Method for detecting impurities 97 and 100 in moxifloxacin hydrochloride

Abstract

The invention provides a method for detecting the content of 6-nitroso octahydro-1H-pyrrolo [4, 3-b] pyridine (impurity 97) and 1-nitroso octahydro-1H-pyrrolo [4, 3-b] pyridine (impurity 100) in moxifloxacin hydrochloride, the method adopts an ultra-high performance liquid chromatography-tandem mass spectrometry method, the adopted mobile phase is an ammonium acetate-methanol solution, and the column temperature is 20-30 DEG C. The detection method has high sensitivity, the quantitation limits of the impurity 97 and the impurity 100 in the moxifloxacin bulk drug are respectively 0.33 ppm and 0.34 ppm, the quantitation limits of the impurity 97 and the impurity 100 in the moxifloxacin tablet are respectively 0.36 ppm and 0.37 ppm, and according to related guidance principles and methodology verification, the related requirements are met, and the detection method has good clinical application prospects. The method provided by the invention can be used for detecting the contents of the two impurities in a moxifloxacin hydrochloride raw material medicine and a moxifloxacin hydrochloride tablet.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for detecting the impurity content in a sample by liquid chromatography-mass spectrometry. More specifically, it relates to a method for detecting the content of 6-nitrosooctahydro-1H-pyrrolo[4,3-b]pyridine (Impurity 97) and 1-nitrosooctahydro-1H-pyrrolo[4,3-b]pyridine (Impurity 100) in moxifloxacin hydrochloride by ultra-high performance liquid chromatography-tandem mass spectrometry. Background Art

[0002] Moxifloxacin hydrochloride, chemically named: 1-cyclopropyl-6-fluoro-7-((4aS,7aS)-1H-[3,4-b]hexahydropyridino[1,2-a]pyrrol-6(2H))-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride, has the following structural formula: , Moxifloxacin hydrochloride is a broad-spectrum quinolone antibiotic developed by Bayer Healthcare. It was first launched in Germany in September 1999 and approved by the FDA for marketing in the United States in December of the same year. It is an 8-methoxyfluoroquinolone antibacterial drug with broad-spectrum antibacterial activity and strong bactericidal effect. During the synthesis of moxifloxacin hydrochloride, there may be impurities such as 6-nitrosooctahydro-1H-pyrrolo[4,3-b]pyridine (hereinafter referred to as Impurity 97), with the following structural formula: , and impurity 1-nitrosooctahydro-1H-pyrrolo[4,3-b]pyridine (hereinafter referred to as Impurity 100), with the following structural formula: , Therefore, it is necessary to study the residues of Impurity 97 and Impurity 100 in moxifloxacin hydrochloride raw materials and moxifloxacin hydrochloride tablets. However, there is no publicly reported detection method for the above two impurities. Due to the requirement of controlling the quality of drugs throughout the process from the source, it is very necessary to detect the content of Impurity 97 and Impurity 100 to ensure that the content meets the requirements. Therefore, it is necessary to develop a corresponding detection method to improve the detection sensitivity, meet the detection needs, and fill the blank of the existing technology. Summary of the Invention

[0003] In view of the blank in the prior art, the present invention provides a method for detecting the content of 6-nitrosooctahydro-1H-pyrrolo[4,3-b]pyridine (referred to as Impurity 97) and 1-nitrosooctahydro-1H-pyrrolo[4,3-b]pyridine (referred to as Impurity 100) that may exist during the synthesis of moxifloxacin hydrochloride by liquid chromatography-tandem mass spectrometry. The detection method is ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLCMS / MS), which has high sensitivity and meets the detection requirements.

[0004] A method for detecting impurities 97 and 100 in moxifloxacin hydrochloride provided by the present invention is characterized in that UHPLC-MS / MS is used for detection, and the method comprises the following steps: (1) Solution preparation: Prepare a reference stock solution, a reference solution, a test solution, and a sensitivity solution respectively; the reference stock solution is a mixed solution of 6-nitrosooctahydro-1H-pyrrolo[4,3-b]pyridine (impurity 97) and 1-nitrosooctahydro-1H-pyrrolo[4,3-b]pyridine (impurity 100); the test solution is a moxifloxacin hydrochloride sample solution.

[0005] (2) Injection: After the ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) instrument system is stable, inject the blank solution, the reference solution, and the test solution for detection respectively, record the chromatogram, and the detection conditions are as follows: Chromatographic column: α1-acid glycoprotein-bonded silica gel as the filler, the mobile phase is an ammonium acetate-methanol solution, and gradient elution is carried out.

[0006] The impurity 97 is 6-nitrosooctahydro-1H-pyrrolo[4,3-b]pyridine, and the impurity 100 is 1-nitrosooctahydro-1H-pyrrolo[4,3-b]pyridine; The preparation method of the reference stock solution is: accurately measure the impurity 97 stock solution and the impurity 100 stock solution respectively, place them in a volumetric flask, dilute to the scale with a diluent, and shake well.

[0007] The preparation method of the reference solution is: accurately measure an appropriate amount of the reference stock solution and place it in a volumetric flask, dilute to the scale with a diluent, and shake well.

[0008] The diluent is an acetonitrile solution.

[0009] The blank solution is a diluent.

[0010] The preparation method of the test solution is: accurately weigh a certain amount of the test sample, place it in a volumetric flask, dissolve it with a diluent and dilute to the scale, and shake well.

[0011] The preparation method of the sensitivity solution is: accurately measure a certain amount of the reference solution and place it in a volumetric flask, dilute to the scale with a diluent, and shake well.

[0012] The instruments used are an ultra-high performance liquid chromatograph, a triple quadrupole mass spectrometer equipped with APCI, and an electronic analytical balance.

[0013] The filler of the chromatographic column used is α1-acid glycoprotein-bonded silica gel, and the chromatographic column is Daicel chiralpak® AGP, with a specification of 4.0 mm×100 mm, 5.0 μm.

[0014] The mobile phase described above includes mobile phase A and mobile phase B. Mobile phase A is an ammonium acetate solution, and mobile phase B is methanol. The chromatographic elution method is gradient elution, and the gradient elution process is shown in Table 1 below:

[0015] The mass spectrometry conditions are shown in Table 2:

[0016] The contents of 6-nitrosooctahydro-1H-pyrrolo[4,3-b]pyridine (Impurity 97) and 1-nitrosooctahydro-1H-pyrrolo[4,3-b]pyridine (Impurity 100) in moxifloxacin hydrochloride of the test sample are calculated according to the following formula: Results

[0017] Where: R U : The peak areas measured for each component in the chromatogram of the test solution; R S : The average peak areas for each component in the chromatogram of the reference solution; C S : The concentration (ng / ml) of each component in the reference solution in the form of hydrochloride; C U : The concentration (mg / ml) of the test solution; M1: The molecular weight of Impurity 97 (or Impurity 100): 155.20; M2: The molecular weight of the hydrochloride of Impurity 97 (or Impurity 100): 191.66.

[0018] The detection method described above has been verified methodologically and meets the relevant requirements. The test results are shown in Table 3 below:

[0019]

[0020] The method provided by the present invention for detecting the contents of 6-nitrosooctahydro-1H-pyrrolo[4,3-b]pyridine (Impurity 97) and 1-nitrosooctahydro-1H-pyrrolo[4,3-b]pyridine (Impurity 100) in moxifloxacin hydrochloride adopts ultra-high performance liquid chromatography-tandem mass spectrometry. The mobile phase used is ammonium acetate-methanol solution. This detection method has high sensitivity. The limits of quantification of Impurity 97 and Impurity 100 in moxifloxacin raw material drug are 0.33 ppm and 0.34 ppm respectively, and the limits of quantification of Impurity 97 and Impurity 100 in moxifloxacin tablets are 0.36 ppm and 0.37 ppm respectively. According to relevant guiding principles, by strictly controlling the impurity content, the limits of moxifloxacin Impurity 97 and moxifloxacin Impurity 100 in moxifloxacin hydrochloride raw material drug are 3.4 ppm, and the limits of moxifloxacin Impurity 97 and moxifloxacin Impurity 100 in moxifloxacin tablets calculated based on the labeled amount of moxifloxacin are 3.75 ppm. Moreover, the method is verified by items such as system suitability, specificity, precision, limit of quantification, LOQ precision, limit of detection, etc., and all meet the relevant requirements. The method provided by the present invention can be used for detecting the contents of the above two impurities in moxifloxacin hydrochloride. Description of the Drawings

[0021] Figure 1 Typical chromatogram of the reference solution in Example 2 Figure 2 Chromatogram of the moxifloxacin hydrochloride raw material drug positioning solution in Example 3 Figure 3 Chromatogram of the moxifloxacin tablet positioning solution in Example 3 Figure 4 Chromatogram of Precision-1 (moxifloxacin hydrochloride raw material drug) in Example 4 Figure 5 Chromatogram of Precision-1 (moxifloxacin hydrochloride tablet) in Example 4 Figure 6 Chromatogram of LOQ-1 in Example 5 Figure 7 Chromatogram of LOD in Example 5 Figure 8 Typical chromatogram of the linear solution-100% in Example 6 Figure 9 Linear graph of the peak area of Impurity 97 in Example 6 Figure 10 Linear graph of the peak area of Impurity 100 in Example 6 Figure 11 Chromatogram of the solution stability - test solution (moxifloxacin hydrochloride raw material drug) - 37h in Example 8 Figure 12 Chromatogram of the solution stability - test solution (moxifloxacin hydrochloride tablet) - 38h in Example 8 Figure 13 Solution stability in Example 8 - Reference solution - Spectrogram at 45 h Figure 14 Solution stability in Example 8 - Selective solution (Moxifloxacin hydrochloride bulk drug) - Spectrogram at 38 h Figure 15 Solution stability - Selective solution (Moxifloxacin hydrochloride tablets) - Spectrogram at 39 h Figure 16 Localization spectrogram of impurity 97 and impurity 100 in Comparative Example 1 Figure 17 Poor resolution of impurity 97 and impurity 100 in Comparative Example 2 Detailed implementation manners

[0022] The present invention will be further illustrated by specific examples below. It should be understood that: the examples of the present invention are only used to illustrate the present invention, rather than to limit the present invention. Technical solutions obtained by making simple improvements to the present invention on the basis of the technical solutions of the present invention or by using conventional means or components for equivalent replacement all fall within the protection scope of the present invention. Impurity 97 and impurity 100 described in the present invention are 6 - nitrosooctahydro - 1H - pyrrolo[4,3 - b]pyridine and 1 - nitrosooctahydro - 1H - pyrrolo[4,3 - b]pyridine respectively. Information such as the instruments, chromatographic columns, reagents, reference substances, test substances, etc. used in the examples of the present invention are shown in Tables 4, 5, and 6.

[0023]

[0024]

[0025]

[0026] Detection method of Example 1 (1) Detection conditions and reagents Instruments and conditions: Instruments: Ultra - high performance liquid chromatograph: Model: Agilent 1290 + Agilent 6470, chromatographic column: chiralpak® AGP, 4.0 mm×100 mm, 5.0 μm, triple quadrupole mass spectrometer equipped with APCI, electronic analytical balance, gradient elution conditions are shown in Table 1, and mass spectrometer conditions are shown in Table 2.

[0027] Reagents and reference substances: Acetonitrile: HPLC grade and above; Water: Ultra - pure water; Methanol: HPLC grade and above; Ammonium acetate: AR grade and above; Moxifloxacin hydrochloride impurity 97 hydrochloride reference substance: Purchased externally; Moxifloxacin hydrochloride impurity 100 hydrochloride reference substance: Purchased externally (2) Test operation Solution preparation: Diluent: Acetonitrile: Water = 50:50 (V / V); Blank solution: Diluent; Impurity 97 stock solution: Take about 10 mg of reference substance of impurity 97 hydrochloride, weigh accurately, place in a 10 ml volumetric flask, dissolve and dilute to the mark with diluent, and shake well; accurately measure 740 μl of the above solution, place in a 50 ml volumetric flask, and dilute to the mark with diluent, and shake well; Impurity 100 stock solution: Take about 10 mg of reference substance of impurity 100 hydrochloride, weigh accurately, place in a 10 ml volumetric flask, dissolve and dilute to the mark with diluent, and shake well; accurately measure 740 μl of the above solution, place in a 50 ml volumetric flask, and dilute to the mark with diluent, and shake well; Reference substance stock solution: Accurately measure 1 ml each of impurity 97 stock solution and impurity 100 stock solution, place in a 20 ml volumetric flask, and dilute to the mark with diluent, and shake well; Reference substance solution: Accurately measure 500 μl of reference substance stock solution and place in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well; Sensitivity solution: Accurately measure 1 ml of reference substance solution and place in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well; Test solution (moxifloxacin hydrochloride): Take about 88 mg of the test substance, weigh accurately, place in a 10 ml volumetric flask, dissolve and dilute to the mark with diluent, and shake well; Test solution (moxifloxacin hydrochloride tablets): Take 5 tablets of the test substance (containing 2 g of moxifloxacin), place in a 50 ml volumetric flask, add about 20 ml of diluent, ultrasonicate for 5 min to dissolve, dilute to the mark with diluent, shake well, filter the supernatant through a 0.22 μm filter membrane, take 2.0 ml of the continued filtrate and place in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well; Injection and data processing: After the system is stable, inject 1 needle of blank solution (inject 2 - 3 needles if necessary), 1 needle of sensitivity solution, 6 needles of reference substance solution, 1 needle of blank solution, and 1 needle of test solution, and record the chromatogram; The S / N value of each component in the sensitivity solution should not be less than 10; the resolution between the two components in the first needle of reference substance solution should not be less than 1.5; the RSD of the peak areas of each component in the 6 needles of reference substance solution should not be greater than 10.0%; The content of impurity 97 and impurity 100 in moxifloxacin hydrochloride is calculated according to the following formula: Result , where: R U : The measured peak area of each component in the chromatogram of the test solution; R S : The average peak area of each component in the chromatogram of the 6 needles of reference substance solution; C S : The concentration (ng / ml) of each component in the reference substance solution in the form of hydrochloride; CU : Concentration of the test solution (mg / ml); M1: Molecular weight of impurity 97 (or impurity 100): 155.20; M2: Molecular weight of hydrochloride of impurity 97 (or impurity 100): 191.66.

[0028] Example 2 System Suitability System suitability is achieved by evaluating the S / N value of each component in the sensitivity solution, the resolution of the reference solution, and the RSD of the peak areas of each component in the 6 - injection reference solution. It is required that the S / N value of each component in the sensitivity solution should not be less than 10; the resolution between the two components in the first - injection reference solution should not be less than 1.5; the RSD of the peak areas of each component in the 6 - injection reference solution should not be greater than 10.0%.

[0029] Solution Preparation: Diluent: Acetonitrile: Water = 50:50 (V / V); Blank Solution: Diluent; Stock Solution of Impurity 97: Take about 10 mg of the reference substance of hydrochloride of impurity 97, weigh accurately, place it in a 10 - ml volumetric flask, dissolve and dilute to the mark with the diluent, and shake well; accurately measure 740 μl of the above solution, place it in a 50 - ml volumetric flask, and dilute to the mark with the diluent, and shake well; Stock Solution of Impurity 100: Take about 10 mg of the reference substance of hydrochloride of impurity 100, weigh accurately, place it in a 10 - ml volumetric flask, dissolve and dilute to the mark with the diluent, and shake well; accurately measure 740 μl of the above solution, place it in a 50 - ml volumetric flask, and dilute to the mark with the diluent, and shake well; Stock Solution of Reference Substances: Accurately measure 1 ml each of the stock solution of impurity 97 and the stock solution of impurity 100, place them in a 20 - ml volumetric flask, dilute to the mark with the diluent, and shake well; Reference Solution: Accurately measure 500 μl of the stock solution of reference substances and place it in a 10 - ml volumetric flask, dilute to the mark with the diluent, and shake well; Sensitivity Solution: Accurately measure 1 ml of the reference solution and place it in a 10 - ml volumetric flask, dilute to the mark with the diluent, and shake well; Injection: After the system is stable, according to the detection method of Example 1, inject 1 needle of the sensitivity solution and 6 needles of the reference solution, and record the chromatograms. The conversion results according to the formula are shown in Tables 7 and 8 as follows:

[0030]

[0031] Example 3 Specificity The specificity of the method is achieved by determining whether the blank solution interferes with the detection of each component and the resolution between each component in the selectivity solution and the adjacent impurity peaks. It is required that the blank solution should not interfere with the detection of each component, and the resolution between each component in the selectivity solution and the adjacent impurity peaks should be not less than 1.5.

[0032] Solution preparation: Diluent: Acetonitrile: Water = 50:50 (V / V); Blank solution: Diluent; Impurity 97 stock solution: Take about 10 mg of reference substance of impurity 97 hydrochloride, weigh accurately, place it in a 10 ml volumetric flask, dissolve and dilute to the mark with diluent, and shake well; accurately measure 740 μl of the above solution, place it in a 50 ml volumetric flask, and dilute to the mark with diluent, and shake well; Impurity 100 stock solution: Take about 10 mg of reference substance of impurity 100 hydrochloride, weigh accurately, place it in a 10 ml volumetric flask, dissolve and dilute to the mark with diluent, and shake well; accurately measure 740 μl of the above solution, place it in a 50 ml volumetric flask, and dilute to the mark with diluent, and shake well; Reference substance stock solution: Accurately measure 1 ml each of impurity 97 stock solution and impurity 100 stock solution, place them in a 20 ml volumetric flask, and dilute to the mark with diluent, and shake well; Preparation of impurity 97 localization solution and impurity 100 localization solution: Accurately measure 1 ml each of impurity 97 stock solution and impurity 100 stock solution, place them in a 20 ml volumetric flask, and dilute to the mark with diluent, and shake well; then accurately measure 500 μl of the above solution respectively, place them in a 10 ml volumetric flask, and dilute to the mark with diluent, and shake well; Preparation of mixed reference substance solution: See the preparation of the reference substance solution in Example 1; Moxifloxacin hydrochloride localization solution (test solution): See the preparation of the test solution in Example 1; Moxifloxacin hydrochloride tablet localization solution (test solution): See the preparation of the test solution in Example 1; Moxifloxacin hydrochloride selectivity solution: Take about 88 mg of the test substance, weigh accurately, place it in a 10 ml volumetric flask, accurately measure 500 μl of the reference substance stock solution, place it in this volumetric flask, dissolve and mix well. The concentration of moxifloxacin hydrochloride is 8.8290 mg / ml, the concentration of impurity 97 is 29.4290 ng / ml, and the concentration of impurity 100 is 29.5244 ng / ml; Moxifloxacin Hydrochloride Tablets Selectivity Solution: Take 5 tablets of the test sample, accurately measure 625 μl of the impurity 97 stock solution and 625 μl of the impurity 100 stock solution, place them in a 50-ml volumetric flask, add about 20 ml of the diluent, sonicate for 5 min to dissolve, dilute to the mark with the diluent, shake well, take the supernatant and filter it through a 0.22-μm filter membrane. Take 2.0 ml of the subsequent filtrate and place it in a 10-ml volumetric flask, dilute to the mark with the diluent, shake well. The concentration of moxifloxacin hydrochloride is 8 mg / ml, the concentration of impurity 97 is 29.4290 ng / ml, and the concentration of impurity 100 is 29.5244 ng / ml; Injection: After the system is stable, according to the detection method of Example 1, inject 1 needle of the blank solution, 1 needle of the moxifloxacin hydrochloride API positioning solution (test sample solution), 1 needle of the moxifloxacin hydrochloride tablets positioning solution (test sample solution), 1 needle of the impurity 97 positioning solution, 1 needle of the impurity 100 positioning solution, 1 needle of the mixed reference solution, 1 needle of the moxifloxacin hydrochloride API selectivity solution, and 1 needle of the moxifloxacin hydrochloride tablets selectivity solution, and record the chromatogram. The conversion results according to the formula are shown in Table 9 below:

[0033] Example 4 Precision Repeatability is achieved by measuring the RSD of the determination results of each component in 6 test sample solutions (spiked). It is required that the RSD of the determination results of each component in 6 test sample solutions (spiked) should not be greater than 10.0%.

[0034] Solution Preparation: Diluent: Acetonitrile: Water = 50:50 (V / V); Blank Solution: Diluent; Impurity 97 Stock Solution: See the preparation of the impurity 97 stock solution under the system suitability of Example 2; Impurity 100 Stock Solution: See the preparation of the impurity 100 stock solution under the system suitability of Example 2; Reference Stock Solution: See the preparation of the reference stock solution under the system suitability of Example 2; Reference Solution: See the preparation of the reference solution under the system suitability of Example 2; Precision determination solution (moxifloxacin hydrochloride raw material): Refer to the preparation of the selective solution in Example 3, and sequentially prepare Precision-1, with the obtained moxifloxacin hydrochloride concentration of 8.8130 mg / ml, impurity 97 concentration of 29.0809 ng / ml, and impurity 100 concentration of 29.8039 ng / ml; Precision-2, with the obtained moxifloxacin hydrochloride concentration of 8.9050 mg / ml, impurity 97 concentration of 29.0809 ng / ml, and impurity 100 concentration of 29.8039 ng / ml; Precision-3, with the obtained moxifloxacin hydrochloride concentration of 8.8690 mg / ml, impurity 97 concentration of 29.0809 ng / ml, and impurity 100 concentration of 29.8039 ng / ml; Precision-4, with the obtained moxifloxacin hydrochloride concentration of 8.8380 mg / ml, impurity 97 concentration of 29.0809 ng / ml, and impurity 100 concentration of 29.8039 ng / ml; Precision-5, with the obtained moxifloxacin hydrochloride concentration of 8.7910 mg / ml, impurity 97 concentration of 29.0809 ng / ml, and impurity 100 concentration of 29.8039 ng / ml; Precision-6, with the obtained moxifloxacin hydrochloride concentration of 8.8440 mg / ml, impurity 97 concentration of 29.0809 ng / ml, and impurity 100 concentration of 29.8039 ng / ml; Precision determination solution (moxifloxacin hydrochloride tablets): Refer to the preparation of the selective solution in Example 3, and sequentially prepare Precision-1, with the obtained moxifloxacin hydrochloride concentration of 8 mg / ml, impurity 97 concentration of 29.0809 ng / ml, and impurity 100 concentration of 29.8039 ng / ml; Precision-2: with the obtained moxifloxacin hydrochloride concentration of 8 mg / ml, impurity 97 concentration of 29.0809 ng / ml, and impurity 100 concentration of 29.8039 ng / ml; Precision-3: with the obtained moxifloxacin hydrochloride concentration of 8 mg / ml, impurity 97 concentration of 29.0809 ng / ml, and impurity 100 concentration of 29.8039 ng / ml; Precision-4: with the obtained moxifloxacin hydrochloride concentration of 8 mg / ml, impurity 97 concentration of 29.0809 ng / ml, and impurity 100 concentration of 29.8039 ng / ml; Precision-5: with the obtained moxifloxacin hydrochloride concentration of 8 mg / ml, impurity 97 concentration of 29.0809 ng / ml, and impurity 100 concentration of 29.8039 ng / ml; Precision-6: with the obtained moxifloxacin hydrochloride concentration of 8 mg / ml, impurity 97 concentration of 29.0809 ng / ml, and impurity 100 concentration of 29.8039 ng / ml; Injection: After the system is stable, inject Precision-1, Precision-2, Precision-3, Precision-4, Precision-5, and Precision-6 one by one according to the detection method in Example 1, and record the chromatograms. The conversion results according to the formula are shown in Tables 10, 11, 12, and 13 as follows:

[0035]

[0036]

[0037]

[0038] Detection Limit and Quantification Limit in Example 5 The detection limit is obtained by detecting that the ratio of its response signal to noise is not less than 3:1; the quantification limit is obtained by detecting that the ratio of its response signal to noise is not less than 10:1. At the LOQ concentration level, 6 portions of the quantification limit test solution are repeatedly examined. The LOQ of moxifloxacin hydrochloride 各组分 should not be greater than 1.02 ppm, S / N ≥ 10; the LOQ of moxifloxacin hydrochloride tablets 各组分 should not be greater than 1.13 ppm, S / N ≥ 10; the RSD of the peak area per unit concentration of each component in 6 portions of the LOQ solution should not be greater than 20.0%; the S / N of each component in the LOD solution should not be less than 3, and LOD < LOQ.

[0039] Solution Preparation: Diluent: Acetonitrile: Water = 50:50 (V / V); Blank Solution: Diluent; Impurity 97 Stock Solution: See the preparation of the impurity 97 stock solution under the system suitability in Example 2; Impurity 100 Stock Solution: See the preparation of the impurity 100 stock solution under the system suitability in Example 2; Reference Solution: See the preparation of the reference solution under the system suitability in Example 2; Sensitivity Solution: See the preparation of the sensitivity solution under the system suitability in Example 2; LOQ Solution: Accurately measure 1.0 ml of the reference solution, place it in a 10-ml volumetric flask, dilute it to the mark with the diluent, and shake well. The resulting concentration of impurity 97 is 2.9081 ng / ml, and the concentration of impurity 100 is 2.9804 ng / ml; LOD Solution: Accurately measure 3.0 ml of the LOQ solution and transfer it to a 10-ml volumetric flask. Dilute it to the mark with the diluent and mix well. The concentration of impurity 97 in the resulting solution is 0.8724 ng / ml, and the concentration of impurity 100 is 0.9841 ng / ml. Injection: After the system stabilizes, inject one needle each of LOQ-1, LOQ-2, LOQ-3, LOQ-4, LOQ-5, LOQ-6, and LOD solution according to the detection method in Example 1, and record the chromatograms. The conversion results according to the formula are shown in Tables 14, 15, and 16 below:

[0040]

[0041]

[0042] Example 6 Linearity and Range Take 6 points evenly within the range of LOQ concentration to 150% of the limit concentration. Use the concentration as the abscissa and the peak area as the ordinate to plot a curve. It is required that each component should be linear within the range of LOQ concentration to 150% of the limit concentration, and the square of the linear correlation coefficient R (R 2 ) should be not less than 0.99, and the absolute value of the y-axis intercept should be within 25% of the peak area at 100% concentration.

[0043] Solution Preparation Diluent: Acetonitrile: Water = 50:50 (V / V); Blank Solution: Diluent; Impurity 97 Stock Solution: See the preparation of the impurity 97 stock solution under the system suitability in Example 2; Impurity 100 Stock Solution: See the preparation of the impurity 100 stock solution under the system suitability in Example 2; Reference Substance Stock Solution: See the preparation of the reference substance stock solution under the system suitability in Example 2; Reference Substance Solution: See the preparation of the reference substance solution under the system suitability in Example 2; Linear LOQ Solution -1, Linear LOQ Solution -2, Linear LOQ Solution -3: See the preparation of LOQ Solution -1, LOQ Solution -2, and LOQ Solution -3 under the quantitative limit and detection limit in Example 5 respectively; Linear solutions: Linear solution - 50%: Accurately measure 250 μl of the reference stock solution, transfer it to a 10-ml volumetric flask, dilute it to the mark with the diluent, and mix well. The concentration of impurity 97 is 14.5405 ng / ml, and the concentration of impurity 100 is 14.9020 ng / ml; following this method, successively prepare Linear solution - 80%: The concentration of impurity 97 is 23.2647 ng / ml, and the concentration of impurity 100 is 23.8431 ng / ml; Linear solution - 100%: The concentration of impurity 97 is 29.0809 ng / ml, and the concentration of impurity 100 is 29.8039 ng / ml; Linear solution - 120%: The concentration of impurity 97 is 34.8971 ng / ml, and the concentration of impurity 100 is 35.7647 ng / ml; Linear solution - 150%: The concentration of impurity 97 is 43.6214 ng / ml, and the concentration of impurity 100 is 44.7059 ng / ml; Injection: After the system is stable, following the detection method in Example 1, inject 1 injection of Linear solution - LOQ - 1, Linear solution - LOQ - 2, and Linear solution - LOQ - 3 respectively, and inject 3 injections of Linear solution - 50%, Linear solution - 80%, Linear solution - 100%, Linear solution - 120%, and Linear solution - 150% respectively, and record the chromatograms. The conversion results according to the formula are shown in Tables 17 and 18 as follows:

[0044]

[0045] Example 7 Accuracy (Recovery Rate) Accuracy is achieved through the recovery rate between the measured concentration and the theoretical concentration of the analyte and the total RSD of the recovery rate (n = 9). It is required that the recovery rate of each component in the accuracy solutions added with LOQ concentration, 100% limit concentration, and 150% limit concentration should be between 70.0% and 130.0%, and the total RSD of the recovery rate (n = 9) should not be greater than 20.0%.

[0046] Solution preparation: Diluent: Acetonitrile:Water = 50:50 (V / V); Blank solution: Diluent; Accuracy solution - 100% - 1: See Precision - 1 under the precision item in Example 4; Accuracy solution 100% - 2: See Precision - 2 under the precision item in Example 4; Accuracy solution 100% - 3: See Precision - 3 under the precision item in Example 4; Accuracy Solution - 150% - 1: Weigh approximately 88 mg of the test substance accurately. Pipette precisely 300 μl of the reference substance stock solution into a 10 - ml volumetric flask, dilute to the mark with the diluent, and mix well. The resulting concentration of moxifloxacin hydrochloride is 8.8490 mg / ml, the concentration of impurity 97 is 44.5872 ng / ml, and the concentration of impurity 100 is 44.3215 ng / ml; successively prepare Accuracy Solution - 150% - 2, with the resulting concentration of moxifloxacin hydrochloride being 8.8520 mg / ml, the concentration of impurity 97 being 43.6214 ng / ml, and the concentration of impurity 100 being 44.7059 ng / ml; Accuracy Solution - 150% - 3: The resulting concentration of moxifloxacin hydrochloride is 8.8050 mg / ml, the concentration of impurity 97 is 44.1434 ng / ml, and the concentration of impurity 100 is 44.2865 ng / ml; Preparation of Accuracy Solution: Prepared with reference to the preparation method of the selectivity solution in Example 3. Successively prepare Accuracy Solution LOQ - 1, with the resulting concentration of moxifloxacin hydrochloride being 2.5205 mg / ml and the concentration of impurity 99 being 0.8599 ng / ml; Accuracy Solution LOQ - 2, with the resulting concentration of moxifloxacin hydrochloride being 2.5165 mg / ml and the concentration of impurity 99 being 0.8641 ng / ml; Accuracy Solution LOQ - 3, with the resulting concentration of moxifloxacin hydrochloride being 2.5145 mg / ml and the concentration of impurity 99 being 0.8473 ng / ml; Injection: After the system is stable, inject one injection each of Accuracy Solution 100% - 1, Accuracy Solution 100% - 2, Accuracy Solution 100% - 3, Accuracy Solution LOQ - 1, Accuracy Solution LOQ - 2, Accuracy Solution LOQ - 3, Accuracy Solution 150% - 1, Accuracy Solution 150% - 2, and Accuracy Solution 150% - 3 under the detection conditions of Example 1, and record the chromatograms. The conversion results according to the formula are shown in Tables 19, 20, 21, and 22 as follows:

[0047]

[0048]

[0049]

[0050] Example 8 Durability Examine the variation law of the detection results with time when the reference substance solution, test substance solution, and selectivity solution are injected after being placed at room temperature for a period of time, so as to provide a reference for the placement time of the reference substance solution and test substance solution during detection.

[0051] Requirement: Compared with the reference solution at 0 hr, during the room temperature investigation of the reference solution, the recovery rates of each component are between 80.0% and 120.0%, and there is no obvious change trend, then the reference solution is stable during the room temperature investigation; If the test component is detected in the test solution at 0 hr, within a certain period of time when the test solution is placed at room temperature, the change value of the measurement result is within 20% of the limit, and there is no obvious change trend, then the test solution is stable during the room temperature investigation; if the test component is not detected in the test solution at 0 hr, and no components are detected after the test solution is placed at room temperature for a certain period of time, then the test solution is stable during the room temperature investigation; During a certain period of time when the selectivity solution is placed at room temperature, the recovery rates of each component are between 80.0% and 120.0%, and there is no obvious change trend, then the selectivity solution is stable during the room temperature investigation.

[0052] Solution preparation: Diluent: Acetonitrile: Water = 50:50 (V / V); Blank solution: Diluent; Solution stability - Preparation of impurity 97 stock solution and impurity 100 stock solution: See the preparation of impurity 97 stock solution and impurity 100 stock solution under the system suitability in Example 3; Solution stability - Preparation of reference stock solution: See the preparation of reference stock solution under the specificity in Example 3; Solution stability - Preparation of reference solution: See the preparation of the mixed reference solution under the specificity in Example 3. The obtained reference solution - 1: The concentration of impurity 97 is 29.7248 ng / ml, and the concentration of impurity 100 is 29.5477 ng / ml; reference solution - 2: The concentration of impurity 97 is 29.4290 ng / ml, and the concentration of impurity 100 is 29.5244 ng / ml; reference solution - 3: The concentration of impurity 97 is 30.0844 ng / ml, and the concentration of impurity 100 is 29.6729 ng / ml; reference solution - 4: The concentration of impurity 97 is 29.4290 ng / ml, and the concentration of impurity 100 is 29.5244 ng / ml; Solution stability - Preparation of test solution (moxifloxacin hydrochloride raw material): See the moxifloxacin hydrochloride positioning solution under the specificity in Example 3. The obtained test solution - 1 has a concentration of 8.8480 mg / ml, test solution - 2 has a concentration of 8.8430 mg / ml, test solution - 3 has a concentration of 8.8140 mg / ml, and test solution - 4 has a concentration of 8.8430 mg / ml; Solution stability - Preparation of test solution (moxifloxacin hydrochloride tablets): See the moxifloxacin hydrochloride tablet positioning solution under the specificity in Example 3. The obtained test solutions all have a concentration of 8 mg / ml; Solution Stability - Selective Solution Preparation (Moxifloxacin Hydrochloride Bulk Drug): See the selective solution of moxifloxacin hydrochloride under the specificity item in Example 3. The obtained selective solution - 1: the concentration of moxifloxacin hydrochloride is 8.7980 mg / ml, the concentration of impurity 97 is 29.7248 ng / ml, and the concentration of impurity 100 is 29.5477 ng / ml; selective solution - 2: the concentration of moxifloxacin hydrochloride is 8.8290 mg / ml, the concentration of impurity 97 is 29.4290 ng / ml, and the concentration of impurity 100 is 29.5244 ng / ml; selective solution - 3: the concentration of moxifloxacin hydrochloride is 8.8360 mg / ml, the concentration of impurity 97 is 30.0844 ng / ml, and the concentration of impurity 100 is 29.6729 ng / ml; selective solution - 4: the concentration of moxifloxacin hydrochloride is 8.8290 mg / ml, the concentration of impurity 97 is 29.4290 ng / ml, and the concentration of impurity 100 is 29.5244 ng / ml; Solution Stability - Selective Solution Preparation (Moxifloxacin Hydrochloride Tablets): See the selective solution of moxifloxacin hydrochloride tablets under the specificity item in Example 3. The obtained selective solution - 1: the concentration of moxifloxacin hydrochloride is 8 mg / ml, the concentration of impurity 97 is 29.7248 ng / ml, and the concentration of impurity 100 is 29.5477 ng / ml; selective solution - 2: the concentration of moxifloxacin hydrochloride is 8 mg / ml, the concentration of impurity 97 is 29.4290 ng / ml, and the concentration of impurity 100 is 29.5244 ng / ml; selective solution - 3: the concentration of moxifloxacin hydrochloride is 8 mg / ml, the concentration of impurity 97 is 30.0844 ng / ml, and the concentration of impurity 100 is 29.6729 ng / ml; selective solution - 4: the concentration of moxifloxacin hydrochloride is 8 mg / ml, the concentration of impurity 97 is 29.4290 ng / ml, and the concentration of impurity 100 is 29.5244 ng / ml; Injection: After the system is stable, according to the detection conditions in Example 1, inject the reference solution - 0h, reference solution - 19h, reference solution - 33h, reference solution - 38h, test solution - 0h, test solution - 19h, test solution - 32h, test solution - 37h, selective solution - 0h, selective solution - 19h, selective solution - 33h, and selective solution - 38h, one injection each, and record the chromatograms. The conversion results according to the formula are shown in Tables 23, 24, 25, 26, and 27 as follows:

[0053]

[0054]

[0055]

[0056]

[0057] Comparative Example 1 (1)Detection conditions and reagents Instruments and conditions: Instrument: Ultra high performance liquid chromatography - tandem mass spectrometer: Model: Agilent1290&Agilent QQQ, Agilent QQQ is equipped with an ESI(+)MS detector; Chromatographic column: Agilent Eclipse Plus C18 RRHD 3.0×150mm, 1.8µm; Agilent Eclipse Plus C18 RRHD 3.0×150mm, 1.8µm, electronic analytical balance, gradient elution conditions are shown in Table 28, and mass spectrometer conditions are shown in Table 29.

[0058]

[0059]

[0060] Reagents and reference substances: Methanol: HPLC; Ultra - pure water: HPLC; Formic acid: HPLC; Acetonitrile: HPLC; Moxifloxacin hydrochloride impurity 97 hydrochloride reference substance: purchased externally; Moxifloxacin hydrochloride impurity 100 hydrochloride reference substance: purchased externally.

[0061] (2)Test operation Solution preparation: Diluent: Acetonitrile; Blank solution: Diluent; Moxifloxacin hydrochloride impurity 97 hydrochloride stock solution: Accurately weigh about 20mg of moxifloxacin hydrochloride impurity 97 hydrochloride reference substance and place it in a 200ml volumetric flask, dissolve it with diluent and dilute to the mark, shake well; Accurately measure 100µl of the above solution and place it in a 100ml volumetric flask, dilute it to the mark with diluent, and shake well; Moxifloxacin hydrochloride impurity 100 hydrochloride stock solution: Accurately weigh about 20mg of moxifloxacin hydrochloride impurity 100 hydrochloride reference substance and place it in a 200ml volumetric flask, dissolve it with diluent and dilute to the mark, shake well; Accurately measure 100µl of the above solution and place it in a 100ml volumetric flask, dilute it to the mark with diluent, and shake well; Reference substance solution: Accurately measure 300ul each of moxifloxacin hydrochloride impurity 97 hydrochloride stock solution and moxifloxacin hydrochloride impurity 100 hydrochloride stock solution and place them in a 10ml volumetric flask, dilute it to the mark with diluent, and shake well; Test solution (moxifloxacin hydrochloride): Accurately weigh about 80 mg of moxifloxacin hydrochloride sample and place it in a 100 ml volumetric flask. Dissolve it with the diluent and dilute to the mark, then shake well; Test solution (moxifloxacin hydrochloride tablets): Accurately weigh about 80 mg of moxifloxacin hydrochloride tablets sample and place it in a 100 ml volumetric flask. Dissolve it with the diluent and dilute to the mark, then shake well; Injection and data processing: After the system is stable, inject 1 needle of blank solution, 5 needles of reference solution, and 1 needle of test solution, and record the chromatogram. The RSD of the peak areas of each component in the 5 needles of test solution should not be greater than 10.0%.

[0062] The contents of impurity 97 and impurity 100 in moxifloxacin hydrochloride are calculated according to the following formula: Results , where: R U : The measured peak area of each component in the chromatogram of the test solution; R S : The average peak area of each component in the chromatogram of 6 needles of reference solution; C S : The concentration (ng / ml) of each component in the reference solution in the form of hydrochloride; C U : The concentration (mg / ml) of the test solution; M1: The molecular weight of impurity 97 (or impurity 100): 155.20; M2: The molecular weight of impurity 97 (or impurity 100) hydrochloride: 191.66.

[0063] Result analysis: Using a common C18 chromatographic column or a C18-phenyl column cannot separate impurity 97 and impurity 100. Figure 16 It can be seen from the localization map that the retention times of impurity 97 and impurity 100 on the common C18 chromatographic column almost overlap and there is no separation effect. At the same time, to separate impurity 97 and impurity 100, a specific chromatographic column is required: such as α1-acid glycoprotein-bonded silica gel, Agilent Poroshell 120 Chiral-T chromatographic column, both of which can achieve effective separation.

[0064] Comparative example 2 (1) Detection conditions and reagents Instruments and conditions: Instruments: Ultra-high performance liquid chromatography-quadrupole high-resolution electrostatic field orbitrap mass spectrometry; ME 204T / 02 electronic balance; The chromatographic column is ACE Excel 1.7 C18-PFP (100 mm × 2.1 mm, 1.7 μm), the column temperature is 55 °C, the flow rate is 0.3 mL / min, the mobile phase is A: 0.1% formic acid-aqueous solution; B: 0.1% formic acid-methanol solution, the mobile phase gradient conditions are shown in Table 1, the injection volume is 5 μL, and the gradient elution conditions are shown in Table 30.

[0065]

[0066] Reagents and reference substances: Reference substance for moxifloxacin hydrochloride impurity 97: purchased externally; Reference substance for moxifloxacin hydrochloride impurity 100: purchased externally; Methanol: for HPLC; Ultra-pure water: for HPLC; Formic acid: for HPLC.

[0067] (2)Test operation Solution preparation: Reference substance solution: Weigh appropriate amounts of reference substances for moxifloxacin hydrochloride impurity 97 and moxifloxacin hydrochloride impurity 100 respectively, accurately weigh them, add methanol to each for volume fixation, and prepare stock solutions of moxifloxacin hydrochloride impurity 97 and moxifloxacin hydrochloride impurity 100 with concentrations of 1 mg / mL respectively. Take 100 μL of the stock solutions of moxifloxacin hydrochloride impurity 97 and moxifloxacin hydrochloride impurity 100 respectively, place them in a 10 mL volumetric flask, dilute with methanol and fix the volume to obtain a stock solution of the mixed reference substance solution, and then dilute with methanol to obtain a mixed reference substance solution with a concentration of 1 μg / mL; Test solution (moxifloxacin hydrochloride): Take a 10 mL volumetric flask, weigh 300 mg of moxifloxacin hydrochloride, dissolve it in methanol-water (50:50), vortex for 5 min, and fix the volume to obtain a test solution of moxifloxacin hydrochloride with a concentration of 30.0 mg / mL; Test solution (moxifloxacin hydrochloride tablets): Take a 10 mL volumetric flask, weigh 300 mg of moxifloxacin hydrochloride tablets, dissolve it in methanol-water (50:50), vortex for 5 min, and fix the volume to obtain a test solution of moxifloxacin hydrochloride with a concentration of 30.0 mg / mL.

[0068] Injection and data processing: After the system is stable, inject 1 needle of blank solution, 5 needles of reference substance solution, and 1 needle of test solution, and record the chromatograms. The RSD of the peak areas of each component in the 5 needles of reference solution should not be greater than 10.0%.

[0069] The contents of impurity 97 and impurity 100 in moxifloxacin hydrochloride are calculated according to the following formula: Results , where: R U : The measured peak areas of each component in the chromatogram of the test solution; R S : The average peak areas of each component in the chromatogram of the 6 needles of reference substance solution; C S : The concentration (ng / ml) of each component in the reference substance solution in the form of hydrochloride; C U: Concentration of the test solution (mg / ml); M1: Molecular weight of impurity 97 (or impurity 100): 155.20; M2: Molecular weight of hydrochloride of impurity 97 (or impurity 100): 191.66.

[0070] Result analysis: Using a normal C18 chromatographic column or a C18-phenyl column cannot separate impurity 97 and impurity 100. Figure 17 It can be seen that the retention times of impurity 97 and impurity 100 on the normal C18 chromatographic column almost overlap, and there is no separation effect. At the same time, specific chromatographic columns are required to separate impurity 97 and impurity 100, such as α1-acid glycoprotein-bonded silica gel and Agilent Poroshell 120 Chiral-T chromatographic column, both of which can achieve effective separation. The chromatographic column chiral AGP used in this scheme is a chromatographic column filled with α1-acid glycoprotein-bonded silica gel, which is more suitable for the simultaneous detection and separation of impurity 97 and impurity 100 compared with the comparative example.

Claims

1. A method for detecting impurities 97 and 100 in moxifloxacin hydrochloride, characterized in that: The sample was detected by ultra-high performance liquid chromatography, and the mobile phase used was ammonium acetate-methanol solution. The impurity 97 was 6-nitrosooctahydro-1H-pyrrolo[4,3-b]pyridine, and the impurity 100 was 1-nitrosooctahydro-1H-pyrrolo[4,3-b]pyridine.

2. The detection method according to claim 1, characterized in that: The liquid chromatography is ultra-high performance liquid chromatography, and the mass spectrometry is tandem mass spectrometry.

3. The detection method according to claim 1, characterized in that: The mobile phase is ammonium acetate-methanol solution, wherein the ammonium acetate solution is mobile phase A, methanol is mobile phase B, and the chromatographic elution method is gradient elution. The gradient elution process is shown in the following table: 。 4. The detection method according to any one of claims 1 to 3, characterized in that: The detection method comprises the following steps: (1) solution preparation: preparing a reference solution and a test solution respectively, wherein the reference solution is a mixed solution of 6-nitrosooctahydro-1H-pyrrolo[4,3-b]pyridine hydrochloride and 1-nitrosooctahydro-1H-pyrrolo[4,3-b]pyridine hydrochloride; the test solution is a moxifloxacin hydrochloride sample solution; (2) injection: after the ultra-high performance liquid chromatography-tandem mass spectrometry instrument system is stable, injecting a blank solution, a reference solution and a test solution respectively for detection, and recording a spectrum; the blank solution is acetonitrile.

5. The detection method according to claim 4, characterized in that: The concentration of the reference solution is 20 ng / ml to 40 ng / ml, and the concentration of the test solution is 8 mg / ml to 10 mg / ml.

6. The detection method according to claim 3, characterized in that: The concentration of ammonium acetate in the mobile phase A is 10 mM.

7. The detection method according to any one of claims 1 to 3, 5 to 6, characterized in that: The filler of the chromatographic column used is α1-acid glycoprotein bonded silica gel.

8. The detection method according to claim 7, characterized in that: The chromatographic column is Daicel chiral pak® AGP, with specifications of 4.0 mm×100 mm and 5.0 μm.

9. The detection method according to claim 7, characterized in that: The injection volume in the detection method was 1 μl and the column temperature was 25°C.

10. The detection method according to claim 4, characterized in that: The preparation method of the reference substance solution is as follows: accurately weighing an appropriate amount of reference substances 6-nitrosooctahydro-1H-pyrrolo[4,3-b]pyridine hydrochloride and 1-nitrosooctahydro-1H-pyrrolo[4,3-b]pyridine hydrochloride and placing them in a volumetric flask, adding a diluent to dissolve and dilute to the scale, shaking well, and obtaining a reference substance reserve solution; accurately measuring an appropriate amount of the reference substance reserve solution and placing it in a volumetric flask, adding a diluent to dilute to the scale, shaking well, and obtaining the reference substance reserve solution.

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