High performance liquid detection method for separating and detecting cefdil intermediates and impurities

Abstract

The invention relates to a high performance liquid detection method for separating and detecting cefdil intermediates and impurities, a reversed-phase high performance liquid chromatography method is adopted to detect a sample to be detected, qualitative or quantitative analysis is carried out according to a chromatographic result, methanol and water are used as mobile phases, a special bonding C18 octadecylsilane bonded silica gel chromatographic column is adopted, and the content of the cefdil intermediates and the impurities in the cefdil intermediates and the impurities in the cefdil intermediates and the impurities in the cefdil intermediates is detected. According to the present invention, the elution condition and the detection condition of the high performance liquid chromatography are scientifically screened and optimized, the effective separation and detection of the cefdil synthesis key intermediate and the related substances thereof are achieved, the separation effect is good, and the detection method has advantages of high accuracy, good repeatability, high sensitivity, good specificity, simple operation and high efficiency. The medicine quality control is effectively performed, the adverse reaction possibly occurring in the clinical treatment of the medicine is reduced, and the effectiveness and the safety of the medication of the patient are ensured.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical analysis, and particularly to a high performance liquid chromatography method for separating and detecting the starting materials of cefiderocol. Background Art

[0002] Cefiderocol is a novel siderophore cephalosporin developed by Shionogi & Co., Ltd. of Japan and is used for the treatment of Gram-negative bacterial infections. As a novel siderophore cephalosporin, cefiderocol can penetrate the outer cell membrane of Gram-negative pathogens (including multi-drug resistant strains), and it has the unique ability to overcome all carbapenem resistances. The structure of cefiderocol is shown in Formula I:

[0003]

[0004] 2-chloro-3,4-di-p-methoxybenzyloxy-N-(2-(1-pyrrolidino)ethyl)benzamide is an intermediate for the synthesis of cefiderocol (hereinafter simply referred to as cefiderocol intermediate C), and its structural formula is shown in Formula II:

[0005]

[0006] The synthesis route of cefiderocol intermediate C is as follows:

[0007]

[0008] In this synthesis route, the product cefiderocol intermediate C will contain various related substances, including unreacted raw materials, process intermediates, etc.

[0009] The detection methods for cefiderocol intermediate are not recorded in the current pharmacopoeias of various countries. At present, there is no specific report on the detection methods for cefiderocol intermediate and its related substances in the relevant literature.

[0010] Therefore, there is currently a lack of a detection method for cefiderocol intermediate C and its related substances. Summary of the Invention

[0011] The purpose of the present invention is to provide a high performance liquid chromatography detection method for separating and detecting cefiderocol intermediate and impurities. Using this method, various impurities can be effectively separated, and the detection method has high accuracy, good repeatability, high sensitivity, good specificity, simple operation and high efficiency.

[0012] The purpose of the present invention can be achieved by the following technical solutions:

[0013] A high performance liquid chromatography detection method for separating and detecting cefiderocol intermediate and impurities, wherein the impurities are selected from impurity A, impurity B and / or impurity C, and the specific steps are as follows:

[0014] S1. Mix methanol and water to obtain a blank solution;

[0015] S2. Mix cefidercol intermediate C reference substance, impurity A reference substance, impurity B reference substance, and impurity C reference substance with methanol and water to obtain a system suitability solution;

[0016] S3. Mix the test sample, methanol, and water to obtain a test solution;

[0017] S4. Take the blank solution prepared in step S1, the system suitability solution prepared in step S2, and the test solution prepared in step S3 for high performance liquid chromatography detection to obtain the chromatographic information of the test solution and the system suitability solution.

[0018] Among them, the cefidercol intermediate C has the structural formula shown in Formula II:

[0019]

[0020] The impurity A has the structural formula shown in Formula III:

[0021]

[0022] The impurity B has the structural formula shown in Formula IV:

[0023]

[0024] The impurity C has the structural formula shown in Formula V:

[0025]

[0026] Furthermore, in step S1, the volume ratio of methanol to water is 30:70.

[0027] Furthermore, in step S2, the preparation method of the system suitability solution is as follows:

[0028] Mix impurity A reference substance, impurity B reference substance, and impurity C reference substance with diluent methanol to prepare a related substances stock solution with a concentration of 100 μg / ml;

[0029] Take 25 mg of cefidercol intermediate C in a 50 ml volumetric flask, add 1.25 ml of the related substances stock solution, and dilute to volume with diluent water to obtain the system suitability solution.

[0030] Furthermore, in step S3, the test sample contains cefidercol intermediate C and / or impurities.

[0031] Furthermore, in step S4, the detection conditions of the high performance liquid chromatography are as follows:

[0032] Chromatographic column: The stationary phase is octadecylsilyl-bonded silica gel. The column temperature of the chromatographic column is 26°C - 37°C. The mobile phase is a mixed solution of water - methanol.

[0033] The elution mode is gradient elution. The flow rate of the mobile phase is 0.5 mL / min - 1.4 mL / min.

[0034] The detection wavelength of the ultraviolet detector is 190 nm - 300 nm, and the injection volume is 1 - 30 μL.

[0035] Furthermore, the octadecylsilyl-bonded silica gel chromatographic column is selected from the octadecylsilyl-bonded silica gel columns produced by any one of ThermoFisher, YMC, Phenomenex, ES, Merck, Agilent, Kromasil, Agela or Techmate.

[0036] Furthermore, the octadecylsilyl-bonded silica gel chromatographic column is selected from any one or a combination of more than one of Eclipse Plus C18, Kromasil 100-5-C18, Kromasil Eternity-5-C18, Kromasil Eternity XT-10-C18, Kromasil 100-10-C18, Kromasil 300-5-C18, YMC Triat C18, YMC ODS C18, YMC-Pack C18, Phenomenex kinetex C18, Titank C18, ES-C18, Epic C18, ZORBAX SB-C18, ZORBAX300SB-C18, Poroshell 120EC-C18, XDB-C18, TC-C18, Extend-C18, Bonshell C18, BonshellASB C18, Venusil HLP C18, Venusil C18 Plus, Venusil XBP C18(A), Venusil XBP C18(B), Venusil MP C18, Innoval Neo XD C18, Techmate C18-ST, Techmate C18-STⅡ, Techmate CI8 UG or Agilent Microspher C18.

[0037] As a preferred technical solution, the model of the chromatographic column is ShimNex UP C18, 150 mm × 4.6 mm, 3.5 μm.

[0038] As a preferred technical solution, the column temperature of the chromatographic column is 28°C - 35°C, preferably 30 - 32°C.

[0039] Furthermore, the mobile phase is prepared as follows:

[0040] Mobile phase A is obtained by ultrasonicating distilled water for 5 min, and mobile phase B is obtained by ultrasonicating HPLC-grade methanol for 5 min. The mobile phase is obtained by mixing mobile phase A and mobile phase B in a volume ratio of 95 - 5:5 - 95.

[0041] As a preferred technical solution, the flow rate of the mobile phase is 0.9 mL / min - 1.2 mL / min, preferably 0.6 mL / min - 1.0 mL / min.

[0042] As a preferred technical solution, the ultraviolet detector is a DAD full-wavelength scanning ultraviolet detector.

[0043] As a preferred technical solution, the detection wavelength of the ultraviolet detector is 200 nm - 250 nm, preferably 210 - 254 nm.

[0044] As a preferred technical solution, the injection volume is 1 - 15 μL, preferably 1 - 10 μL.

[0045] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0046] (1) By scientifically screening and optimizing the elution conditions and detection conditions of high-performance liquid chromatography, the present invention realizes the effective separation and detection of 4 difficult-to-separate compounds in the synthesis process of cefiderocol intermediate C. The separation effect is good, and the detection method has high accuracy, good repeatability, high sensitivity, good specificity, simple and efficient operation. It effectively controls the drug quality, reduces the possible adverse reactions in drug clinical trials and treatments, and ensures the effectiveness and safety of patient medication. The method of the present invention takes a short time, can save time costs, improve economic benefits, and is suitable for industrialized and large-scale production. The mobile phase of the present invention is easy to prepare and can be prepared according to conventional methods, avoiding the problem of needing to readjust the mobile phase ratio due to changes in the sample retention time.

[0047] (2) The resolution represents the degree of separation between adjacent peaks. The larger the R value, the better the separation of adjacent components. Generally speaking, when R < 1, there is partial overlap between the two peaks; when R = 1.0, the resolution can reach 98%; when R = 1.5, the resolution can reach 99.7%. Usually, R = 1.5 is used as the standard for complete separation of adjacent components. Therefore, the method of the present invention has strong specificity, and the relevant substances do not interfere with each other with the blank solvent, and the resolution between the peaks of each component is greater than 1.5; the method for detecting relevant substances under the chromatographic conditions of the present invention has high sensitivity, and the signal-to-noise ratio is greater than 3; when there are minor changes in the chromatographic conditions, it does not affect the separation of the peaks of each component, and the durability of this method is good.

[0048] (3) Through linearity and range experiments, the results show that there is a good linear relationship within the range where each impurity is located; through the recovery rate experiment, the results show that the average recovery rates of each impurity are within 85% - 115%, and the RSDs are within 10%. The detection method of the present invention has good accuracy. Description of the Drawings

[0049] Figure 1 It is the liquid chromatogram of the blank solvent in Comparative Example 1;

[0050] Figure 2 It is the liquid chromatogram of the mixed solution in Example 1;

[0051] Figure 3 It is the liquid chromatogram of impurity A in Example 2;

[0052] Figure 4 It is the liquid chromatogram of impurity B in Example 2;

[0053] Figure 5 It is the liquid chromatogram of impurity C in Example 2;

[0054] Figure 6 It is the liquid chromatogram of cefiderocol intermediate C in Example 2;

[0055] Figure 7 It is the schematic diagram of the linear experiment result of cefiderocol intermediate C in Example 3. Detailed Embodiments

[0056] The present invention will be described in detail below with reference to the drawings and specific embodiments. These embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation manners and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments.

[0057] Unless otherwise specified, the raw materials, reagents or devices used in the following embodiments can all be obtained from conventional commercial channels or can be obtained by existing known methods.

[0058] In the following examples, unless otherwise specified, when the present invention relates to the percentage between liquids, the percentage is volume / volume percentage; when the present invention relates to the percentage between a liquid and a solid, the percentage is volume / weight percentage; when the present invention relates to the percentage between a solid and a liquid, the percentage is weight / volume percentage; and the rest are weight / weight percentages.

[0059] The following will, with reference to the accompanying drawings, elaborate on some embodiments of the present invention. Without conflict, the following embodiments and the features in the embodiments can be combined with each other.

[0060] Example 1

[0061] This example provides a high-performance liquid chromatography detection method for separating and detecting cefiderocol intermediates and impurities, where the impurities are selected from Impurity A, Impurity B, and / or Impurity C. The specific steps are as follows:

[0062] Liquid chromatography instrument: Shimadzu LC-20A;

[0063] Chromatographic column: ShimNex UP C18, 150 mm × 4.6 mm, 3.5 μm;

[0064] Column temperature: 30 °C;

[0065] Mobile phase: Gradient elution with water - methanol, and the elution program is shown in Table 1:

[0066] Table 1 Elution program of water - methanol gradient

[0067]

[0068] Flow rate: 0.6 mL / min;

[0069] Detector: Ultraviolet detector;

[0070] Detection wavelength: 254 nm;

[0071] Solvent: Methanol : water = 30 : 70;

[0072] Injection volume: 1 μL;

[0073] Mixed solution: Cefiderocol intermediate C, reference substance of Impurity A, reference substance of Impurity B, and reference substance of Impurity C, dissolved and diluted with acetonitrile to prepare a mixed solution containing approximately 25 μg of each per 1 mL;

[0074] The chromatographic results are as Figure 2 shown: The analysis method described in this example can effectively separate cefiderocol intermediates and their related impurity systems, and has many advantages such as good specificity, high sensitivity, convenient operation, and appropriate analysis time.

[0075] Example 2

[0076] This example provides a specificity test for a high-performance liquid chromatography detection method for separating and detecting cefiderocol intermediates and impurities. The specific steps are as follows:

[0077] Liquid chromatograph: Shimadzu LC-20A;

[0078] Chromatographic column: ShimNex UP C18, 150 mm × 4.6 mm, 3.5 μm;

[0079] Column temperature: 30 °C;

[0080] Mobile phase: Gradient elution with water - methanol. The elution program is shown in Table 2:

[0081] Table 2 Elution program of water - methanol gradient

[0082]

[0083] Flow rate: 0.6 mL / min;

[0084] Detector: UV detector;

[0085] Detection wavelength: 254 nm;

[0086] Solvent: Methanol: Water = 30:70;

[0087] Injection volume: 1 μL;

[0088] Positioning solution: Dissolve the cefiderocol intermediate C, reference substances of impurity A, impurity B, and impurity C in acetonitrile respectively and dilute them into reserve solutions with appropriate concentrations, and then dilute them with the mobile phase to prepare solutions containing approximately 10 μg per 1 mL;

[0089] Mixed solution: Cefiderocol intermediate C, reference substances of impurity A, impurity B, and impurity C, dissolve them in acetonitrile and dilute to prepare a mixed solution containing approximately 25 μg per 1 mL for each;

[0090] The chromatographic results of the mixed solution are shown in Table 3 and Figures 3 to 6 as follows. The minimum resolution between each impurity peak and between the main component and the adjacent impurity peak is 1.819, greater than 1.5, and the signal - to - noise ratio is greater than 3, indicating good method specificity.

[0091] Table 3 Chromatographic results of the mixed solution

[0092]

[0093] Example 3

[0094] This example provides a linear range test for a high-performance liquid chromatography detection method for separating and detecting cefiderocol intermediates and impurities. The specific steps for preparing the linear solutions are as follows:

[0095] 30% linear solution: Take about 15 mg of cefiderocol intermediate C, weigh it precisely, place it in a 100-ml volumetric flask, dissolve it with 0.5 ml of diluent, and then dilute it to the mark with diluent. Shake well.

[0096] 60% linear solution: Take about 15 mg of cefiderocol intermediate C, weigh it precisely, place it in a 50-ml volumetric flask, dissolve it with 0.5 ml of diluent, and then dilute it to the mark with diluent. Shake well.

[0097] 80% linear solution: Take about 20 mg of cefiderocol intermediate C, weigh it precisely, place it in a 50-ml volumetric flask, dissolve it with 0.5 ml of diluent, and then dilute it to the mark with diluent. Shake well.

[0098] 100% linear solution: Take about 25 mg of cefiderocol intermediate C, weigh it precisely, place it in a 50-ml volumetric flask, dissolve it with 0.5 ml of diluent, and then dilute it to the mark with diluent. Shake well.

[0099] 120% linear solution: Take about 30 mg of cefiderocol intermediate C, weigh it precisely, place it in a 50-ml volumetric flask, dissolve it with 0.5 ml of diluent, and then dilute it to the mark with diluent. Shake well.

[0100] 160% linear solution: Take about 40 mg of cefiderocol intermediate C, weigh it precisely, place it in a 50-ml volumetric flask, dissolve it with 0.5 ml of diluent, and then dilute it to the mark with diluent. Shake well.

[0101] Quantitation limit solution: Precisely measure 1 ml of the 100% linear solution into a 100-ml volumetric flask, dilute it to the mark with diluent, shake well. Then precisely measure 3 ml of the above solution into a 100-ml volumetric flask, dilute it to the mark with diluent, and shake well to obtain the solution.

[0102] The results are as Figure 7 shown. The linear curve is y = 13.6578x + 52.3097, R 2 = 0.99999, and the linearity is good when the sample concentration is in the range of 150 μg / ml to 800.4 μg / ml.

[0103] Example 4

[0104] This example provides a quantitation limit and detection limit test for a high-performance liquid chromatography detection method for separating and detecting cefiderocol intermediates and impurities. The specific steps are as follows:

[0105] Quantitation Limit Solution: Accurately pipette 1 mL each of the reserve solution of the impurity reference substance and the reserve solution of the reference substance of Intermediate C of cefiderocol into a 100-mL volumetric flask, dilute to the mark with the diluent, shake well. Then accurately pipette 3 mL of the above solution into a 100-mL volumetric flask, dilute to the mark with the diluent, and shake well to obtain the solution.

[0106] Detection Limit Solution: Accurately pipette 3 mL of the quantitation limit solution into a 10-mL volumetric flask, dilute to the mark with the diluent, and shake well to obtain the solution.

[0107] Inject the blank solution (diluent), system suitability solution, test solution, quantitation limit solution, and detection limit solution into a high performance liquid chromatograph for detection respectively, and record the chromatogram.

[0108] Table 4 Results of the quantitation limit and detection limit tests for the high performance liquid chromatography detection method

[0109]

[0110] The results are shown in Table 4: When this method is applied to the detection of related substances in the cefiderocol intermediate and impurities, the detection time is short, and the resolution between impurity peaks is good. The resolution between impurity peaks is all ≥6, and the system suitability meets the requirements.

[0111] Comparative Example 1

[0112] This comparative example provides an HPLC separation and detection method for Intermediate C of cefiderocol, reference substance of impurity A, reference substance of impurity B, and reference substance of impurity C. The specific steps are as follows:

[0113] Liquid chromatograph: Shimadzu LC-20A;

[0114] Chromatographic column: ShimNex UP C18, 150 mm × 4.6 mm, 3.5 μm;

[0115] Column temperature: 30 °C;

[0116] Mobile phase: Gradient elution with tetrahydrofuran - acetonitrile. The elution program is shown in Table 5:

[0117] Table 5 Elution program of the tetrahydrofuran - acetonitrile gradient

[0118]

[0119] Flow rate: 1.0 mL / min;

[0120] Detector: Ultraviolet detector;

[0121] Detection wavelength: 254 nm;

[0122] Solvent: Methanol: Water = 30:70;

[0123] Sample volume: 1 μL;

[0124] Mixed solution: 1 mL of the positioning solution of cefiderocol intermediate C, reference substance of impurity A, reference substance of impurity B, and reference substance of impurity C, diluted with the solvent to a mixed solution with a concentration of about 100 μg / mL;

[0125] The chromatographic results are as Figure 1 shown: The gradient elution with tetrahydrofuran - acetonitrile cannot effectively separate and detect cefiderocol intermediate C and its related substance impurities, and cannot be used to control the product quality.

[0126] In the present invention, the resolution of the cefiderocol intermediate reference substance and the reference substances of impurity A, impurity B, and impurity C is greater than 1.5, and the theoretical plate number of the cefiderocol intermediate reference substance and the reference substances of impurity A, impurity B, and impurity C is greater than 10,000. The method has strong specificity, and each related substance does not interfere with the blank solvent.

[0127] The above description of the embodiments is to enable those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative labor. Therefore, the present invention is not limited to the above embodiments, and the improvements and modifications made by those skilled in the art without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. A high performance liquid phase detection method for separating and detecting cefiderocol intermediates and impurities, characterized in that: The impurities are selected from impurity A, impurity B and / or impurity C, and the specific steps are as follows: S1, mixing methanol and water to obtain a blank solution; S2, mixing the cefiderocol intermediate C reference substance, the impurity A reference substance, the impurity B reference substance, the impurity C reference substance with methanol and water to obtain a system suitability solution; S3, mixing the sample to be tested, methanol and water to obtain a test solution; S4, taking the blank solution prepared in step S1, the system suitability solution prepared in step S2, and the test solution prepared in step S3 for high performance liquid chromatography to obtain chromatographic information of the test solution and the system suitability solution, Wherein, the cefiderocol intermediate C has the structural formula shown in Formula II: The impurity A has the structural formula shown in Formula III: The impurity B has the structural formula shown in Formula IV: The impurity C has a structural formula shown in Formula V:

2. A high performance liquid phase detection method for separating and detecting cefiderocol intermediates and impurities according to claim 1, characterized in that: In step S1, the volume ratio of methanol to water is 30:

70.

3. A high performance liquid phase detection method for separating and detecting cefiderocol intermediates and impurities according to claim 1, characterized in that: In step S2, the system suitability solution is prepared as follows: The impurity A reference substance, the impurity B reference substance, the impurity C reference substance and the diluent methanol were mixed to prepare a related substance stock solution with a concentration of 100 μg / ml; Take 25 mg of Cefiderocol intermediate C in a 50 ml volumetric flask, add 1.25 ml of the related substance stock solution, and dilute to volume with diluent water to obtain a system suitability solution.

4. A high performance liquid phase detection method for separating and detecting cefiderocol intermediates and impurities according to claim 1, characterized in that: In step S3, the sample to be tested contains cefiderocol intermediate C and / or impurities.

5. A high performance liquid phase detection method for separating and detecting cefiderocol intermediates and impurities according to claim 1, characterized in that: In step S4, the detection conditions of the high performance liquid chromatography are as follows: Chromatographic column: The stationary phase is octadecylsilane bonded silica gel, the column temperature is 26℃-37℃, and the mobile phase is a mixed solution of water and methanol. The elution method is gradient elution, and the flow rate of the mobile phase is 0.5mL / min-1.4mL / min. The detection wavelength of the UV detector is 190nm-300nm, and the injection volume is 1-30μL.

6. A high performance liquid phase detection method for separating and detecting cefiderocol intermediates and impurities according to claim 5, characterized in that: The octadecylsilane bonded silica gel chromatographic column is selected from octadecylsilane bonded silica gel columns produced by any one of ThermoFisher, YMC, Phenomenex, ES, Merck, Agilent, Kromasil, Agela or Techmate.

7. A high performance liquid phase detection method for separating and detecting cefiderocol intermediates and impurities according to claim 6, characterized in that: The model of the chromatographic column is ShimNex UP C18, 150 mm × 4.6 mm, 3.5 μm; The column temperature of the chromatographic column is 28°C-35°C.

8. A high performance liquid phase detection method for separating and detecting cefiderocol intermediates and impurities according to claim 5, characterized in that: The mobile phase was prepared as follows: Distilled water was ultrasonicated for 5 minutes to obtain mobile phase A, HPLC grade methanol was ultrasonicated for 5 minutes to obtain mobile phase B, and mobile phase A and mobile phase B were mixed in a volume ratio of 95-5:5-95 to obtain a mobile phase.

9. A high performance liquid phase detection method for separating and detecting cefiderocol intermediates and impurities according to claim 5, characterized in that: The flow rate of the mobile phase is 0.9 mL / min-1.2 mL / min.

10. A high performance liquid phase detection method for separating and detecting cefiderocol intermediates and impurities according to claim 5, characterized in that: The detection wavelength of the ultraviolet detector is 200nm-250nm; The injection volume is 1-15 μL.

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