Method for detecting nitrosamine impurities in terbutaline sulfate aerosol inhalation solution

Abstract

The invention belongs to the technical field of analysis and detection, and relates to a method for detecting nitrosamine impurities in a terbutaline sulfate aerosol inhalation solution. The method comprises the following steps: diluting a terbutaline sulfate aerosol inhalation solution to obtain a test solution; detecting the test solution by adopting an ultra-high performance liquid chromatography-tandem mass spectrometry method; wherein the chromatographic conditions are as follows: a filling agent of a chromatographic column is octadecylsilane chemically bonded silica; the mobile phase A is an ammonium acetate aqueous solution; the mobile phase B is acetonitrile; the nitrosamine impurity is N-nitroso-terbutaline, and the nitrosamine impurity is N-nitroso-terbutaline. The method for detecting the N-nitroso-terbutaline impurities in the terbutaline sulfate aerosol inhalation solution by adopting the ultra-high performance liquid chromatography-tandem mass spectrometry technology is proposed for the first time, the method is high in selectivity, the quantitation limit is as low as 1.5% of the limit, the detection limit is as low as 0.5% of the limit, detection can be completed only in 10 minutes, and the method is suitable for large-scale popularization and application. Powerful guarantee is provided for effectively controlling the medicine quality and guaranteeing the medicine safety.

Description

Technical Field

[0001] The present invention belongs to the technical field of analytical detection, and relates to a method for detecting nitrosamine impurities in terbutaline sulfate inhalation solution. Background Art

[0002] Any discussion of the prior art throughout the specification should not be construed as an admission that such prior art is widely known or forms part of the common general knowledge in the art.

[0003] Terbutaline Sulfate is a selective β2 receptor agonist and is widely used in the treatment of asthma and chronic obstructive pulmonary disease (COPD). Terbutaline sulfate inhalation solution is one of the commonly used administration methods in clinical practice, with advantages such as rapid onset and accurate dosage. Respiratory drugs such as terbutaline sulfate inhalation solution act directly on lung tissue. Compared with the oral administration route, impurities may have a more direct impact on sensitive tissues. Therefore, controlling the potential carcinogen N-Nitroso Terbutaline in such preparations has special significance for health protection, and establishing a highly sensitive detection method is crucial for ensuring patient safety.

[0004]

[0005] Nitrosamine compounds are classified as Group 2A potential carcinogens by the IARC (International Agency for Research on Cancer). They have been widely detected in drugs, foods, and the environment. Since 2018, several drugs such as valsartan (NDMA), ranitidine (NDMA), and rifampicin (MNP) have been globally recalled due to excessive nitrosamines, causing significant public health and economic losses. Given that long-term inhalation preparations can directly deposit impurities in lung tissue, both the FDA's September 2024 "Control of Nitrosamine Impurities in Human Drugs (Rev.2)" and EMA / ICH M7 (R2) require risk assessment, confirmatory testing, and control for all drug dosage forms (including inhalation solutions). In the CPCA prediction table, the structurally related impurity of terbutaline sulfate, N-Nitroso Terbutaline, is classified as Category 5, and the acceptable intake (AI) = 1500 ng day-1. If the maximum daily dose of the inhalation solution is 15 mg, then the impurity control limit is 0.01% (100 ppm). Due to the long-term use of inhalation preparations and their targeting of the lungs, higher requirements are placed on the detection sensitivity and method robustness. Therefore, developing a sub-ppm–ppb level detection method and incorporating it into life cycle quality management is the key to preventing future recalls and ensuring the safety of drugs for patients with chronic respiratory diseases.

[0006] Currently, in the quality standards of terbutaline sulfate raw materials and related preparations in major pharmacopoeias worldwide, such as the United States Pharmacopeia (USP), European Pharmacopoeia (EP), British Pharmacopoeia (BP), and Chinese Pharmacopoeia (ChP), the detection method for N-nitroso-terbutaline impurities is not included. In addition, through literature retrieval, no research reports on the detection methods for nitrosamine impurities in terbutaline sulfate and its preparations have been found.

[0007] Traditional analytical detection methods often have disadvantages such as insufficient sensitivity and poor selectivity when faced with the detection of low-content impurities, making it difficult to meet the accurate detection requirements for trace N-nitroso-terbutaline impurities. Especially for complex pharmaceutical preparation matrices, traditional methods often struggle to effectively separate and identify target impurities. With the increasing requirements of global drug regulatory agencies for the control of nitrosamine impurities, many drugs previously considered safe are facing re-evaluation. Highly sensitive detection methods are crucial for meeting this evolving regulatory requirement, enabling pharmaceutical manufacturers to maintain compliance in an increasingly strict regulatory environment and having significant forward-looking value.

[0008] Therefore, developing a highly sensitive and selective detection method is crucial for ensuring the safety of terbutaline sulfate nebulization solution. This not only helps improve the level of drug quality control but also provides important technical support for the formulation of quality standards for related preparations. Summary of the Invention

[0009] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide a detection method for nitrosamine impurities in terbutaline sulfate nebulization solution, which can quickly, sensitively, and accurately detect N-nitroso-terbutaline impurities in terbutaline sulfate nebulization solution.

[0010] The detection method of the present invention not only meets regulatory requirements but also has a preventive safeguard function. In the life cycle management of drugs, factors such as changes in raw material sources, production process adjustments, or storage conditions can all lead to changes in the impurity profile. This method provides a technical means to promptly detect potential risks, enabling pharmaceutical manufacturers to take measures before problems expand, avoiding possible batch recalls and patient risks.

[0011] Specifically, the present invention aims to solve at least one of the following technical problems: existing analytical methods are difficult to meet the accurate detection requirements for trace N-nitroso-terbutaline impurities; in complex preparation matrices, the selectivity for detecting low-content impurities is insufficient; there is a lack of a reliable method to determine whether the content of N-nitroso-terbutaline impurities in terbutaline sulfate nebulization solution meets strict safety limit requirements.

[0012] The detection limit of the method provided by the present invention is equivalent to 0.5% of the limit (when the limit is 0.01%, the detection limit is 0.00005%), and the quantitation limit is equivalent to 1.5% of the limit (when the limit is 0.01%, the quantitation limit is 0.00015%). The method of the present invention is very sensitive and far superior to the minimum requirements of regulations for impurity control. It can be used to accurately detect trace N-nitroso-terbutaline impurities, thus ensuring the safety and compliance of drugs, achieving rapid and accurate detection of N-nitroso-terbutaline, a nitrosamine impurity in terbutaline sulfate inhalation solution, and providing technical support for effectively controlling drug quality and ensuring drug safety.

[0013] Specifically, the present invention provides the following technical solutions.

[0014] The detection method of the present invention is not only applicable to the quality control of finished products, but also can be used in multiple links such as the detection of intermediates, the acceptance control of raw materials, and the stability study during the production process, providing comprehensive technical support for the quality management of the entire drug life cycle. This multi-link application ability greatly enhances the practical value of the method, especially in the context of increasing attention to the source management of drug quality today.

[0015] In the first aspect of the present invention, a method for detecting nitrosamine impurities in terbutaline sulfate inhalation solution is provided, comprising the following steps:

[0016] After diluting the terbutaline sulfate inhalation solution, a test solution is obtained;

[0017] The test solution is detected by ultra-high performance liquid chromatography-tandem mass spectrometry;

[0018] Among them, the chromatographic conditions are: the filler of the chromatographic column is octadecylsilane-bonded silica gel; mobile phase A is an aqueous ammonium acetate solution; mobile phase B is acetonitrile;

[0019] The nitrosamine impurity is N-nitroso-terbutaline, and its chemical name is N-(tert-butyl)-N-(2-(3,5-dihydroxyphenyl)-2-hydroxyethyl)nitrous amide, and its structural formula is as follows:

[0020]

[0021] In some embodiments, the diluent for diluting the terbutaline sulfate inhalation solution is a sulfuric acid-water-methanol solution.

[0022] In some embodiments, in the diluent, the volume percentage of methanol is 10-30%, preferably 15%.

[0023] In some embodiments, the diluent is a 0.005 mol / L sulfuric acid water-methanol solution, wherein the volume ratio of the sulfuric acid aqueous solution to methanol is 85:15.

[0024] This diluent formulation can not only ensure the full dissolution of the main drug and impurities, but also inhibit the hydrolysis or degradation reaction of N-nitroso-terbutaline impurities through a weakly acidic environment, thereby improving the stability and accuracy of the analysis results.

[0025] Compared with the common methanol-water or pure organic solvent dilution systems in the prior art, the introduction of a low-concentration sulfuric acid component in the diluent of the present invention can effectively adjust the pH of the test sample, keep N-nitroso-terbutaline chemically stable before analysis, and avoid degradation or transformation. In addition, this dilution system is fully compatible with subsequent chromatographic and mass spectrometric analysis conditions, avoiding the introduction of systematic errors or analysis delays due to condition changes, simplifying the overall operation process, and improving the method adaptability.

[0026] Experimental results show that, compared with the diluent system without an acid regulator, under the conditions of using the diluent formulation of the present invention, the peak area of N-nitroso-terbutaline is more stable, the retention time shift is smaller, and the signal decline amplitude within 24 hours is significantly reduced, indicating that this system has good impurity stability maintenance ability in the sample pretreatment stage.

[0027] In some embodiments, in the test sample solution, the concentration of terbutaline sulfate is 50-150 μg / mL, preferably 100 μg / mL.

[0028] In some embodiments, in mobile phase A, the concentration of ammonium acetate is 2 mmol / L, which is beneficial to the mass spectrometry ionization response.

[0029] Compared with the common mobile phase systems of phosphate buffer and methanol in the prior art, the present invention preferably adopts a mobile phase system composed of 2 mmol / L ammonium acetate aqueous solution and acetonitrile. This system has better mass spectrometry compatibility, higher signal-to-noise ratio, and more stable retention behavior, and can achieve lower detection limits and shorter analysis times while maintaining good separation.

[0030] Experimental results show that when using the traditional phosphate-methanol mobile phase system, the response signal of N-nitroso-terbutaline in the negative ion detection mode is significantly unstable, the retention time is prone to shift, and signal suppression is observed in some samples, seriously affecting the accuracy and reproducibility of the detection results.

[0031] In addition, ammonium formate is also used as a buffer salt in some existing technologies. However, in the negative ion electrospray ionization mode, the ammonium formate system may cause ion suppression effects, resulting in unstable response signals for structurally sensitive nitrosamine impurities such as N-nitroso-terbutaline. At the same time, its strong acidic environment may also cause degradation of impurities during the injection process, thus affecting the reliability of the results.

[0032] In contrast, the ammonium acetate buffer system used in the present invention is milder and more mass spectrometry-friendly, which can significantly improve the detection sensitivity in the negative ion mode, maintain the structural stability of N-nitroso-terbutaline impurities, and ensure the reproducibility of retention time and the integrity of peak shape.

[0033] In some embodiments, the gradient elution program is as follows:

[0034] From 0 to 3 minutes, mobile phase A decreases from 85% to 70%, and mobile phase B increases from 15% to 30%;

[0035] From 3 to 6 minutes, mobile phase A decreases from 70% to 40%, and mobile phase B increases from 30% to 60%;

[0036] From 6 to 8 minutes, mobile phase A is maintained at 40%, and mobile phase B is maintained at 60%;

[0037] From 8 to 8.1 minutes, mobile phase A increases from 40% to 85%, and mobile phase B decreases from 60% to 15%;

[0038] From 8.1 to 10 minutes, mobile phase A is maintained at 85%, and mobile phase B is maintained at 15%.

[0039] The above percentages are volume percentages.

[0040] In these embodiments, the elution program of the present invention adopts a five-stage gradient design. Maintaining a relatively high proportion of the aqueous phase in the initial stage helps to retain strongly polar impurities; gradually increasing the proportion of acetonitrile in the middle stage can effectively elute hydrophobic impurities and compress the retention time interval; quickly restoring the initial conditions and maintaining system stability in the final stage to meet the requirements of the next injection.

[0041] Compared with some existing methods that use fixed ratios or slowly changing gradients, resulting in long analysis cycles and broadened peak shapes, the gradient design of the present invention can complete the separation and detection of the target substance within 10 minutes, and has good reproducibility of retention time and peak shape symmetry, improving the detection efficiency and method reliability.

[0042] The experimental results show that under this program, the retention time of N-nitroso-terbutaline impurity is stably around 3.6 min, and the signal-to-noise ratio is better than that of conventional isocratic elution or other gradient elution methods with gradually changing ratios, demonstrating the application advantages of this elution program in the rapid separation and detection of trace impurities.

[0043] In some embodiments, the chromatographic conditions are as follows: the chromatographic column is packed with octadecylsilyl-bonded silica gel; the chromatographic column is Hypersil GOLD, 100 mm × 2.1 mm, 1.9 μm; the flow rate is 0.4 mL / min, the column temperature is 30 °C, and the injection volume is 10 μL.

[0044] In some embodiments, during the detection process, the mass spectrometry conditions are as follows: the ion source is an electrospray ion source, the scanning mode is the negative ion mode, and the detection mode is multiple reaction monitoring MRM.

[0045] In some embodiments, the mass spectrometer is a triple quadrupole mass spectrometer, and after gradient elution, it is detected by a triple quadrupole mass spectrometer.

[0046] In some embodiments, the mass spectrometry conditions are as follows: the IS voltage is -4500 V; the curtain gas CUR is 40 psi; the nebulizer GAS1 is 50 psi; the auxiliary gas GAS2 is 50 psi; the ion source temperature is 500 °C; the collision gas: 8; Q1 is 253.1 Da, Q3 is 172.8 Da, DP is -118 V, and CE is -24 V.

[0047] The detection method of the present invention has high selectivity and can effectively distinguish N-nitroso-terbutaline impurity from other substances; it has high sensitivity, the detection limit is as low as 0.5% of the limit, and the quantitation limit is as low as 1.5% of the limit. That is, when the limit is 0.01%, the detection limit is as low as 0.00005%, and the quantitation limit is as low as 0.00015%. The method of the present invention is very sensitive, far superior to the minimum requirements of regulations for impurity control, and can be used for precise detection of trace N-nitroso-terbutaline impurity; the detection speed is fast, and a complete analysis can be completed in only 10 minutes; the sample pretreatment is simple, and only dilution is required for analysis, reducing the errors that may be introduced during the sample treatment process.

[0048] In the second aspect of the present invention, there is provided a method for determining the content of N-nitroso-terbutaline impurity in terbutaline sulfate inhalation solution, and the method includes:

[0049] Detecting the N-nitroso-terbutaline impurity according to the detection method described in the first aspect above;

[0050] Calculating the content of the N-nitroso-terbutaline by the external standard method.

[0051] In some embodiments, the calculation formula of the external standard method is:

[0052]

[0053] Wherein:

[0054] Au represents the area of N-nitroso-terbutaline in the test solution;

[0055] As represents the area of N-nitroso-terbutaline in the reference solution;

[0056] Cs represents the concentration of N-nitroso-terbutaline in the reference solution, ng / mL;

[0057] Cu represents the concentration of the test solution calculated based on the labeled amount (calculated as terbutaline sulfate), ng / mL.

[0058] By calculating the impurity content by the external standard method, the interference that may be brought by the need to add an internal standard substance in the internal standard method is avoided; the calculation method is simple and direct, and is easy to apply in the actual quality control process; the results are accurate and reliable. In the range of 50% - 150% of the limit concentration, the recovery rate of N-nitroso-terbutaline is between 85% - 115%, meeting the precision requirements of drug quality control.

[0059] In the third aspect of the present invention, a quality control method for a solution for nebulized inhalation of terbutaline sulfate is provided, and the method includes:

[0060] Detecting the content of N-nitroso-terbutaline impurity in the solution for nebulized inhalation of terbutaline sulfate by using the detection method described in the first aspect above;

[0061] Comparing the detection result with a preset limit value to determine whether the quality of the solution for nebulized inhalation of terbutaline sulfate is qualified.

[0062] In some embodiments, the preset limit value is 0.01%.

[0063] The present invention combines the detection method with the quality standard to form a complete quality control system; the preset limit value meets the latest requirements of international regulatory agencies for nitrosamine impurities, ensuring product safety; the quality control method is easy to operate, the result judgment is clear, and it is convenient to be actually applied in production and quality control.

[0064] In the fourth aspect of the present invention, an application of the detection method described in the first aspect above in the quality control of a solution for nebulized inhalation of terbutaline sulfate is provided.

[0065] Applying the detection method to the field of quality control provides technical support for the factory inspection and market sampling inspection of drugs; helps to improve the overall quality level and safety of the solution for nebulized inhalation of terbutaline sulfate products.

[0066] In the fourth aspect of the present invention, there is provided an application of the detection method described in the first aspect above in the production process control of terbutaline sulfate nebulization solution.

[0067] Applying the detection method to the production process control can detect possible impurity risks at an early stage of production; help adjust production process parameters in a timely manner, reduce the risk of defective products; improve production efficiency and reduce quality control costs; and provide impurity control technical support for the whole life cycle of pharmaceutical production enterprises.

[0068] The beneficial effects obtained by one or more of the above embodiments of the present invention are as follows:

[0069] The present invention provides a method for detecting the nitrosamine impurity N-nitroso-terbutaline in terbutaline sulfate nebulization solution. The present invention first proposes to use ultra-high performance liquid chromatography-tandem mass spectrometry technology to analyze and detect the nitrosamine impurity N-nitroso-terbutaline in terbutaline sulfate nebulization solution, providing a strong guarantee for effectively controlling the quality of drugs and ensuring the safety of drugs.

[0070] Using the ultra-high performance liquid chromatography-tandem mass spectrometry technology of the present invention to detect the nitrosamine impurity N-nitroso-terbutaline in terbutaline sulfate nebulization solution has high selectivity, good method specificity, a low limit of quantitation of 1.5% of the limit, and a low limit of detection of 0.5% of the limit (that is, when the limit is 0.01%, the limit of detection is as low as 0.00005%, and the limit of quantitation is as low as 0.00015%). The detection can be completed in only 10 minutes. The method has high sensitivity and can quickly and accurately detect the content of trace nitrosamine impurity N-nitroso-terbutaline in the sample, effectively reducing the analysis cost.

[0071] The present invention also verifies the accuracy of the nitrosamine impurity in the range of 50% to 150% of the limit concentration (0.01%). The recovery rate of N-nitroso-terbutaline is between 85% and 115%, and the accuracy is good. The linearity of N-nitroso-terbutaline in the range of 1.5% to 200% of the limit concentration is also verified. In the above range, r>0.999 for N-nitroso-terbutaline, and the linearity is good.

[0072] Experiments have proved that the detection method of the present invention has good specificity, accuracy, precision and durability, and can meet the daily detection requirements of N-nitroso-terbutaline impurities in terbutaline sulfate inhalation solution, providing an important technical means for drug safety evaluation and quality control. The detection method of the present invention is easy to operate and has a short analysis time, significantly reducing the detection cost and time compared with traditional methods. In a large-scale production environment, this efficiency improvement can be translated into significant economic benefits and improve the timeliness of drug release. According to industry data, the improvement of quality control efficiency can reduce the drug inventory turnover time and lower the operating cost of enterprises.

[0073] In summary, the present invention not only solves the technical problem of detecting N-nitroso-terbutaline impurities in terbutaline sulfate inhalation solution, but also can be widely applied to drug quality control and production process control, having important practical application value. Brief Description of the Drawings

[0074] The accompanying drawings forming a part of this application are used to provide a further understanding of this application. The schematic embodiments and descriptions thereof of this application are used to explain this application and do not constitute an improper limitation of this application. Hereinafter, the embodiments of this application will be described in detail with reference to the accompanying drawings, where:

[0075] Figure 1 It is the chromatogram of the blank solution of Example 1 of the present invention.

[0076] Figure 2 It is the chromatogram of the reference solution of N-nitroso-terbutaline impurities of Example 1 of the present invention.

[0077] Figure 3 It is the chromatogram of the test solution of Example 1 of the present invention.

[0078] Figure 4 It is the chromatogram of the system suitability solution of Example 1 of the present invention. Detailed Description of the Embodiments

[0079] The present application will be further described in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present application and not to limit its scope. The experimental methods without specifying specific conditions in the embodiments are usually carried out according to conventional conditions or the conditions recommended by the manufacturer.

[0080] Unless otherwise defined, all professional terms and scientific terms used in this application should have the meanings familiar to those skilled in the art. Unless otherwise specified, the reagents or raw materials used in this application can be obtained through conventional channels and used according to the conventional methods in the art or the product specifications. In addition, any content similar or equivalent to the described methods or materials can be applied to the methods of this application. The preferred embodiments and materials described in this application are only for illustrative purposes.

[0081] General scheme of the detection method of this application

[0082] The method for analyzing nitrosamine impurities in terbutaline sulfate inhalation solution provided by this application is based on ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) technology, and adopts a strategy combining reverse-phase chromatography with selective mass spectrometry detection. This method is applicable to the trace detection and quantitative analysis of N-nitroso-terbutaline impurities in terbutaline sulfate inhalation solution.

[0083] 1. Sample pretreatment strategy

[0084] This method uses a simple dilution strategy for sample pretreatment to avoid complex extraction and purification processes. The sample of terbutaline sulfate inhalation solution is diluted to an appropriate concentration with a 0.005 mol / L sulfuric acid water-methanol (85:15) mixed solution. This solvent system can effectively dissolve the sample and is compatible with the subsequent chromatographic separation conditions. At the same time, the addition of sulfuric acid helps to improve the stability of the sample solution and prevent the degradation of N-nitroso-terbutaline impurities in the solution.

[0085] 2. Chromatographic separation principle

[0086] This method adopts the principle of reverse-phase chromatography and uses the interaction between a non-polar stationary phase and a medium-polar mobile phase to achieve efficient separation of target impurities. The chromatographic column uses octadecylsilane-bonded silica gel (C18) as the packing material, with specific specifications of Hypersil GOLD, 100 mm×2.1 mm, 1.9 μm. Such a stationary phase has appropriate hydrophobicity and is particularly suitable for separating terbutaline sulfate and its nitrosamine impurities.

[0087] 3. Mobile phase system design

[0088] The mobile phase adopts a binary gradient system:

[0089] Mobile phase A is a 2 mmol / L ammonium acetate aqueous solution, which provides an appropriate buffer environment and promotes the electrospray ionization process;

[0090] Mobile phase B is acetonitrile, which has moderate elution strength and good MS compatibility.

[0091] This combination of mobile phases can not only meet the chromatographic separation requirements but also ensure the sensitivity of mass spectrometry detection.

[0092] 4. Gradient elution strategy

[0093] An elaborate multi - stage gradient elution program is adopted: in the initial stage, a relatively high proportion of the aqueous phase is maintained (A = 85:15); subsequently, the proportion of the organic phase is gradually increased to A = 40:60 to enhance the elution ability for hydrophobic impurities; finally, it quickly returns to the initial conditions and equilibrates the system. This gradient design optimizes the analysis time while ensuring sufficient separation, and the entire analysis process only takes 10 minutes.

[0094] 5. Mass Spectrometry Detection Technology and Parameters

[0095] This method uses a triple quadrupole mass spectrometer. Through the negative ion mode of the electrospray ionization source (ESI) and in combination with the multiple reaction monitoring (MRM) technology, highly sensitive and selective detection of target impurities is achieved. The key mass spectrometry parameters are set as follows:

[0096] IS voltage: - 4500V

[0097] Curtain gas (CUR): 40 psi

[0098] Nebulizer gas (GAS1): 50 psi

[0099] Auxiliary gas (GAS2): 50 psi

[0100] Ion source temperature: 500 °C

[0101] Collision gas: 8

[0102] Mass analysis: Q1 is 253.1 Da, Q3 is 172.8 Da

[0103] Declustering potential (DP): - 118V

[0104] Collision energy (CE): - 24V

[0105] After these parameters are optimized, the best signal intensity and sensitivity can be provided, and the detection limit is as low as 0.00005% (equivalent to 0.5% of the limit).

[0106] 6. General Operating Procedures

[0107] The implementation of this detection method generally includes the following steps:

[0108] (1) System preparation: Install the chromatographic column into the UPLC - MS / MS system and equilibrate the chromatographic column to a stable baseline according to the set conditions.

[0109] (2) Solution preparation:

[0110] Prepare the diluent: 0.005 mol / L sulfuric acid - water - methanol (85:15) solution.

[0111] Prepare the N - nitroso - terbutaline reference substance solution: Make its concentration 10 ng / mL.

[0112] Prepare the test solution: Dilute the terbutaline sulfate inhalation solution to approximately 100 μg / mL.

[0113] Prepare the system suitability solution: Containing approximately 100 μg / mL of terbutaline sulfate and approximately 10 ng / mL of N-nitroso-terbutaline.

[0114] (3) System suitability test: Inject the system suitability solution and confirm that the following key parameters meet the requirements:

[0115] The signal-to-noise ratio of the N-nitroso-terbutaline impurity peak should be high enough (≥10);

[0116] The peak shape is good and the retention time is stable.

[0117] (4) Sample analysis: Inject the test solution and the reference solution for analysis and record the chromatogram.

[0118] (5) Data processing: Calculate the content of N-nitroso-terbutaline impurity based on the external standard method; the calculation formula is as follows:

[0119]

[0120] Where:

[0121] A U Represents the peak area of N-nitroso-terbutaline in the test solution;

[0122] A S Represents the peak area of N-nitroso-terbutaline in the reference solution;

[0123] C S Represents the concentration of N-nitroso-terbutaline in the reference solution, ng / mL;

[0124] C U Represents the concentration of the test solution calculated based on the labeled amount (calculated as terbutaline sulfate), ng / mL.

[0125] 7. Method robustness parameter range

[0126] Through systematic method validation experiments, within the following ranges of condition changes, this method can still maintain good accuracy and repeatability and has good robustness:

[0127] Flow rate: 0.35 - 0.45 mL / min

[0128] Column temperature: 25 - 35 °C

[0129] Concentration of ammonium acetate in mobile phase A: 1.8 - 2.2 mmol / L

[0130] Initial composition of the mobile phase (A): 83:17 - 87:13

[0131] Methanol content in the diluent: 13 - 17%

[0132] Within this parameter range, key performance indicators (such as recovery rate, precision, and detection sensitivity, etc.) remain stable. Among them, the spiked recovery rate is maintained between approximately 99.0% and 104.1%, the precision RSD is less than 2.5%, and the deviation of the impurity retention time does not exceed 0.2 min, showing good method stability and adaptability. This indicates that this method still has good repeatability and reliability when the chromatographic conditions change within a small range, and has robustness in practical applications. In specific applications, it is recommended to confirm that the system performance meets the requirements through system suitability testing before use.

[0133] In summary, this application has established a set of UPLC - MS / MS analysis strategies with simple operation, high sensitivity, and good stability, which is suitable for the trace detection of N - nitrosoterbutaline impurities. Based on the above general scheme, the present invention has comprehensively evaluated the performance characteristics of the method through a series of verification experiments. The following further illustrates the present invention with specific examples to show the application effect and various performance indicators of this method under actual conditions. These examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention.

[0134] Example 1 Specificity

[0135] 1 - 1 Solution preparation

[0136] The molecular formula of N - nitrosoterbutaline is C 12 H 18 N2O4. This reference substance is purchased from CATO Corporation, Cat No.: C4X - 118368.

[0137] Diluent (blank solution): Measure 0.6 mL of sulfuric acid, 2000 mL of water, and 353 mL of methanol, and mix well.

[0138] N - nitrosoterbutaline impurity reference substance solution: Weigh an appropriate amount of N - nitrosoterbutaline impurity reference substance accurately, dissolve it with the diluent and quantitatively dilute it to make a solution of 10 ng / mL.

[0139] Test solution: Take an appropriate amount of terbutaline sulfate inhalation solution, dissolve it with the diluent and quantitatively dilute it to make a solution containing approximately 100 μg of terbutaline sulfate per 1 mL.

[0140] System suitability solution: Take an appropriate amount of terbutaline sulfate inhalation solution, add an appropriate amount of N-nitroso-terbutaline impurity, and dilute with a diluent to prepare a mixed solution containing about 100 μg of terbutaline sulfate and 10 ng of N-nitroso-terbutaline per 1 mL.

[0141] 1-2 Chromatographic and mass spectrometric conditions

[0142] Chromatographic column: Hypersil GOLD, 100 mm × 2.1 mm, 1.9 μm;

[0143] Mobile phase:

[0144] Mobile phase A is an aqueous solution of 2 mmol / L ammonium acetate;

[0145] Mobile phase B is acetonitrile;

[0146] Elution method: Gradient elution, the program is as follows:

[0147] Time (min) Mobile phase A (%) Mobile phase B (%) 0～3 85～70 15～30 3～6 70～40 30～60 6～8 40 60 8～8.1 40～85 60～15 8.1～10 85 15

[0148] Flow rate 0.4 mL / min;

[0149] Column temperature is 30 °C;

[0150] Injection volume is 10 μL.

[0151] Ion source: ESI source, negative ion scan;

[0152] Scanning mode: MRM acquisition mode;

[0153] IS voltage: 4500 V(-);

[0154] Curtain gas CUR is 40 psi; nebulizer GAS1 is 50 psi; auxiliary gas GAS2 is 50 psi; ion source temperature is 500 °C; collision gas: 8;

[0155] Mass spectrometry parameters:

[0156] Q1 (Da) Q3 (Da) DP (V) CE (V) Remarks 253.1 172.8 -118 -24 Quantification

[0157] 1-3 Experimental conclusions:

[0158] Precisely measure the above solution, inject it into an ultra-high performance liquid chromatography-mass spectrometry instrument, record the chromatogram, and the results are shown in Table 1. Figures 1 to 4 .

[0159] Table 1 System suitability and impurity localization results

[0160]

[0161] Figure 1The chromatogram of a blank solution of 0.005 mol / L sulfuric acid water - methanol (85:15) is shown. The baseline in the chromatogram is stable and there are no significant interference signals. Particularly importantly, there are no interference peaks in the retention time region of N-nitroso-terbutaline (about 3.56 minutes). This proves that the solvent system of this method will not interfere with the detection of the target impurity, laying the foundation for the specificity of the method. Figure 2 The chromatogram of a reference solution of N-nitroso-terbutaline at a concentration of 10 ng / mL is shown. A clear and sharp peak is shown at 3.56 minutes on the chromatogram, which is exactly the characteristic peak of N-nitroso-terbutaline. The peak shape is symmetrical and the baseline separation is good, indicating that the selected chromatographic conditions are suitable for the analysis of this substance. This retention time is recorded in Table 1 as a precise positioning reference for the target impurity. Figure 3 The chromatogram of terbutaline sulfate inhalation solution (about 100 μg / mL) is shown. It can be observed from the chromatogram that there is no obvious chromatographic peak at the expected retention time of N-nitroso-terbutaline (3.56 minutes). This indicates that the content of N-nitroso-terbutaline in the tested sample is lower than the detection limit of the method or does not contain this impurity. This result also proves that the method can accurately reflect the impurity situation in the actual sample without being interfered by the main components of the sample. Figure 4 The chromatogram of the system suitability solution (containing about 100 μg / mL of terbutaline sulfate and about 10 ng / mL of N-nitroso-terbutaline) is shown. The characteristic peak of N-nitroso-terbutaline is shown at 3.63 minutes in the chromatogram. The appearance of this peak proves that even in a complex sample matrix, this method can still accurately detect the target impurity. The retention time of the peak in the system suitability solution (3.63 minutes) is slightly different from the retention time in the reference solution (3.56 minutes). This small difference is common in analytical chemistry and may be caused by the sample matrix effect, which does not affect the reliability of the analysis results.

[0162] Based on the above results, the following conclusions can be drawn: 1) The method has good specificity: The blank solution and the matrix of the test solution will not interfere with the detection of N-nitroso-terbutaline, and the target impurity can be accurately located and detected. 2) The separation effect is satisfactory: The peak shape of N-nitroso-terbutaline is good, without obvious tailing phenomenon, and is fully separated from other possible impurity peaks. 3) The reproducibility is stable: The retention time of N-nitroso-terbutaline in different solutions is stable, indicating good performance of the chromatographic system. 4) The detection sensitivity is high: A significant chromatographic peak is produced by N-nitroso-terbutaline at a concentration of 10 ng / mL in the reference solution, and the signal-to-noise ratio is high, indicating that the method has sufficient sensitivity to meet the requirements of trace analysis.

[0163] In summary, the specificity experiment results of Example 1 prove that the UPLC-MS / MS detection method of the present invention has good specificity, can specifically detect N-nitroso-terbutaline impurities in terbutaline sulfate nebulization solution, is not interfered by solvents and sample matrices, and meets the requirements of drug quality control. These results together prove that this method has good specificity and can specifically detect N-nitroso-terbutaline impurities in terbutaline sulfate nebulization solution, meeting the basic requirements of drug quality control methods.

[0164] Example 2 Quantitation limit and detection limit

[0165] 2-1 Solution preparation

[0166] Take an appropriate amount of N-nitroso-terbutaline impurity reference substance, weigh it accurately, and prepare a stock solution with an appropriate concentration. Through serial dilution, a series of solutions with concentration gradients are prepared. The concentration when the signal-to-noise ratio S / N≈3 is taken as the detection limit, and the concentration when S / N≈10 is taken as the quantitation limit. The concentration when the signal-to-noise ratio S / N≈3 is taken as the detection limit, and the concentration when S / N≈10 is taken as the quantitation limit.

[0167] 2-2 The chromatographic and mass spectrometric conditions are the same as those in Example 1.

[0168] 2-3 Conclusion:

[0169] Precisely measure the above solutions, inject them into the ultra-high performance liquid chromatography-mass spectrometry instrument, record the chromatograms, and the results are shown in Table 2.

[0170] Table 2 Test results of quantitation limit and detection limit

[0171]

[0172] Conclusion: This method shows extremely high sensitivity. The detection limit is 0.05 ng / mL, which is 0.5% of the limit (0.01%), and the signal-to-noise ratio is 4.6 at this time; the quantitation limit is 0.15 ng / mL, which is 1.5% of the limit, and the signal-to-noise ratio is 12.5 at this time. These results indicate that the detection sensitivity of this method is far better than the general requirements for drug impurity control (the quantitation limit generally requires to be lower than 10% of the limit), can meet the trace analysis requirements of N-nitroso-terbutaline impurities in terbutaline sulfate nebulization solution, and provides a reliable analysis means for drug safety evaluation.

[0173] Example 3 Linearity and range experiment

[0174] 3-1 Solution preparation

[0175] Based on the limit of quantitation determined in Example 2 (equivalent to 1.5% of the limit), an appropriate amount of N-nitroso-terbutaline impurity reference substance was accurately weighed and a series of concentration solutions were prepared to cover the range from the limit of quantitation (1.5%) to 200% of the limit for linear relationship evaluation.

[0176] The chromatographic and mass spectrometric conditions in 3-2 were the same as those in Example 1.

[0177] 3-3 Conclusion:

[0178] An accurate volume of the above solution was taken and injected into the ultra-high performance liquid chromatography-mass spectrometry instrument, and the chromatogram was recorded. The results are shown in Table 3. A linear regression equation was made with the concentration as the abscissa and the peak area as the ordinate.

[0179] Table 3 Linear experiment results of N-nitroso-terbutaline impurity

[0180] Component Linear equation r N-Nitroso-terbutaline y = 35181x + 2879.1 0.9998

[0181] Conclusion: In the concentration range from the limit of quantitation determined in Example 2 (0.15 ng / mL, equivalent to 1.5% of the limit) to 200% of the limit (20 ng / mL), an excellent linear relationship was shown between the peak area and concentration of N-nitroso-terbutaline impurity, and the correlation coefficient r = 0.9998. This result proves that the method has good linear response characteristics in a relatively wide concentration range, fully meets the requirements of the drug impurity analysis method (r > 0.99), can accurately quantify N-nitroso-terbutaline impurities at different concentration levels, and provides reliable analytical support for drug quality control.

[0182] Example 4 Accuracy experiment

[0183] Accuracy was investigated through a recovery test, and the research covered the limit concentration range of 50% - 150%.

[0184] 4-1 Solution preparation

[0185] N-nitroso-terbutaline impurity stock solution (100 μg / mL): An appropriate amount of N-nitroso-terbutaline impurity reference substance was taken, accurately weighed, dissolved and diluted to a solution of 100 μg / mL with 0.005 mol / L sulfuric acid water-methanol (85:15) solution.

[0186] N-nitroso-terbutaline impurity stock solution (0.25 μg / mL): 0.25 mL of the N-nitroso-terbutaline impurity stock solution (100 μg / mL) was accurately measured and placed in a 100 mL volumetric flask, diluted to the mark with 0.005 mol / L sulfuric acid water-methanol (85:15) solution, and shaken well.

[0187] Reference solution: Accurately measure 1 mL of the N-nitrosoterbutaline impurity stock solution (0.25 μg / mL), transfer it to a 25-mL volumetric flask, dilute it to the mark with a 0.005 mol / L sulfuric acid-water-methanol (85:15) solution, and mix well.

[0188] Accuracy solution: Accurately measure 1 mL of each of the terbutaline sulfate inhalation solution (2 mL: 5 mg), transfer them to 25-mL volumetric flasks respectively. Accurately measure 0.5 mL, 1.0 mL, and 1.5 mL of the N-nitrosoterbutaline impurity stock solution (100 ng / mL) respectively, dissolve and dilute them to the mark with the diluent, and mix well. Prepare 3 replicates in parallel for each spiked concentration level.

[0189] 4-2 The chromatographic and mass spectrometric conditions are the same as those in Example 1.

[0190] 4-3 Conclusion:

[0191] Accurately measure the above solutions, inject them into an ultra-high performance liquid chromatography-mass spectrometry instrument, record the chromatograms, and calculate by the external standard method based on the peak areas. The accuracy results are shown in Table 4.

[0192] Table 4 Accuracy test results

[0193]

[0194] Conclusion: The accuracy of this method was evaluated at three spiked levels of 50%, 100%, and 150% of the limit. The recovery rate range of the N-nitrosoterbutaline impurity was 98.9% - 105.6%, and the RSD value was 2.13%, fully meeting the acceptance criteria for drug impurity analysis methods (usually requiring the recovery rate to be between 85% and 115%). These results indicate that the method has excellent accuracy and precision, can accurately determine the N-nitrosoterbutaline impurity at different concentration levels in the terbutaline sulfate inhalation solution, and meets the requirements of drug quality control. It should be noted that in all test samples, the background amount was 0.000, indicating that the content of the N-nitrosoterbutaline impurity in the test sample was lower than the detection limit, which further proves the good specificity of the method.

[0195] Example 5 Precision experiment

[0196] 5-1 Solution preparation

[0197] Accurately measure 1 mL of the terbutaline sulfate inhalation solution, transfer it to a 25-mL volumetric flask, add 1.0 mL of the N-nitrosoterbutaline impurity stock solution (0.25 μg / mL), dissolve and dilute it to the mark with the diluent, and mix well. Prepare 6 replicates in parallel.

[0198] Reference solution: The same as in Example 4.

[0199] 5-2 The chromatographic and mass spectrometric conditions were the same as those in Example 1.

[0200] 5-3 Conclusion:

[0201] Accurately measure an appropriate amount of the above solution, inject it into an ultra-high performance liquid chromatography-mass spectrometry instrument, record the chromatogram, calculate the impurity content, and the precision results are shown in Table 5.

[0202] Table 5 Precision test results

[0203] Sample N-Nitroso-terbutaline (%) 1 0.010 2 0.010 3 0.010 4 0.010 5 0.009 6 0.010 Average (%) 0.010 RSD (%) 2.50

[0204] Conclusion: The test results of 6 replicated sample solutions prepared in parallel showed that the RSD value of the impurity content of N-nitroso-terbutaline was 2.50%, far lower than the 10% usually required for impurity analysis methods. This indicates that this method has excellent precision, can provide highly consistent measurement results, and meets the requirements of drug quality control for the reliability of analysis methods. The good repeatability of the method provides a strong guarantee for the accurate quantification of impurity content and ensures the credibility of measurement data. This result is consistent with the small RSD value (2.13%) observed in Example 4, further confirming the stability and reliability of the method.

[0205] Example 6 Solution stability experiment

[0206] 6-1 Solution preparation

[0207] Take the test solution and reference solution in Example 1 and inject them for detection at different time points.

[0208] 6-2 The chromatographic and mass spectrometric conditions were the same as those in Example 1.

[0209] 6-3 Conclusion:

[0210] Accurately measure an appropriate amount of the above solution, inject it into an ultra-high performance liquid chromatography-mass spectrometry instrument, record the chromatogram, and the solution stability results are shown in Table 6.

[0211] Table 6 Solution stability test results

[0212]

[0213] Conclusion: The test solution and the reference solution remain stable within 12 hours at room temperature. The RSD value of the area of N-nitroso-terbutaline in the reference solution was 2.70%, indicating that the impurity reference did not degrade significantly during the test. The N-nitroso-terbutaline impurity was not detected in the test solution at each time point, indicating that the content of this impurity in the original sample was extremely low (below the detection limit) on the one hand, and on the other hand, it also proved that no new N-nitroso-terbutaline was generated under the test conditions. These results confirmed that the solutions used in this method remained stable within the time required for the analysis process, ensuring the reliability and accuracy of the analysis results, and facilitating the application of this method for routine analysis in actual quality control.

[0214] Example 7 Robustness Test

[0215] Robustness refers to the tolerance of the determination result to be unaffected by minor changes in the chromatographic conditions. The spiked test solution (system suitability solution), test solution, and reference solution were prepared in the same manner as in Example 1. Based on the detection conditions of Example 1, the robustness of the detection method was determined under the conditions of changing temperature, flow rate, and the initial ratio of mobile phase B. The determination results are shown in Table 7.

[0216] Table 7 Results of Robustness Test

[0217]

[0218] Conclusion: When the chromatographic conditions were changed (column temperature ±5°C, flow rate ±0.05 mL / min, initial ratio of mobile phase B ±2%), the method performance remained stable. Under all test conditions, the blank solution and the test solution did not interfere with the determination of the N-nitroso-terbutaline impurity, and this impurity was not detected in the test sample. The recovery rate of the spiked test sample was in the range of 99.0% - 104.1%, fully meeting the requirements of method validation. The retention time of N-nitroso-terbutaline changed slightly under different conditions (3.31 - 3.82 minutes), but the change range was small and did not affect the accurate identification of the compound. These results proved that this method had good robustness, could adapt to minor fluctuations in chromatographic conditions in actual applications, ensured the reliability and consistency of the analysis results, and provided guarantee for the application of the method under different laboratory and instrument conditions.

[0219] In summary, the method for detecting N-nitroso-terbutaline impurity in terbutaline sulfate nebulization solution provided by the present invention using ultra-high performance liquid chromatography-tandem mass spectrometry has excellent comprehensive performance: the specificity test proves that the method can specifically identify the target impurity without being interfered by the sample matrix; the sensitivity evaluation shows that the detection limit and quantification limit reach 0.5% and 1.5% of the limit respectively, far superior to the conventional requirements; the linear range is from the quantification limit to 200% of the limit, and the correlation coefficient r = 0.9998; the recoveries at three concentration levels in the accuracy test are 98.9%-105.6%, and the RSD is only 2.13%; the RSD value in the precision test is 2.50%, far lower than the usually required 10%; the solution remains stable within 12 hours at room temperature; the robustness test proves that the method still maintains reliable performance under minor changes in chromatographic conditions. This highly sensitive, highly selective, fast (only 10 minutes) and easy-to-operate method provides strong support for the quality control and safety evaluation of terbutaline sulfate nebulization solution, effectively solving the challenges of detecting nitrosamine impurities in the prior art. Through systematic methodology verification, it is proved that this method meets the requirements of drug impurity analysis and is reliably applicable to the fields of drug R & D and quality control.

[0220] The above are only the preferred embodiments of the present application and are not used to limit the scope of the present application. Although the present application has been described in detail with reference to the above embodiments, those skilled in the art can still make various modifications or equivalent replacements of some technical features after reading this specification. Any modification, equivalent replacement or improvement made within the spirit and principle of the present application shall be regarded as falling within the protection scope of the present application.

Claims

1. A method for detecting nitrosamine impurities in terbutaline sulfate inhalation solution, characterized in that, It includes the following steps: After diluting the terbutaline sulfate solution for inhalation nebulization, a test solution is obtained; The test solution is detected by ultra-high performance liquid chromatography-tandem mass spectrometry; Among them, the chromatographic conditions are: the filler of the chromatographic column is octadecylsilane-bonded silica gel; mobile phase A is an aqueous ammonium acetate solution; mobile phase B is acetonitrile; The nitrosamine impurity is N-nitroso-terbutaline.

2. The detection method according to claim 1, characterized in that, The diluent for diluting the terbutaline sulfate solution for inhalation nebulization is a sulfuric acid-water-methanol solution; Preferably, in the diluent, the volume percentage of methanol is 10-30%, preferably 15%; Preferably, the diluent is a 0.005mol / L sulfuric acid-water-methanol solution, in which the volume ratio of the sulfuric acid aqueous solution to methanol is 85:15; Preferably, in the test solution, the concentration of terbutaline sulfate is 50-150μg / mL, preferably 100μg / mL; Preferably, in mobile phase A, the concentration of ammonium acetate is 2mmol / L.

3. The detection method according to claim 1, wherein The gradient elution program is: 0-3min, mobile phase A decreases from 85% to 70%, and mobile phase B increases from 15% to 30%; 3-6min, mobile phase A decreases from 70% to 40%, and mobile phase B increases from 30% to 60%; 6-8min, mobile phase A maintains 40%, and mobile phase B maintains 60%; 8-8.1min, mobile phase A increases from 40% to 85%, and mobile phase B decreases from 60% to 15%; 8.1-10min, mobile phase A maintains 85%, and mobile phase B maintains 15%, The above percentages are volume percentages.

4. The detection method according to claim 1, wherein The chromatographic conditions are: the chromatographic column uses octadecylsilane-bonded silica gel as the filler; the chromatographic column is HypersilGOLD, 100mm×2.1mm, 1.9μm; the flow rate is 0.4mL / min, the column temperature is 30℃, and the injection volume is 10μL.

5. The detection method according to claim 1, wherein, During the detection process, the mass spectrometry conditions are: the ion source is an electrospray ion source, the scanning mode is negative ion mode, and the detection mode is multiple reaction monitoring MRM; Preferably, the mass spectrometer is a triple quadrupole mass spectrometer, and after gradient elution, it is detected by a triple quadrupole mass spectrometer; Preferably, the mass spectrometry conditions are: the IS voltage is -4500V; the curtain gas CUR is 40psi; the nebulizer GAS1 is 50psi; the auxiliary gas GAS2 is 50psi; the ion source temperature is 500℃; the collision gas: 8; Q1 is 253.1Da, Q3 is 172.8Da, DP is -118V, and CE is -24V.

6. The method according to claim 1, wherein The detection limit of the method is equivalent to 0.5% of the impurity limit, and the quantitation limit is equivalent to 1.5% of the limit.

7. A method for determining the content of N-nitroso-terbutaline impurity in terbutaline sulfate inhalation solution, characterized in that, The method includes: Detecting the N-nitroso-terbutaline impurity according to the detection method described in any one of claims 1 to 6; Calculating the content of the N-nitroso-terbutaline by the external standard method.

8. A quality control method for terbutaline sulfate inhalation solution, characterized in that, The method includes: Detecting the content of the N-nitroso-terbutaline impurity in the terbutaline sulfate solution for inhalation nebulization by the detection method described in any one of claims 1 to 6; Comparing the detection result with a preset limit value to judge whether the quality of the terbutaline sulfate solution for inhalation nebulization is qualified; Preferably, the preset limit value is 0.01%.

9. Use of the detection method according to any one of claims 1 to 6 in quality control of terbutaline sulfate inhalation solution.

10. Use of the detection method according to any one of claims 1 to 6 in process control of terbutaline sulfate inhalation solution.

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