Method for separating and detecting impurities in metronidazole-ursodeoxycholic acid conjugate and application thereof

Abstract

The invention provides a method for separating and detecting impurities in a metronidazole-ursodesoxycholic acid conjugate and application of the metronidazole-ursodesoxycholic acid conjugate. The method comprises the following steps: preparing a metronidazole-ursodesoxycholic acid conjugate solution to obtain a test solution; performing high performance liquid chromatography detection on the test solution by adopting a high performance liquid chromatograph with an electrified spray detector; wherein the chromatographic conditions of the high performance liquid chromatography detection are as follows: a mobile phase is a mixed solution of a 0.1% formic acid aqueous solution, methanol and acetonitrile in a volume ratio of 40: 35: 25; and the power law of the electrospray detector is 1.4. According to the application, an electrospray detector is adopted for detection, so that the problem of low sensitivity under an ultraviolet detector due to few conjugated systems in an ursodesoxycholic acid structure and large terminal absorption interference is solved; the electrospray detector serves as a mass type detector, the detection of the electrospray detector is irrelevant to the molecular structure of a detected substance, the response value of the detected substance is determined by the absolute mass of a detected sample, and the detection requirements of ursodesoxycholic acid and metronidazole can be met.

Description

Technical Field

[0001] The present application relates to the technical field of drug analysis, and in particular to a method for separating and detecting impurities in a metronidazole-ursodeoxycholic acid conjugate and its use. Background Art

[0002] Metronidazole (MTZ) is a nitroimidazole antibiotic with excellent anti-Clostridium difficile activity. However, it is easy to develop drug resistance during use, leading to recurrence of Clostridium difficile infection. In the past decade, metronidazole-derived conjugates have shown potential applications in research. Ursodeoxycholic acid (UDCA) can maintain the homeostasis of intestinal flora. Studies have shown that some bile acid compounds have a good regulatory effect on the germination of Clostridium difficile spores and the growth of Clostridium difficile.

[0003] Metronidazole-ursodeoxycholic acid conjugate (UDCA-MTZ), numbered I-2, has a chemical name of 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl (R)-4-((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[α]phenanthrene-17-yl)pentanoate. Studies have shown that the inhibitory activity of metronidazole-ursodeoxycholic acid conjugate against Clostridium difficile is better than that of metronidazole, and can be used to treat or inhibit Clostridium difficile infection.

[0004] In the prior art, there are no literature reports on the detection of raw materials in metronidazole-ursodeoxycholic acid conjugates; in view of this, the present application proposes a detection method that can be used to control the content of metronidazole and ursodeoxycholic acid, the starting materials of the reaction, during the preparation process, and can reduce the interference of potential parent compounds when studying the in vivo behavior of the conjugate, which is conducive to exploring the antibacterial mechanism of the conjugate. Summary of the invention

[0005] The purpose of the present application is to provide a method for separating and detecting impurities in a metronidazole-ursodeoxycholic acid conjugate and its use.

[0006] To achieve the above objectives, the present application proposes the following technical solutions:

[0007] In a first aspect, the present invention provides a method for separating and detecting impurities in a metronidazole-ursodeoxycholic acid conjugate, the method comprising:

[0008] Prepare a metronidazole-ursodeoxycholic acid conjugate solution to obtain a test solution;

[0009] The test solution was subjected to HPLC detection using a HPLC instrument with an electrospray detector;

[0010] Wherein, the chromatographic conditions of the high performance liquid chromatography detection include:

[0011] The mobile phase was a mixed solution of 0.1% formic acid aqueous solution, methanol, and acetonitrile in a volume ratio of 40:35:25;

[0012] The power ratio of the electrospray detector was 1.4.

[0013] As an embodiment, the elution mode of the mobile phase is isocratic elution.

[0014] As an embodiment, the chromatographic column used in the high performance liquid chromatography detection is Ultimate XB-C18.

[0015] As an embodiment, the column temperature of the chromatographic column is 35°C.

[0016] As an embodiment, the atomization temperature of the electrospray detector is 35°C.

[0017] As an embodiment, the flow rate of the mobile phase is 1.0 mL / min.

[0018] As an embodiment, the preparation of the metronidazole-ursodeoxycholic acid conjugate solution comprises:

[0019] Take the metronidazole-ursodeoxycholic acid conjugate, add methanol to dissolve it, and dilute it with a solvent to obtain a metronidazole-ursodeoxycholic acid conjugate solution;

[0020] Wherein, the solvent is a mixed solution of 0.1% formic acid aqueous solution, methanol and acetonitrile in a volume ratio of 40:35:25.

[0021] As an embodiment, the sampling frequency of the electrospray detector is 10 Hz.

[0022] As an embodiment, the filtration constant of the electrospray detector is 10s.

[0023] In a second aspect, the embodiments of the present application provide use of the method described in the first aspect in product quality control of metronidazole-ursodeoxycholic acid conjugate.

[0024] The embodiments of the present application have at least the following beneficial effects:

[0025] The present application uses an electrospray detector for detection, which solves the problem of low sensitivity under ultraviolet detectors due to the small number of conjugated systems in the structure of ursodeoxycholic acid and large interference from terminal absorption; as a mass detector, the electrospray detector has nothing to do with the molecular structure of the substance being tested, and the response value of the substance being tested is determined by the absolute mass of the sample being tested, which can take into account the detection requirements of ursodeoxycholic acid and metronidazole. The present application uses high performance liquid chromatography to effectively separate and detect the related substances metronidazole and ursodeoxycholic acid at the same time, and has a wider detection range.

[0026] Additional aspects and advantages of the present application will be partially given in the following description, which will become apparent from the following description, or will be understood through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the chromatogram of test condition 1 in Example 1;

[0028] Figure 2 is the chromatogram of test condition 2 in Example 1;

[0029] Figure 3 is the chromatogram of test condition 3 in Example 1;

[0030] Figure 4 is the chromatogram of blank solvent in Example 2;

[0031] Figure 5 is the chromatogram of the system suitability solution in Example 2;

[0032] Figure 6 is the chromatogram of the oxidative degradation blank solution in Example 3;

[0033] Figure 7 The chromatograms of the acid degradation and alkali degradation blank solutions in Example 3 are shown;

[0034] Figure 8 is the chromatogram of the acid degradation solution in Example 3;

[0035] Fig. 9 is the chromatogram of the alkaline degradation solution in Example 3;

[0036] Fig.10 is the chromatogram of the oxidative degradation solution in Example 3;

[0037] Fig.11 is the chromatogram of the high temperature degradation solution in Example 3;

[0038] Fig.12 It is the chromatogram under the quantitative limit of metronidazole in Example 4;

[0039] Fig.13 The chromatogram at the quantitative limit of ursodeoxycholic acid in Example 4;

[0040] Fig.14 It is the chromatogram under the detection limit of metronidazole in Example 4;

[0041] Fig.15 is a chromatogram at the detection limit of ursodeoxycholic acid in Example 4;

[0042] Fig.16 is the linear regression curve of metronidazole in Example 5;

[0043] Fig.17 is the linear regression curve of ursodeoxycholic acid in Example 5;

[0044] Fig.18 The chromatogram is the precision in Example 6. DETAILED DESCRIPTION

[0045] The technical solutions in the embodiments will be described clearly and completely below in combination with the embodiments and drawings of the present application. Obviously, the embodiments described below are only part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0046] It should be understood that when used in this specification and the appended claims, the terms "include" and "comprises" indicate the presence of described features, integers, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or combinations thereof.

[0047] It should also be understood that the terms used in this specification of the embodiments of the present application are only for the purpose of describing specific embodiments and are not intended to limit the embodiments of the present application. As used in the specification of the embodiments of the present application and the appended claims, unless the context clearly indicates otherwise, the singular forms of "a", "an" and "the" are intended to include plural forms.

[0048] The method for separating and detecting impurities in the metronidazole-ursodeoxycholic acid conjugate and its use in the embodiments of the present application will be described in detail below.

[0049] First, the method of this embodiment is described.

[0050] method

[0051] This embodiment provides a method for separating and detecting impurities in a metronidazole-ursodeoxycholic acid conjugate, the method comprising:

[0052] (1) preparing a metronidazole-ursodeoxycholic acid conjugate solution to obtain a test solution;

[0053] (2) Performing high performance liquid chromatography on the test solution using a high performance liquid chromatograph equipped with an electrospray ionization detector;

[0054] Wherein, the chromatographic conditions of the high performance liquid chromatography detection include:

[0055] The mobile phase was a mixed solution of 0.1% formic acid aqueous solution, methanol, and acetonitrile in a volume ratio of 40:35:25;

[0056] The power ratio of the electrospray detector was 1.4.

[0057] In this embodiment, the structure of the metronidazole-ursodeoxycholic acid conjugate (No. I-2) is as follows:

[0058]

[0059] Among them, when synthesizing the metronidazole-ursodeoxycholic acid conjugate using metronidazole and ursodeoxycholic acid as the reaction starting materials, a certain amount of metronidazole and ursodeoxycholic acid will remain; and since the metronidazole-ursodeoxycholic acid conjugate is better than metronidazole and ursodeoxycholic acid in inhibiting the activity of Clostridium difficile, the residues of metronidazole and ursodeoxycholic acid will affect the purity of the metronidazole-ursodeoxycholic acid conjugate (regarded as impurities), thereby affecting the inhibitory effect of the metronidazole-ursodeoxycholic acid conjugate on the activity of Clostridium difficile.

[0060] The structural formulas of metronidazole and ursodeoxycholic acid are shown in Table 1 below.

[0061] Table 1:

[0062]

[0063] It can be understood that this implementation configures the test solution and uses a high-performance liquid chromatograph with an electrospray detector to perform high-performance liquid chromatography on the test solution; wherein, the use of an electrospray detector for detection solves the problem of low sensitivity under ultraviolet detectors due to the small number of conjugated systems in the structure of ursodeoxycholic acid and large interference from terminal absorption; as a mass detector, the electrospray detector has nothing to do with the molecular structure of the analyte, and the response value of the analyte is determined by the absolute mass of the sample being detected, which can take into account the detection requirements of ursodeoxycholic acid and metronidazole. This application uses high-performance liquid chromatography to effectively separate and detect the related substances metronidazole and ursodeoxycholic acid at the same time, and has a wider detection range.

[0064] Specifically, the chromatographic conditions of the present application use a power rate of 1.4, which greatly improves the nonlinear response of each compound in the target range and provides each compound with a suitable signal-to-noise ratio. It is understandable that the electrospray detector is a nonlinear detector, and changing the power rate can linearize the detector signal, and the instrument allows the power rate to be changed within the range of 1.0 to 2.0. The present application investigated the power rate and measured the same sample at different power rate values. It was found that as the power rate increases, the signal-to-noise ratio (S / N) of each compound increases accordingly, and the signal-to-noise ratio of each compound reaches a maximum when the power rate reaches 2.0. However, too high a power rate can cause data distortion, and the closer it is to 1, the better the correlation is within the linear range. Therefore, when the present application selects a power rate of 1.4, the linear response of each compound within the target range observed is good.

[0065] At the same time, the present application selects a mobile phase composition of 0.1% formic acid aqueous solution-methanol-acetonitrile = 40:35:25 (v / v / v) for detection, and each compound can obtain good separation effect, repeatability and perfect peak shape; the electrospray ionization detector is based on the atomization-aerosol principle, requiring that the mobile phase additive must be volatile, and non-volatile additives such as phosphates should be avoided; the selection of the mobile phase must take into account the properties of metronidazole and ursodeoxycholic acid at the same time.

[0066] The present application comparatively investigates the systems of 0.1% formic acid aqueous solution-acetonitrile, 0.1% formic acid-methanol and 0.1% formic acid-methanol-acetonitrile. When the mobile phase is a binary mixture of 0.1% formic acid aqueous solution and acetonitrile, the peak position of metronidazole coincides with the position of the solvent peak. When low concentration of metronidazole is measured, the blank solvent peak interferes with the calculation of the metronidazole peak area. When the mobile phase is a binary mixture of 0.1% formic acid aqueous solution and methanol, ursodeoxycholic acid appears as a front extension peak. When the composition of the mobile phase is a ternary mixture of 0.1% formic acid aqueous solution, methanol and acetonitrile, 0.1% formic acid aqueous solution-methanol-acetonitrile = 40:35:25 (v / v / v) is finally selected after continuous adjustment of the ratio to ensure that each compound has a suitable retention time and a good peak shape.

[0067] It can be understood that the 0.1% formic acid aqueous solution in this embodiment refers to a formic acid aqueous solution with a concentration of 0.1%.

[0068] The relevant steps of the above method will be further described below.

[0069] In step (1), the preparation of the metronidazole-ursodeoxycholic acid conjugate solution comprises:

[0070] Take the metronidazole-ursodeoxycholic acid conjugate, add methanol to dissolve it, and dilute it with a solvent to obtain a metronidazole-ursodeoxycholic acid conjugate solution;

[0071] Wherein, the solvent is a mixed solution of 0.1% formic acid aqueous solution, methanol and acetonitrile in a volume ratio of 40:35:25.

[0072] Exemplarily, 16 mg of metronidazole-ursodeoxycholic acid conjugate is taken and placed in a 2 mL volumetric flask, 1 mL of methanol is added to dissolve it, and a solvent is added to dilute the volume to make a solution containing 8 mg per 1 mL, which is used as the test solution.

[0073] In the detection process of step (2), the elution mode of the mobile phase is isocratic elution; specifically, the flow rate of the mobile phase is 1.0 mL / min.

[0074] As a preferred embodiment, the chromatographic column used for high performance liquid chromatography detection is Ultimate XB-C18, and the column temperature of the chromatographic column is 35°C.

[0075] As a preferred embodiment, the atomization temperature of the electrospray detector is 35° C., the sampling frequency of the electrospray detector is 10 Hz, and the filtration constant of the electrospray detector is 10 s.

[0076] The present application selects a low atomization temperature (35°C) to give each compound a higher sensitivity; the atomization temperature affects the formation of analyte particles. Since the formation of particles is based on volatility, higher temperatures usually lead to reduced signal intensity of semi-volatile substances; the present application evaluates sensitivity at low atomization temperatures (35°C) and high atomization temperatures (55°C). As the evaporation temperature increases, the signal will significantly weaken, so a low atomization temperature (35°C) is selected to ensure the sensitivity of detection and analysis.

[0077] In summary, the high performance liquid chromatography used in this application can effectively separate and detect the related substances metronidazole and ursodeoxycholic acid at the same time, and has a wider detection range.

[0078] Next, the use of the above method in the quality control of metronidazole-ursodeoxycholic acid conjugate products will be described.

[0079] use

[0080] It is understandable that when synthesizing the metronidazole-ursodeoxycholic acid conjugate using metronidazole and ursodeoxycholic acid as the starting materials for the reaction, a certain amount of metronidazole and ursodeoxycholic acid will remain; and since the metronidazole-ursodeoxycholic acid conjugate is better than metronidazole and ursodeoxycholic acid in inhibiting Clostridium difficile activity, the residues of metronidazole and ursodeoxycholic acid will affect the purity of the metronidazole-ursodeoxycholic acid conjugate (considered as impurities), and further affect the inhibitory effect of the metronidazole-ursodeoxycholic acid conjugate on Clostridium difficile activity. Therefore, when producing the metronidazole-ursodeoxycholic acid conjugate using metronidazole and ursodeoxycholic acid as raw materials, the residual amount of metronidazole and ursodeoxycholic acid in the metronidazole-ursodeoxycholic acid conjugate should be limited.

[0081] As mentioned above, the method provided in this embodiment can effectively separate and detect the related substances metronidazole and ursodeoxycholic acid at the same time; therefore, it can meet the screening needs of related substances in the metronidazole-ursodeoxycholic acid conjugate, and can provide good technical support for the quality control of the metronidazole-ursodeoxycholic acid conjugate, indicating that the detection method has better application prospects in the quality control of the metronidazole-ursodeoxycholic acid conjugate.

[0082] The present application will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate / explain the present application and are not used to limit the scope of the present application.

[0083] In the following examples, the materials, reagents and instruments used can be purchased from commercial sources unless otherwise specified.

[0084] It should be noted that in the following examples, unless otherwise specified, the instruments, reagents, and methods and conditions for detection using a high performance liquid chromatograph are as follows:

[0085] instrument:

[0086] Thermo Fisher Scientific Dionex Ultimate3000 high performance liquid chromatograph, equipped with the following: CoronaVeoRS electrospray detector, quaternary pump, constant temperature column oven, autosampler, temperature control plate heating system, Empower data processing system;

[0087] Mettler Toledo XS205 1 / 100,000 analytical balance, Millipore ultrapure water machine.

[0088] Reagents:

[0089] Metronidazole (content 100%, reference substance of China Food and Drug Inspection Institute);

[0090] Ursodeoxycholic acid (content 99.6%, reference substance of China Food and Drug Administration);

[0091] Metronidazole-ursodeoxycholic acid conjugate (content>98%, homemade in our laboratory);

[0092] Formic acid (chromatographic grade, Maclean);

[0093] Methanol (chromatographic grade, Honeywell);

[0094] Acetonitrile (chromatographic grade, Honeywell).

[0095] Solvent: 0.1% formic acid aqueous solution-methanol-acetonitrile = 40:35:25 (v / v / v) as blank solvent;

[0096] Impurity reference substance stock solution: accurately weigh about 10 mg of metronidazole and ursodeoxycholic acid reference substances respectively, place in a 10 mL volumetric flask, add methanol to dissolve and dilute to the scale, respectively as known impurity reference substance stock solutions;

[0097] Impurity reference solution: accurately measure 80 μL of metronidazole reference stock solution and 64 μL of ursodeoxycholic acid reference stock solution, place in a 2 mL volumetric flask, add solvent to dilute to the mark, and shake well to obtain the impurity reference solution;

[0098] System suitability solution: Take about 16 mg of metronidazole-ursodeoxycholic acid conjugate reference substance, weigh it, put it in a 2 mL volumetric flask, add 1 mL of methanol to dissolve it, accurately add 80 μL of metronidazole reference substance stock solution and 64 μL of ursodeoxycholic acid reference substance stock solution, add solvent to dilute to the scale, shake well, and make a solution containing 8 mg of conjugate, 40 μg of metronidazole and 32 μg of ursodeoxycholic acid per 1 mL.

[0099] Test solution: Take about 16 mg of metronidazole-ursodeoxycholic acid conjugate, place it in a 2 mL volumetric flask, add 1 mL of methanol to dissolve it, and dilute it with solvent to make a solution containing about 8 mg per 1 mL, which is used as the test solution;

[0100] Control solution: Accurately measure 0.1 mL of the test solution, place it in a 10 mL volumetric flask, and dilute to the mark with solvent as a 1% self-control solution;

[0101] Sensitivity solution: Accurately weigh an appropriate amount of metronidazole-ursodeoxycholic acid conjugate reference substance, add 2 mL of methanol to dissolve it, and add solvent to dilute it into a solution containing approximately 15 μg per 1 mL, which is used as the sensitivity solution.

[0102] The chromatographic column is Ultimate XB-C18, with specifications of 4.6mm*250mm*5μm; the column temperature is 35℃; the mobile phase elution mode is isocratic elution, with a flow rate of 1.0mL / min; the injection volume is 5μL;

[0103] The atomization temperature of the electrospray detector is 35°C; the power rate of the electrospray detector is 1.4; the sampling frequency of the electrospray detector is 10 Hz; and the filter constant of the electrospray detector is 10 s.

[0104] Example 1

[0105] This example tests the influence of mobile phase composition on the separation of related substances, and the specific process is as follows:

[0106] (1) Prepare the test mixed stock solution: accurately weigh about 3 mg of metronidazole and ursodeoxycholic acid reference substance respectively, place them in the same 2 mL volumetric flask, and add methanol to dissolve to the scale;

[0107] (2) Preparation of test solution: Take about 2 mg of metronidazole-ursodeoxycholic acid conjugate reference substance, weigh it, place it in a 2 mL volumetric flask, add 0.5 mL of methanol to dissolve it, accurately add 1 mL of the test mixed stock solution (1) above, add solvent to dilute to the scale, shake well, and obtain the solution;

[0108] Take the above solution (2) and inject it into the liquid chromatograph under test conditions 1 to 3 respectively, and record the chromatogram. The test results are shown in Table 2 and Figure 1-Figure 3 shown.

[0109] Table 2:

[0110]

[0111] in, Figure 1 is the chromatogram of test condition 1 in Example 1; Figure 2 is the chromatogram of test condition 2 in Example 1; Figure 3 This is the chromatogram of test condition 3 in Example 1.

[0112] Combined with Table 2 and Figure 1-Figure 3 The test results show that the mobile phase system of 0.1% formic acid aqueous solution-methanol-acetonitrile is suitable for the detection of metronidazole-ursodeoxycholic acid conjugate-related substances. After continuous adjustment of the ratio, the mobile phase ratio was finally determined to be 0.1% formic acid aqueous solution-methanol-acetonitrile = 40:35:25 (volume ratio).

[0113] Example 2

[0114] This example conducts a system suitability test for the detection of metronidazole-ursodeoxycholic acid conjugate related substances, and the specific process is as follows:

[0115] (1) Prepare a blank solvent using 0.1% formic acid aqueous solution-methanol-acetonitrile = 40:35:25 (v / v / v) as the blank solvent;

[0116] (2) Prepare system suitability solution: take about 16 mg of metronidazole-ursodeoxycholic acid conjugate reference substance, weigh it, place it in a 2 mL volumetric flask, add 1 mL of methanol to dissolve it, accurately add 80 μL of metronidazole reference substance stock solution and 64 μL of ursodeoxycholic acid reference substance stock solution, add solvent to dilute to the scale, shake well, and prepare a solution containing 8 mg of conjugate, 40 μg of metronidazole and 32 μg of ursodeoxycholic acid per 1 mL.

[0117] Take the above solutions (1) and (2) respectively, inject them into the liquid chromatograph, record the chromatogram, and the test results are shown in Table 3 and Figure 4-Figure 5 .

[0118] Table 3:

[0119]

[0120] in, Figure 4 is the chromatogram of blank solvent; Figure 5 It is the chromatogram of the system suitability solution in Example 2. The above results show that the solvent peak has no interference with the inspection of conjugate-related substances; the order of peaks in the system suitability solution is metronidazole, ursodeoxycholic acid, and conjugate, and the attribution of each peak is clear. The system suitability test results can meet the experimental requirements.

[0121] Example 3

[0122] This example conducts a forced degradation test on the detection of related substances of metronidazole-ursodeoxycholic acid conjugate, and the specific process is as follows:

[0123] (1) Oxidative degradation blank solution: take 0.4 mL of 30% hydrogen peroxide solution into a 2 mL volumetric flask, place at room temperature for 3 h, and dilute to the mark with solvent;

[0124] (2) Acid degradation and alkali degradation blank solution: Take 1 mol·L -1 0.4 mL of hydrochloric acid solution was placed at room temperature for 3 h, and 1 mol·L -1 Neutralize with 0.4 mL of sodium hydroxide solution and dilute to scale with solvent;

[0125] (3) Acid degradation solution: Take about 16 mg of the sample and place it in a 2 mL volumetric flask. Add 1 mol·L -1 0.4 mL of hydrochloric acid solution was placed at room temperature for 3 h, and 1 mol·L -1 Neutralize with 0.4 mL of sodium hydroxide solution, add 0.6 mL of methanol to dissolve, and dilute to the mark with solvent;

[0126] (4) Alkaline degradation solution: take about 16 mg of the sample and place it in a 2 mL volumetric flask. Add 1 mol·L -1 0.4 mL of sodium hydroxide solution was placed at room temperature for 3 h, and 1 mol·L -1 Neutralize with 0.4 mL of hydrochloric acid solution, add 0.6 mL of methanol to dissolve, and dilute to the mark with solvent;

[0127] (5) Oxidative degradation solution: take about 16 mg of the sample, place it in a 2 mL volumetric flask, add 0.4 mL of 30% hydrogen peroxide solution, leave it at room temperature for 3 h, add 0.8 mL of methanol to dissolve it, and dilute it to the mark with solvent;

[0128] (6) High temperature degradation solution: take about 16 mg of the sample, place it in a 2 mL volumetric flask, heat it in a 58 °C oven for 3 h, cool it to room temperature, add 1 mL of methanol to dissolve it, and add solvent to make up to the mark;

[0129] Take the above solutions (1), (2), (3), (4), (5) and (6) respectively, inject them into the liquid chromatograph, record the chromatogram, and the results are shown in Figure 6-Figure 11 ; Figure 6 is the chromatogram of the oxidative degradation blank solution in Example 3; Figure 7 The chromatograms of the acid degradation and alkali degradation blank solutions in Example 3 are shown; Figure 8 is the chromatogram of the acid degradation solution in Example 3; Fig. 9 is the chromatogram of the alkaline degradation solution in Example 3; Fig.10 is the chromatogram of the oxidative degradation solution in Example 3; Fig.11This is the chromatogram of the high temperature degradation solution in Example 3.

[0130] Combination Figure 6-Figure 11 It can be seen from the chromatogram that the metronidazole-ursodeoxycholic acid conjugate is relatively stable under high temperature and oxidative conditions, but unstable under strong acidic and alkaline conditions. Metronidazole and ursodeoxycholic acid are the main degradation impurities, and other unknown impurities are also found to be produced. Therefore, under the chromatographic conditions of this embodiment, the conjugate can be effectively separated from the degradation products produced under various destruction conditions, and the degradation products have no interference with the detection of various impurities of related substances.

[0131] Example 4

[0132] This example conducts quantitative limit and detection limit tests on the detection of metronidazole-ursodeoxycholic acid conjugate related substances. The specific process is as follows:

[0133] (1) Stock solutions of impurity reference substances: accurately weigh about 10 mg of metronidazole and ursodeoxycholic acid reference substances respectively, place them in a 10 mL volumetric flask, add methanol to dissolve and dilute to the mark, and use them as stock solutions of known impurity reference substances respectively;

[0134] The impurity reference substance stock solution in (1) was diluted step by step with solvent, injected into the liquid chromatograph, and the chromatogram was recorded. The concentration with a signal-to-noise ratio (S / N) of about 10 was taken as the limit of quantification (LOQ), and the concentration with a signal-to-noise ratio (S / N) of about 3 was taken as the limit of detection (LOD). The test results are shown in Table 4 and Figure 12-Figure 15 shown.

[0135] Table 4:

[0136]

[0137]

[0138] in, Fig.12 It is the chromatogram under the quantitative limit of metronidazole in Example 4; Fig.13 The chromatogram at the quantitative limit of ursodeoxycholic acid in Example 4; Fig.14 It is the chromatogram under the detection limit of metronidazole in Example 4; Fig.15 This is the chromatogram at the detection limit of ursodeoxycholic acid in Example 4.

[0139] According to the test results, the concentration of metronidazole is 8.4880μg / mL, and the signal-to-noise ratio can reach 10:1; the concentration of ursodeoxycholic acid is 6.8380μg / mL, and the signal-to-noise ratio can reach 10:1; the concentration of metronidazole is 4.7745μg / mL, and the signal-to-noise ratio can reach 3:1; the concentration of ursodeoxycholic acid is 3.6820μg / mL, and the signal-to-noise ratio can reach 3:1.

[0140] Example 5

[0141] This example conducts a linear test on the detection of related substances of metronidazole-ursodeoxycholic acid conjugate, and the specific process is as follows:

[0142] (1) Stock solutions of impurity reference substances: accurately weigh about 10 mg of metronidazole and ursodeoxycholic acid reference substances respectively, place them in a 10 mL volumetric flask, add methanol to dissolve and dilute to the mark, and use them as stock solutions of known impurity reference substances respectively;

[0143] Take appropriate amounts of metronidazole reference substance stock solution and ursodeoxycholic acid reference substance stock solution in (1), place them in a 10 mL volumetric flask, and dilute to the mark with solvent to make approximately 80 μg mL -1 and 65 μg mL -1 The corresponding single component stock solution was diluted to 5 concentrations by adding solvent, injected into the liquid chromatograph, and the chromatogram was recorded. The mass concentration (μg·mL -1 ) as the horizontal axis, the peak area as the vertical axis, and linear regression was performed; the test results are shown in Table 5 and Figure 16-Figure 17 .

[0144] Table 5:

[0145] name Regression equation Correlation coefficient <![CDATA[Range μg·mL -1 > Metronidazole y=104147x-788287 0.9913 7.90～79.1 Ursodeoxycholic acid y=359299x-993334 0.9994 6.84～68.4

[0146] According to Table 5 and Figure 16-Figure 17 The results showed that the linear range of each impurity met the determination requirements.

[0147] Example 6

[0148] This example conducts a precision test on the detection of related substances of metronidazole-ursodeoxycholic acid conjugate, and the specific process is as follows:

[0149] (1) Stock solutions of impurity reference substances: accurately weigh about 10 mg of metronidazole and ursodeoxycholic acid reference substances respectively, place them in a 10 mL volumetric flask, add methanol to dissolve them and dilute to the mark with solvent, respectively, as stock solutions of known impurity reference substances;

[0150] Accurately measure 4 mL of metronidazole reference substance stock solution and 3.2 mL of ursodeoxycholic acid reference substance stock solution in (1), place them in the same 100 mL volumetric flask, add solvent to make up to the mark; inject into liquid chromatograph, record the chromatogram, inject 6 times continuously, measure the peak area, and the test results are as follows: Fig.18 shown.

[0151] The test results showed that the RSD% of the peak area of ​​metronidazole was 2.5, and the RSD% of the peak area of ​​ursodeoxycholic acid was 2.1, indicating that the precision of this example was good.

[0152] Example 7

[0153] This example conducts a recovery test on the detection of related substances of metronidazole-ursodeoxycholic acid conjugate, and the specific process is as follows:

[0154] (1) Stock solutions of impurity reference substances: accurately weigh about 10 mg of metronidazole and ursodeoxycholic acid reference substances respectively, place them in a 10 mL volumetric flask, add methanol to dissolve and dilute to the mark, and use them as stock solutions of known impurity reference substances respectively.

[0155] Accurately weigh an appropriate amount of the test sample (metronidazole and ursodeoxycholic acid are both <LOD (0.1%)) about 16 mg, place it in a 2 mL volumetric flask, add 1 mL of methanol to dissolve, accurately add 64 μL of metronidazole reference stock solution and 51 μL of ursodeoxycholic acid reference stock solution; 80 μL of metronidazole reference stock solution and 64 μL of ursodeoxycholic acid reference stock solution; 3 portions of 96 μL of metronidazole reference stock solution and 77 μL of ursodeoxycholic acid reference stock solution, dilute to the scale with solvent, shake well, and prepare 80%, 100% and 200% (low, medium and high) three concentration levels of test solutions. The test results of metronidazole are shown in Table 6, and the test results of ursodeoxycholic acid are shown in Table 7.

[0156] Table 6: Metronidazole recovery data

[0157]

[0158]

[0159] Table 7: Ursodeoxycholic Acid Recovery Data

[0160]

[0161] According to the results in Table 6 and Table 7, the recoveries and RSD values ​​of metronidazole and ursodeoxycholic acid met the test requirements.

[0162] Example 8

[0163] In this example, the related substances of the test samples prepared by different scale synthesis processes were determined according to the detection of related substances of metronidazole-ursodeoxycholic acid conjugate. The specific process is as follows:

[0164] About 16 mg of metronidazole-ursodeoxycholic acid conjugate test sample prepared by different scale synthesis processes were accurately weighed, placed in a 2 mL volumetric flask, dissolved in 1 mL of methanol, and diluted with solvent to make a solution containing about 8 mg per 1 mL as the test sample solution; injected into the liquid chromatograph, and the chromatogram was recorded and the contents of metronidazole and ursodeoxycholic acid were calculated by the external standard method, and the content of unknown impurities was calculated by the principal component self-reference method. The results are shown in Table 8.

[0165] Table 8:

[0166]

[0167] The above results show that the detection method of metronidazole-ursodeoxycholic acid conjugate related substances provided in this example has good versatility and can meet the requirements of quality control.

[0168] In summary, this embodiment uses an electrospray detector for detection, which solves the problem of low sensitivity under ultraviolet detectors due to the small number of conjugated systems in the structure of ursodeoxycholic acid and large interference from terminal absorption; as a mass detector, the electrospray detector has nothing to do with the molecular structure of the analyte, and the response value of the analyte is determined by the absolute mass of the sample being detected, which can take into account the detection requirements of ursodeoxycholic acid and metronidazole. This embodiment uses high performance liquid chromatography to effectively separate and detect related substances metronidazole and ursodeoxycholic acid at the same time, and has a wider detection range.

[0169] The technical solutions provided in the embodiments of the present application are introduced in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the embodiments of the present application. The description of the above embodiments is only applicable to help understand the principles of the embodiments of the present application. At the same time, for those skilled in the art, according to the embodiments of the present application, there will be changes in the specific implementation methods and application scopes. In summary, the contents of this specification should not be understood as limiting the present application.

Claims

1. A method for separating and detecting impurities in a metronidazole-ursodeoxycholic acid conjugate, characterized in that: The method comprises: Prepare a metronidazole-ursodeoxycholic acid conjugate solution to obtain a test solution; The test solution was subjected to HPLC detection using a HPLC instrument with an electrospray detector; Wherein, the chromatographic conditions of the high performance liquid chromatography detection include: The mobile phase was a mixed solution of 0.1% formic acid aqueous solution, methanol, and acetonitrile in a volume ratio of 40:35:25; The power ratio of the electrospray detector was 1.

4.

2. The method according to claim 1, characterized in that The elution mode of the mobile phase is isocratic elution.

3. The method according to claim 1, characterized in that: The chromatographic column used in the high performance liquid chromatography detection is Ultimate XB-C18.

4. The method according to claim 3, characterized in that The column temperature of the chromatographic column is 35°C.

5. The method according to claim 1, characterized in that The nebulization temperature of the electrospray detector was 35°C.

6. The method according to claim 1, characterized in that The flow rate of the mobile phase was 1.0 mL / min.

7. The method according to claim 1, characterized in that The preparation of the metronidazole-ursodeoxycholic acid conjugate solution comprises: Take the metronidazole-ursodeoxycholic acid conjugate, add methanol to dissolve it, and dilute it with a solvent to obtain a metronidazole-ursodeoxycholic acid conjugate solution; Wherein, the solvent is a mixed solution of 0.1% formic acid aqueous solution, methanol and acetonitrile in a volume ratio of 40:35:

25.

8. The method according to claim 1, characterized in that The sampling frequency of the electrospray detector was 10 Hz.

9. The method according to claim 1, characterized in that: The filtration constant of the electrospray detector is 10 s.

10. Use of the method according to any one of claims 1 to 9 in the quality control of metronidazole-ursodeoxycholic acid conjugate products.

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