Method for detecting related substances of moxifloxacin hydrochloride bulk drug

Abstract

The invention relates to the technical field of drug analysis and detection, in particular to a method for detecting related substances in moxifloxacin hydrochloride bulk drugs. According to the detection method provided by the invention, the separation efficiency of the known impurity (impurity F) can be improved, and the separation degree between the main component and the adjacent impurity can reach 1.5 or above; in addition, a solvent formula is optimized, so that a test sample is more stable, and the detection method is more accurate; the detection accuracy is improved, and the recovery rate is controlled within the range of 93-102%.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis and detection technology, specifically to a method for detecting related substances in moxifloxacin hydrochloride raw material. Background Technology

[0002] Moxifloxacin hydrochloride is a fourth-generation quinolone antibiotic that works by inhibiting bacterial DNA topoisomerases and is widely used to treat infectious diseases of the respiratory and urinary tracts. Related substances in the active pharmaceutical ingredient (including synthetic byproducts and degradation products generated during storage) may affect drug safety and efficacy; therefore, establishing specific and sensitive methods for detecting related substances is a crucial aspect of drug quality control.

[0003] According to the Chinese Pharmacopoeia and international drug registration requirements (such as ICH Q3A), active pharmaceutical ingredients (APIs) require qualitative identification and quantitative control of relevant substances. The accuracy and reliability of the testing methods are directly related to the production quality of the drug and the safety of clinical use.

[0004] Currently, high-performance liquid chromatography (HPLC) is mainly used to detect related substances in moxifloxacin hydrochloride raw material. However, moxifloxacin hydrochloride undergoes a photo-oxidation reaction under light, which makes the test sample unstable, resulting in low accuracy of related substance detection. Summary of the Invention

[0005] The purpose of this invention is to provide a method for detecting related substances in moxifloxacin hydrochloride raw material. The detection method provided by this invention provides a more stable test sample and higher detection accuracy.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for detecting related substances in moxifloxacin hydrochloride raw material, comprising the following steps:

[0008] The sample to be tested is dissolved to obtain a test solution; the solvent used for dissolution includes tetrabutylammonium hydrogen sulfate, potassium dihydrogen phosphate, phosphoric acid, water, triethylamine, anhydrous sodium sulfite and methanol;

[0009] The test solution was analyzed by high performance liquid chromatography, and the content of related substances in moxifloxacin hydrochloride raw material was obtained by using either the main component self-comparison method or the impurity external standard method.

[0010] The impurities mentioned include impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H and impurity SM-6;

[0011]

[0012]

[0013] The conditions for the liquid chromatography detection include: mobile phase A is a buffer solution, and mobile phase B is methanol; the buffer solution includes tetrabutylammonium hydrogen sulfate, potassium dihydrogen phosphate, water, phosphoric acid, and triethylamine;

[0014] The flow rates of mobile phases A and B are 1.3 mL / min;

[0015] The elution method is gradient elution; the gradient elution procedure is as follows:

[0016] 0.0–30 min: The volume percentage of the mobile phase A decreases uniformly from 75% to 68%;

[0017] 30–45 min: The volume percentage of the mobile phase A decreases uniformly from 68% to 45%;

[0018] 45–60 min: The volume percentage of the mobile phase A is 45%;

[0019] 60–61 min: The volume percentage of the mobile phase A increases uniformly from 45% to 75%;

[0020] 61–75 min: The volume percentage of the mobile phase A is 75%.

[0021] The detection wavelengths are 293nm and 248nm.

[0022] Preferably, the chromatographic column of the liquid chromatography is packed with phenylsilane-bonded silica gel; the column temperature is 40°C; and the injection volume is 20 μL.

[0023] Preferably, the chromatographic column is an Agilent Eclipse XDB-Phenyl column.

[0024] Preferably, the detection wavelength of impurities A, B, C, D, E and F is 293 nm.

[0025] Preferably, the detection wavelength for impurities G, H, and SM-6 is 248 nm.

[0026] Preferably, the volume ratio of the mixture of tetrabutylammonium hydrogen sulfate, potassium dihydrogen phosphate, phosphoric acid, water, triethylamine and anhydrous sodium sulfite in the dissolving solvent to methanol is 20:80.

[0027] Preferably, the mass ratio of tetrabutylammonium hydrogen sulfate to potassium dihydrogen phosphate is 0.5:1; the mass ratio of potassium dihydrogen phosphate to anhydrous sodium sulfite is 100:2; and the mass ratio of potassium dihydrogen phosphate to the volume of phosphoric acid is 1g:2mL.

[0028] Preferably, the mass ratio of tetrabutylammonium hydrogen sulfate to potassium dihydrogen phosphate in mobile phase A is 0.5:1; the mass ratio of potassium dihydrogen phosphate to the volume ratio of phosphoric acid is 1g:2mL; and the pH value of mobile phase A is 2.5.

[0029] Preferably, when using the external standard method, the impurity G, impurity H, and impurity SM-6 are calculated using Equation 2:

[0030]

[0031] In Equation 2: X i --The content of impurities G, H, and SM-6 in the test sample, in %;

[0032] A X --Peak areas of impurities G, H, and SM-6 in the test solution;

[0033] m -- Weight of the test sample, mg;

[0034] f is calculated from equation 1.

[0035] In Equation 1: f -- response factor of impurities G, H, and SM-6 in the reference solution;

[0036] C R --Concentrations of impurities G, H, and SM-6 in the reference solution, in μg / mL;

[0037] A R --Peak areas of impurities G, H, and SM-6 in the reference solution.

[0038] Preferably, impurities A, B, C, D, E, and F are calculated using Equation 3:

[0039]

[0040] In Equation 3: X t --The content of single impurities A, B, C, D, E, and F in the test sample, in %;

[0041] A X --The peak area of ​​impurities A, B, C, D, E, and F in the test solution;

[0042] A R --Peak area of ​​the main component in the control solution;

[0043] F -- Relative correction factor for each impurity, where impurities A, C, D, and F are 1; impurity B is 1.4; and impurity E is 3.5.

[0044] The detection method provided by this invention can improve the separation efficiency of known impurities (impurity F), and the separation degree between the main component and adjacent impurities can reach more than 1.5, which is higher than the separation degree in the prior art. In addition, this invention optimizes the solvent formulation, making the test sample more stable and the detection method more accurate; it improves the detection accuracy and controls the recovery rate in the range of 93% to 102%. Attached Figure Description

[0045] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0046] Figure 1 The linear equation diagram of moxifloxacin;

[0047] Figure 2 The linear equation diagram for impurity A;

[0048] Figure 3 The linear equation diagram for impurity B;

[0049] Figure 4 The graph shows the linear equation for impurity C.

[0050] Figure 5 The linear equation diagram for impurity D;

[0051] Figure 6 The graph shows the linear equation for impurity E.

[0052] Figure 7 The graph shows the linear equation for impurity F;

[0053] Figure 8 The graph shows the linear equation for impurity G;

[0054] Figure 9 Linear equation plot for impurity SM-6;

[0055] Figure 10 The graph shows the linear equation for impurity H;

[0056] Figure 11 This is a spectrum for testing the suitability of the moxifloxacin hydrochloride system. Detailed Implementation

[0057] This invention provides a method for detecting related substances in moxifloxacin hydrochloride raw material, comprising the following steps:

[0058] Dissolve the sample to be tested to obtain the sample solution;

[0059] The sample solution was analyzed by high performance liquid chromatography, and the content of related substances in the moxifloxacin hydrochloride raw material was obtained by either the main component self-comparison method or the impurity external standard method.

[0060] The present invention dissolves the sample to be tested to obtain the sample solution.

[0061] In one embodiment of the present invention, the solvent for dissolving the material comprises tetrabutylammonium bisulfate, potassium dihydrogen phosphate, phosphoric acid, water, triethylamine, anhydrous sodium sulfite, and methanol. In another embodiment, the mass ratio of tetrabutylammonium bisulfate to potassium dihydrogen phosphate can be 0.5:1; the mass ratio of potassium dihydrogen phosphate to anhydrous sodium sulfite can be 100:2; and the mass-to-volume ratio of potassium dihydrogen phosphate to phosphoric acid can be 1 g:2 mL. The volume ratio of the mixture of tetrabutylammonium bisulfate, potassium dihydrogen phosphate, phosphoric acid, water, triethylamine, and anhydrous sodium sulfite to methanol in the solvent for dissolving the material can be 20:80.

[0062] After obtaining the sample solution to be tested, the present invention uses high performance liquid chromatography to detect the sample solution to be tested, and uses the main component self-comparison method or the impurity external standard method to obtain the content of related substances in the moxifloxacin hydrochloride raw material.

[0063] In one embodiment of the present invention, the conditions for liquid chromatography detection include: mobile phase A is a buffer solution, and mobile phase B is methanol; the buffer solution includes tetrabutylammonium hydrogen sulfate, potassium dihydrogen phosphate, water, phosphoric acid, and triethylamine; the mass ratio of tetrabutylammonium hydrogen sulfate to potassium dihydrogen phosphate in mobile phase A is 0.5:1; the mass ratio of potassium dihydrogen phosphate to phosphoric acid volume is 1 g: 2 mL; the pH value of mobile phase A is 2.5; the flow rate of mobile phases A and B is 1.3 mL / min; the elution method is gradient elution; the gradient elution procedure is shown in Table 1.

[0064] Table 1 Gradient elution program

[0065] Time (min) Mobile phase A (%) Mobile phase B (%) 0 75 25 30 68 32 45 45 55 60 45 55 61 75 25 75 75 25

[0066] The detection wavelengths are 293nm and 248nm.

[0067] In one embodiment of the present invention, the detection wavelength of impurity A, impurity B, impurity C, impurity D, impurity E and impurity F is preferably 293 nm; the detection wavelength of impurity G, impurity H and impurity SM-6 is preferably 248 nm.

[0068] In one embodiment of the present invention, the chromatographic column used for liquid chromatography detection is packed with phenylsilane-bonded silica gel. Specifically, the chromatographic column can be an Agilent Eclipse XDB-Phenyl column; the column temperature can be 40°C; and the injection volume can be 20 μL.

[0069] As one embodiment of the present invention, the impurities include impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H and impurity SM-6, the structural formulas of which are shown in Table 2.

[0070] The relevant substance testing was conducted in accordance with the draft pharmacopoeia of moxifloxacin hydrochloride. The EP (European Pharmacopoeia) standard for moxifloxacin hydrochloride controls N-methylmoxifloxacin (impurity F) as a known impurity. Furthermore, through reaction mechanism analysis, recrystallization using ethanol-water solution in the preparation process of moxifloxacin hydrochloride raw material may lead to the formation of N-methylmoxifloxacin. Therefore, the source of N-methylmoxifloxacin was identified as a process impurity and investigated. The relevant impurities correspond to the impurities in the draft Chinese Pharmacopoeia, EP (European Pharmacopoeia), USP2021 (United States Pharmacopeia), and USP41 (tablets) (United States Pharmacopeia) standards as follows:

[0071] Table 2 List of Organic Impurities in Raw Materials

[0072]

[0073]

[0074] In addition, in this invention, the relevant substances are determined according to high performance liquid chromatography (Chinese Pharmacopoeia 2020 edition, Part IV, General Chapter 0512). Specifically, 20 μl each of blank solution (i.e., solvent under section 1.2.1), sensitivity solution, reference solution, reference standard solution, system suitability solution and test solution are accurately measured and injected into the liquid chromatograph, and the chromatograms are recorded. Impurity G, impurity SM-6 and impurity H in the reference standard solution are eluted in sequence.

[0075] Limit requirements: If there is a chromatographic peak in the chromatogram of the test solution at 248 nm that has the same retention time as impurity G, impurity H, and impurity SM-6, the amount of impurity G and impurity H shall be ≤0.1% and the amount of impurity SM-6 shall be ≤0.15% based on peak area using the external standard method.

[0076] At 293 nm, if there are impurity peaks in the chromatogram of the test solution, the peak areas of impurities A, B, C, D, E, and F (N-methylmoxifloxacin) calculated by multiplying by correction factors (see Table 2) shall not exceed the main peak area of ​​the control solution (0.1%). The peak areas of other individual impurities shall not exceed the main peak area of ​​the control solution (0.1%). The sum of the peak areas of all impurities (calculated by external standard method for known impurities at 248 nm and by corrected peak areas for all impurities at 293 nm) shall not exceed 0.15%. Peaks with corrected peak areas less than 0.5 times the main peak area of ​​the control solution shall be ignored (0.05%).

[0077] Table 3. Relative retention times and correction factors for each impurity.

[0078] impurities Relative retention time Relative correction factor A 1.08 1 B 1.23 1.4 C 1.33 1 D 1.41 1 E 1.60 3.5 F 0.91 1

[0079] In this invention, when the external standard method is used, the impurities G, H, and SM-6 are calculated using Equation 2:

[0080]

[0081] In Equation 2: X i -The content of impurities G, H, and SM-6 in the test sample, in %;

[0082] A X --Peak areas of impurities G, H, and SM-6 in the test solution;

[0083] m -- Weight of the test sample, mg;

[0084] f is calculated from equation 1.

[0085] In Equation 1: f - the response factor of impurities G, H, and SM-6 in the reference solution;

[0086] C R --Concentrations of impurities G, H, and SM-6 in the reference solution, in μg / mL;

[0087] A R --Peak areas of impurities G, H, and SM-6 in the reference solution.

[0088] In this invention, impurities A, B, C, D, E, and F are calculated using Equation 3:

[0089]

[0090] In Equation 3: X t --Content of impurities A, B, C, D, E, F and other unknown single impurities in the test sample, %;

[0091] A X --The peak areas of impurities A, B, C, D, E, F and other unknown single impurities in the test solution;

[0092] A R --Peak area of ​​the main component in the control solution;

[0093] F -- Relative correction factor for each impurity, data are shown in Table 2, and all other impurities are 1.0.

[0094] In this invention, the total amount of impurities is calculated according to Equation 4:

[0095]

[0096] In Equation 4: X 总 --Total impurities in the test sample, %;

[0097] ∑Xi -- the sum of known impurities in the test sample (248nm and 293nm), %;

[0098] ∑At -- The sum of the peak areas of unknown impurities in the test solution (293nm).

[0099] To further illustrate the present invention, the following detailed description of the invention's solutions, in conjunction with the accompanying drawings and embodiments, is provided, but should not be construed as limiting the scope of protection of the present invention.

[0100] The source of moxifloxacin hydrochloride in the examples was: Reference standard: China National Institutes for Food and Drug Control, batch number: 510140-202003.

[0101] Example 1

[0102] 1. Detection conditions and solution preparation

[0103] 1.1 Chromatographic and detection conditions:

[0104] The chromatographic column was an Agilent Eclipse XDB-Phenyl (4.6 mm × 250 mm, 5 μm); the column temperature was 40 °C.

[0105] Buffer solution (mobile phase A): Dissolve 0.5 g of tetrabutylammonium hydrogen sulfate and 1.0 g of potassium dihydrogen phosphate in 500 mL of water, add 2 mL of phosphoric acid, dilute with water to 1000 mL, and adjust the pH to 2.5 with triethylamine; use methanol as mobile phase B, with a flow rate of 1.3 mL / min; the elution program is shown in Table 1, and the injection volume is 20 μL.

[0106] The detection wavelengths are 293nm and 248nm.

[0107] 1.2 Solution preparation:

[0108] 1.2.1 Solvent: Dissolve 0.5 g of tetrabutylammonium hydrogen sulfate and 1.0 g of potassium dihydrogen phosphate in 500 mL of water. Add 2 mL of phosphoric acid and dilute with water to 1000 mL. Adjust the pH to 2.5 with triethylamine. Add 20 mg of anhydrous sodium sulfite. Mix the resulting mixture with methanol (volume ratio of mixture to methanol is 20:80).

[0109] 1.2.2 Test solution: Take an appropriate amount of moxifloxacin hydrochloride test sample, accurately weigh it, dissolve and dilute it with solvent to prepare a solution containing about 1 mg of moxifloxacin hydrochloride per 1 mL.

[0110] 1.2.3 Control Solution: Accurately measure 1 mL of the test solution and place it in a 100 mL volumetric flask. Dilute to the mark with solvent and mix well. Take 1 mL of this solution and place it in a 10 mL volumetric flask. Dilute to the mark with solvent and mix well to prepare a solution containing approximately 1 μg of moxifloxacin hydrochloride per mL, which will serve as the control solution.

[0111] 1.2.4 Mixed reference solution of impurities G, H and SM-6: Take appropriate amounts of impurity G reference standard, impurity H reference standard and impurity SM-6 reference standard respectively, weigh accurately, add appropriate amount of methanol to dissolve, and dilute quantitatively with solvent to prepare a mixed solution containing about 1 μg each of impurity G, impurity H and impurity SM-6 per 1 mL.

[0112] The specific preparation method is as follows:

[0113] Stock solutions of impurity G and impurity SM-6: Weigh approximately 10 mg of impurity G and impurity SM-6 reference standards accurately, place them in 10 mL volumetric flasks respectively, dissolve and dilute to the mark with methanol, and shake well.

[0114] Impurity H stock solution: Weigh approximately 10 mg of impurity H reference standard accurately, place it in a 10 mL volumetric flask, add 1 mL of dimethyl sulfoxide to dissolve it, dilute to the mark with methanol, and shake well.

[0115] Mixed stock solution of impurities G, SM-6, and H: Accurately measure 1 mL each of the stock solutions of impurities G, SM-6, and H, place them in a 100 mL volumetric flask, dilute to the mark with solvent, and mix well.

[0116] Mixed reference solution of impurities G, SM-6, and H: Accurately measure 1 mL of the mixed impurity stock solution into a 10 mL volumetric flask, dilute to the mark with solvent, and mix well.

[0117] System suitability solution: Take an appropriate amount of moxifloxacin hydrochloride peak positioning reference (including moxifloxacin hydrochloride, impurity A, impurity B, impurity C, impurity D, impurity E), impurity F (N-methylmoxifloxacin), impurity G, impurity SM-6, and impurity H, accurately weigh them, place them in a 10 mL volumetric flask, add solvent to dissolve and dilute to prepare a mixed solution containing approximately 1 mg of moxifloxacin hydrochloride peak positioning reference and 1 μg each of impurities A, B, C, D, E, F (N-methylmoxifloxacin), G, SM-6, and H per mL.

[0118] Sensitivity solution: Accurately measure 2 mL of the control solution, place it in a 10 mL volumetric flask, dilute to the mark with solvent, and shake well.

[0119] 2. Related Matter Methodology Validation

[0120] 2.1 System Applicability

[0121] System applicability requirements:

[0122] In the 293 nm system suitability solution chromatogram, impurities F (N-methylmoxifloxacin), moxifloxacin, impurities A, B, C, D, and E elute sequentially. In the 248 nm system suitability solution chromatogram, impurities G, SM-6, and H elute sequentially. The resolution between each peak should meet the requirements. In the sensitivity solution chromatogram, the signal-to-noise ratio of the main component peak should be greater than 10.

[0123] Accurately measure 20 μL of the system suitability solution and inject it into the liquid chromatograph. Inject the sample 6 times consecutively and record the chromatogram. The experimental results are shown in Tables 4 to 8.

[0124] Table 4 Results of the investigation of peak areas of various impurities in the solution for system suitability

[0125]

[0126] Table 5 Results of the retention time study of various impurities in the solution for system suitability.

[0127]

[0128]

[0129] Table 6. Results of the theoretical plate number investigation for various impurities in the solution for system suitability.

[0130] 293nm 1 2 3 4 5 6 F 11906 11916 12009 12060 11985 12134 Moxifloxacin 9486 9466 9457 9324 9369 9422 A 17028 17274 17163 16963 16960 17304 B 15259 14759 14806 14569 14620 14835 C 17565 18012 17270 16681 17191 17459 D 18703 18397 18644 18419 18239 18473 E 65584 67292 64560 67225 65847 67971 248nm 1 2 3 4 5 6 G 296391 297821 295432 296621 298319 293225 SM-6 229225 224383 225791 226549 223722 228543 H 189924 189721 190043 188651 189456 188664

[0131] Table 7 Results of the investigation of tailing factors of various impurities in the system suitability solution

[0132] 293nm 1 2 3 4 5 6 F 0.88 0.9 0.88 0.89 0.89 0.9 Moxifloxacin 0.77 0.77 0.77 0.77 0.77 0.77 A 1.02 1.03 1.02 1.03 1.03 1.01 B 1.08 1.07 1.08 1.07 1.07 1.07 C 0.96 0.99 0.97 0.98 0.99 0.98 D 0.97 0.96 0.96 0.95 0.94 0.94 E 0.98 0.98 0.97 0.97 0.98 0.97 248nm 1 2 3 4 5 6 G 1.08 1.06 1.08 1.07 1.08 1.07 SM-6 1.08 1.08 1.08 1.08 1.07 1.08 H 1.07 1.07 1.07 1.08 1.08 1.07

[0133] Table 8 Results of the system suitability study on the separation degree of various impurities in the solution.

[0134] 293nm 1 2 3 4 5 6 F - - - - - - Moxifloxacin 3.13 3.15 3.13 3.17 3.14 3.15 A 2.61 2.59 2.62 2.64 2.62 2.61 B 5.18 5.17 5.18 5.16 5.18 5.17 C 3.35 3.33 3.37 3.35 3.38 3.32 D 2.05 2.05 2.06 2.08 2.05 2.04 E 6.57 6.59 6.54 6.52 6.55 6.57 248nm 1 2 3 4 5 6 G - - - - - - SM-6 8.17 8.21 8.16 8.19 8.18 8.16 H 5.57 5.59 5.57 5.56 5.59 5.58

[0135] Conclusion: The separation degree between the main peak and adjacent impurities is greater than 1.5, and the peak area RSD is less than 2.0%, which meets the system applicability requirements.

[0136] 2.2 Specificity 2.2.1 Impurity Localization Test

[0137] Accurately weigh approximately 10 mg each of impurity A, impurity B, impurity C, impurity D, impurity E, and impurity F reference standards, place them in separate 10 mL volumetric flasks, add an appropriate amount of diluent (1.2.1 solvent), shake to dissolve and dilute to the mark, and shake well to prepare the reference standard stock solutions for each impurity.

[0138] Impurity G reference standard stock solution: Weigh approximately 10 mg of impurity G reference standard accurately, place it in a 10 mL volumetric flask, add an appropriate amount of methanol, shake to dissolve and dilute to the mark, shake well to obtain impurity G reference standard stock solution.

[0139] Impurity H reference standard stock solution: Weigh approximately 10 mg of impurity H reference standard accurately, place it in a 10 mL volumetric flask, add 1 mL of dimethyl sulfoxide, shake to dissolve, and dilute to the mark with methanol. Shake well to obtain impurity H reference standard stock solution.

[0140] Stock solution of impurity SM-6 reference standard: Weigh approximately 10 mg of impurity SM-6 reference standard accurately, place it in a 10 mL volumetric flask, add methanol, shake to dissolve, and dilute to the mark with methanol. Shake well to obtain stock solution of impurity SM-6 reference standard.

[0141] Mixed solution of impurity reference standards: Accurately measure 1 mL each of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, and impurity SM-6 reference standard stock solution, place them in the same 100 mL volumetric flask, dilute to the mark with diluent, and shake well to obtain the mixed solution of impurity reference standards.

[0142] Accurately weigh 10 mg of moxifloxacin hydrochloride and place it in a 10 mL volumetric flask. Accurately add 1 mL of the impurity reference standard mixed solution, dilute to the mark with diluent, and shake well. This solution is used as the system suitability solution.

[0143] Accurately measure 5 μl of stock solutions of impurities A, B, C, D, E, F, G, H, and SM-6 reference standards, and 20 μl of system suitability solution, and inject them into the liquid chromatograph, respectively, and record the chromatograms.

[0144] Judgment criteria: The retention times of impurities in each solution are basically consistent; the solvent peak must not interfere with impurity detection; and the resolution between the main peak of the system suitability solution and adjacent impurity peaks must not be less than 1.5. The test results are shown in Table 9 below.

[0145] Table 9. Test results of system suitability solutions and solutions for identifying various impurities.

[0146]

[0147] Conclusion: The retention times of each impurity peak in the impurity localization solution are basically consistent with those in the system suitability solution. The solvent peak does not interfere with the detection of impurities. The resolution between the main peak and adjacent impurity peaks in the system suitability solution is greater than 1.5, which meets the requirements.

[0148] 2.2.2 Forced Degradation Test

[0149] Table 10 Forced Degradation Test Methods

[0150]

[0151] Accurately measure 20 mL of the solution after the forced degradation treatment and inject it into the liquid chromatograph. Record the chromatogram. The results are shown in the table below:

[0152] Table 11 Results of Forced Degradation Test

[0153]

[0154]

[0155] Conclusion: After forced degradation, the results showed that the separation degree between the impurity peaks and the main peak under each degradation condition was >1.5 at both wavelengths, indicating good separation. The impurity peaks increased significantly under each degradation condition, but the DAD detector results showed that the main peak had good purity and did not contain other impurity peaks. The sample showed a significant increase in impurities under acid and oxygen degradation conditions, indicating that this product is sensitive to acid and oxygen, and attention should be paid to this during storage or preparation.

[0156] 2.2.3 Material Balance Test

[0157] Material balance studies were conducted on the above specificity tests. In tests where no changes in impurities were observed, the total peak area showed no significant change. However, in degradation tests where changes in impurities were observed, the total peak area was calculated using the normalized method based on the peak area. The results are shown in the table below:

[0158] Table 12 Material Balance Results of Destructive Testing

[0159] sample Total peak area Material balance (%) The test sample was not damaged. 188078262 - Acid degradation of the test sample 1 183208309 98.1 Acid degradation of the test sample 2 195969067 98.0 Alkali degradation of the test sample 1 176704365 97.4 Alkali degradation of the test sample 2 181433849 96.9 Oxidative degradation of the test sample 1 175817358 100.5 Oxidative degradation of the test sample 2 178004900 99.9 10 days of high temperatures 183542176 99.3 30 days of high temperatures 192074032 98.2 10 days of sunlight 185421796 99.5 30 days of sunlight 190217683 100.4 10 days of high humidity 186705421 99.7 30 days of high humidity 189054219 99.5

[0160] Conclusion: As can be seen from the above data, the total peak area of ​​this product under various degradation conditions is basically known, and the material balance is maintained.

[0161] 3. Solution stability

[0162] Test solution: 100% spiked solution under the "Accuracy" section.

[0163] The relative standard deviation (RSD) of the changes in the peak areas of the principal component and each impurity should be less than 3%.

[0164] Determination: Accurately inject 20 μL of the above test solution into the liquid chromatograph at 0 h, 3 h, 6 h, 9 h, 12 h, 15 h, 18 h, 20 h, 22 h, and 24 h, record the chromatograms, and the detection results are shown in the table below:

[0165] Table 13 Results of solution stability test

[0166]

[0167] Conclusion: The concentrations of all impurities in the test solution were all within the 0.1% level. The RSD values ​​of the peak areas of the main component and each impurity were between 0.12% and 1.94%, and the RSD values ​​of the main component and each impurity met the requirements. No new impurities were found in the test solution, and the test solution was stable within 24 hours.

[0168] 2.4 Linear Range

[0169] 2.4.1 Solution Preparation

[0170] Solvent: Same as 1.2.1.

[0171] Stock solutions of moxifloxacin hydrochloride, impurity A, impurity B, impurity C, impurity D, impurity E, and impurity F: Weigh 10 mg each of moxifloxacin hydrochloride, impurity A, impurity B, impurity C, impurity D, impurity E, and impurity F accurately, place them in 10 mL volumetric flasks, dissolve and dilute to the mark with solvent, and shake well.

[0172] The preparation of impurity G, impurity SM-6 stock solution and impurity H stock solution is the same as in 1.2.4.

[0173] Accurately measure 1 mL each of the linear mixed stock solutions of moxifloxacin hydrochloride, impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity SM-6, and impurity H, place them in the same 100 mL volumetric flask, dilute with solvent and bring to the mark, mix well, and the content is 10 μg / mL.

[0174] Take the linear mixed stock solution and prepare linearity test solutions for each impurity according to the table below.

[0175]

[0176]

[0177] Table 14 Preparation of Linear Solutions

[0178] 2.4.2 Measurement

[0179] Accurately measure linear test solutions of each concentration and inject them into the liquid chromatograph. Record the chromatograms and perform linear regression analysis with concentration on the x-axis and peak area on the y-axis to plot a standard curve. The correlation coefficient r should not be less than 0.995; the absolute value of the y-intercept should be within 25% of the 100% response value. The test results are shown in Tables 15-24. Figures 1-10 .

[0180] Table 15 Results of Moxifloxacin Linear Detection

[0181] Solution serial number percentage(%) Concentration (μg / mL) Peak area Limit of Quantification 20％ 0.2050 47643 ① 50％ 0.5124 121361 ② 100％ 1.0248 242425 ③ 150％ 1.5372 363628 ④ 200％ 2.0496 497325

[0182] Linear equation: y = 239387x + 573, linear regression coefficient r = 0.9997, intercept percentage: 0.2%.

[0183] Conclusion: Moxifloxacin (293 nm) showed good linearity in the concentration range of 0.2050–2.0496 μg / mL, which is equivalent to 20%–200% of the unknown impurity limit.

[0184] Table 16 Linear detection results of impurity A

[0185] Solution serial number percentage(%) Concentration (μg / mL) Peak area Limit of Quantification 20％ 0.2052 52198 ① 50％ 0.5053 129996 ② 100％ 1.0105 255991 ③ 150％ 1.5158 383087 ④ 200％ 2.0211 512982

[0186] Linear equation: y = 252327x + 1782, linear regression coefficient r = 1.000, intercept percentage: 0.7%. Conclusion: Impurity A (293nm) shows good linearity in the concentration range of 0.2052–2.0211 μg / mL, equivalent to 20%–200% of the unknown impurity limit.

[0187] Table 17 Results of linear detection of impurity B

[0188] Solution serial number percentage(%) Concentration (μg / mL) Peak area Limit of Quantification 20％ 0.2052 38679 ① 50％ 0.5130 89175 ② 100％ 1.0260 168350 ③ 150％ 1.5390 258852 ④ 200％ 2.0520 336808

[0189] Linear equation: y = 164006x + 2655.6, linear regression coefficient r = 0.9997, intercept percentage: 1.6%. Conclusion: Impurity B (293nm) shows good linearity in the concentration range of 0.2052–2.0520 μg / mL, equivalent to 20%–200% of the unknown impurity limit.

[0190] Table 18 Results of linear detection of impurity C

[0191] Solution serial number percentage(%) Concentration (μg / mL) Peak area Limit of Quantification 20％ 0.2108 42482 ① 50％ 0.5269 105899 ② 100％ 1.0538 211908 ③ 150％ 1.5807 318862 ④ 200％ 2.1076 426826

[0192] Linear equation: y = 203248x - 1854, linear regression coefficient r = 1.000, intercept percentage: 0.9%. Conclusion: Impurity C (293nm) showed good linearity in the concentration range of 0.2108 to 2.1076 μg / mL, which is equivalent to 20% to 200% of the unknown impurity limit.

[0193] Table 19 Results of linear detection of impurity D

[0194] Solution serial number percentage(%) Concentration (μg / mL) Peak area Limit of Quantification 20％ 0.2061 65911 ① 50％ 0.5153 135899 ② 100％ 1.0306 261908 ③ 150％ 1.5459 400862 ④ 200％ 2.0612 516826

[0195] Linear equation: y = 253471x + 1602.6, linear regression coefficient r = 0.9990, intercept percentage: 0.6%.

[0196] Conclusion: Impurity D (293 nm) showed good linearity in the concentration range of 0.2061–2.0612 μg / mL, which is equivalent to 20%–200% of the limit for unknown impurities.

[0197] Table 20 Results of linear detection of impurity E

[0198] Solution serial number percentage(%) Concentration (μg / mL) Peak area Limit of Quantification 20％ 0.2102 14853 ① 50％ 0.5254 35632 ② 100％ 1.0508 70564 ③ 150％ 1.5763 110896 ④ 200％ 2.1017 148528

[0199] Linear equation: y = 70600x - 892.17, linear regression coefficient r = 0.9996, intercept percentage: 1.3%.

[0200] Conclusion: Impurity E (293 nm) showed good linearity in the concentration range of 0.2102–2.1017 μg / mL, which is equivalent to 20%–200% of the limit for unknown impurities.

[0201] Table 21 Linear detection results of impurity F

[0202] Solution serial number percentage(%) Concentration (μg / mL) Peak area Limit of Quantification 20％ 0.2044 42241 ① 50％ 0.5111 105652 ② 100％ 1.0221 211704 ③ 150％ 1.5332 317856 ④ 200％ 2.0442 423308

[0203] Linear equation: y = 206141x + 1482.2, linear regression coefficient r = 1.000, intercept percentage: 0.7%.

[0204] Conclusion: Impurity F (293 nm) showed good linearity in the concentration range of 0.2044–2.0442 μg / mL, which is equivalent to 20%–200% of the limit for unknown impurities.

[0205] Table 22 Results of linear detection of impurity G

[0206] Solution serial number percentage(%) Concentration (μg / mL) Peak area Limit of Quantification 20％ 0.2020 47003 ① 50％ 0.5051 118307 ② 100％ 1.0102 236013 ③ 150％ 1.5153 354220 ④ 200％ 2.0205 472826

[0207] Linear equation: y = 234018x - 190, linear regression coefficient r = 1.000, intercept percentage: 0.1%.

[0208] Conclusion: Impurity G (248 nm) showed good linearity in the concentration range of 0.2020–2.0205 μg / mL, which is equivalent to 20%–200% of the limit for unknown impurities.

[0209] Table 23 Linearity Detection Results of Impurity SM-6

[0210] Solution serial number percentage(%) Concentration (μg / ml) Peak area Limit of Quantification 20％ 0.2065 31535 ① 50％ 0.5163 78089 ② 100％ 1.0326 158177 ③ 150％ 1.5489 236266 ④ 200％ 2.0651 316454

[0211] Linear equation: y = 153307x - 529.07, linear regression coefficient r = 1.000, intercept percentage: 0.3%. Conclusion: Impurity SM-6 (248nm) showed good linearity in the concentration range of 0.20265–2.0651 μg / mL, equivalent to 20%–200% of the unknown impurity limit.

[0212] Table 24 Results of linear detection of impurity H

[0213] Solution serial number percentage(%) Concentration (μg / mL) Peak area Limit of Quantification 20％ 0.2077 20880 ① 50％ 0.5193 52850 ② 100％ 1.0386 104899 ③ 150％ 1.5579 157049 ④ 200％ 2.0772 209898

[0214] Linear equation: y = 100921x + 106.45, linear regression coefficient r = 1.000, intercept percentage: 0.1%. Conclusion: Impurity H (248nm) shows good linearity in the concentration range of 0.2077 to 2.0772 μg / mL, which is equivalent to 20% to 200% of the limit for unknown impurities.

[0215] 2.4.3 Correction Factor

[0216] 2.4.3.1 Calculation results of correction factor

[0217] Table 25 Calculation results of 293nm correction factor

[0218] Impurity Name Slope of linear equation Correction factor calculation value EP USP Final value of correction factor Impurity A 252327 0.95 1.0 1.0 1.0 Impurity B 164006 1.46 1.4 1.41 1.4 Impurity C 203248 1.18 1.0 1.0 1.0 Impurity D 253471 0.94 1.0 1.0 1.0 Impurity E 70600 3.39 3.5 3.45 3.5 impurity F 206141 1.16 1.0 1.0 1.0 Moxifloxacin 239387 / / / /

[0219] Conclusion: The correction factors for impurities A, B, C, D, E, and F are basically consistent with the correction factors for the relevant impurities specified in EP and USP. Therefore, it is finally determined that the correction factors for the relevant impurities specified in EP shall be used for calculation.

[0220] 2.4.3.2 Accuracy of the correction factor

[0221] The test results under the repeatability item were used to compare the calculation results of the principal component self-control method and the impurity external standard method with correction factors to confirm the accuracy of the correction factors. The specific results are shown in the table below:

[0222] Table 26 Comparison of results from the correction factor method and the external standard method

[0223]

[0224] Conclusion: The above results show that the results of the two calculation methods are basically consistent, with no significant differences.

[0225] 2.5 Limit of detection and limit of quantitation

[0226] 2.5.1 Solvent: Same as 1.2.1.

[0227] 2.5.2 Moxifloxacin hydrochloride stock solution: Weigh 10 mg of moxifloxacin hydrochloride reference standard accurately, place it in a 10 mL volumetric flask, dissolve and dilute to the mark with solvent, and shake well.

[0228] 2.5.3 Impurity Stock Solution: Accurately weigh approximately 10 mg of impurity A reference standard, place it in a 10 mL volumetric flask, dissolve and dilute to the mark with solvent, and shake well. The stock solutions for impurities B, C, D, E, and F are prepared in the same manner as the impurity A stock solution.

[0229] 2.5.4 The preparation of impurity G, impurity SM-6 stock solution and impurity H stock solution is the same as in 1.2.4.

[0230] For the limit of quantitation solution, take appropriate amounts of the above stock solutions and dilute quantitatively according to a ratio of the intensity of the target peak to the noise peak signal of not less than 10:1. Prepare 6 replicates.

[0231] Take appropriate amounts of the above stock solutions and quantitatively dilute them according to the intensity ratio of the peak to be measured to the noise peak signal of not less than 3:1.

[0232] Accurately measure 20 μl of the limit of quantitation (LOQ) solution and limit of detection (LOD) solution, inject them into the liquid chromatograph, and record the chromatogram. The signal-to-noise ratio (SNR) of each impurity's LOQ should be no less than 10:1; for six LOQ solutions of the same concentration, the RSD value of the peak area of ​​each analyte should not exceed 10%; the SNR of each impurity's LOD should be no less than 3:1. The test results are shown in the table below:

[0233] Table 27 Results of determination of quantitation limits and detection limits for each impurity.

[0234]

[0235]

[0236] Table 28 Results of repeatability tests at the limit of quantitation

[0237] name 1 2 3 4 5 6 average value RSD% Impurity A 52001 52984 51339 52176 51898 50262 51777 1.8 Moxifloxacin 47589 48243 46998 47121 49082 46222 47543 2.1 Impurity B 38664 37525 38112 36254 39505 38385 38074 2.9 Impurity C 42396 42882 43439 44001 42988 41950 42943 1.7 Impurity D 65231 64410 65012 63358 62624 64991 64271 1.6 Impurity E 14688 14233 14052 14222 14505 14476 14363 1.6 impurity F 42352 41159 42287 43202 44009 42544 42592 2.2 Impurity G 46989 47201 46854 46544 46825 47300 46952 0.6 Impurity SM-6 31264 30599 32222 31463 30879 32411 31473 2.3 impurity H 20769 20998 21455 22004 21052 20653 21155 2.4

[0238] Conclusion: The signal-to-noise ratio of the quantitation limit for each impurity was not less than 10:1; the RSD values ​​of the peak areas of each analyte in the six quantitation limit solutions of the same concentration were all between 0.6% and 2.9%; the signal-to-noise ratio of the detection limit for each impurity was not less than 3:1, which met the validation requirements.

[0239] 2.6 Precision

[0240] 2.6.1 Repeatability

[0241] 2.6.1.1 Solvent is the same as in 1.2.1.

[0242] 2.6.1.2 Moxifloxacin hydrochloride stock solution: Same as 2.5.2.

[0243] Control solution: Same as 1.2.3.

[0244] 2.6.1.3 Impurity stock solution: Same as 2.5.3.

[0245] 2.6.1.4 The preparation of impurity G, impurity SM-6 stock solution and impurity H stock solution is the same as in 1.2.4.

[0246] 2.6.1.5 Impurity Mixed Stock Solution: Accurately measure 1 mL of each of the stock solutions of impurities A, B, C, D, E, F, G, SM-6, and H, place them in a 100 mL volumetric flask, dilute to the mark with solvent, and mix well.

[0247] 2.6.1.6 Spiked Solution: Accurately weigh 10 mg of this product and place it in a 10 mL volumetric flask. Add an appropriate amount of solvent to dissolve it. Accurately add 1 mL of the impurity stock solution, dilute to the mark with solvent, and shake well. Prepare 6 replicates.

[0248] Take 20 μl each of the blank solution (solvent under section 1.2.1), the control solution, and the spiked solution, and inject them separately into the liquid chromatograph, recording the chromatograms. The RSD values ​​of the six analytes should not exceed 3%. The detection results (impurities A, B, C, D, E, and F are quantified using the self-reference method; impurities G, SM-6, and H are quantified using the external standard method) are shown in the table below:

[0249] Table 29 Repeatability Test Results

[0250] name 1 2 3 4 5 6 average value RSD% Impurity A% 0.11 0.11 0.10 0.10 0.11 0.11 0.11 2.0 Impurities B% 0.11 0.11 0.11 0.10 0.11 0.11 0.11 1.2 Impurities C% 0.10 0.10 0.10 0.10 0.11 0.10 0.10 2.8 Impurities D% 0.10 0.11 0.10 0.11 0.10 0.10 0.10 2.2 Impurities E% 0.10 0.10 0.09 0.10 0.10 0.10 0.10 2.7 Impurities F% 0.11 0.11 0.11 0.11 0.11 0.11 0.11 2.2 Impurities G% 0.10 0.10 0.10 0.10 0.10 0.10 0.10 2.4 Impurities SM-6% 0.12 0.12 0.12 0.12 0.12 0.12 0.12 1.4 Impurities H% 0.10 0.10 0.10 0.10 0.10 0.10 0.10 2.7

[0251] Conclusion: The RSD values ​​of impurities in the six spiked test solutions ranged from 1.2% to 2.8%, none of which exceeded 3%, and the repeatability met the requirements.

[0252] 2.6.2 Reproducibility

[0253] Following the repeatability test method, 12 spiking solutions were prepared for the same batch of samples on different dates, using different instruments, in different laboratories, and by different operators. The results (impurities A, B, C, D, E, and F were quantified using the self-control method, and impurities G, SM-6, and H were quantified using the external standard method) are detailed in the table below. The RSD value of the 12 analytes must not exceed 6%.

[0254] Table 30 Reproducibility Test Results

[0255]

[0256] Conclusion: The RSD values ​​of impurity content in the 12 spiked test solutions ranged from 1.9% to 5.8%, none of which exceeded 6%, and the reproducibility met the requirements.

[0257] 2.7 Accuracy

[0258] The solvent is the same as in 1.2.1.

[0259] 2.7.1 Test solution: Weigh 10 mg of this product accurately, place it in a 10 mL volumetric flask, add solvent to dissolve and dilute to the mark, and mix well.

[0260] 2.7.2 The preparation of the stock solutions for impurities A, B, C, D, E, and F is the same as in 2.5.3.

[0261] 2.7.3 The impurity mixed stock solution is the same as 2.6.1.5.

[0262] 2.7.4 Accurately measure 1 mL of the impurity mixed stock solution into a 10 mL volumetric flask, dilute to the mark with solvent, and mix well.

[0263] For the limit of quantitation (LOQ) solution, accurately weigh 10 mg of this product and place it in a 10 mL volumetric flask. Add an appropriate amount of solvent to dissolve the product, accurately add 0.2 mL of the impurity stock solution, dilute to the mark with solvent, and shake well. This is the limit of quantitation (LOQ) solution. Prepare three parallel solutions.

[0264] For the 50% spiked solution, accurately weigh 10 mg of this product and place it in a 10 mL volumetric flask. Add an appropriate amount of solvent to dissolve it, accurately add 0.5 mL of the impurity stock solution, dilute to the mark with solvent, shake well, and repeat to prepare 3 portions.

[0265] For a 100% spiked solution, accurately weigh 10 mg of this product and place it in a 10 mL volumetric flask. Add an appropriate amount of solvent to dissolve the product, then accurately add 1 mL of the impurity stock solution. Dilute to the mark with solvent and mix well. Repeat to prepare 3 portions.

[0266] For the 150% spiked solution, accurately weigh 10 mg of this product and place it in a 10 mL volumetric flask. Add an appropriate amount of solvent to dissolve it, then accurately add 1.5 mL of the impurity stock solution. Dilute to the mark with solvent and shake well. Repeat to prepare 3 portions.

[0267] The above solutions were injected into the liquid chromatograph, and the chromatograms were recorded. The recoveries and average recoveries at the quantitation limit concentrations were both 75%–120%, and the recoveries and average recoveries at concentrations of 50%–150% were both 80%–120%; the RSD value of the recoveries should not exceed 6%. The detection results (impurities A, B, C, D, E, and F were quantified using their own reference method; impurities G, SM-6, and H were quantified using the external standard method) are as follows:

[0268] Table 31 Results of Impurity A Recovery Rate Test

[0269]

[0270] Table 32 Results of Impurity B Recovery Rate Test

[0271]

[0272]

[0273] Table 33 Results of Impurity C Recovery Rate Test

[0274]

[0275] Table 34 Results of Impurity D Recovery Rate Test

[0276]

[0277] Table 35 Results of Impurity E Recovery Rate Test

[0278]

[0279]

[0280] Table 36 Results of Impurity F Recovery Rate Test

[0281]

[0282] Table 37 Results of Impurity G Recovery Rate Test

[0283]

[0284] Table 38 Results of the SM-6 Impurity Recovery Test

[0285]

[0286]

[0287] Table 39 Results of Impurity H Recovery Rate Test

[0288]

[0289] Conclusion: As shown in the table above, the average recoveries of each impurity at their quantitation limit concentration levels are between 98.1% and 100.6%, and the average recoveries at the limit concentration levels are between 93.2% and 101.9%, with RSDs not exceeding 6%. This method has good accuracy.

[0290] 2.8 Method Durability

[0291] Solvents, control solutions, and spiking solutions shall be prepared in accordance with section 2.6.1.

[0292] Sensitivity solution: Accurately measure 2 mL of the control solution, place it in a 10 mL volumetric flask, dilute to the mark with solvent, and shake well.

[0293] Accurately measure the blank solution (solvent under section 1.2.1), sensitivity solution, control solution, and spiked solution, inject them into the liquid chromatograph, and record the chromatogram.

[0294] The robustness of the method was assessed by examining the resolution of each impurity in the chromatogram of the spiked solution and the determination of the content of each impurity in the spiked solution of the same concentration after minor variations in chromatographic conditions. The changes in conditions and the determination results are shown in the table below. The resolution between the peaks of each component was not less than 1.5, and the content of each impurity in the spiked solution of the same concentration was basically consistent with the results under normal conditions.

[0295] Table 40 Range of Durability Test Parameters

[0296]

[0297] Table 41 Durability Test Results

[0298]

[0299]

[0300] Conclusion: After minor changes in chromatographic conditions, the resolution between the peaks of each component was not less than 1.5. The content of each impurity in the spiked solution of the same concentration was basically consistent with the detection results under normal conditions, indicating good robustness.

[0301] 3. Summary of Methodological Validation of Related Matter

[0302] This analytical method is used for the quantitative determination of impurities. According to the Guidelines for Validation of Analytical Methods in Drug Quality Standards (General Chapter 9101, Part IV of the Chinese Pharmacopoeia 2020), the validation of related substances methodologies includes system suitability, specificity, solution stability, limit of detection, limit of quantitation, linear range, precision, accuracy, and robustness testing.

[0303] Table 42 Summary of Methodological Validation Related to Matter

[0304]

[0305]

[0306] 4. Technical Effects

[0307] The related substance detection method provided by this invention combines a chromatographic column packed with phenylsilane-bonded silica gel (Agilent Eclipse XDB-Phenyl, 4.6 × 250 mm, 5 μm) with methanol-pH 2.5 buffer solution flow and employs gradient elution to achieve the following technical effects:

[0308] High sensitivity: The detection limit is as low as 0.07 μg / mL, which can effectively detect trace residues of impurities in moxifloxacin hydrochloride, and the sensitivity is improved by 2 times compared with traditional methods;

[0309] High resolution: This method, by selecting chromatographic columns and chromatographic conditions, enables moxifloxacin hydrochloride to achieve baseline separation from adjacent impurity peaks, with a resolution greater than 1.5, resulting in more accurate detection results.

[0310] More stable test solution: This method makes the test solution more stable by adding anhydrous sodium sulfite to the solvent, thereby achieving more accurate detection results.

[0311] Controlling impurity F as a known impurity: This method controls impurity F (N-methylmoxifloxacin) as a known impurity, allowing for a better examination of how this impurity changes during stability testing.

[0312] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for detecting related substances in moxifloxacin hydrochloride raw material, characterized in that, Includes the following steps: The sample to be tested is dissolved to obtain a test solution; the solvent used for dissolution includes tetrabutylammonium hydrogen sulfate, potassium dihydrogen phosphate, phosphoric acid, water, triethylamine, anhydrous sodium sulfite and methanol; The test solution was analyzed by high performance liquid chromatography, and the content of related substances in moxifloxacin hydrochloride raw material was obtained by using either the main component self-comparison method or the impurity external standard method. The impurities mentioned include impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H and impurity SM-6; The conditions for the liquid chromatography detection include: mobile phase A is a buffer solution, and mobile phase B is methanol; the buffer solution includes tetrabutylammonium hydrogen sulfate, potassium dihydrogen phosphate, water, phosphoric acid, and triethylamine; The flow rates of mobile phases A and B are 1.3 mL / min; The elution method is gradient elution; the gradient elution procedure is as follows: 0.0–30 min: The volume percentage of the mobile phase A decreases uniformly from 75% to 68%; 30–45 min: The volume percentage of the mobile phase A decreases uniformly from 68% to 45%; 45–60 min: The volume percentage of the mobile phase A is 45%; 60–61 min: The volume percentage of the mobile phase A increases uniformly from 45% to 75%; 61–75 min: The volume percentage of the mobile phase A is 75%. The detection wavelengths are 293nm and 248nm.

2. The detection method according to claim 1, characterized in that, The liquid chromatography column is packed with phenylsilane-bonded silica gel; the column temperature is 40℃; and the injection volume is 20μL.

3. The detection method according to claim 1, characterized in that, The chromatographic column used was an Agilent Eclipse XDB-Phenyl column.

4. The detection method according to claim 1, characterized in that, The detection wavelength for impurities A, B, C, D, E, and F is 293 nm.

5. The detection method according to claim 1, characterized in that, The detection wavelength for impurities G, H, and SM-6 is 248 nm.

6. The detection method according to claim 1, characterized in that, The volume ratio of the mixture of tetrabutylammonium bisulfate, potassium dihydrogen phosphate, phosphoric acid, water, triethylamine and anhydrous sodium sulfite in the solvent used for dissolving to methanol is 20:

80.

7. The detection method according to claim 6, characterized in that, The mass ratio of tetrabutylammonium bisulfate to potassium dihydrogen phosphate is 0.5:1; the mass ratio of potassium dihydrogen phosphate to anhydrous sodium sulfite is 100:2; and the mass ratio of potassium dihydrogen phosphate to the volume ratio of phosphoric acid is 1g:2mL.

8. The detection method according to claim 1, characterized in that, The mass ratio of tetrabutylammonium bisulfate to potassium dihydrogen phosphate in mobile phase A is 0.5:1; the mass ratio of potassium dihydrogen phosphate to the volume ratio of phosphoric acid is 1 g: 2 mL; and the pH value of mobile phase A is 2.

5.

9. The detection method according to claim 1, characterized in that, When using the external standard method, impurity G, impurity H, and impurity SM-6 are calculated using Equation 2: In Formula 2: Xi -- the content of impurities G, H, and SM-6 in the test sample, %; A X --Peak areas of impurities G, H, and SM-6 in the test solution; m -- Weight of the test sample, mg; f is calculated from equation 1. In Equation 1: f -- response factor of impurities G, H, and SM-6 in the reference solution; C R --Concentrations of impurities G, H, and SM-6 in the reference solution, in μg / mL; A R --Peak areas of impurities G, H, and SM-6 in the reference solution.

10. The detection method according to claim 1, characterized in that, Impurities A, B, C, D, E, and F are calculated using Equation 3: In Equation 3: X t --The content of single impurities A, B, C, D, E, and F in the test sample, in %; A X --The peak area of ​​impurities A, B, C, D, E, and F in the test solution; A R --Peak area of ​​the main component in the control solution; F -- Relative correction factor for each impurity, where impurities A, C, D, and F are 1; impurity B is 1.4; and impurity E is 3.5.

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