Method for rapidly detecting genotoxic impurity hydroxylamine hydrochloride by HPLC-UV (High Performance Liquid Chromatography-Ultraviolet) method

Abstract

The invention relates to the technical field of pharmaceutical analysis, and provides a method for rapidly detecting a genotoxic impurity hydroxylamine hydrochloride by applying an HPLC-UV (High Performance Liquid Chromatography-Ultraviolet) method, which is used for detecting hydroxylamine hydrochloride in moxidectin by adopting the HPLC-UV method. During detection, a derivatization reagent and an organic acid solution are added for a derivatization reaction when a sample solution containing hydroxylamine hydrochloride is prepared. According to the technical scheme, the problems of low detection recovery rate and low detection accuracy when the content of the genotoxic impurity hydroxylamine hydrochloride in moxidectin is detected in the prior art are solved.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to a method for rapidly detecting genotoxic impurity hydroxylamine hydrochloride by using an HPLC-UV method. Background Art

[0002] Moxidectin, also known as moxidectin, is a macrolide antibiotic produced by fermentation of Streptomyces, which is widely used to treat and prevent internal and external parasitic infections in animals. Impurity levels are one of the important factors affecting the effectiveness and safety of drugs, and hydroxylamine hydrochloride is a key raw material for the synthesis of APIs, generally used as a reducing agent. Hydroxylamine hydrochloride is used in the synthesis process of moxidectin to participate in redox reactions, and it is very likely to remain in the final product. Since hydroxylamine hydrochloride is a genotoxic impurity, it is mutagenic and carcinogenic. Therefore, establishing a practical and feasible method for controlling the potential genotoxic impurity hydroxylamine hydrochloride detection is of great significance to improving the quality of moxidectin and ensuring drug safety.

[0003] High performance liquid chromatography (HPLC) is widely used in the field of drug analysis due to its fast analysis speed, high separation efficiency, and high detection sensitivity. Since hydroxylamine hydrochloride itself has no ultraviolet absorption, it is impossible to achieve quantitative detection of it in the raw material of moxidectin by directly using the HPLC-UV method. In the patent applications with publication numbers CN115876930A and CN118225941A, benzaldehyde is used as a derivatization reagent. In order to reduce the interference of the sample matrix, extraction or centrifugation is first used, and then derivatization is performed to obtain the test solution. However, such a sample processing method makes the recovery rate of the detection method low, and it is impossible to quickly and efficiently perform quantitative detection of hydroxylamine hydrochloride.

[0004] In view of this, a method for quickly and efficiently detecting the content of hydroxylamine hydrochloride in moxidectin raw materials is proposed, which is of great significance for the quality control of moxidectin raw materials. Summary of the invention

[0005] The invention provides a method for quickly detecting hydroxylamine hydrochloride, a genotoxic impurity, by using an HPLC-UV method, which solves the problems of low detection recovery rate and low detection accuracy when detecting the content of hydroxylamine hydrochloride, a genotoxic impurity in moxidectin in the related art.

[0006] The technical solution of the present invention is as follows: The present invention provides a method for rapidly detecting the genotoxic impurity hydroxylamine hydrochloride by using the HPLC-UV method, and the hydroxylamine hydrochloride in moxidectin is detected by using the HPLC-UV method; During the detection, a derivatization reagent and an organic acid solution are added to the sample solution containing hydroxylamine hydrochloride to carry out a derivatization reaction.

[0007] As a further technical solution, the derivatization reagent includes a p-nitrobenzaldehyde solution; the organic acid solution includes one of a formic acid solution, an acetic acid solution, and a phosphoric acid solution; The temperature of the derivatization reaction is 50-60° C. and the time is 10-20 min.

[0008] As a further technical solution, when the organic acid solution is an acetic acid solution, the mass fraction of the acetic acid solution is 0.5% to 1.5%; The concentration of the p-nitrobenzaldehyde solution is 0.9-1.1 mg / mL.

[0009] As a further technical solution, the sample solution containing hydroxylamine hydrochloride includes a test solution and a reference solution.

[0010] As a further technical solution, the method for preparing the test solution comprises the following steps: adding a derivatization reagent and an organic acid solution to a moxidectin stock solution to perform a derivatization reaction to obtain a test solution; The preparation method of the reference substance solution comprises the following steps: adding a derivatization reagent and an organic acid solution to a hydroxylamine hydrochloride stock solution, performing a derivatization reaction, and obtaining a reference substance solution.

[0011] As a further technical solution, a diluent is added after the derivatization reaction.

[0012] The diluent is a mixed solution of acetonitrile and water, and the volume fraction of acetonitrile in the mixed solution is 70% to 75%; The volume ratio of the moxidectin stock solution to the diluent is 1:8-10.

[0013] As a further technical solution, the method for preparing the moxidectin stock solution comprises the following steps: dissolving moxidectin in a solvent to obtain a moxidectin stock solution with a concentration of 10 mg / mL; The preparation method of the hydroxylamine hydrochloride stock solution comprises the following steps: dissolving hydroxylamine hydrochloride in a solvent to obtain a hydroxylamine hydrochloride stock solution with a concentration of 0.04 mg / mL.

[0014] As a further technical solution, when the moxidectin is dissolved in a solvent, the solvent is acetonitrile.

[0015] As a further technical solution, when the hydroxylamine hydrochloride is dissolved in a solvent, the solvent is a mixed solution of acetonitrile and water, and the volume ratio of the acetonitrile to water is 3-4:1.

[0016] As a further technical solution, the following steps are included: the sample solutions containing hydroxylamine hydrochloride are tested respectively by HPLC-UV method, the chromatogram is recorded, and the content of hydroxylamine hydrochloride is calculated by peak area according to external standard method.

[0017] As a further technical solution, when the test solution is prepared, the volume ratio of the moxidectin stock solution, the derivatization reagent, and the organic acid solution is 1 mL: 1 mL: 18~23 µL; Preferably, the volume ratio of moxidectin stock solution, derivatization reagent, and organic acid solution is 1 mL:1 mL:20 µL.

[0018] As a further technical solution, when the reference solution is prepared, the volume ratio of the hydroxylamine hydrochloride stock solution, the derivatization reagent, and the organic acid solution is 1 mL: 1 mL: 18~23 µL; Preferably, the volume ratio of the hydroxylamine hydrochloride stock solution, the derivatization reagent, and the organic acid solution is 1 mL:1 mL:20 µL.

[0019] As a further technical solution, during the HPLC-UV detection, the chromatographic column is an Angilent SB C18 column with a specification of 150×4.6 mm; The column temperature is 25-35°C, the flow rate is 0.8-1.2 mL / min, and the detection wavelength is 300 nm; The mobile phase is trifluoroacetic acid solution and acetonitrile, and the volume ratio of the trifluoroacetic acid solution to acetonitrile is 7:3-3:1; The mass fraction of trifluoroacetic acid in the trifluoroacetic acid solution is 0.03% to 0.05%.

[0020] As a further technical solution, during the HPLC-UV detection, the filler is octadecylsilane bonded silica gel.

[0021] In the present invention, the maximum absorption wavelength of p-nitrobenzaldehyde is 265nm, while the maximum absorption wavelength of moxidectin and its related substances is 240nm, and the maximum absorption wavelength of hydroxylamine hydrochloride derivatives is 300nm. Both ends of p-nitrobenzaldehyde are electron-withdrawing groups, which hinder each other. After derivatization, the aldehyde becomes an oxime, and the electron-withdrawing ability is reduced. The electrons as a whole move closer to the benzene ring and the nitro group, the conjugation is enhanced, and the ultraviolet absorption moves to the long wave, which is consistent with the maximum absorption wavelength of 300nm of the hydroxylamine hydrochloride derivatives. Therefore, the maximum absorption wavelength of 300nm of the hydroxylamine hydrochloride derivatives is selected as the detection wavelength, and the interference caused by the sample matrix of moxidectin and its related substances can be eliminated at this wavelength.

[0022] In the present invention, an Angilent SB C18 chromatographic column is used, and the method is suitable for a low organic phase and a low pH mobile phase, thereby enhancing the retention of highly polar compounds. At the same time, a trifluoroacetic acid solution and an acetonitrile mobile phase are used, and at a detection wavelength of 280 to 320 nm, the symmetry of the chromatographic peak is improved, and the sensitivity and accuracy of the detection are improved.

[0023] The working principle and beneficial effects of the present invention are: 1. The present invention reduces the sample extraction and centrifugation processes by performing a pre-column derivatization reaction on a sample solution containing hydroxylamine hydrochloride, thereby making the derivatization reaction simple and quick to operate, and the generated derivatives are highly stable, thereby avoiding damage to the chromatographic column and the instrument due to unstable decomposition of compound molecules, and improving the accuracy of the detection results of the genotoxic impurity hydroxylamine hydrochloride; wherein, during the derivatization reaction, a derivatization reagent and an organic acid solution are added to react with hydroxylamine hydrochloride to generate hydroxylamine hydrochloride derivatives, which ultimately improves the recovery rate of hydroxylamine hydrochloride; 2. The present invention can quantitatively detect the genotoxic impurity hydroxylamine hydrochloride, thereby effectively tracking and monitoring the quality changes of the finished product reaction process, and has great application value in the quality control of drug research and development and production. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0025] Figure 1 It is the chromatogram of the reference substance solution in Example 1; Figure 2 The chromatogram of the test solution in Example 1; Figure 3 It is the blank solution chromatogram in Example 1; Figure 4 This is the linear relationship diagram of the reference substance in Example 1. DETAILED DESCRIPTION The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] In the following examples and comparative examples, the content of moxidectin was 99.1%, which was homemade; The content of hydroxylamine hydrochloride was 99.0%, which was purchased from Tianjin Guangfu Fine Chemical Research Institute.

[0027] Example 1 A method for rapidly detecting genotoxic impurity hydroxylamine hydrochloride by using HPLC-UV method, wherein hydroxylamine hydrochloride in moxidectin is detected by using HPLC-UV method; during the detection, a derivatization reagent and an organic acid solution are added to the sample solution containing hydroxylamine hydrochloride for derivatization reaction; wherein the derivatization reagent comprises a p-nitrobenzaldehyde solution and the organic acid solution is an acetic acid solution; the detection is as follows: Column: Angilent SB C18, 150×4.6 mm, 5 μm; Filler: octadecylsilane bonded silica gel; Mobile phase: by volume percentage, 75% trifluoroacetic acid and 25% acetonitrile with a mass fraction of 0.05%; Column temperature: 30°C; Detection wavelength: 300nm; Flow rate: 1.0 mL / min; Injection volume: 20 μL; The preparation method of the reference substance stock solution comprises the following steps: accurately weighing 10 mg of the hydroxylamine hydrochloride reference substance, dissolving it in 250 mL of a mixed solution (the volume ratio of acetonitrile to water is 3:1), shaking well, and obtaining a hydroxylamine hydrochloride stock solution with a concentration of 0.04 mg / mL for standby use; The preparation method of the reference substance solution comprises the following steps: accurately measuring 1 mL of the reference substance stock solution, adding 1 mL of a 1 mg / mL p-nitrobenzaldehyde solution and mixing for 15 minutes, after uniform mixing, adding 20 µL of a 1% acetic acid solution, reacting for 10 minutes in a water bath at 60°C, adding 8 mL of a diluent (a mixed solution of acetonitrile and water, with a volume fraction of 75% acetonitrile), shaking well, and setting aside; The preparation method of the test sample stock solution comprises the following steps: accurately weighing 100 mg of moxidectin, dissolving it in 10 mL of acetonitrile, and shaking it well to obtain a moxidectin stock solution with a concentration of 10 mg / mL for use; The preparation method of the test solution comprises the following steps: accurately measuring 1 mL of the test stock solution, adding 1 mL of a 1 mg / mL p-nitrobenzaldehyde solution and mixing for 15 minutes, after mixing evenly, adding 20 µL of a 1% acetic acid solution, reacting for 10 minutes in a 60°C water bath, adding 8 mL of a diluent (a mixed solution of acetonitrile and water, with a volume fraction of 75% acetonitrile), shaking well, and setting aside; Determination: Take the test solution and the reference solution and inject them separately, and record the chromatogram; The chromatogram of the reference solution is as follows Figure 1 As shown, the chromatogram of the test solution is Figure 2 shown.

[0028] Example 2 A method for rapidly detecting genotoxic impurity hydroxylamine hydrochloride by using HPLC-UV method, wherein hydroxylamine hydrochloride in moxidectin is detected by using HPLC-UV method; during the detection, a derivatization reagent and an organic acid solution are added to the sample solution containing hydroxylamine hydrochloride for derivatization reaction; wherein the derivatization reagent comprises a p-nitrobenzaldehyde solution and the organic acid solution is an acetic acid solution; the detection is as follows: Column: Angilent SB C18, 150×4.6 mm, 5 μm; Filler: octadecylsilane bonded silica gel; Mobile phase: 75% trifluoroacetic acid and 25% acetonitrile with a mass fraction of 0.03% by volume; Column temperature: 25°C; Detection wavelength: 300nm; Flow rate: 0.8 mL / min; Injection volume: 20 μL; The preparation method of the reference substance stock solution comprises the following steps: accurately weighing 10 mg of the hydroxylamine hydrochloride reference substance, dissolving it in 250 mL of a mixed solution (the volume ratio of acetonitrile to water is 4:1), shaking well, and obtaining a hydroxylamine hydrochloride stock solution with a concentration of 0.04 mg / mL for standby use; The preparation method of the reference substance solution comprises the following steps: accurately measuring 1 mL of the reference substance stock solution, adding 1 mL of a 0.9 mg / mL p-nitrobenzaldehyde solution and mixing for 15 minutes, after uniform mixing, adding 20 µL of a 0.5% acetic acid solution, reacting for 20 minutes in a water bath at 50°C, adding 10 mL of a diluent (a mixed solution of acetonitrile and water, with a volume fraction of 70% acetonitrile), shaking well, and setting aside; The preparation method of the test sample stock solution comprises the following steps: accurately weighing 100 mg of moxidectin, dissolving it in 10 mL of acetonitrile, and shaking it well to obtain a moxidectin stock solution with a concentration of 10 mg / mL for use; The preparation method of the test solution includes the following steps: accurately measure 1 mL of the test stock solution, add 1 mL of 0.9 mg / mL p-nitrobenzaldehyde solution and mix for 15 minutes. After mixing evenly, add 20 µL of 0.5% acetic acid solution, react in a 50°C water bath for 20 minutes, add 10 mL of diluent (a mixed solution of acetonitrile and water, with a volume fraction of 70% acetonitrile), shake well, and set aside.

[0029] Example 3 A method for rapidly detecting genotoxic impurity hydroxylamine hydrochloride by using HPLC-UV method, wherein hydroxylamine hydrochloride in moxidectin is detected by using HPLC-UV method; during the detection, a derivatization reagent and an organic acid solution are added to the sample solution containing hydroxylamine hydrochloride for derivatization reaction; wherein the derivatization reagent comprises a p-nitrobenzaldehyde solution and the organic acid solution is an acetic acid solution; the detection is as follows: Column: Angilent SB C18, 150×4.6 mm, 5 μm; Filler: octadecylsilane bonded silica gel; Mobile phase: 75% trifluoroacetic acid and 25% acetonitrile with a mass fraction of 0.03% by volume; Column temperature: 35°C; Detection wavelength: 300nm; Flow rate: 1.2 mL / min; Injection volume: 20 μL; The preparation method of the reference substance stock solution comprises the following steps: accurately weighing 10 mg of the hydroxylamine hydrochloride reference substance, dissolving it in 250 mL of a mixed solution (the volume ratio of acetonitrile to water is 4:1), shaking well, and obtaining a hydroxylamine hydrochloride stock solution with a concentration of 0.04 mg / mL for standby use; The preparation method of the reference substance solution comprises the following steps: accurately measuring 1 mL of the reference substance stock solution, adding 1 mL of a 1.1 mg / mL p-nitrobenzaldehyde solution and mixing for 20 minutes, after uniform mixing, adding 20 µL of a 1.5% acetic acid solution, reacting for 20 minutes in a water bath at 50°C, adding 9 mL of a diluent (a mixed solution of acetonitrile and water, with a volume fraction of 70% acetonitrile), shaking well, and setting aside; The preparation method of the test sample stock solution comprises the following steps: accurately weighing 100 mg of moxidectin, dissolving it in 10 mL of acetonitrile, and shaking it well to obtain a moxidectin stock solution with a concentration of 10 mg / mL for use; The preparation method of the test solution includes the following steps: accurately measure 1 mL of the test stock solution, add 1 mL of 1.1 mg / mL p-nitrobenzaldehyde solution and mix for 20 minutes, after mixing evenly, add 20 µL of 1.5% acetic acid solution, react in a 50°C water bath for 20 minutes, add 9 mL of diluent (a mixed solution of acetonitrile and water, with a volume fraction of 70% acetonitrile), shake well, and set aside.

[0030] Example 4 The only difference between this embodiment and embodiment 1 is that in this embodiment, in the preparation method of the reference solution and the test solution, the amount of acetic acid solution added is 15 μL.

[0031] Example 5 The only difference between this embodiment and embodiment 1 is that in this embodiment, in the preparation method of the reference solution and the test solution, the amount of acetic acid solution added is 25 μL.

[0032] Example 6 The only difference between this embodiment and embodiment 1 is that in this embodiment, in the preparation method of the reference solution and the test solution, the amount of acetic acid solution added is 18 μL.

[0033] Example 7 The only difference between this embodiment and embodiment 1 is that in this embodiment, in the preparation method of the reference solution and the test solution, the amount of acetic acid solution added is 23 μL.

[0034] Example 8 The only difference between this embodiment and embodiment 1 is that, in this embodiment, the volume ratio of trifluoroacetic acid with a mass fraction of 0.03% and acetonitrile in the mobile phase is 7:3.

[0035] Example 9 The only difference between this embodiment and embodiment 1 is that in this embodiment, in the mobile phase, the volume ratio of trifluoroacetic acid with a mass fraction of 0.03% and acetonitrile is 1.5:1.

[0036] Example 10 The only difference between this embodiment and embodiment 1 is that in this embodiment, in the mobile phase, the volume ratio of trifluoroacetic acid with a mass fraction of 0.03% and acetonitrile is 4:1.

[0037] Embodiment 11 The only difference between this embodiment and embodiment 1 is that, in this embodiment, in the preparation method of the reference solution and the test solution, the 1% acetic acid solution by mass is replaced by an equal amount of 1% sodium carbonate solution by mass.

[0038] Example 12 The only difference between this embodiment and embodiment 1 is that in this embodiment, in the preparation method of the reference solution and the test solution, the 1 mg / mL p-nitrobenzaldehyde solution is replaced by an equal amount of 1 mg / mL benzaldehyde solution.

[0039] The recovery rates of hydroxylamine hydrochloride of Examples 1 to 12 are shown in Table 1: Table 1 Recovery rate of hydroxylamine hydrochloride in Examples 1 to 12

[0040] As can be seen from Table 1, compared with Examples 11 to 12, the recovery rate of hydroxylamine hydrochloride in Example 1 is significantly improved, indicating that during the derivatization reaction, when p-nitrobenzaldehyde solution and acetic acid solution are present at the same time, the recovery rate of hydroxylamine hydrochloride can be improved. The recovery rate of hydroxylamine hydrochloride in Examples 4 to 5 is low, and the recovery rate of hydroxylamine hydrochloride in Examples 1 and 6 to 7 is high. Comparison shows that when the volume ratio of the reference substance stock solution, p-nitrobenzaldehyde solution, and acetic acid solution is 1mL:1mL:18~23µL, the recovery rate of hydroxylamine hydrochloride meets the requirements. Compared with Examples 9 to 10, the recovery rates of hydroxylamine hydrochloride in Examples 1 and 8 are improved, indicating that during the HPLC-UV detection process, the volume ratio of trifluoroacetic acid with a mass fraction of 0.03% and acetonitrile in the mobile phase is 7:3~3:1, which can improve the recovery rate of hydroxylamine hydrochloride.

[0041] Experimental Example 1 Methodological Verification The detection method of Example 1 was subjected to specificity test, solution stability test, accuracy test, linearity test, precision test and durability test.

[0042] 1. Specificity test Blank solvent: Accurately measure 1 mL of diluent (a mixed solution of acetonitrile and water, with a volume fraction of 75% acetonitrile), add 1 mL of 1 mg / mL p-nitrobenzaldehyde solution and mix for 15 minutes. After mixing evenly, add 20 µL of 1% acetic acid solution, react in a water bath at 60°C for 10 minutes, add 8 mL of diluent, shake well, and set aside. The blank solvent chromatogram is shown in the figure below: Figure 3 As shown; Reference substance solution: accurately measure 1 mL of reference substance stock solution, add 1 mL of 1 mg / mL p-nitrobenzaldehyde solution and mix for 15 min. After mixing evenly, add 20 µL of 1% acetic acid solution and react in a water bath at 60°C for 10 min. Add 8 mL of diluent (a mixed solution of acetonitrile and water with a volume fraction of 75% acetonitrile), shake well and set aside.

[0043] Test sample stock solution: Accurately weigh 100 mg of moxidectin, dissolve it in 10 mL of acetonitrile, shake well to obtain a moxidectin stock solution with a concentration of 10 mg / mL, and set aside.

[0044] Table 2 Specificity test results

[0045] The results showed that the blank solvent had no interference with the detection, and there was no interference from other peaks in the chromatogram of the test solution, indicating that this method had good specificity.

[0046] 2 Solution stability test The reference solution and the test solution were placed at room temperature, sampled every 2 hours, and the changes in the main peak area were counted.

[0047] The test was carried out according to the test conditions in Example 1, and the test results are shown in Table 3: Table 3 Solution stability test results

[0048] It can be concluded from Table 3 that both the reference solution and the test solution have good stability within 12 hours.

[0049] 3 Accuracy test The above-mentioned reference solution and test solution were used to carry out the spike recovery test. The test results are shown in Table 4: Table 4 Spiked recovery results

[0050] As can be seen from Table 4, in the range of 50% to 150%, the recovery rates were all within the range of 95% to 105%, the average recovery rate was 98.86%, and RSD ≤ 10%, indicating that the accuracy of this method was good.

[0051] 4 Linearity test Hydroxylamine hydrochloride was prepared into a series of reference solutions with concentrations of 10%, 20%, 50%, 70%, 100%, 150% and 200%, respectively. The concentration (C) was used as the abscissa and the peak area (A) was used as the ordinate for linear regression. The regression equation was Y=182481X+110.82, (R 2 =0.9992), indicating a good linear correlation. The linear regression curve is shown in Figure 4 shown.

[0052] 5 Precision test The same batch of samples were taken and analyzed by different analysts on different dates using different equipment and different chromatographic columns. The RSD of the recovery rate of hydroxylamine hydrochloride in the 6 solutions prepared by the two analysts was calculated and the results are recorded in Table 5: Table 5 Precision test results

[0053] The results showed that the RSD (n=6) of the recovery rate of hydroxylamine hydrochloride was less than 5%, indicating that the precision of this method was good.

[0054] 6 Durability test Take the hydroxylamine hydrochloride reference solution, change the column temperature ±5℃, flow rate ±0.1mL / min, detection wavelength ±2nm, and examine the changes in the retention time and theoretical plate number of hydroxylamine hydrochloride. The results are shown in Table 6: Table 6 Durability test results

[0055] The results show that this method has good durability within a certain range.

[0056] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for rapidly detecting genotoxic impurity hydroxylamine hydrochloride using HPLC-UV method, characterized in that: The hydroxylamine hydrochloride in moxidectin was detected by HPLC-UV method; During the detection, a derivatization reagent and an organic acid solution are added to the sample solution containing hydroxylamine hydrochloride to carry out a derivatization reaction.

2. The method for rapid detection of genotoxic impurity hydroxylamine hydrochloride by HPLC-UV method according to claim 1, characterized in that: The derivatization reagent includes a p-nitrobenzaldehyde solution; the organic acid solution includes one of a formic acid solution, an acetic acid solution, and a phosphoric acid solution; The temperature of the derivatization reaction is 50-60° C. and the time is 10-20 min.

3. The method for rapid detection of genotoxic impurity hydroxylamine hydrochloride by HPLC-UV method according to claim 2, characterized in that: When the organic acid solution is an acetic acid solution, the mass fraction of the acetic acid solution is 0.5% to 1.5%; The concentration of the p-nitrobenzaldehyde solution is 0.9-1.1 mg / mL.

4. The method for rapid detection of genotoxic impurity hydroxylamine hydrochloride by HPLC-UV method according to claim 1, characterized in that: The sample solution containing hydroxylamine hydrochloride comprises a test solution and a reference solution.

5. The method for rapid detection of genotoxic impurity hydroxylamine hydrochloride by HPLC-UV method according to claim 4, characterized in that: The method for preparing the test solution comprises the following steps: adding a derivatization reagent and an organic acid solution to a moxidectin stock solution to perform a derivatization reaction to obtain a test solution; The preparation method of the reference substance solution comprises the following steps: adding a derivatization reagent and an organic acid solution to a hydroxylamine hydrochloride stock solution, performing a derivatization reaction, and obtaining a reference substance solution.

6. The method for rapid detection of genotoxic impurity hydroxylamine hydrochloride by HPLC-UV method according to claim 5, characterized in that: The method for preparing the moxidectin stock solution comprises the following steps: dissolving moxidectin in a solvent to obtain a moxidectin stock solution with a concentration of 10 mg / mL; The preparation method of the hydroxylamine hydrochloride stock solution comprises the following steps: dissolving hydroxylamine hydrochloride in a solvent to obtain a hydroxylamine hydrochloride stock solution with a concentration of 0.04 mg / mL.

7. The method for rapid detection of genotoxic impurity hydroxylamine hydrochloride by HPLC-UV method according to claim 1, characterized in that: The following steps are involved: The sample solutions containing hydroxylamine hydrochloride were tested respectively by HPLC-UV method, the chromatograms were recorded, and the content of hydroxylamine hydrochloride was calculated by peak area according to external standard method.

8. The method for rapid detection of genotoxic impurity hydroxylamine hydrochloride by HPLC-UV method according to claim 5, characterized in that: When preparing the test solution, the volume ratio of the moxidectin stock solution, the derivatization reagent, and the organic acid solution is 1 mL: 1 mL: 18-23 µL; Preferably, the volume ratio of moxidectin stock solution, derivatization reagent, and organic acid solution is 1 mL:1 mL:20 µL.

9. The method for rapid detection of genotoxic impurity hydroxylamine hydrochloride by HPLC-UV method according to claim 5, characterized in that: When preparing the reference solution, the volume ratio of the hydroxylamine hydrochloride stock solution, the derivatization reagent, and the organic acid solution is 1 mL: 1 mL: 18-23 µL; Preferably, the volume ratio of the hydroxylamine hydrochloride stock solution, the derivatization reagent, and the organic acid solution is 1 mL:1 mL:20 µL.

10. A method for rapid detection of genotoxic impurity hydroxylamine hydrochloride using HPLC-UV method according to claim 1, characterized in that: During the HPLC-UV detection, the chromatographic column is an Angilent SB C18 column with a specification of 150×4.6 mm; The column temperature is 25-35°C, the flow rate is 0.8-1.2 mL / min, and the detection wavelength is 300 nm; The mobile phase is trifluoroacetic acid solution and acetonitrile, and the volume ratio of the trifluoroacetic acid solution to acetonitrile is 7:3-3:1; The mass fraction of trifluoroacetic acid in the trifluoroacetic acid solution is 0.03% to 0.05%.

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