technology.

[0071] Furthermore, the capsules tested in the present invention are solid dosage forms, and the complexity of the matrix presents three major technical challenges:

[0072] 1. Interference of excipients: The capsules contain polar excipients such as hydroxypropyl cellulose, lactose, and silicon dioxide. The extract contains polar excipients and pH changes, which cause peak changes. The present invention solves the interference caused by matrix effect by screening diluents and mobile phases.

[0073] 2. Extraction efficiency and accuracy: Capsules are affected by excipients, impurities, and filler particles. Pre-processing, including dissolution, extraction, and filtration, is required. Solvent selection and extraction method selection are also crucial. Effective dissolution is essential for accurate detection. However, extraction is not required in CN119985822A.

[0074] 3. Solution stability: The pH of the capsule extract fluctuates due to excipients. The present invention stabilizes the pH by controlling the pH of the mobile phase and diluent, so that the reference solution remains stable for 5 months and the test solution remains stable for more than 48 hours, laying the foundation for the accurate detection of the active ingredient content in nemonoxacin malate capsules.

[0075] Furthermore, the technical problems and solutions in the technical solutions of CN119985822A and the present invention belong to different technical branches and have different technical requirements. In pursuit of isomer separation, CN119985822A sacrifices detection efficiency, cost, accuracy, and repeatability, resulting in costs and environmental impacts such as a long analysis time of 15 minutes, a complex mobile phase, and the use of the highly toxic reagent tetrahydrofuran. To address rapid quality control in industrial production, the present invention balances resolution (typically >1.5 is sufficient), detection efficiency (<8 minutes), cost (the organic phase ratio is only half that of CN119985822A), environmental pollution (no tetrahydrofuran), accuracy, and precision. This difference in technical objectives results in the two methods belonging to different technical branches: CN119985822A belongs to the "impurity control field," while the present invention belongs to the "content determination field," and they are not directly comparable.

[0076] 2) The core key technical means of the technical solutions provided by the present invention are different from those provided by the prior art; a comparative analysis is provided using CN119985822A as an example.

[0077] The HPLC conditions adopted in the present invention include "a chromatographic column using an Inersustain C18 (4.6×150 mm or 4.6×125 mm, 5 μm) and a mobile phase consisting of a triethylamine phosphoric acid aqueous solution and acetonitrile in a volume ratio of (74-78):(22-26); the volume fractions of triethylamine and phosphoric acid in the triethylamine phosphoric acid aqueous solution are both 0.3-0.6 vol%." However, in CN119985822A, "a chromatographic column using a Welch Ultimate LP-C18 (4.6×150 mm, 5 μm) and a mobile phase consisting of a 0.1% trifluoroacetic acid aqueous solution and methanol-tetrahydrofuran (97.5:2.5) in a volume ratio of 55:45" are used. Therefore, the present invention differs from CN119985822A in core key technical means such as the chromatographic column and mobile phase, and accurate detection of the active ingredient content in nemonoxacin malate capsules cannot be achieved without adopting the core key means defined in the present invention.

[0078] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples where specific conditions are not specified are generally measured in accordance with national standards. If there are no corresponding national standards, then the methods are carried out in accordance with general international standards, conventional conditions, or the conditions recommended by the manufacturer.

[0079] Nemonofloxacin malate capsules involved in the following examples and comparative examples were purchased from Xinchang Pharmaceutical Factory of Zhejiang Medicine Co., Ltd., and their theoretical content should be 90.0% to 110.0% of the labeled amount.

[0080] The calculation method of the number of theoretical plates involved in the following examples and comparative examples is as follows: N in the United States Pharmacopoeia (USP) 2 (N is the number of theoretical plates, is the retention time of nemonoxacin, and w is the half-peak width of the nemonoxacin peak).

[0081] Example 1

[0082] This example provides a method for detecting the content of the active ingredient in nemonofloxacin malate capsules, which comprises the following steps:

[0083] Step (1), prepare solution:

[0084] Step (1.1), prepare triethylamine phosphoric acid solution: take 5 ml of triethylamine and 5 ml of phosphoric acid, dilute to 1000 ml with water, and obtain a triethylamine phosphoric acid solution with a volume fraction of 0.5 vol% of triethylamine and phosphoric acid respectively;

[0085] Step (1.2), prepare the diluent: mix the triethylamine phosphoric acid aqueous solution obtained in the above (1.1) and acetonitrile in a volume ratio of 76:24;

[0086] Step (1.3), prepare the test solution: take an appropriate amount of the contents of nemonoxacin malate capsules, accurately weigh them, add an appropriate amount of diluent (the specific composition of the diluent should be consistent with the subsequent mobile phase), sonicate (ultrasonic power 500W, ultrasonic frequency 50Hz) for 3 minutes to dissolve, quantitatively dilute with diluent to make a solution containing 0.2 mg of the active ingredient nemonoxacin per 1 ml, and filter;

[0087] Step (1.4), prepare reference solution: take an appropriate amount of nemonoxacin malate reference substance, accurately weigh it, dissolve it in a solvent (the specific composition of the diluent should be consistent with the subsequent mobile phase), and quantitatively dilute it to make a solution containing 0.2 mg of nemonoxacin per 1 ml;

[0088] Step (2), using high performance liquid chromatography to detect the content of the nemonoxacin malate capsules:

[0089] Step (2.1), HPLC conditions:

[0090] Mobile phase: composed of the triethylamine phosphoric acid solution obtained in (1.1) above and acetonitrile in a volume ratio of 76:24.

[0091] Chromatographic column: Inersustain C18 4.6×150mm, 5μm;

[0092] Detection wavelength: 254nm;

[0093] Column temperature: 30 °C;

[0094] Flow rate: 1.0 ml / min;

[0095] Injection volume: 5 μl;

[0096] Step (2.2), detection operation: Measure the test solution and reference solution, inject them into the liquid chromatograph respectively, detect according to the isocratic elution procedure, and record the chromatogram.

[0097] The typical chromatogram obtained in this example is the chromatogram of the test solution. Figure 1 As shown by Figure 1 It can be seen that the separation between the main peak of the active ingredient and the adjacent impurity peak is greater than 1.5, and the impurity peaks, diluents, blank excipients and other non-active ingredients (non-active ingredients all peak before 3.5 minutes) do not interfere with the main peak of the active ingredient; the theoretical plate number of the main peak of the active ingredient in the chromatogram of the test solution is 5920; and the tailing factor of the nemonoxacin peak is 1.2, and the content detection result (calculated by the external standard method, the calculation formula is as follows) is 100.7%, which is consistent with the theoretical feeding result, and the content detection accuracy is high; the RSD (relative standard deviation) of 6 copies is 0.15%, and the repeatability is good; among them,

[0098] External standard method calculation formula

[0099] %;

[0100] Where:

[0101] C 对 is the concentration of the reference solution, mg / ml;

[0102] A 对 is the main peak area of ​​the reference solution;

[0103] A 样 is the main peak area in the test solution;

[0104] V 供 is the dilution factor of the test solution;

[0105] W 供 is the sample weight of the test product, mg;

[0106] The average filling quantity is the average filling quantity of 20 capsules, mg;

[0107] 250 means the labeled amount is 250 mg.

[0108] Example 2

[0109] This example provides a method for detecting the content of the active ingredient in nemonofloxacin malate capsules, which comprises the following steps:

[0110] Step (1), prepare solution:

[0111] Step (1.1), preparing a triethylamine phosphoric acid solution: taking 3 ml of triethylamine and 3 ml of phosphoric acid, diluting to 1000 ml with water to obtain the triethylamine phosphoric acid solution in which the volume fractions of triethylamine and phosphoric acid are both 0.3 vol%;

[0112] Step (1.2), prepare the diluent: mix the triethylamine phosphoric acid aqueous solution obtained in (1.1) above and acetonitrile in a volume ratio of 74:26;

[0113] Step (1.3), prepare the test solution: take an appropriate amount of the contents of nemonoxacin malate capsules, accurately weigh them, add an appropriate amount of diluent (the specific composition of the diluent should be consistent with the subsequent mobile phase), sonicate (ultrasonic power 500W, ultrasonic frequency 50Hz) for 3 minutes to dissolve, quantitatively dilute with diluent to make a solution containing 0.2 mg of the active ingredient nemonoxacin per 1 ml, and filter;

[0114] Step (1.4), prepare reference solution: take an appropriate amount of nemonoxacin malate reference substance, accurately weigh it, add diluent (the specific composition of the diluent should be consistent with the subsequent mobile phase), dissolve it, and quantitatively dilute it to make a solution containing 0.2 mg of nemonoxacin per 1 ml;

[0115] Step (2), using high performance liquid chromatography to detect the content of the nemonoxacin malate capsules:

[0116] Step (2.1), HPLC conditions:

[0117] Mobile phase: The mobile phase is composed of the triethylamine phosphoric acid solution obtained in (1.1) above and acetonitrile in a volume ratio of 74:26.

[0118] Column: Inertsustain C18, 4.6 × 125 mm, 5 μm;

[0119] Detection wavelength: 252nm;

[0120] Column temperature: 28°C;

[0121] Flow rate: 0.9 ml / min;

[0122] Injection volume: 3ul.

[0123] The typical chromatogram obtained in this example is the chromatogram of the test solution. Figure 2 As shown by Figure 2It can be seen that the separation between the main peak of the active ingredient and the adjacent impurity peaks is greater than 1.5, and the impurity peaks, diluents, blank excipients and other non-active ingredients (non-active ingredients all peak before 3.5 minutes) do not interfere with the main peak of the active ingredient; in the chromatogram of the test solution, the theoretical plate number is 5969 calculated based on nemonoxacin; and the tailing factor of the nemonoxacin peak is 1.2, the content detection result is 99.4%, which is consistent with the theoretical feeding result, and the content detection accuracy is high; RD (relative deviation) is 0.04%, and the repeatability is good.

[0124] Example 3

[0125] This example provides a method for detecting the content of the active ingredient in nemonofloxacin malate capsules, which comprises the following steps:

[0126] Step (1), prepare solution:

[0127] Step (1.1), preparing a triethylamine phosphoric acid solution: taking 6 ml of triethylamine and 6 ml of phosphoric acid, diluting to 1000 ml with water to obtain the triethylamine phosphoric acid solution in which the volume fractions of triethylamine and phosphoric acid are both 0.6 vol%;

[0128] Step (1.2), prepare the diluent: mix the triethylamine phosphoric acid aqueous solution obtained in the above (1.1) and acetonitrile in a volume ratio of 78:22;

[0129] Step (1.3), prepare the test solution: take an appropriate amount of the contents of nemonoxacin malate capsules, accurately weigh them, add an appropriate amount of diluent (the specific composition of the diluent should be consistent with the subsequent mobile phase), sonicate (ultrasonic power 500W, ultrasonic frequency 50Hz) for 3 minutes to dissolve, quantitatively dilute with diluent to make a solution containing 0.2 mg of the active ingredient nemonoxacin per 1 ml, and filter;

[0130] Step (1.4), prepare reference solution: take an appropriate amount of nemonoxacin malate reference substance, accurately weigh it, add diluent (the specific composition of the diluent should be consistent with the subsequent mobile phase), dissolve it, and quantitatively dilute it to make a solution containing 0.2 mg of nemonoxacin per 1 ml;

[0131] Step (2), using high performance liquid chromatography to detect the content of the nemonoxacin malate capsules:

[0132] Step (2.1), HPLC conditions:

[0133] Mobile phase: The mobile phase is composed of the triethylamine phosphoric acid solution obtained in the above (1.1) and acetonitrile in a volume ratio of 78:22.

[0134] Column: Inersustain C18, 4.6 × 150 mm, 5 μm;

[0135] Detection wavelength: 256nm;

[0136] Column temperature: 32°C;

[0137] Flow rate: 1.1 ml / min;

[0138] Injection volume: 8 μl.

[0139] The typical chromatogram obtained in this example is the chromatogram of the test solution. Figure 3 As shown by Figure 3 It can be seen that the separation between the main peak of the active ingredient and the adjacent impurity peak is greater than 1.5, and the non-active ingredients such as the impurity peaks, diluents and blank excipients do not interfere with the main peak of the active ingredient; in the chromatogram of the test solution, the theoretical plate number calculated based on nemonoxacin is 7957; and the tailing factor of the nemonoxacin peak is 1.2, and the content detection result is 100.3%, which is consistent with the theoretical feeding result, and the content detection accuracy is high; RD (relative deviation) is 0.15%, and the repeatability is good.

[0140] Example 4

[0141] This example provides a method for detecting the content of the active ingredient in nemonofloxacin malate capsules, which comprises the following steps:

[0142] Step (1), prepare solution:

[0143] Step (1.1), preparing a triethylamine phosphoric acid solution: taking 5 ml of triethylamine and 5 ml of phosphoric acid, diluting to 1000 ml with water to obtain the triethylamine phosphoric acid solution in which the volume fractions of triethylamine and phosphoric acid are both 0.5 vol%;

[0144] Step (1.2), prepare the diluent: mix the triethylamine phosphoric acid aqueous solution obtained in (1.1) above and acetonitrile in a volume ratio of 76:24;

[0145] Step (1.3), prepare the test solution: take an appropriate amount of the contents of nemonoxacin malate capsules, accurately weigh them, add an appropriate amount of diluent (the specific composition of the diluent should be consistent with the subsequent mobile phase), manually shake for 3 minutes or ultrasonically extract (ultrasonic power: 500W, ultrasonic frequency: 50Hz) for 3-10 minutes to dissolve, quantitatively dilute with diluent to make a solution containing 0.2 mg of the active ingredient nemonoxacin per 1 ml, and filter;

[0146] Step (1.4), prepare reference solution: take an appropriate amount of nemonoxacin malate reference substance, accurately weigh it, add diluent (the specific composition of the diluent should be consistent with the subsequent mobile phase), dissolve it, and quantitatively dilute it to make a solution containing 0.2 mg of nemonoxacin per 1 ml;

[0147] Step (2), using high performance liquid chromatography to detect the content of the nemonoxacin malate capsules:

[0148] Step (2.1), HPLC conditions:

[0149] Mobile phase: The mobile phase is composed of the triethylamine phosphoric acid solution obtained in (1.1) above and acetonitrile in a volume ratio of 76:24.

[0150] Column: Inersustain C18, 4.6 × 150 mm, 5 μm;

[0151] Detection wavelength: 254nm;

[0152] Column temperature: 30°C;

[0153] Flow rate: 1.0 ml / min;

[0154] Injection volume: 5 μl.

[0155] The results obtained from the measurement in this example are shown in Table 1 below.

[0156] Table 1

[0157]

[0158] According to the analysis of the above test results, the content detected by directly shaking the sample is far lower than that by ultrasonic extraction. There is no difference in the content between 3 and 10 minutes of ultrasonic extraction, and the extraction capacity is equivalent. In order to improve the efficiency of sample preparation, ultrasonic 3 minutes is used as the extraction method to meet the detection needs.

[0159] Comparative Example 1

[0160] This example provides a method for detecting the content of the active ingredient in nemonoxacin malate capsules. The only difference from Example 1 is that the chromatographic column is adjusted to Waters DELTA PAK 100A C18, 3.9×150 mm, 5 μm.

[0161] The typical chromatogram obtained in this example is the chromatogram of the test solution. Figure 4 As shown by Figure 4 It can be seen that under the conditions of this chromatographic column, the nemonoxacin peak in the chromatogram of the test solution is severely tailed, with a tailing factor of 4.3.

[0162] Comparative Example 2

[0163] This example provides a method for detecting the content of the active ingredient in nemonoxacin malate capsules. The only difference from Example 1 is that the mobile phase is composed of triethylamine aqueous solution and acetonitrile in a volume ratio of 70:30.

[0164] The typical chromatogram obtained in this example is the chromatogram of the test solution. Figure 5 As shown by Figure 5 It can be seen that under the conditions of this chromatographic column, there is a solvent effect on the elution position of the nemonoxacin peak in the chromatogram of the test solution.

[0165] Comparative Example 3

[0166] This example provides a method for detecting the content of the active ingredient in nemonoxacin malate capsules. The only difference from Example 1 is that the mobile phase is composed of 0.01 mol / L potassium dihydrogen phosphate solution and acetonitrile in a volume ratio of 76:24.

[0167] The typical chromatogram obtained in this example is the chromatogram of the test solution. Figure 6 As shown by Figure 6 It can be seen that under the conditions of this chromatographic column, the chromatogram of the test solution has a tailing peak of nemonoxacin, and the tailing factor is 1.6.

[0168] Comparative Example 4

[0169] This example provides a method for detecting the content of the active ingredient in nemonoxacin malate capsules. The only difference from Example 1 is that the triethylamine phosphoric acid solution is prepared by adjusting the amount of triethylamine and 5 ml of phosphoric acid to 1000 ml with water to obtain the triethylamine phosphoric acid solution.

[0170] The typical chromatogram obtained in this example is the chromatogram of the test solution. Figure 7 As shown by Figure 7 It can be seen that under the conditions of this chromatographic column, the chromatogram of the test solution has a tailing peak of nemonoxacin, and the tailing factor is 1.5.

[0171] Test Case

[0172] In order to verify the scientificity and rationality of the detection method for the content of the active ingredient in the nemonoxacin malate capsules provided by the present invention, this method (taking the detection condition parameters provided in Example 1 as an example) was methodologically validated in accordance with the "Chinese Pharmacopoeia" (2020 edition, Part IV 9101 Drug Quality Standard Analytical Method Validation Guidelines). The validation items included system suitability (injection precision), specificity, quantitative limit and detection limit, accuracy, linearity and range, precision (repeatability and intermediate precision), solution stability, filter membrane and durability, etc. The test results showed that the above test items all met the requirements.

[0173] Some test results are shown below:

[0174] 1) System suitability results: The diluent did not interfere with the detection of the main component. In the chromatogram of the reference solution, the theoretical plate number calculated based on the nemonoxacin peak was greater than 2000, and the tailing factor of the nemonoxacin peak was 1.2. Reference solution 1 was injected continuously for 5 times. The RSD of the retention time of the main component peak was less than 1.0%, and the RSD of the peak area was less than 2.0%. Reference solution 2 was injected continuously for 2 times, and the response factor recovery rate was 99.9% relative to the average response factor of reference solution 1, ranging from 98.0% to 102.0%.

[0175] 2) Linearity and range results: In the concentration range of 0.1313 mg / ml to 0.3938 mg / ml, the peak area of ​​the principal component showed a good linear relationship with the concentration. The linear equation was y = 4,557,741.3277 x + 3,872.6759, the linear correlation coefficient r was 1.0000, and the absolute value of the Y-axis intercept was 0.32% of the 100% limit concentration response value. These results indicate that this method has a good linear relationship between peak area and concentration.

[0176] 3) The accuracy test results are shown in Table 2:

[0177] Table 2

[0178]

[0179] Three replicates of the test sample solutions at 50%, 100%, and 150% concentrations were prepared in parallel. The recoveries were calculated using the external standard method and were all within the 98% to 102% range. The RSD% of the recoveries at each concentration level was less than 2.0%, and the RSD of all nine recoveries was less than 2.0%. This demonstrates the accuracy of this method.

[0180] 4) Durability test results are shown in Table 3:

[0181] Table 3

[0182]

[0183] After fine-tuning the chromatographic conditions, the ratios of the sample solution content under each condition to the original conditions were between 0.98 and 1.02, meeting the system suitability requirements. This demonstrates that this method has strong anti-interference capabilities and excellent durability.

[0184] In summary, the embodiments of the present invention provide a method for detecting the content of the active ingredient in nemonofloxacin malate capsules. The present invention provides a method for detecting the content of the active ingredient in nemonofloxacin malate capsules for the first time. The inventors have found that selecting a specific chromatographic column and a specific mobile phase for isocratic elution can effectively avoid the interference of non-active ingredients such as impurities and excipients in nemonofloxacin malate capsules, improve the accuracy of the detection of the active ingredient content in nemonofloxacin malate capsules, and achieve accurate quantification of the content of nemonofloxacin malate capsules, thus filling the gap in the prior art.

[0185] Various embodiments of the present invention may be presented in the form of a range; it should be understood that the description in a range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention; therefore, the range description should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within the range. For example, the description of a range from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within the range, such as 1, 2, 3, 4, 5, and 6, regardless of the range. In addition, whenever a numerical range is indicated herein, it is intended to include any cited numeral (fractional or integer) within the indicated range.

[0186] The foregoing description is intended only to provide specific embodiments of the present invention, which will enable those skilled in the art to understand and implement the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not intended to be limited to the embodiments shown herein, but is intended to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A method for detecting the content of active ingredient in Nemonofloxacin Malate Capsules, characterized in that: The detection method comprises the following steps: The content of the active ingredient nemofloxacin in the nemofloxacin malate capsules was detected by high performance liquid chromatography; The conditions of the high performance liquid chromatography method include: Column: Inersustain C18, 4.6 × 150 mm, 5 μm or Inertsustain C18, 4.6 × 125 mm, 5 μm; Mobile phase: The mobile phase is composed of triethylamine phosphoric acid aqueous solution and acetonitrile in a volume ratio of (74-78): (22-26); the volume fractions of triethylamine and phosphoric acid in the triethylamine phosphoric acid aqueous solution are both 0.3-0.6 vol%; Elution mode: isocratic elution.

2. The method for detecting the content of the active ingredient in nemofloxacin malate capsules according to claim 1, characterized in that: The mobile phase consists of triethylamine phosphoric acid aqueous solution and acetonitrile in a volume ratio of 76:

24.

3. The method for detecting the content of the active ingredient in nemofloxacin malate capsules according to claim 1, characterized in that: The volume fractions of triethylamine and phosphoric acid in the triethylamine phosphoric acid aqueous solution are both 0.5 vol %.

4. The method for detecting the active ingredient content in nemofloxacin malate capsules according to any one of claims 1 to 3, characterized in that: The conditions of the high performance liquid chromatography also include: Detection wavelength: 252~256 nm; Column temperature: 28~32℃; Flow rate: 0.9~1.1 ml / min; Injection volume: 3~8μl; Diluent: composed of the triethylamine phosphoric acid aqueous solution and acetonitrile in a volume ratio of (74~78): (22~26).

5. The method for detecting the content of active ingredient in Nemonofloxacin Malate Capsules according to claim 4, characterized in that: The detection wavelength is 254 nm; and / or, the column temperature: 30°C; and / or, the flow rate: 1.0 ml / min; And / or, the injection volume: 5 μl; And / or, the diluent is composed of the triethylamine phosphoric acid aqueous solution and acetonitrile in a volume ratio of 76:

24.

6. The method for detecting the content of the active ingredient in nemofloxacin malate capsules according to claim 5, characterized in that: When preparing the solution, the diluent used should be consistent with the mobile phase.

7. The method for detecting the content of active ingredient in Nemonofloxacin Malate Capsules according to claim 6, characterized in that: The solution includes a test solution and a reference solution.

8. The method for detecting the content of active ingredient in Nemonofloxacin Malate Capsules according to claim 7, characterized in that: The test results meet the following requirements: the separation between the main peak of the active ingredient and the adjacent impurity peaks is greater than 1.5, each impurity peak does not interfere with the main peak of the active ingredient, and the tailing factor of the main peak of the active ingredient is ≤1.2; The impurities include impurity A, impurity B, impurity C, impurity D and impurity E; The chemical structural formula of the impurity A is shown below: ； The chemical structural formula of the impurity B is as follows: ； The chemical structural formula of the impurity C is shown below: ； The chemical structural formula of the impurity D is as follows: ； The chemical structural formula of the impurity E is shown below: 。 9. The method for detecting the content of the active ingredient in nemofloxacin malate capsules according to claim 8, characterized in that: The theoretical plate number of the main peak of the active ingredient in the chromatogram of the test solution is not less than 2000.

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