Method for detecting hydroxylamine hydrochloride in azithromycin

Abstract

The invention provides a method for detecting hydroxylamine hydrochloride in azithromycin, and belongs to the technical field of pharmaceutical analysis. The detection method adopts ultraviolet detector type high performance liquid chromatography for detection, and specifically comprises the following steps: (1) preparation of a test solution: taking to-be-detected azithromycin and anhydrous sodium acetate, dissolving the azithromycin and anhydrous sodium acetate with an alcohol organic solvent, and then adding acetic acid and benzaldehyde for derivatization to obtain the test solution; and (2) injecting the test solution into a high performance liquid chromatograph for detection, and recording a chromatogram. According to the detection method disclosed by the invention, quantitative detection of hydroxylamine hydrochloride in azithromycin can be realized by adopting an ultraviolet detector type high performance liquid chromatograph, the responsivity is high, and the universality is good. Besides, the detection method is good in separation effect on hydroxylamine hydrochloride in azithromycin, good in detection specificity, high in sensitivity and low in detection cost, so that the detection method is very suitable for quantitative detection of hydroxylamine hydrochloride in azithromycin and has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to a method for detecting hydroxylamine hydrochloride in azithromycin. Background Art

[0002] Azithromycin is a widely used semi-synthetic macrolide antibiotic with a broad antimicrobial spectrum, strong antimicrobial activity, high bioavailability, wide tissue distribution, and a long half-life. It is used to treat a variety of bacterial infections. In the production of azithromycin, hydroxylamine hydrochloride is an important raw material used to synthesize intermediates such as azithromycin oxime. Hydroxylamine hydrochloride is genotoxic and mutagenic, and it may remain in the azithromycin reaction process, adversely affecting the quality and safety of azithromycin. Therefore, detecting and controlling the residual amount of hydroxylamine hydrochloride in azithromycin is crucial for ensuring drug safety, efficacy, and quality.

[0003] Currently, ion chromatography, liquid chromatography electrochemical detection, and redox titration are the main methods used to detect hydroxylamine hydrochloride in azithromycin. However, ion chromatography requires high instrument costs and a relatively complex operation. Furthermore, hydroxylamine hydrochloride is unstable in aqueous solution, and sample solutions must be prepared fresh before use. This introduces certain inconveniences into practical operations and increases the complexity and cost of detection. Liquid chromatography electrochemical detection requires a specialized detector, resulting in high instrument costs and complex operation. Redox titration suffers from weak specificity, low sensitivity, and is easily affected by subjective factors, leading to poor parallelism when performing parallel sample analyses.

[0004] With the continuous development of pharmaceutical technology, the requirements for determination methods are becoming increasingly higher. In addition, the market demand for azithromycin is constantly increasing, and the quality control of azithromycin has also received greater attention. Therefore, how to quickly, accurately, and simply analyze the content of hydroxylamine hydrochloride in azithromycin to meet the needs of industry development is a major issue currently faced. Developing a method for detecting hydroxylamine hydrochloride in azithromycin that is simple to operate, highly sensitive, accurate, and low in cost is of great significance for ensuring the quality and safety of azithromycin drugs. Summary of the Invention

[0005] The object of the present invention is to provide a method for detecting hydroxylamine hydrochloride in azithromycin.

[0006] The present invention provides a method for detecting hydroxylamine hydrochloride in azithromycin, which adopts ultraviolet detector high performance liquid chromatography for detection, and specifically comprises the following steps:

[0007] (1) Preparation of test solution: Dissolve the azithromycin to be tested and anhydrous sodium acetate in an alcoholic organic solvent, then add acetic acid and benzaldehyde and mix well to perform derivatization, and then dilute to volume with an alcoholic organic solvent to obtain a test solution;

[0008] (2) The test solution was injected into a high performance liquid chromatograph for detection. The chromatographic conditions were as follows:

[0009] Chromatographic column: octadecylsilane bonded silica gel as filler; mobile phase A is phosphate buffer, and mobile phase B is acetonitrile.

[0010] Further,

[0011] In step (1), the concentration of azithromycin in the test solution is 20-30 mg / ml;

[0012] and / or, in step (1), the concentration of anhydrous sodium acetate in the test solution is 1 to 5 mg / ml;

[0013] And / or, in step (1), the mass volume ratio of azithromycin and acetic acid is 1 g: 10-20 ml;

[0014] And / or, in step (1), the mass volume ratio of azithromycin and benzaldehyde is 1 g:30-60 μl.

[0015] Further,

[0016] In step (1), the concentration of azithromycin in the test solution is 20 mg / ml;

[0017] And / or, in step (1), the concentration of anhydrous sodium acetate in the test solution is 3 mg / ml;

[0018] And / or, in step (1), the mass volume ratio of azithromycin and acetic acid is 1g:15ml;

[0019] And / or, in step (1), the mass volume ratio of azithromycin and benzaldehyde is 1 g:40 μl.

[0020] Further,

[0021] In step (1), the alcohol organic solvent used in the process of dissolving with an alcohol organic solvent is methanol and 80% methanol aqueous solution;

[0022] And / or, in step (1), the alcoholic organic solvent used in the process of fixing the volume with an alcoholic organic solvent is an 80% methanol aqueous solution;

[0023] And / or, in step (1), the derivatization condition is incubation at 70-90° C. for 30-60 min.

[0024] Preferably, the mass-to-volume ratio of azithromycin to methanol is 1 g:10 ml.

[0025] Furthermore, in step (2), the chromatographic conditions are as follows:

[0026] Chromatographic column: octadecylsilane bonded silica gel as filler; mobile phase A is phosphate buffer, mobile phase B is acetonitrile; flow rate is 1-5 ml / min; detection wavelength is 250-260 nm; injection volume is 5-15 μl; column temperature is 30-50°C; elution mode is gradient elution;

[0027] Preferably, the injection volume is 5 μl; and / or the flow rate is 1.5 ml / min; and / or the detection wavelength is 254 nm; and / or the column temperature is 40° C.

[0028] Furthermore, the conditions of the gradient elution are as follows:

[0029] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 75 25 15 75 25 17 20 80 30 20 80 32 75 25 40 75 25 .

[0030] Preferably, the chromatographic column is CERIL-Column C18 (250 mm×4.6 mm, 5 μm).

[0031] Preferably, the phosphate buffer solution is prepared by weighing 1.36 g of potassium dihydrogen phosphate, dissolving it in water and diluting it to 1000 ml, and adjusting the pH value to 2.5 with phosphoric acid.

[0032] Furthermore, the detection method further comprises preparing a reference solution, and injecting the reference solution into a high performance liquid chromatograph for detection; the preparation method of the reference solution comprises the following steps:

[0033] Hydroxylamine hydrochloride and anhydrous sodium acetate were weighed, dissolved in an alcoholic organic solvent, and then acetic acid and benzaldehyde were added and mixed to perform derivatization. The volume was then fixed with an alcoholic organic solvent to obtain a reference solution.

[0034] Further,

[0035] In the reference solution, the concentration of hydroxylamine hydrochloride is 0.00001 to 0.0001 mg / ml;

[0036] and / or, in the reference solution, the concentration of anhydrous sodium acetate is 0.0001 to 0.0005 mg / ml;

[0037] and / or, in the reference solution, the mass volume ratio of hydroxylamine hydrochloride to acetic acid is 0.001 mg: 3-5 ml;

[0038] And / or, in the reference solution, the mass volume ratio of hydroxylamine hydrochloride to benzaldehyde is 0.1 mg: 1-5 ml.

[0039] Further,

[0040] In the reference solution, the concentration of hydroxylamine hydrochloride is 0.00004 mg / ml;

[0041] and / or, in the reference solution, the concentration of anhydrous sodium acetate is 0.0003 mg / ml;

[0042] and / or, in the reference solution, the mass volume ratio of hydroxylamine hydrochloride to acetic acid is 0.001 mg:3.75 ml;

[0043] And / or, in the reference solution, the mass volume ratio of hydroxylamine hydrochloride to benzaldehyde is 0.1 mg:1 ml.

[0044] Furthermore, the alcohol organic solvent used in the process of dissolving with an alcohol organic solvent is methanol and 80% methanol aqueous solution;

[0045] And / or, the alcoholic organic solvent used in the process of fixing the volume with an alcoholic organic solvent is an 80% methanol aqueous solution;

[0046] And / or, the derivatization condition is incubating at 70-90° C. for 30-60 min.

[0047] Preferably, the mass volume ratio of the hydroxylamine hydrochloride and methanol is 1 mg: 0.5-10 ml.

[0048] The present invention has achieved the following beneficial effects:

[0049] The present invention provides a method for detecting hydroxylamine hydrochloride in azithromycin. This method utilizes a high-performance liquid chromatograph with an ultraviolet detector to quantitatively detect hydroxylamine hydrochloride in azithromycin, exhibiting high responsiveness and good universality. Furthermore, the method exhibits excellent separation effect for hydroxylamine hydrochloride in azithromycin, high detection specificity, high sensitivity, and low detection cost. Therefore, the method is highly suitable for the quantitative detection of hydroxylamine hydrochloride in azithromycin and has promising application prospects.

[0050] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0051] The following is a detailed description of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-mentioned content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 The blank solution chromatogram.

[0053] Figure 2 This is the system suitability chromatogram. DETAILED DESCRIPTION

[0054] The raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.

[0055] The present invention selects a high performance liquid chromatography (HPLC) ultraviolet detector for detection.

[0056] Example 1: Detection method of hydroxylamine hydrochloride in azithromycin according to the present invention

[0057] (1) Chromatographic conditions:

[0058] Chromatographic column: CERIL-Column C18 (250 mm × 4.6 mm, 5 μm);

[0059] Flow rate: 1.5 ml / min;

[0060] Detection wavelength: 254nm;

[0061] Injection volume: 5 μl;

[0062] Column temperature: 40°C;

[0063] Mobile phase A: phosphate buffer, prepared by weighing 1.36 g of potassium dihydrogen phosphate, dissolving it in water and diluting it to 1000 ml. Adjust the pH to 2.5 with phosphoric acid.

[0064] Mobile phase B: acetonitrile;

[0065] Elution mode: gradient elution;

[0066] Elution conditions: The specific conditions are shown in Table 1.

[0067] Table 1. Gradient elution conditions

[0068] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 75 25 15 75 25 17 20 80 30 20 80 32 75 25 40 75 25

[0069] (2) Preparation of reference solution: Accurately weigh 20 mg of hydroxylamine hydrochloride powder and 150 mg of anhydrous sodium acetate into a 50 ml volumetric flask, add 10 ml of methanol and 10 ml of 80% methanol aqueous solution, sonicate to dissolve, and dilute to the mark with 80% methanol aqueous solution. Then accurately pipette 1 ml of this solution into a 100 ml volumetric flask, dilute to the mark with 80% methanol aqueous solution, and then accurately pipette 1 ml of this solution into a 100 ml volumetric flask, add 15 ml of acetic acid and 40 μl of benzaldehyde, shake well, incubate in a 70°C water bath for 30 minutes, cool to room temperature, and dilute to the mark with 80% methanol aqueous solution to obtain the reference solution.

[0070] (3) Preparation of test solution: Accurately weigh 1 g of azithromycin powder and 150 mg of anhydrous sodium acetate into a 50 ml volumetric flask, add 10 ml of methanol and 10 ml of 80% methanol aqueous solution, dissolve by ultrasonication, add 15 ml of acetic acid and 40 μl of benzaldehyde, shake well, and place in a 70°C water bath for 30 minutes. Cool to room temperature and dilute to the mark with 80% methanol aqueous solution to obtain the test solution.

[0071] (4) Determination: Accurately measure 5 μl of the reference solution and the test solution, inject them into the liquid chromatograph, record the chromatogram, and calculate the content of hydroxylamine hydrochloride in the test sample by the peak area according to the external standard method.

[0072] Based on the International Council for Harmonization (ICH) M7 guidelines, the residual limit of hydroxylamine hydrochloride in drugs should not exceed 2 ppm.

[0073] Example 2, Methodological Study

[0074] 1. System suitability test and specificity

[0075] The chromatographic conditions and the preparation methods of the reference solution and the test solution were the same as those in Example 1. At the same time, a blank solution (excluding azithromycin) was prepared according to the preparation method of the test solution.

[0076] Judgment criteria: Accurately measure 5 μl of each of the reference solution, test solution, and blank solution, inject them into the liquid chromatograph, and record the chromatogram. The blank solution should not interfere with the detection of the main peak.

[0077] Test results ( Figure 1 and Figure 2 ) It was found that the blank solution did not interfere with the detection of the main peak. The detection method of the present invention has good system applicability and specificity.

[0078] 2. Repeatability

[0079] Prepare 6 test sample solutions and 2 reference sample solutions according to the method described in Example 1.

[0080] Accurately measure 5 μl of the reference solution and the test solution, inject them into the liquid chromatograph respectively, record the chromatogram, and calculate the content of hydroxylamine hydrochloride by the peak area according to the external standard method.

[0081] Acceptability criteria: The absolute deviation of the results of 6 test solution should not exceed 0.03%.

[0082] The test results showed that the absolute value deviation of the hydroxylamine hydrochloride content results of the 6 test solutions was 0, indicating that the detection method of the present invention has good repeatability.

[0083] 3. Limit of quantification and limit of detection

[0084] Prepare one reference solution according to the method described in Example 1, take the reference solution and dilute it step by step, accurately pipette 5 μl of each solution, inject it into the liquid chromatograph, and record the chromatogram. When the reference peak signal-to-noise (S / N) ratio is about 3, the concentration is the detection limit, and when the reference peak signal-to-noise (S / N) ratio is about 10, the concentration is the quantification limit.

[0085] Solution preparation:

[0086] Take 1 ml of the reference solution, place it in a 2 ml volumetric flask, add 80% methanol aqueous solution to dilute to the scale, shake well, and make a solution containing approximately 0.02128585 μg of hydroxylamine hydrochloride per 1 ml, which is used as the reference solution B1.

[0087] Take 1 ml of the reference solution, place it in a 5 ml volumetric flask, add 80% methanol aqueous solution to dilute to the scale, shake well, and make a solution containing approximately 0.00851434 μg of hydroxylamine hydrochloride per 1 ml, which is used as the reference solution B2.

[0088] Take 1 ml of reference solution B2, place it in a 2 ml volumetric flask, add 80% methanol aqueous solution to dilute to the scale, shake well, and make a solution containing approximately 0.00425717 μg of hydroxylamine hydrochloride per 1 ml, which is used as reference solution B3.

[0089] After testing, it was determined that the detection limit of hydroxylamine hydrochloride was 0.02128585 ng and the quantification limit was 0.0425717 ng.

[0090] 4. Linearity and range

[0091] Accurately weigh 20 mg of hydroxylamine hydrochloride powder and 150 mg of anhydrous sodium acetate into a 50 ml volumetric flask. Add 10 ml of methanol and 10 ml of 80% methanol aqueous solution, sonicate to dissolve, and dilute to the mark with 80% methanol aqueous solution. Then, accurately pipette 1 ml of this solution into a 100 ml volumetric flask, add 15 ml of acetic acid and 40 μl of benzaldehyde, shake well, and place in a 70°C water bath for 30 minutes. Cool to room temperature, and dilute to the mark with 80% methanol aqueous solution to obtain a solution containing 4 μg / ml of hydroxylamine hydrochloride, which is reference solution a.

[0092] Take the quantitative limit solution under "3. Quantitative limit and detection limit" as the linear solution 1

[0093] Accurately measure 0.5 ml of reference solution a and place it in a 100 ml volumetric flask. Add diluent to dilute to the mark and shake well to prepare a solution containing approximately 0.02 μg of hydroxylamine hydrochloride per ml, which is used as linear solution 2.

[0094] Accurately measure 0.75 ml of reference solution a and place it in a 100 ml volumetric flask. Add diluent to dilute to the mark and shake well to prepare a solution containing approximately 0.03 μg of hydroxylamine hydrochloride per ml, which is used as linear solution 3.

[0095] Accurately measure 1 ml of reference solution a and place it in a 100 ml volumetric flask. Add diluent to dilute to the mark and shake well to prepare a solution containing approximately 0.04 μg of hydroxylamine hydrochloride per 1 ml, which is used as linear solution 4.

[0096] Accurately measure 1.5 ml of reference solution a and place it in a 100 ml volumetric flask. Add diluent to dilute to the mark and shake well to prepare a solution containing approximately 0.06 μg of hydroxylamine hydrochloride per ml, which is used as linear solution 5.

[0097] Take 5 μl of each of the above solutions and inject them into a liquid chromatograph. Record the chromatogram and perform linear regression analysis using concentration versus peak area. Report the linear equation and linear correlation coefficient. The linear correlation coefficient R is required to be greater than 0.99. From the concentration series that meets this linear requirement, select the highest and lowest concentrations to constitute the analytical range.

[0098] After testing, the linear equation of hydroxylamine hydrochloride of this detection method is y=33.778x+0.0822, the correlation coefficient R is 1.00, and the linear range is 0.00851434μg / ml to 0.06385755μg / ml.

[0099] 5. Stability of test solution and reference solution

[0100] Assay: 5 μl of each of the reference solution and the test solution prepared in Example 1 were injected into a high-performance liquid chromatograph at five random time points between 0 h and 25 h, and the chromatograms were recorded. For the test solution, the ratio of the content at each time point to the content at time zero was calculated. For the reference solution, the ratio of the peak area at each time point to the peak area at time zero was calculated.

[0101] Acceptable criteria: The ratio of the result at time zero to the result at each subsequent time point should be within the range of 0.95-1.05 (Note: If the result at a certain time point in the middle no longer meets the requirements, the subsequent time points may no longer be confirmed).

[0102] The test results showed that the reference solution had good stability within 24 hours, and the test solution had good stability within 24 hours.

[0103] The above methodological study obtained the results shown in Table 2.

[0104] Table 2. Methodological study results

[0105]

[0106]

[0107] After research, it was found that the system applicability and specificity, repeatability, quantitative limit, detection limit, linearity, range and stability of the method for detecting hydroxylamine hydrochloride in azithromycin of the present invention all met the relevant provisions of the current edition of the "Chinese Pharmacopoeia".

[0108] The beneficial effects of the present invention are demonstrated below through specific test examples.

[0109] Experimental Example 1: Study of different injection volumes

[0110] According to the chromatographic conditions described in Example 1, injection volumes of 5 μl, 10 μl, 15 μl and 50 μl were selected for detection.

[0111] Preparation of reference solution: Accurately weigh 1 g of hydroxylamine hydrochloride powder and 150 mg of anhydrous sodium acetate into a 50 ml volumetric flask, add 10 ml of methanol and 10 ml of 80% methanol aqueous solution, dissolve by ultrasonication, add 15 ml of acetic acid and 25 mg of p-dimethylaminobenzaldehyde, shake well, and place in a 70°C water bath for 30 minutes. Cool to room temperature and dilute to the mark with 80% methanol aqueous solution to obtain the reference solution.

[0112] Preparation of test solution: Accurately weigh 1 g of azithromycin powder and 150 mg of anhydrous sodium acetate into a 50 ml volumetric flask, add 10 ml of methanol and 10 ml of 80% methanol aqueous solution, ultrasonically dissolve, add 15 ml of acetic acid and 25 mg of p-dimethylaminobenzaldehyde, shake well, incubate in a 70°C water bath for 30 minutes, cool to room temperature, and dilute to the mark with 80% methanol aqueous solution to obtain the test solution.

[0113] After accurately measuring the reference solution and the test solution according to different injection volumes, they were injected into the liquid chromatograph, and the chromatograms were recorded. The content of hydroxylamine hydrochloride in azithromycin was then calculated based on the peak area. The response values under different injection volume conditions are shown in Table 3. Test sample 1 and test sample 2 in Table 3 are two parallel experiments of the above test solution.

[0114] Table 3. Response values under different injection volume conditions

[0115]

[0116] The results in Table 3 show that using dimethylaminobenzaldehyde as the derivatization agent, the response was irregular between different injection volumes, and the repeatability of the response within the same injection volume was poor. Theoretically, injections of 10μl, 15μl, and 50μl should double or show an incremental trend compared to the 5μl injection. However, the results in the table above show an irregular increase, and no peak is observed at all with the 50μl injection. There are two possible reasons for these results: 1) the derivatization agent is not suitable, resulting in different substances emitting peaks at the same position; 2) the injection volume of the substance is too large, resulting in overload and no response.

[0117] In order to confirm whether the substance does not produce a peak due to overloading, a pure reference substance is used to conduct a quantitative study.

[0118] Test Example 2: Study on the concentration of reference substance (injection volume 5 μl)

[0119] The chromatographic conditions described in Example 1 were followed, and an injection volume of 5 μl was selected for detection. The test solution was prepared in the same manner, and the effect of different reference substance concentrations on the response was studied.

[0120] Preparation of test solution: Accurately weigh 1 g of azithromycin powder and 150 mg of anhydrous sodium acetate into a 50 ml volumetric flask, add 10 ml of methanol and 10 ml of 80% methanol aqueous solution, ultrasonically dissolve, add 15 ml of acetic acid and 25 mg of p-dimethylaminobenzaldehyde, shake well, incubate in a 70°C water bath for 30 minutes, cool to room temperature, and dilute to the mark with 80% methanol aqueous solution to obtain the test solution.

[0121] Preparation of reference solution ①: Accurately weigh 1 g of hydroxylamine hydrochloride powder and 150 mg of anhydrous sodium acetate into a 50 ml volumetric flask, add 10 ml of methanol and 10 ml of 80% methanol aqueous solution, and after ultrasonic dissolution, add 15 ml of acetic acid and 25 mg of p-dimethylaminobenzaldehyde, shake well, and incubate in a 70°C water bath for 30 minutes. Cool to room temperature, and then dilute to the mark with 80% methanol aqueous solution to obtain a reference solution ① with a concentration of 20 mg / ml.

[0122] Preparation of Reference Solution ②: Accurately weigh 20 mg of hydroxylamine hydrochloride powder and 150 mg of anhydrous sodium acetate into a 50 mL volumetric flask. Add 10 mL of methanol, sonicate, and dilute to the mark with 80% methanol. Then, accurately pipette 1 mL of this solution into a 100 mL volumetric flask, dilute to the mark with 80% methanol, and accurately pipette 1 mL of this solution into a 100 mL volumetric flask. Add 15 mL of acetic acid and 25 mg of p-dimethylaminobenzaldehyde, shake well, and incubate in a 70°C waterbath for 30 minutes. Cool to room temperature, and then dilute to the mark with 80% methanol. This yields Reference Solution ② with a concentration of 0.00004 mg / mL.

[0123] After accurately measuring different reference solution and test solution, each solution was injected into a liquid chromatograph, and the chromatogram was recorded. The hydroxylamine hydrochloride content in azithromycin was then calculated based on the peak area. The response values detected using different reference solution solutions are shown in Table 4. In Table 4, Test 1 and Test 2 represent two parallel experiments using the above test solution solutions.

[0124] Table 4. Response value results of different reference solution tests

[0125]

[0126] Table 4 shows significant discrepancies between the test sample results for different reference concentrations. A 500,000-fold decrease in reference concentration from 20 mg / ml to 0.00004 mg / ml should have resulted in a 500,000-fold decrease in the test sample results. However, the test sample results were only reduced by approximately 1,100-fold. Reasons for this are: ① The hydroxylamine hydrochloride concentration was too high, affecting the response; ② The derivatization agent was inappropriate.

[0127] In summary, the possible influencing factors of the entire experimental process are: ① the detection concentration of hydroxylamine hydrochloride should not be too high; ② the derivatization agent has a significant impact on the test results.

[0128] Test Example 3: Study of Derivatizing Agents

[0129] The detection was performed using an injection volume of 5 μl according to the chromatographic conditions described in Example 1. The derivatization agents in the test solution and the reference solution were replaced, and a low concentration reference solution was used.

[0130] Preparation of test solution: Accurately weigh 1 g of azithromycin powder and 150 mg of anhydrous sodium acetate into a 50 ml volumetric flask, add 10 ml of methanol and 10 ml of 80% methanol aqueous solution, dissolve by ultrasonication, add 15 ml of acetic acid and 40 μl of benzaldehyde, shake well, incubate in a 70°C water bath for 30 minutes, cool to room temperature, and dilute to the mark with 80% methanol aqueous solution to obtain the test solution.

[0131] Preparation of reference solution: Accurately weigh 20 mg of hydroxylamine hydrochloride powder and 150 mg of anhydrous sodium acetate into a 50 mL volumetric flask. Add 10 mL of methanol and 10 mL of 80% methanol solution. Dissolve by ultrasonication and dilute to the mark with 80% methanol solution. Then, accurately pipette 1 mL of this solution into a 100 mL volumetric flask, dilute to the mark with 80% methanol solution, and accurately pipette 1 mL of this solution into a 100 mL volumetric flask. Add 15 mL of acetic acid and 40 μl of benzaldehyde, shake well, and incubate in a 70°C water bath for 30 minutes. Cool to room temperature, and dilute to the mark with 80% methanol solution to obtain a reference solution with a concentration of 0.00004 mg / mL.

[0132] After accurately measuring each reference solution and test solution, inject them into a liquid chromatograph, record the chromatogram, and calculate the hydroxylamine hydrochloride content in azithromycin based on the peak area. The response values are shown in Table 5. Test 1 and Test 2 in Table 5 represent two parallel experiments using the above test solution solutions.

[0133] Table 5. Response value results

[0134]

[0135] As shown in Table 5, when the test sample preparation method remains unchanged and the derivatization agent is replaced while the reference sample concentration is reduced, the results are close to those in the control standard. Therefore, the optimal method for detecting hydroxylamine hydrochloride in azithromycin according to the present invention is the method described in Example 1.

[0136] In summary, the present invention provides a method for detecting hydroxylamine hydrochloride in azithromycin. This method can achieve quantitative detection of hydroxylamine hydrochloride in azithromycin using a high-performance liquid chromatograph with an ultraviolet detector, with high responsiveness and good universality. In addition, the detection method of the present invention has good separation effect for hydroxylamine hydrochloride in azithromycin, good detection specificity, high sensitivity, and low detection cost. Therefore, it is very suitable for the quantitative detection of hydroxylamine hydrochloride in azithromycin and has good application prospects.

Claims

1. A method for detecting hydroxylamine hydrochloride in azithromycin, characterized in that: It is detected by high performance liquid chromatography with ultraviolet detector, which includes the following steps: (1) Preparation of test solution: Dissolve the azithromycin to be tested and anhydrous sodium acetate in an alcoholic organic solvent, then add acetic acid and benzaldehyde and mix well to perform derivatization, and then dilute to volume with an alcoholic organic solvent to obtain a test solution; (2) The test solution was injected into a high performance liquid chromatograph for detection. The chromatographic conditions were as follows: Chromatographic column: octadecylsilane bonded silica gel as filler; mobile phase A is phosphate buffer, and mobile phase B is acetonitrile.

2. The detection method according to claim 1, wherein: In step (1), the concentration of azithromycin in the test solution is 20-30 mg / ml; and / or, in step (1), the concentration of anhydrous sodium acetate in the test solution is 1 to 5 mg / ml; And / or, in step (1), the mass volume ratio of azithromycin and acetic acid is 1 g: 10-20 ml; And / or, in step (1), the mass volume ratio of azithromycin and benzaldehyde is 1 g:30-60 μl.

3. The detection method according to claim 2, wherein: In step (1), the concentration of azithromycin in the test solution is 20 mg / ml; And / or, in step (1), the concentration of anhydrous sodium acetate in the test solution is 3 mg / ml; And / or, in step (1), the mass volume ratio of azithromycin and acetic acid is 1g:15ml; And / or, in step (1), the mass volume ratio of azithromycin and benzaldehyde is 1 g:40 μl.

4. The detection method according to claim 1, wherein: In step (1), the alcohol organic solvent used in the process of dissolving with an alcohol organic solvent is methanol and 80% methanol aqueous solution; And / or, in step (1), the alcoholic organic solvent used in the process of fixing the volume with an alcoholic organic solvent is an 80% methanol aqueous solution; and / or, in step (1), the derivatization condition is incubation at 70-90° C. for 30-60 min; Preferably, the mass-to-volume ratio of azithromycin to methanol is 1 g:10 ml.

5. The detection method according to claim 1, wherein: In step (2), the chromatographic conditions are as follows: Chromatographic column: octadecylsilane bonded silica gel as filler; mobile phase A is phosphate buffer, mobile phase B is acetonitrile; flow rate is 1-5 ml / min; detection wavelength is 250-260 nm; injection volume is 5-15 μl; column temperature is 30-50°C; elution mode is gradient elution; Preferably, the injection volume is 5 μl; and / or the flow rate is 1.5 ml / min; and / or the detection wavelength is 254 nm; and / or the column temperature is 40° C.

6. The detection method according to claim 5, characterized in that: The conditions of the gradient elution are as follows: 。 7. The detection method according to claim 1, wherein: The detection method further includes preparing a reference solution, and injecting the reference solution into a high performance liquid chromatograph for detection; the preparation method of the reference solution includes the following steps: Hydroxylamine hydrochloride and anhydrous sodium acetate were weighed, dissolved in an alcoholic organic solvent, and then acetic acid and benzaldehyde were added and mixed to perform derivatization. The volume was then fixed with an alcoholic organic solvent to obtain a reference solution.

8. The detection method according to claim 7, wherein: In the reference solution, the concentration of hydroxylamine hydrochloride is 0.00001 to 0.0001 mg / ml; and / or, in the reference solution, the concentration of anhydrous sodium acetate is 0.0001 to 0.0005 mg / ml; and / or, in the reference solution, the mass volume ratio of hydroxylamine hydrochloride to acetic acid is 0.001 mg: 3-5 ml; And / or, in the reference solution, the mass volume ratio of hydroxylamine hydrochloride to benzaldehyde is 0.1 mg: 1-5 ml.

9. The detection method according to claim 8, wherein: In the reference solution, the concentration of hydroxylamine hydrochloride is 0.00004 mg / ml; and / or, in the reference solution, the concentration of anhydrous sodium acetate is 0.0003 mg / ml; and / or, in the reference solution, the mass volume ratio of hydroxylamine hydrochloride to acetic acid is 0.001 mg:3.75 ml; And / or, in the reference solution, the mass volume ratio of hydroxylamine hydrochloride to benzaldehyde is 0.1 mg:1 ml.

10. The detection method according to claim 7, wherein: The alcohol organic solvent used in the process of dissolving with an alcohol organic solvent is methanol and 80% methanol aqueous solution; And / or, the alcoholic organic solvent used in the process of fixing the volume with an alcoholic organic solvent is an 80% methanol aqueous solution; and / or, the derivatization condition is incubating at 70-90° C. for 30-60 min; Preferably, the mass volume ratio of the hydroxylamine hydrochloride and methanol is 1 mg: 0.5-10 ml.

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