Method for detecting hydroxylamine hydrochloride in azithromycin dry suspension

Abstract

The application discloses a hydroxylamine hydrochloride detection method in azithromycin dry suspension, and relates to the trace detection field. The method uses isoamyl aldehyde as a derivatizing agent, generates isoamyl aldoxime by performing derivatization treatment on a sample to be measured, and then uses N-nitrosodiisopropylamine as an internal standard substance to quantitatively determine the isoamyl aldoxime by using liquid chromatography-tandem mass spectrometry. The detection method adopts an isoamyl aldehyde derivatizing agent-N-nitrosodiisopropylamine internal standard substance system, can correct detection errors caused by factors such as matrix effect, pretreatment loss and derivatization efficiency fluctuation, and improves the accuracy and sensitivity of the trace hydroxylamine hydrochloride detection result, thereby guaranteeing the safety of the medicine.

Description

Technical Field

[0001] This application relates to the field of trace detection, and in particular to a method for detecting hydroxylamine hydrochloride in azithromycin dry suspension. Background Technology

[0002] Azithromycin dry suspension is an oral formulation of a macrolide antibiotic, widely used clinically due to its ease of administration to children and patients with swallowing difficulties. In the synthesis process of this drug, hydroxylamine hydrochloride is often used as a condensing agent or is present in some starting materials, potentially leading to its residue as a process impurity in the final product. Because hydroxylamine hydrochloride has certain potential genotoxic and cytotoxic effects, establishing a sensitive and accurate analytical method to monitor its content in azithromycin dry suspension is crucial for ensuring medication safety.

[0003] Currently, for the detection of hydroxylamine hydrochloride in azithromycin raw materials, existing studies have employed external standard-liquid chromatography-tandem mass spectrometry (LC-MS / MS) for qualitative and quantitative determination. However, hydroxylamine hydrochloride itself is highly polar and water-soluble, exhibiting extremely weak retention on reversed-phase chromatographic columns. More importantly, its molecular structure results in extremely poor UV absorption and mass spectrometry ionization efficiency, making direct high-sensitivity detection difficult. To overcome this technical bottleneck, existing technologies such as CN111812249A and CN114994212A often employ derivatization reagents such as benzaldehyde and nitrobenzaldehyde (e.g., 2-nitrobenzaldehyde, p-nitrobenzaldehyde) for sample pretreatment. Through derivatization, the polarity of hydroxylamine hydrochloride is significantly reduced, improving its retention behavior and separation efficiency in liquid chromatography and endowing it with good UV absorption characteristics. Furthermore, the derivatization products also greatly enhance its ionization efficiency in the mass spectrometry ion source, thus meeting the detection requirements.

[0004] While the external standard method offers advantages such as ease of operation and the elimination of the need for an internal standard, it is only suitable for samples containing a high concentration of the active ingredient (e.g., azithromycin with a content greater than 70%). Its limitations are significant when dealing with complex matrix formulations like azithromycin dry suspensions (where the effective azithromycin content is typically only 2.4-4.8%). The large amounts of sugars such as sucrose and lactose, inorganic salts such as citrate and phosphate, and high-molecular-weight polymers such as xanthan gum and sodium carboxymethyl cellulose in these formulations, compared to high-purity azithromycin, produce strong and unpredictable matrix effects in LC-MS analysis, significantly interfering with and inhibiting the ionization efficiency of the target analyte. Furthermore, the complex sample pretreatment process, especially the derivatization reaction step, is susceptible to fluctuations in efficiency due to factors such as temperature, time, and pH. In addition, the unavoidable loss of the target analyte during sample transfer and filtration introduces significant measurement errors. Conventional external standard methods are insufficient to effectively correct for systematic and random errors caused by factors such as strong matrix effect, pretreatment loss and fluctuations in derivatization efficiency, ultimately resulting in unsatisfactory accuracy and sensitivity of the method. Summary of the Invention

[0005] This application provides a method for detecting hydroxylamine hydrochloride in azithromycin dry suspension, which can correct for detection errors caused by matrix effects, pretreatment losses, and fluctuations in derivatization efficiency, thereby improving the accuracy and sensitivity of the detection results and ensuring drug safety.

[0006] In a first aspect, this application provides a method for detecting hydroxylamine hydrochloride in azithromycin dry suspension, characterized in that isovaleraldehyde is used as a derivatizing agent to derivatize the sample to be tested to generate isovaleraldehyde oxime, and then N-nitrosodiisopropylamine is used as an internal standard to quantitatively determine isovaleraldehyde oxime by liquid chromatography-tandem mass spectrometry.

[0007] In any of the above technical solutions, the derivatization process includes: dissolving the derivatizing agent in a solvent to prepare a derivatizing agent solution with a concentration of 30-80 μg / mL; preparing the sample to be tested into a solution, adding the derivatizing agent solution, and derivatizing at 40-50℃ for 20-30 min to obtain the sample.

[0008] In any of the above technical solutions, liquid chromatography detection is performed in accordance with General Chapter 0512 of Part IV of the Chinese Pharmacopoeia 2020 Edition, and mass spectrometry detection is performed in accordance with General Chapter 0431 of Part IV of the Chinese Pharmacopoeia 2020 Edition.

[0009] This application's technical solution resolves the fundamental contradiction of the difficulty in synergistic use of existing derivatization processes and internal standard methods. Existing technologies often use benzaldehyde or nitrobenzaldehyde as derivatizing agents. These agents and their derivatized products (benzaldehyde oxime) possess conjugated structures, exhibiting strong UV absorption and relatively high mass spectrometric responses. However, the aromatic ring structure in these derivatizing agents results in low polarity and strong hydrophobicity in their derivatized products, leading to long retention times in chromatography. This makes it difficult to match them with internal standards that maintain high consistency in chromatographic behavior and ionization efficiency. When faced with the extremely complex matrix of azithromycin dry suspension, the matrix effects and process losses introduced by the large amounts of sugars, salts, and polymers in the sample during sample pretreatment and LC-MS analysis are random and unpredictable. Without an internal standard undergoing a relatively consistent extraction, derivatization, chromatographic separation, and ionization process, it is impossible to effectively correct the errors generated during this process. Using internal standards with significantly different properties may even introduce new errors. Therefore, traditional methods are limited by the mismatch in physicochemical properties between derivatized products and potential internal standards, and often employ external standard methods that lack error correction, thus sacrificing the precision and accuracy of the method.

[0010] The core of this application lies in the highly synergistic derivatization-internal standard system of isovaleraldehyde and N-nitrosodiisopropylamine. Isovaleraldehyde, as a derivatizing agent, produces isovaleraldehyde oxime, which shares key structural similarities with the internal standard N-nitrosodiisopropylamine. Both are small nitrogen-containing molecules with a moderate carbon number, lack strong conjugated systems, and exhibit similar moderate polarity and hydrophobicity. This structural convergence directly translates into convergence in chromatographic behavior and mass spectrometry response. In chromatographic separation, isovaleraldehyde oxime and N-nitrosodiisopropylamine can achieve good separation on a standard C18 column while having very similar retention times. This indicates that the matrix environment they experience during pretreatment, the matrix interference they encounter on the column, and the ionization environment they experience in the mass spectrometry ion source are highly consistent. N-nitrosodiisopropylamine can thus correct systematic and random errors caused by matrix effects, pretreatment losses, and fluctuations in derivatization efficiency throughout the entire process from sample derivatization to instrument detection with extremely high precision, thereby enabling this application to achieve high-precision and high-accuracy quantification.

[0011] In any of the above technical solutions, the single injection detection time for the liquid chromatography-tandem mass spectrometry quantitative determination is 7 to 9 minutes.

[0012] Compared to macromolecules containing benzene ring structures such as benzaldehyde and nitrobenzaldehyde, isovaleraldehyde reacts with hydroxylamine hydrochloride with high efficiency. The derivative (isovaleraldehyde oxime) has a moderate retention time in the chromatographic column, resulting in fast mass spectrometry response and high sensitivity, which can effectively reduce the detection time (the detection time for a single injection of benzaldehyde is about 20 to 30 minutes).

[0013] In any of the above technical solutions, the solvent is ammonia water with a concentration of 20-30 wt%.

[0014] The derivatization reaction of hydroxylamine hydrochloride and isovaleraldehyde is a reversible reaction. In the context of trace analysis, this reverse reaction leads to fluctuations in the concentration of the target derivative, directly affecting the accuracy and repeatability of low-level detection, becoming a key factor limiting method performance. To address the detection requirements of trace hydroxylamine hydrochloride in azithromycin dry suspension, this application sets the derivatization reaction system in a 20–30 wt% ammonia solution. This strongly alkaline environment significantly accelerates the reaction rate by enhancing the nucleophilic activity of the hydroxylamine anion, shifting the reaction equilibrium towards the formation of isovaleraldehyde oxime. This mechanism is particularly important in trace detection scenarios, effectively suppressing the reverse reaction that is prone to occur due to extremely low derivative concentrations. It ensures efficient and complete derivatization even under mild conditions of 40–50°C, minimizing fluctuations in derivatization efficiency and target analyte loss due to reaction equilibrium issues, and significantly improving the sensitivity, recovery rate, and method reproducibility of trace-level detection.

[0015] In any of the above technical solutions, the chromatographic column of the liquid chromatography uses octadecylsilane-bonded silica gel as the packing material, and the column temperature is 35-45℃.

[0016] In any of the above technical solutions, the liquid chromatography uses 0.1% formic acid water as mobile phase A and acetonitrile as mobile phase B.

[0017] In any of the above technical solutions, the elution rate of the liquid chromatography is 0.3 mL / min, and the elution gradient changes as follows: 0~0.8min: 90%A→90%A, 10%B→10%B; 0.8~2min: 90%A→5%A, 10%B→95%B; 2~6min: 5%A→5%A, 95%B→95%B; 6~6.1min: 5%A→90%A, 95%B→10%B; 6.1~8min:90%A→90%A, 10%B→10%B.

[0018] In any of the above technical solutions, in the mass spectrometry detection, the qualitative ion pair of isovaleraldehyde oxime is 102.07→43.15 m / z, and the quantitative ion pair is 102.07→46.4 m / z.

[0019] In any of the above technical solutions, in the mass spectrometry detection, the qualitative ion pair of N-nitrosodiisopropylamine is m / z 131.1→89.1, and the quantitative ion pair is m / z 131.1→47.1.

[0020] In any of the above technical solutions, the mass spectrometry detection uses an ESI source in positive ion mode, an ion source temperature of 500℃, a curtain gas of 30psi, a nebulizing gas of 50psi, an auxiliary nebulizing gas of 55psi, and an ion spray voltage of 5500V.

[0021] In summary, this application has the following beneficial effects: This application provides a high-precision method for detecting hydroxylamine hydrochloride residues in azithromycin dry suspension. The method uses isovaleraldehyde as a derivatization reagent to convert the highly polar and poorly sensitive hydroxylamine hydrochloride into the easily analyzable isovaleraldehyde oxime; simultaneously, N-nitrosodiisopropylamine is introduced as an internal standard. This internal standard is highly similar to the derivatized product in chemical properties and chromatographic behavior, thus accurately correcting for strong matrix effects caused by complex matrices, as well as unavoidable target analyte loss and efficiency fluctuations during sample pretreatment and derivatization. Through this synergistic design, this method transforms the traditional, easily interfered external standard detection into stable, error-correctable internal standard quantification, significantly improving analytical reliability. This provides technical support for the accurate monitoring of trace genotoxic impurities of hydroxylamine hydrochloride in azithromycin dry suspension, and is of great significance for ensuring drug safety. Attached Figure Description

[0022] Figure 1 This is the chromatogram of the blank solution in Example 1; Figure 2 This is the chromatogram of the reference solution in Example 1; Figure 3 This is the chromatogram of the sample solution to be tested in Example 1; Figure 4 This is the chromatogram of the limit-of-quantity solution in Example 4; Figure 5 This is the chromatogram of the detection limit solution in Example 4; Figure 6 The chromatogram of the limit-of-quantitation solution in Comparative Example 1 is shown. Figure 7 The chromatogram of the solution with the detection limit in Comparative Example 1 is shown. Figure 8 The chromatogram of the limit-of-quantitation solution in Comparative Example 2 is shown. Figure 9 This is the chromatogram of the detection limit solution in Comparative Example 2. Detailed Implementation

[0023] Example The instruments, reagents and pharmaceuticals used in this application are summarized in Table 1-4: Table 2. Information on the samples to be tested Table 2. Information on Reference Standards Table 3. Information on Main Reagents Table 4. Instrument Information Example 1: Detection of the sample to be tested Internal standard stock solution: Accurately measure 0.1 mL of N-nitrosodiisopropylamine, place it in a 50 mL volumetric flask, dilute to the mark with ultrapure water, and shake well.

[0024] 25% ammonia solution: Accurately measure 0.75 mL of ultrapure water and place it in a centrifuge tube. Then accurately measure 0.25 mL of ammonia solution and shake well.

[0025] Derivatizing agent solution: Accurately measure 0.14 mL of 25% ammonia water and place it in a 200 mL volumetric flask containing ultrapure water. Then accurately measure 12.5 μL of isovaleraldehyde, dilute to the mark with ultrapure water, and shake well.

[0026] Blank solution: Accurately measure 1 mL of methanol and place it in a 20 mL volumetric flask. Accurately measure 2 mL of internal standard stock solution and accurately add 4 mL of derivatizing agent solution. Dilute to the mark with ultrapure water and shake well to obtain the blank solution.

[0027] Test sample solution: Take an appropriate amount of azithromycin dry suspension (containing about 8 mg of azithromycin), accurately weigh it, place it in a centrifuge tube, accurately add 2 mL of methanol, shake well, centrifuge at 4400 rpm for 4 min, accurately measure 1 mL of the supernatant, place it in a 20 mL volumetric flask, accurately add 2 mL of internal standard stock solution (containing 20 ng / mL of NDIPA), accurately add 4 mL of derivatizing agent solution, dilute to the mark with ultrapure water, shake well, and the solution is ready.

[0028] Reference solution: Take an appropriate amount of hydroxylamine hydrochloride, dissolve it in methanol first, and then dilute it with ultrapure water to prepare a solution containing approximately 120 ng of hydroxylamine hydrochloride per 1 mL, which is used as the reference stock solution. Accurately measure 1 mL of methanol, place it in a 20 mL volumetric flask, accurately add 2 mL of the reference stock solution, accurately add 2 mL of the internal standard stock solution, accurately add 4 mL of the derivatizing agent solution, dilute to the mark with ultrapure water, and shake well to prepare the reference solution.

[0029] Derivatization: Take 2 mL each of blank solution, reference solution and test sample solution, derivatize in an oven at 45℃ for 25 min, and cool to room temperature for later use.

[0030] The determination was performed using high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0512) and mass spectrometry (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0431), with octadecylsilane-bonded silica gel as the stationary phase (e.g., ACQUITY UPLC HSS T32.1mm×100mm, 1.8μm); 0.1% formic acid water as mobile phase A and acetonitrile as mobile phase B, with gradient elution according to the table below; column temperature was 40℃; flow rate was 0.3mL per minute.

[0031] Mass spectrometry conditions: Accurately measure 10 μL each of the reference solution and the test sample solution, and inject them separately into the liquid chromatography-mass spectrometry (LC-MS) instrument. Record the chromatograms. The reference solution should be injected continuously 6 times, and the relative correction factor (RSD) for hydroxylamine hydrochloride derivatives should not exceed 20.0%. If hydroxylamine is detected in the test sample solution, calculate the relative correction factor using the internal standard method; the RSD should not exceed 60 ppm.

[0032] The formula for calculating peak area using the internal standard method is as follows: Relative correction factor (f) = W 对 ×P 对 ×V 内标 ×A 内标 / V 对 ×A 对 ×W 内标 ×P 内标 In the formula, W 对 : Amount of reference standard; V 对 : Dilution factor of the reference standard; P 对 : Content of reference standard; A 对 : Peak area of ​​the reference standard; W 内标 : Amount of internal standard; V 内标 Internal standard dilution factor; P 内标 : Content of internal standard; A 内标 : Peak area of ​​internal standard.

[0033] Content (ppm) = (f 平均 ×A 供 ×C 内标 ×V 供 / A 内标 ×W 供 )×1000×1000 Where: f_average: average relative correction factor of STD; A_supply: peak area of ​​the sample to be tested; A_internal_standard: peak area of ​​the internal standard; W_supply: amount of sample to be tested; C_internal_standard: concentration of the internal standard; V_supply: final volume of the sample to be tested.

[0034] Inject the blank solution, the test sample solution, and the reference solution into the sample solution for analysis, and record the chromatograms. See the blank solution chromatogram for reference. Figure 1 See the chromatogram of the reference solution. Figure 1 .

[0035] The derivatized solutions were injected and analyzed separately, and the chromatograms were recorded. The chromatogram of the test sample solution is shown in [reference needed]. Figure 3 (1200mg / vial). Results are detailed in Table 5.

[0036] Table 5 Test results of the samples to be tested Example 2: System Applicability The solution after derivatization in Example 1 was injected for analysis, and the reference solution was injected 6 times consecutively to examine the relative correction factor (RSD).

[0037] Requirement: The relative correction factor (RSD) for six consecutive reference solutions should be ≤20.0%.

[0038] Results: The relative correction factor (RSD) for the reference solution was 3.27% for 6 consecutive tests. The results are shown in Table 5.

[0039] Table 6 System Suitability Test Results Conclusion: This method has good system applicability.

[0040] Example 3: Specificity

[0041] Solution preparation: Internal standard stock solution: Accurately measure 0.1 mL of N-nitrosodiisopropylamine, place it in a 50 mL volumetric flask, dilute to the mark with ultrapure water, and shake well.

[0042] 25% ammonia solution: Accurately measure 0.75 mL of ultrapure water and place it in a centrifuge tube. Then accurately measure 0.25 mL of ammonia solution and shake well.

[0043] Derivatizing agent solution: Accurately measure 0.14 mL of 25% ammonia water and place it in a 200 mL volumetric flask containing ultrapure water. Then accurately measure 12.5 μL of isovaleraldehyde, dilute to the mark with ultrapure water, and shake well.

[0044] Blank solution 1: Accurately measure 1 mL of methanol, place it in a 20 mL volumetric flask, dilute to the mark with ultrapure water, and shake well to obtain the blank solution.

[0045] Blank solution 2: Accurately measure 1 mL of methanol and place it in a 20 mL volumetric flask. Accurately measure 2 mL of internal standard stock solution and accurately add 4 mL of derivatizing agent. Dilute to the mark with ultrapure water and shake well to obtain the blank solution.

[0046] Reference stock solution: Accurately weigh approximately 20 mg of hydroxylamine hydrochloride, place it in a 20 mL volumetric flask, dissolve and dilute to the mark with methanol, and shake well; then accurately measure 0.12 mL, place it in a 10 mL volumetric flask, dilute to the mark with methanol, and shake well; then accurately measure 0.5 mL, place it in a 50 mL volumetric flask, dilute to the mark with ultrapure water, and shake well to obtain the reference stock solution.

[0047] Reference solution: Accurately measure 1 mL of methanol and place it in a 20 mL volumetric flask. Accurately add 2 mL of reference stock solution, 2 mL of internal standard stock solution, and 4 mL of derivatizing agent solution. Dilute to the mark with ultrapure water and shake well to obtain the solution.

[0048] Sample solution 1: Weigh approximately 168 mg of azithromycin dry suspension (1200 mg / bottle) accurately, place it in a centrifuge tube, add 2 mL of methanol accurately, shake well, centrifuge at 4400 rpm for 4 min, accurately measure 1 mL of the supernatant, place it in a 20 mL volumetric flask, add 2 mL of internal standard stock solution accurately, add 4 mL of derivatizing agent solution accurately, dilute to the mark with ultrapure water, shake well, and the solution is ready.

[0049] Sample solution 2: Weigh approximately 336 mg of azithromycin dry suspension (300 mg / bottle) accurately, place it in a centrifuge tube, add 2 mL of methanol accurately, shake well, centrifuge at 4400 rpm for 4 min, accurately measure 1 mL of the supernatant, place it in a 20 mL volumetric flask, add 2 mL of internal standard stock solution accurately, add 4 mL of derivatizing agent solution accurately, dilute to the mark with ultrapure water, shake well, and the solution is ready.

[0050] 100% limit spiked sample solution 1: Weigh approximately 168 mg of azithromycin dry suspension (1200 mg / bottle) accurately, place it in a centrifuge tube, add 2 mL of methanol accurately, shake well, centrifuge at 4400 rpm for 4 min, accurately measure 1 mL of the supernatant, place it in a 20 mL volumetric flask, add 2 mL of reference stock solution accurately, add 2 mL of internal standard stock solution accurately, add 4 mL of derivatizing agent solution accurately, dilute to the mark with ultrapure water, shake well, and the solution is ready.

[0051] 100% limit spiked sample solution 2: Weigh approximately 336 mg of azithromycin dry suspension (300 mg / bottle) accurately, place it in a centrifuge tube, add 2 mL of methanol accurately, shake well, centrifuge at 4400 rpm for 4 min, accurately measure 1 mL of the supernatant, place it in a 20 mL volumetric flask, add 2 mL of reference stock solution accurately, add 2 mL of internal standard stock solution accurately, add 4 mL of derivatizing agent solution accurately, dilute to the mark with ultrapure water, shake well, and the solution is ready.

[0052] Derivatization: Take 2 mL each of the blank solution, reference solution, test sample solution and 100% limit spiked test sample solution, derivatize in an oven at 45 ℃ for 25 min, and cool to room temperature to obtain the final product.

[0053] Inject each of the derivatized blank solutions, reference solutions, test sample solutions, and 100% limit spiked test sample solutions separately, and record the chromatograms.

[0054] Requirements: The blank solution should be free of interference, and the test sample solution should not interfere with the detection of hydroxylamine hydrochloride; the recovery rate of hydroxylamine hydrochloride in the 100% limit spiked test sample solution should be between 80.0% and 120.0%.

[0055] Results: No interference was observed in the blank solutions, and no interference was observed in the test sample solutions for the detection of hydroxylamine hydrochloride derivatives. The recoveries of hydroxylamine hydrochloride in 100% limit-spike test sample solutions 1 (azithromycin content 4.8%) and 2 (azithromycin content 2.4%) were 109.98% and 105.66%, respectively. The results are shown in Table 7.

[0056] Table 7 Results of Recovery Test Note: The background level is calculated by multiplying the background content obtained from the sample by the sample weight, and is used for recovery calculation in spiked experiments.

[0057] Conclusion: This method has good specificity.

[0058] Example 4: Limit of Quantification and Limit of Detection Solution preparation: Reference stock solution: Accurately weigh approximately 20 mg of hydroxylamine hydrochloride, place it in a 20 mL volumetric flask, dissolve and dilute to the mark with methanol, and shake well; then accurately measure 0.12 mL, place it in a 10 mL volumetric flask, dilute to the mark with methanol, and shake well; then accurately measure 0.5 mL, place it in a 50 mL volumetric flask, dilute to the mark with ultrapure water, and shake well to obtain the reference stock solution.

[0059] The preparation of the internal standard stock solution, reference solution, and derivatizing agent solution is the same as in Example 1.

[0060] LOQ solution: Accurately measure 1 mL of methanol and place it in a 20 mL volumetric flask. Accurately add 0.2 mL of reference stock solution, 2 mL of internal standard stock solution, and 4 mL of derivatizing agent solution. Dilute to the mark with ultrapure water and shake well.

[0061] LOD solution: Accurately measure 1 mL of methanol and place it in a 20 mL volumetric flask. Accurately add 0.1 mL of reference stock solution, 2 mL of internal standard stock solution, and 4 mL of derivatizing agent solution. Dilute to the mark with ultrapure water and shake well to obtain the solution.

[0062] Derivatization: Take 2 mL of each limit of quantitation solution and limit of detection solution, derivatize in a 45 ℃ oven for 25 min, and cool to room temperature to obtain the final product.

[0063] Prepare limit of quantitation (LOQ) and limit of detection (LOD) solutions separately from the reference standard stock solution. Prepare six parallel LQ solutions and one LOD solution. Inject the LQ and LOD solutions separately and record the chromatograms.

[0064] After derivatization, each limit of quantitation (LOQ) solution and limit of detection (LOD) solution was injected separately, and the chromatograms were recorded (see [link to chromatogram of limit of quantitation solution]). Figure 4 The chromatogram of the detection limit solution is shown in the figure. Figure 5 ).

[0065] Requirements: At the limit of detection concentration, the peak S / N ratio should be ≥3; at the limit of quantitation concentration, the peak S / N ratio should be ≥10; and the relative correction factor RSD of 6 limit of quantitation solutions should be ≤20.0%.

[0066] Results: At the 5% limit concentration (LOC), the S / N ratio of the hydroxylamine hydrochloride derivative was 8.28; at the 10% limit concentration (LOC), the S / N ratio of the hydroxylamine hydrochloride derivative peak was 11.12, and the relative correction factor in the six LOC solutions was 4.41%. The results are shown in Tables 8-9.

[0067] Table 8 Results of Limit of Quantitation and Limit of Detection Tests Table 9 Results of repeatability tests at the limit of quantitation Conclusion: The limit of quantitation (LOQ) of hydroxylamine hydrochloride in this method corresponds to 6.00 ppm for the main component, and the limit of detection (LOD) corresponds to 3.00 ppm for the main component, which meets the sensitivity requirements for detection.

[0068] Example 5: Accuracy

[0069] Take azithromycin dry suspension and design spiked samples at three concentrations: 10% (limit of quantitation), 100%, and 150% of the limit concentration. Make three samples for each concentration, for a total of n=9.

[0070] Solution preparation: The preparation of internal standard stock solution, 25% ammonia solution, derivatizing agent solution, blank solution, reference standard stock solution, reference standard solution, and test sample solution is the same as in the previous examples.

[0071] Limit of Quantitation (LOQ) Spiked Sample Solution: Accurately weigh an appropriate amount of azithromycin dry suspension (approximately 168 mg for a 1200 mg / vial and approximately 336 mg for a 300 mg / vial, both containing approximately 8 mg of azithromycin), place it in a centrifuge tube, accurately add 2 mL of methanol, shake well, centrifuge at 4400 rpm for 4 min, accurately measure 1 mL of the supernatant, place it in a 20 mL volumetric flask, accurately add 0.2 mL of reference stock solution, accurately add 2 mL of internal standard stock solution, accurately add 4 mL of derivatizing agent solution, dilute to the mark with ultrapure water, shake well, and the solution is ready. Prepare 3 parallel aliquots.

[0072] 100% limit-spike analyte solution: Accurately weigh an appropriate amount of azithromycin dry suspension (approximately 168 mg for a 1200 mg / vial and approximately 336 mg for a 300 mg / vial, each containing approximately 8 mg of azithromycin), place it in a centrifuge tube, accurately add 2 mL of methanol, shake well, centrifuge at 4400 rpm for 4 min, accurately measure 1 mL of the supernatant, place it in a 20 mL volumetric flask, accurately add 2 mL of reference stock solution, accurately add 2 mL of internal standard stock solution, accurately add 4 mL of derivatizing agent solution, dilute to the mark with ultrapure water, shake well, and the solution is ready. Prepare three parallel aliquots. Then prepare a 150% limit-spike analyte solution following the same procedure.

[0073] Derivatization: Take 2 mL each of blank solution, reference solution, test sample solution and spiked test sample solution at each limit, derivatize in an oven at 45 ℃ for 25 min, and cool to room temperature to obtain the final product.

[0074] The blank solution, reference solution, test sample solution, and test sample solutions spiked at each limit were injected separately to investigate the recovery rate and RSD of each concentration.

[0075] Requirements: The recovery rate of the spiked sample solution at each limit should be between 80.0% and 120.0%, and the recovery RSD and total RSD should be ≤20.0%.

[0076] Results: For the 1200 mg / vial (azithromycin 4.8%) specification, the recoveries of the spiked test sample solutions at each limit ranged from 97.47% to 108.71%, with recovery RSD and total RSD ≤ 3.52%.

[0077] For the 300 mg / vial (azithromycin 2.4%) test solution, the recoveries of the spiked test solutions at each limit ranged from 90.30% to 102.88%, with recovery RSD and total RSD ≤ 6.51%. See Tables 10-11 for details.

[0078] Table 10 Accuracy test results (specification 1200 mg / bottle) Table 11 Accuracy test results (specification 300 mg / bottle) Conclusion: This method has good accuracy.

[0079] Example 6: Precision

[0080] Repeatability: Following the steps outlined in "Accuracy in Example 5", one part of the reference solution, three parts of the test sample solution, and six parts of the 100% limit spiked test sample solution were prepared. These were injected separately, and the recovery rate and RSD of hydroxylamine hydrochloride in the six 100% limit spiked test sample solutions were investigated.

[0081] Requirements: The recoveries of 6 100% limit-spikeed test sample solutions should be between 80.0% and 120.0%; the RSD of the recoveries of 6 100% limit-spikeed test sample solutions should be ≤20.0%.

[0082] Results: For the 1200 mg / vial (azithromycin 4.8%) specification, the recoveries of the six 100% limit-spikeed test sample solutions should be between 101.36% and 106.66%; the RSD of the recoveries of the six 100% limit-spikeed test sample solutions was 2.15%.

[0083] For the 300 mg / vial (azithromycin 2.4%) specification: the recovery rate of 6 100% limit-spikeed test sample solutions should be between 96.28% and 100.58%; the RSD of the recovery rate of 6 100% limit-spikeed test sample solutions is 1.56%. See Tables 12-13 for details.

[0084] Table 12 Results of repeatability tests (specification 1200 mg / vial) Table 13 Results of repeatability tests (300 mg / vial) (2) Intermediate precision: On different days and by different analysts, following the steps outlined in "Example 5 Accuracy," one control solution, three test sample solutions, and six 100% limit-spike test sample solutions were prepared. These were injected separately, and the recovery rate and RSD of hydroxylamine hydrochloride in the six 100% limit-spike test sample solutions were examined. The total RSD of repeatability and intermediate precision results was also calculated.

[0085] Requirements: The recoveries of the six 100% limit-spikeed test sample solutions should be between 80.0% and 120.0%; the RSD of the recoveries of the six 100% limit-spikeed test sample solutions should be ≤20.0%; and the total RSD of the recoveries of the twelve 100% limit-spikeed test sample solutions should be ≤20.0%.

[0086] Results: For the 1200 mg / vial (azithromycin 4.8%) specification: the recoveries of the 6 100% limit-spike test solutions should be between 102.31% and 110.17%; the RSD of the recoveries of the 6 100% limit-spike test solutions was 2.74%; and the overall RSD of the recoveries of the 12 100% limit-spike test solutions was 2.55%.

[0087] For the 300 mg / vial (azithromycin 2.4%) specification: the recoveries of 6 100% limit-spike test solutions should be between 98.64% and 102.15%; the RSD of the recoveries of the 6 100% limit-spike test solutions was 1.17%; and the overall RSD of the recoveries of the 12 100% limit-spike test solutions was 1.63%. See Tables 14-17 for detailed results.

[0088] Table 14 Precision test results (specification 1200 mg / bottle) Table 15 Intermediate precision test results (specification 1200 mg / vial) Table 16 Precision test results (300 mg / vial) Table 17 Intermediate precision test results (300 mg / vial) Example 7: Linear

[0089] Solution preparation: The preparation of internal standard stock solution, 25% ammonia solution, derivatizing agent solution, blank solution, reference standard stock solution, reference standard solution, and test sample solution is the same as in the previous examples.

[0090] 200% linear solution: Accurately measure 1 mL of methanol into a 20 mL volumetric flask, accurately add 4 mL of reference stock solution, accurately add 2 mL of internal standard stock solution, accurately add 4 mL of derivatizing agent, dilute to the mark with ultrapure water, and shake well. Prepare 150% linear solution, 100% linear solution, 50% linear solution, 25% linear solution, 50% linear solution, and LOQ solution (10% linear solution) sequentially following this procedure.

[0091] Derivatization: Take 2 mL each of blank solution, reference solution and the linear solution prepared above, derivatize in an oven at 45 ℃ for 25 min, and cool to room temperature to obtain the final product.

[0092] After derivatization, blank solution, reference solution, and linear solution were injected separately, and chromatograms were recorded. A linear regression was performed on the peak area ratio (y) against the solution concentration ratio (x) to obtain the regression equation, linear correlation coefficient r, and intercept ratio.

[0093] Requirements: The linear correlation coefficient r should be ≥0.990, the RSD of the relative correction factor response value for each concentration should be ≤20.0%, and the intercept ratio should be ≤25.0%.

[0094] Results: The linear correlation coefficient r in the linear range from the limit of quantitation to 200% was 0.9987, the RSD of the relative correction factor response value for each concentration was 7.74%, and the intercept ratio was 5.23%. The results are shown in Table 18.

[0095] Table 18 Results of Linearity Tests Conclusion: The method for hydroxylamine hydrochloride exhibits good linearity within the linear range of limit of quantitation to 200%.

[0096] Comparative Example Comparative Example 1 differs from Example 1 in that it uses the external standard method for determination, and the specific operation is as follows: Derivatizing agent solution: Accurately measure 0.14 mL of 25% ammonia water and place it in a 200 mL volumetric flask containing ultrapure water. Then accurately measure 12.5 μL of isovaleraldehyde, dilute to the mark with ultrapure water, and shake well.

[0097] Blank solution: Accurately measure 1 mL of methanol and place it in a 20 mL volumetric flask. Accurately measure 2 mL of internal standard stock solution and accurately add 4 mL of derivatizing agent solution. Dilute to the mark with ultrapure water and shake well to obtain the blank solution.

[0098] Test sample solution: Take an appropriate amount of azithromycin dry suspension (containing about 8 mg of azithromycin), accurately weigh it, place it in a centrifuge tube, accurately add 2 mL of methanol, shake well, centrifuge at 4400 rpm for 4 min, accurately measure 1 mL of the supernatant, place it in a 20 mL volumetric flask, accurately add 4 mL of derivatizing agent solution (0.00625% isovaleraldehyde ammonia solution, ammonia concentration of 0.0175%), dilute to the mark with ultrapure water, shake well, and the solution is ready.

[0099] Reference solution: Take an appropriate amount of hydroxylamine hydrochloride, dissolve it in methanol first, and then dilute it with ultrapure water to prepare a solution containing about 120 ng of hydroxylamine hydrochloride per 1 mL as the reference stock solution. Accurately measure 1 mL of methanol, place it in a 20 mL volumetric flask, accurately add 2 mL of the reference stock solution, accurately add 4 mL of the derivatizing agent solution, dilute to the mark with ultrapure water, and shake well to prepare the reference solution.

[0100] Derivatization: Take 2 mL each of blank solution, reference solution and test sample solution, derivatize in an oven at 45℃ for 25 min, and cool to room temperature for later use.

[0101] The determination was performed using high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0512) and mass spectrometry (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0431), with octadecylsilane-bonded silica gel as the stationary phase (e.g., ACQUITY UPLC HSS T3, 2.1 mm × 100 mm, 1.8 μm); 0.1% formic acid aqueous solution as mobile phase A and acetonitrile as mobile phase B, with gradient elution according to the table below; the column temperature was 40 °C; and the flow rate was 0.3 mL per minute.

[0102] Mass spectrometry conditions: Accurately measure 10 μL each of the reference solution and the test sample solution, inject them separately into the liquid chromatography-mass spectrometry (LC-MS) instrument, and record the chromatograms.

[0103] Following the steps of Example 3 above (removing the internal standard stock solution in the steps), the recovery rate of hydroxylamine hydrochloride using the external standard method was determined, and the results are detailed in Table 19.

[0104] Table 19. Results of recovery test for Comparative Example 1 Referring to the steps of Example 4 above (removing the internal standard stock solution from the steps), determine the limit of quantitation of the external standard method described above (see...). Figure 6 ) and detection limit (see Figure 7 The results are detailed in Table 20.

[0105] Table 20. Results of tests on the limit of quantitation and limit of detection in Comparative Example 1 Referring to the steps of Example 5 above (removing the internal standard stock solution in the steps), the accuracy of the external standard method was determined (characterized by the recovery rate RSD and the total RSD), and the results are detailed in Table 21.

[0106] Table 21. Accuracy test results of Comparative Example 1 In summary, the analytical method in Comparative Example 1, which did not use an internal standard, showed a significant decrease in the recovery rate, sensitivity, and accuracy of the analyte compared to Example 1, indicating the important role of internal standards in the analysis and detection of trace amounts of hydroxylamine hydrochloride in complex matrices.

[0107] Comparative Example 2 differs from Comparative Example 1 in that it uses p-nitrobenzaldehyde instead of isovaleraldehyde for the determination. The specific procedure is as follows: Derivatizing agent solution: Accurately measure 0.14 mL of 25% ammonia water and place it in a 200 mL volumetric flask containing ultrapure water. Then accurately measure 12.5 μL of p-nitrobenzaldehyde, dilute to the mark with ultrapure water, and shake well.

[0108] Blank solution: Accurately measure 1 mL of methanol and place it in a 20 mL volumetric flask. Accurately measure 2 mL of internal standard stock solution and accurately add 4 mL of derivatizing agent solution. Dilute to the mark with ultrapure water and shake well to obtain the blank solution.

[0109] Test sample solution: Take an appropriate amount of azithromycin dry suspension (containing about 8 mg of azithromycin), accurately weigh it, place it in a centrifuge tube, accurately add 2 mL of methanol, shake well, centrifuge at 4400 rpm for 4 min, accurately measure 1 mL of the supernatant, place it in a 20 mL volumetric flask, accurately add 4 mL of derivatizing agent solution (0.00625% isovaleraldehyde ammonia solution, ammonia concentration of 0.0175%), dilute to the mark with ultrapure water, shake well, and the solution is ready.

[0110] Reference solution: Take an appropriate amount of hydroxylamine hydrochloride, dissolve it in methanol first, and then dilute it with ultrapure water to prepare a solution containing about 120 ng of hydroxylamine hydrochloride per 1 mL as the reference stock solution. Accurately measure 1 mL of methanol, place it in a 20 mL volumetric flask, accurately add 2 mL of the reference stock solution, accurately add 4 mL of the derivatizing agent solution, dilute to the mark with ultrapure water, and shake well to prepare the reference solution.

[0111] Derivatization: Take 2 mL each of blank solution, reference solution and test sample solution, derivatize in an oven at 45℃ for 25 min, and cool to room temperature for later use.

[0112] The determination was performed by high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0512) and mass spectrometry (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0431), using octadecylsilane-bonded silica gel as the stationary phase (e.g., ZORBAX Eclipse C18, 2.1 mm × 100 mm, 1.8 μm); 0.1% formic acid aqueous solution as mobile phase A and acetonitrile as mobile phase B, with gradient elution according to the table below; column temperature was 40 °C; flow rate was 0.3 mL per minute.

[0113] Mass spectrometry conditions: Accurately measure 10 μL each of the reference solution and the test sample solution, inject them separately into the liquid chromatography-mass spectrometry (LC-MS) instrument, and record the chromatograms.

[0114] Following the steps of Example 3 above (replacing the derivatizing agent isovaleraldehyde with p-nitrobenzaldehyde and removing the internal standard stock solution from the steps), the recovery rate of hydroxylamine hydrochloride using the external standard method was determined, and the results are detailed in Table 22.

[0115] Table 22. Results of recovery test for Comparative Example 1 Referring to the steps of Example 4 above (replacing the derivatizing agent isovaleraldehyde with p-nitrobenzaldehyde and removing the internal standard stock solution from the steps), the limit of quantitation of the external standard method described above was determined (see Example 4). Figure 8 ) and detection limit (see Figure 9 The results are detailed in Table 23.

[0116] Table 23. Results of tests on the limit of quantitation and limit of detection in Comparative Example 1 Referring to the steps of Example 5 above (removing the internal standard stock solution in the steps), the accuracy of the external standard method was determined (characterized by the recovery RSD and total RSD), and the results are detailed in Table 24.

[0117] Table 24. Accuracy test results of Comparative Example 1 In summary, the analytical method of Comparative Example 2, which uses benzaldehyde as a derivatizing agent and does not use an internal standard, showed a further decrease in the recovery rate, sensitivity, and accuracy of the analyte compared to Comparative Example 1. This demonstrates the crucial role of isovaleraldehyde derivatizing agent in the analysis and detection of trace amounts of hydroxylamine hydrochloride in complex matrices.

[0118] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for detecting hydroxylamine hydrochloride in azithromycin dry suspension, characterized in that, Isovallar oxime was generated by derivatizing the sample with isovaleraldehyde as a derivatizing agent, and then quantitatively determined by liquid chromatography-tandem mass spectrometry using N-nitrosodiisopropylamine as an internal standard.

2. The method for detecting hydroxylamine hydrochloride in azithromycin dry suspension according to claim 1, characterized in that, The derivatization process includes: dissolving the derivatizing agent in a solvent to prepare a derivatizing agent solution with a concentration of 40-60 μg / mL; preparing the sample to be tested into a solution, adding the derivatizing agent solution, and derivatizing at 40-50℃ for 20-30 min to obtain the sample.

3. The method for detecting hydroxylamine hydrochloride in azithromycin dry suspension according to claim 1, characterized in that, The single injection detection time for the quantitative determination by liquid chromatography-tandem mass spectrometry is 7–9 min.

4. The method for detecting hydroxylamine hydrochloride in azithromycin dry suspension according to claim 1, characterized in that, The solvent used is ammonia water with a concentration of 20-30 wt%.

5. The method for detecting hydroxylamine hydrochloride in azithromycin dry suspension according to claim 1, characterized in that, The liquid chromatography column is packed with octadecylsilane-bonded silica gel, and the column temperature is 35–45°C.

6. The method for detecting hydroxylamine hydrochloride in azithromycin dry suspension according to claim 1, characterized in that, The liquid chromatography used 0.1% formic acid water as mobile phase A and acetonitrile as mobile phase B.

7. The method for detecting hydroxylamine hydrochloride in azithromycin dry suspension according to claim 5, characterized in that, The elution rate for the liquid chromatography was 0.3 mL / min, and the elution gradient was as follows: 0~0.8min: 90%A→90%A, 10%B→10%B; 0.8~2min: 90%A→5%A, 10%B→95%B; 2~6min: 5%A→5%A, 95%B→95%B; 6~6.1min: 5%A→90%A, 95%B→10%B; 6.1~8min:90%A→90%A, 10%B→10%B.

8. The method for detecting hydroxylamine hydrochloride in azithromycin dry suspension according to claim 1, characterized in that, In mass spectrometry, the qualitative ion pair of isovaleraldehyde oxime was 102.07→43.15 m / z, and the quantitative ion pair was 102.07→46.4 m / z.

9. The method for detecting hydroxylamine hydrochloride in azithromycin dry suspension according to claim 1, characterized in that, In mass spectrometry, the qualitative ion pair of N-nitrosodiisopropylamine was m / z 131.1→89.1, and the quantitative ion pair was m / z 131.1→47.

1.

10. The method for detecting hydroxylamine hydrochloride in azithromycin dry suspension according to claim 1, characterized in that, In mass spectrometry detection, the ion source was an ESI source, positive ion mode, ion source temperature was 500℃, curtain gas was 30psi, nebulizer gas was 50psi, auxiliary nebulizer gas was 55psi, and ion spray voltage was 5500V.

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