Method for detecting nitrosamine impurity in bulk drug prilocaine

Abstract

The invention relates to the technical field of drug detection, in particular to an exclusive, high-sensitivity, anti-interference and simple-operation quantitative detection method capable of simultaneously aiming at three isomeride nitrosamine impurities in a raw material drug prilocaine. According to the method disclosed by the invention, detection analysis is carried out by virtue of a liquid chromatograph-mass spectrometer, and quantitative detection of three nitrosamine impurities which are very close in structure and large in impurity limit requirement difference in the raw material medicine prilocaine is realized by screening liquid chromatography conditions, so that the quality controllability of the raw material medicine is improved, the carcinogenic risk can be reduced from the source, and the method is suitable for industrial production. The medication safety of a patient is ensured.

Description

Technical Field

[0001] This invention belongs to the field of drug detection technology and relates to a method for detecting nitrosamine impurities in the active pharmaceutical ingredient prilocaine. Specifically, it relates to a method for quantitatively detecting three isomers of nitrosamine impurities in the active pharmaceutical ingredient prilocaine using HPLC-MS. Background Technology

[0002] Prilocaine, chemically named N-(2-methylphenyl)-2-(propylamino)propionamide, is a local anesthetic characterized by rapid onset of action, low toxicity, and significant analgesic effect. It is widely used in clinical settings such as surface anesthesia, infiltration anesthesia, and spinal anesthesia. The quality of the active pharmaceutical ingredient (API) prilocaine directly affects the safety and efficacy of the formulation. Therefore, pharmacopoeias of various countries, such as the Chinese Pharmacopoeia, the European Pharmacopoeia, and the United States Pharmacopeia, have imposed strict control requirements on the purity, related substances, and residual solvents of the API prilocaine.

[0003] In recent years, nitrosamine impurities have received significant attention from global drug regulatory agencies due to their strong carcinogenicity. Nitrosamine compounds, such as N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), and N-nitroso-N-methylaniline (NMPA), are classified as Group 1 or Group 2A carcinogens by the International Agency for Research on Cancer (IARC). Even trace amounts of these impurities can pose a potential threat to human health. Studies have shown that the formation of nitrosamine impurities is closely related to the use of precursor substances in the raw material synthesis process, such as secondary amines, tertiary amines, nitrites, or nitrosating agents. The synthetic route of the active pharmaceutical ingredient prilocaine typically involves the reaction of aromatic amine compounds (such as p-toluidine derivatives) with acylation reagents. Improper process control, such as the introduction of secondary amine impurities from raw materials or nitrite contamination in the reaction system, can lead to the formation of nitrosamine impurities in the active pharmaceutical ingredient prilocaine, which may remain in the final product.

[0004] With increasing awareness of drug safety, global regulatory agencies have successively issued restrictions on nitrosamine impurities. For example, the US FDA issued guidance in 2018 requiring risk assessment and control of nitrosamine impurities for high-risk drugs such as sartans; the European Medicines Agency (EMA) subsequently expanded the scope to all active pharmaceutical ingredients containing amine structures, stipulating that the acceptable daily intake (AI) of nitrosamine impurities must be less than 26.5 ng / day (based on lifetime exposure risk); and the China National Medical Products Administration also clarified in the "Technical Guidelines for Research on Nitrosamine Impurities in Chemical Drugs" issued in 2020 that sensitive and accurate detection methods must be established to monitor the content of active pharmaceutical ingredients that may introduce nitrosamines.

[0005] Currently, the main methods for detecting nitrosamine impurities in drugs include gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS / MS), and high-performance liquid chromatography (HPLC). However, for the three isomers of nitrosamine impurities that may be generated in the active pharmaceutical ingredient prilocaine, existing detection methods still have the following limitations:

[0006] 1. Severe matrix interference: The active pharmaceutical ingredient prilocaine may contain unreacted starting materials (such as o-toluidine), intermediates (such as N-acetylopaine), or other process impurities (such as isomers). These components may co-elute with the target nitrosamine impurities under conventional chromatographic conditions, leading to false positives or inaccurate quantification.

[0007] 2. Insufficient Sensitivity: According to the FDA's Recommended Acceptable Intake Limits for Nitrosamine Drug Substance Related Impurities (NDSRIs), the acceptable daily intake (AI) for N2-nitrosopylocaine is 1500 ng / day, and the acceptable daily intake (AI) for N2-nitrosopylocaine impurity D and N2-nitrosopylocaine impurity E is 400 ng / day. Taking lidocaine-prilocaine aerosol as an example (maximum daily dose of 22.5 mg / day), the calculated quality control limit for N2-nitrosopylocaine in the prilocaine raw material is 66 ppm, and the quality control limits for N2-nitrosopylocaine impurity D and N2-nitrosopylocaine impurity E are 17 ppm. The three nitrosamine impurities in the prilocaine raw material have similar structures and significantly different limit requirements, making it difficult for existing methods to meet the detection requirements.

[0008] 3. Poor method universality: Different nitrosamine impurities have significant differences in physicochemical properties (such as boiling point and polarity), making it difficult for a single method to cover multiple impurities simultaneously. A multi-method combination scheme needs to be developed, increasing detection costs and time. Patent CN120446324A discloses a method for detecting nitrosamine compounds in the active pharmaceutical ingredient (API) of lidocaine cream. This method can simultaneously detect two nitrosamine impurities (lidocaine nitrosamine impurity and prilocaine nitrosamine impurity). However, the applicant found that this detection method is not applicable to the detection of the three isomers of prilocaine, the API of this invention. The smaller the chemical structural differences between the impurities to be detected, the higher the requirements for the specificity and sensitivity of the detection method.

[0009] Therefore, developing a specific, highly sensitive, interference-resistant, and easy-to-operate quantitative detection method for three isomers of nitrosamine impurities in the active pharmaceutical ingredient prilocaine is of great significance for ensuring the quality control of pharmaceutical products and meeting domestic and international regulatory requirements. Summary of the Invention

[0010] To overcome the shortcomings of the prior art, this invention provides a quantitative detection method for nitrosamine impurities in the active pharmaceutical ingredient prilocaine. This method, through high-performance liquid chromatography separation and mass spectrometry detection, can achieve efficient and accurate determination of three isomers of nitrosamine impurities in the active pharmaceutical ingredient prilocaine, thereby ensuring the safe and effective use of the final product.

[0011] The names and structural formulas of the three isomers of nitrosamine impurities in the active pharmaceutical ingredient prilocaine of this invention are as follows:

[0012]

[0013] To achieve the above objectives, this invention provides a method for detecting three isomers of nitrosamine impurities in the active pharmaceutical ingredient prilocaine, specifically comprising the following steps:

[0014] (1) Preparation of reference stock solutions: Accurately weigh N2-nitrosopyracocaine reference standard, N2-nitrosopyracocaine impurity D reference standard and N2-nitrosopyracocaine E reference standard, dissolve them in solvent to obtain N2-nitrosopyracocaine reference stock solution, N2-nitrosopyracocaine impurity D reference stock solution and N2-nitrosopyracocaine impurity E reference stock solution;

[0015] (2) Preparation of standard curve solutions: Accurately measure the reference stock solution from step (1), dilute with solvent to obtain N2-nitrosopyracocaine standard curve solution, N2-nitrosopyracocaine impurity D standard curve solution and N2-nitrosopyracocaine impurity E standard curve solution.

[0016] (3) Preparation of test solution: Take an appropriate amount of test sample, weigh it accurately, add solvent to dissolve it, and the solution is obtained.

[0017] (4) Detection and analysis: Accurately measure the N2-nitrosopyracocaine standard curve solution, the N2-nitrosopyracocaine impurity D standard curve solution, the N2-nitrosopyracocaine impurity E standard curve solution and the test solution, and inject them into the liquid chromatography-mass spectrometry instrument respectively, and record the mass spectrum;

[0018] The liquid chromatography conditions were as follows: octadecylsilane-bonded silica gel was used as the stationary phase; mobile phase A was 0.05% formic acid solution, and mobile phase B was acetonitrile.

[0019] The standard curve solution and the test sample solution described in this invention are prepared fresh before use.

[0020] In one specific embodiment of the present invention, the volume ratio of mobile phase A to mobile phase B in the liquid chromatography conditions is 18:82 to 22:78; preferably, the volume ratio of mobile phase A to mobile phase B is 20:80.

[0021] In one specific embodiment of the present invention, the flow rate of the mobile phase under the liquid chromatography conditions is 0.3 to 0.5 ml / min; preferably, the flow rate of the mobile phase is 0.4 ml / min.

[0022] In one specific embodiment of the present invention, the liquid chromatography employs isocratic elution.

[0023] In a specific embodiment of the present invention, in the detection method, the solvent in steps (1), (2) and (3) is selected from one or more of methanol, acetonitrile, and acetone; preferably, the solvent is selected from methanol.

[0024] In a specific embodiment of the present invention, in the detection method, each 1 ml of N2-nitrosopyracocaine reference stock solution in step (1) contains 100 μg of N2-nitrosopyracocaine; each 1 ml of N2-nitrosopyracocaine impurity D reference stock solution contains 100 μg of N2-nitrosopyracocaine impurity D; and each 1 ml of N2-nitrosopyracocaine impurity E reference stock solution contains 100 μg of N2-nitrosopyracocaine impurity E.

[0025] In the detection method of the present invention, the mass spectrometry conditions of the liquid chromatography-mass spectrometry (LC-MS) instrument in step (4) are as follows:

[0026] The ion source was ESI+; the cone gas pressure was 25 psi; the nebulizer gas pressure was 50 psi; the transfer tube temperature was 500℃; the mass spectrometry acquisition time was 6.5–11 min; the scanning mode was MRM; and the ion mode was negative ion.

[0027] The detection method of the present invention firstly achieves effective separation of three isomer nitrosamine impurities in the active pharmaceutical ingredient prilocaine using liquid chromatography-mass spectrometry (LC-MS), and then detects the substances separated by liquid chromatography using mass spectrometry, thereby obtaining the content of the three nitrosamine impurities in the active pharmaceutical ingredient prilocaine.

[0028] The detection method of the present invention also includes plotting a standard curve and calculating a regression equation based on the concentration and the corresponding peak area; if there is a peak in the mass spectrum of the test sample solution that is consistent with the retention time of the three nitrosamine impurities, the impurity concentration is calculated according to the standard curve method.

[0029] In one specific embodiment of the present invention, the method for obtaining the standard curve includes:

[0030] (1) Preparation of standard samples: The three isomers of nitrosamine impurities were diluted with the solvent to prepare standard samples of different concentrations.

[0031] (2) Detection and analysis: Accurately measure the standard samples of different concentrations of the three isomers of nitrosamine impurities in step (1), inject them into the liquid chromatography-mass spectrometry instrument, and record the mass spectrum; calculate the regression equation based on the concentration and the corresponding peak area to obtain the standard curve.

[0032] In the method for obtaining the standard curve described in this invention, in step (2), the chromatographic / mass spectrometric conditions of the liquid chromatography-mass spectrometry (LC-MS) instrument are as follows:

[0033] Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase; the flow rate was selected from 0.3 to 0.5 ml / min; mobile phase A was 0.05% formic acid solution, and mobile phase B was acetonitrile; isocratic elution was performed; wherein the volume ratio of mobile phase A to mobile phase B was 18:82 to 22:78, preferably 20:80.

[0034] Mass spectrometry conditions: ESI+ as the ion source; cone gas pressure 25 psi; nebulizer gas pressure 50 psi; transfer tube temperature 500℃; mass spectrometry acquisition time 6.5–11 min; scanning mode MRM; ion mode negative ion.

[0035] In the method for obtaining the standard curve of the present invention, the solvent in step (1) is selected from one or more of methanol, acetonitrile, and acetone; preferably, the solvent is selected from methanol.

[0036] In a specific embodiment of the present invention, in step (1) of the method for obtaining the standard curve, the concentration range of the standard samples of different concentrations is as follows: each 1 ml of N2-nitrosopropylcaine standard sample contains 66 ng, 330 ng, 660 ng, 990 ng and 1320 ng of N2-nitrosopropylcaine; each 1 ml of N2-nitrosopropylcaine impurity D contains 17 ng, 85 ng, 170 ng, 255 ng and 340 ng of N2-nitrosopropylcaine impurity D; each 1 ml of N2-nitrosopropylcaine impurity E contains 17 ng, 85 ng, 170 ng, 255 ng and 340 ng of N2-nitrosopropylcaine impurity E.

[0037] In the detection method of the present invention, the formula for calculating the content of nitrosamine impurities in the active pharmaceutical ingredient prilocaine is as follows:

[0038] Impurity content (ng / mg) = C × V / M 粉末

[0039] in,

[0040] C represents the corresponding impurity concentration (ng / ml) calculated from the standard curve;

[0041] V represents the dilution volume (ml) of the test sample;

[0042] M 粉末 Indicates the sample weight (mg).

[0043] Compared with the prior art, the detection method of the present invention has the following beneficial effects:

[0044] (1) The detection method of the present invention can quantitatively detect three isomers of nitrosamine impurities in the active pharmaceutical ingredient prilocaine that have very similar structures and different impurity limit requirements, thereby improving the quality controllability of the active pharmaceutical ingredient.

[0045] (2) The present invention can reduce the risk of carcinogenesis from the source, improve the safety of the preparation, and ensure the safety of patients' medication by detecting the three isomers of nitrosamine impurities in prilocaine aerosol. The detection method of the present invention is applicable to the detection of the three isomers of nitrosamine impurities in prilocaine raw material used in lidocaine prilocaine aerosol. It is also applicable to the detection of the three isomers of nitrosamine impurities in prilocaine raw material used in other preparations where the maximum daily dose of prilocaine is less than the maximum daily dose of prilocaine in the above-mentioned aerosol.

[0046] (3) The detection method of the present invention can simultaneously detect the three isomers of nitrosamine impurities of prilocaine, reducing detection costs and time consumption. The development of the detection method of the present invention is of great significance. Attached Figure Description

[0047] Figure 1 The standard curve for N2-nitrosopyroxycaine;

[0048] Figure 2 Standard curve for N2-nitrosopyropropylcaine impurity D;

[0049] Figure 3 The standard curve for N2-nitrosopyramic acid impurity E; Detailed Implementation

[0050] The present invention will now be clearly and completely described with reference to the embodiments. Those skilled in the art should understand that the embodiments described herein are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0051] Screening of liquid chromatography conditions in experimental cases

[0052] 1. To ensure the effective separation of the three isomers of nitrosamine impurities in the active pharmaceutical ingredient prilocaine, the applicant first screened the mobile phase system for liquid chromatography.

[0053] Other chromatographic conditions: octadecylsilane-bonded silica gel as the stationary phase; flow rate of 0.4 ml / min; isocratic elution.

[0054]

[0055] The applicant investigated the effects of three different mobile phase systems on the separation of three isomers of nitrosamine impurities in the active pharmaceutical ingredient prilocaine. The specific results are shown in Table 1.

[0056] Table 1 Results of investigation on different mobile phase systems

[0057]

[0058] As shown in Table 1, under conditions 1 and 2, N2-nitrosopyracocaine impurity D and N2-nitrosopyracocaine impurity E cannot be effectively separated; under condition 3, the three impurities can be effectively separated. Therefore, the liquid chromatography mobile phase system in the detection method of this invention is: 0.05% formic acid solution-acetonitrile.

[0059] 2. In the detection method of the present invention, the liquid chromatography uses 0.05% formic acid solution as mobile phase A and acetonitrile as mobile phase B; furthermore, the applicant has also investigated the volume ratio of mobile phase A and mobile phase B, as well as the flow rate.

[0060] Other chromatographic conditions: Octadecylsilane-bonded silica gel as the stationary phase; isocratic elution.

[0061]

[0062] The applicant investigated the effects of different mobile phase ratios and flow rates on the separation of three isomers of nitrosamine impurities in the active pharmaceutical ingredient prilocaine. Specific results are shown in Tables 2 and 3.

[0063] Table 2 Results under different mobile phase ratios

[0064]

[0065] Table 3 Results under different flow velocities

[0066]

[0067]

[0068] NA indicates that the two segments were not separated.

[0069] As shown in Table 2, under a flow rate of 0.4 ml / min, when the proportion of mobile phase A is less than 18% (Example 2) or greater than 22% (Example 5), neither N2-nitrosopyracocaine impurity D nor N2-nitrosopyracocaine impurity E can be effectively separated. When the proportion of mobile phase A is set in the range of 18% to 22%, the three impurities can be effectively separated. Moreover, the separation effect is best when the proportion of mobile phase A is 20%.

[0070] As shown in Table 3, when the ratio of mobile phase A to mobile phase B is 20:80, neither N2-nitrosopyracocaine impurity D nor N2-nitrosopyracocaine impurity E can be effectively separated when the flow rate is below 0.3 ml / min (Example 6) or above 0.5 ml / min (Example 9). When the flow rate is in the range of 0.3 to 0.5 ml / min, the three impurities can be effectively separated. Moreover, the separation effect is best when the flow rate is 0.4 ml / min.

[0071] In summary, in the liquid chromatography conditions of the present invention, the volume ratio of mobile phase A to mobile phase B is selected from 18:82 to 22:78, preferably 20:80; the flow rate is selected from 0.3 to 0.5 ml / min, preferably 0.4 ml / min. These chromatographic conditions can effectively separate the three isomers of nitrosamine impurities in the active pharmaceutical ingredient prilocaine.

[0072] Example 1: Detection of nitrosamine impurities in prilocaine raw material 1. Detection conditions

[0073] Instrument: Liquid Chromatography-Mass Spectrometry

[0074] Liquid chromatography conditions:

[0075] The column was filled with octadecylsilane-bonded silica gel (YMC-Triart C18 4.0mm×250mm, 3μm recommended); the mobile phase was 0.05% formic acid solution-acetonitrile (volume ratio 20:80); the flow rate was 0.4ml / min; the column temperature was 35℃; and the injection volume was 2μl.

[0076] Mass spectrometry conditions:

[0077] The ion source was ESI+; the cone gas pressure was 25 psi; the nebulizer gas pressure was 50 psi; the transfer tube temperature was 500℃; the mass spectrometry acquisition time was 6.5–11 min; the scanning mode was MRM; and the ion mode was negative ion.

[0078] 2. Testing steps:

[0079] (1) Preparation of reference stock solutions: Accurately weigh appropriate amounts of N2-nitrosopyracocaine reference standard, N2-nitrosopyracocaine impurity D reference standard and N2-nitrosopyracocaine impurity E reference standard, dissolve and dilute with methanol, and prepare N2-nitrosopyracocaine reference stock solution with N2-nitrosopyracocaine content of 100 μg / ml; N2-nitrosopyracocaine impurity D reference stock solution with N2-nitrosopyracocaine impurity D content of 100 μg / ml; N2-nitrosopyracocaine impurity E reference stock solution with N2-nitrosopyracocaine impurity E content of 100 μg / ml;

[0080] (2) Preparation of standard curve solutions: Accurately measure 200 μl, 1000 μl, 2000 μl, 3000 μl and 4000 μl of the reference stock solutions of the three impurities in step (1), place them in different 20 ml volumetric flasks, dilute with methanol to the mark, shake well, and use them as N2-nitrosopyracocaine standard curve solutions 1-5, N2-nitrosopyracocaine impurity D standard curve solutions 1-5 and N2-nitrosopyracocaine impurity E standard curve solutions 1-5;

[0081] (3) Preparation of sensitivity solutions: Accurately measure 5 ml of the standard curve solution 1 of the three impurities in step (2) and place them in different 10 ml volumetric flasks. Dilute with methanol and shake well to obtain N2-nitrosopyracocaine sensitivity solution, N2-nitrosopyracocaine impurity D sensitivity solution and N2-nitrosopyracocaine impurity E sensitivity solution.

[0082] (4) Preparation of test solution: Weigh about 20 mg of test sample accurately, add 2 ml of methanol, and vortex to dissolve.

[0083] (5) Detection and analysis: Accurately measure 2 μl of N2-nitrosopyracocaine standard curve solutions 1-5, N2-nitrosopyracocaine impurity D standard curve solutions 1-5, N2-nitrosopyracocaine impurity E standard curve solutions 1-5, N2-nitrosopyracocaine sensitivity solutions, N2-nitrosopyracocaine impurity D sensitivity solutions, N2-nitrosopyracocaine impurity E sensitivity solutions and the test sample solution, respectively, and inject them into the liquid chromatography-mass spectrometry instrument to record the mass spectra;

[0084] The specific test results are shown in Table 4.

[0085] Table 4 shows the test results for the three batches.

[0086]

[0087] Example 2: Linearity and Range Examination

[0088] (1) Preparation of reference stock solutions: Accurately weigh appropriate amounts of N2-nitrosopyracocaine reference standard, N2-nitrosopyracocaine impurity D reference standard and N2-nitrosopyracocaine impurity E reference standard, dissolve and dilute with methanol, and prepare N2-nitrosopyracocaine reference stock solution with N2-nitrosopyracocaine content of 100 μg / ml; N2-nitrosopyracocaine impurity D reference stock solution with N2-nitrosopyracocaine impurity D content of 100 μg / ml; N2-nitrosopyracocaine impurity E reference stock solution with N2-nitrosopyracocaine impurity E content of 100 μg / ml;

[0089] (2) Preparation of linear stock solutions: Accurately measure 1320 μl of N2-nitrosopyracocaine reference stock solution, 340 μl of N2-nitrosopyracocaine impurity D reference stock solution and 340 μl of N2-nitrosopyracocaine impurity E reference stock solution, place them in 20 ml volumetric flasks, dilute with methanol to the mark, and shake well to obtain linear stock solutions for the three impurities;

[0090] (3) Preparation of linear and range solutions: Accurately measure 200 μl, 1000 μl, 2000 μl, 3000 μl and 4000 μl of the linear stock solution of the three impurities, put them into 20 ml volumetric flasks, dilute with methanol to the mark, shake well, and use as linear and range solutions 1 to 5 of the three impurities.

[0091] (4) Detection and analysis: 2 μl of linear and range solutions 1-5 of the three impurities were accurately measured and injected into the liquid chromatograph-mass spectrometer. The mass spectra were recorded, and linear regression was performed with concentration as the abscissa and the corresponding peak area as the ordinate to plot the standard curve.

[0092] The results of the linearity and range tests are shown in Table 5, and the corresponding standard curves are attached. Figure 1 -Appendix Figure 3 .

[0093] Table 5 Results of Linearity and Range Examination

[0094]

[0095] Based on the above findings, we can conclude that:

[0096] N2-nitrosopyroxycaine showed a good linear relationship between concentration and peak area in the range of 66 ng / ml to 1320 ng / ml, with the linear regression equation being y = 518x + 2.04e 3 The correlation coefficient r = 1.0000; for N2-nitrosopyroxycaine impurity D in the range of 17 ng / ml to 340 ng / ml, the concentration and corresponding peak area showed a linear relationship, with the linear regression equation being y = 1.2e. 3x+341, linear correlation coefficient r=1.0000; N2-nitrosopyroxycaine impurity E in the range of 17ng / ml~340ng / ml, concentration and corresponding peak area show a linear relationship, the linear regression equation is y=1.15e 3 x+915, linear correlation coefficient r=0.9999; indicating that the test method of the present invention has a good linear relationship.

[0097] Example 3: Investigation of detection limit and quantitation limit

[0098] (1) Preparation of reference stock solutions: Accurately weigh appropriate amounts of N2-nitrosopyracocaine reference standard, N2-nitrosopyracocaine impurity D reference standard and N2-nitrosopyracocaine impurity E reference standard, dissolve and dilute with methanol, and prepare N2-nitrosopyracocaine reference stock solution with N2-nitrosopyracocaine content of 100 μg / ml; N2-nitrosopyracocaine impurity D reference stock solution with N2-nitrosopyracocaine impurity D content of 100 μg / ml; N2-nitrosopyracocaine impurity E reference stock solution with N2-nitrosopyracocaine impurity E content of 100 μg / ml;

[0099] (2) Preparation of linear stock solutions: Accurately measure 1320 μl of N2-nitrosopyracocaine reference stock solution, 340 μl of N2-nitrosopyracocaine impurity D reference stock solution and 340 μl of N2-nitrosopyracocaine impurity E reference stock solution, place them in 20 ml volumetric flasks, dilute with methanol to the mark, and shake well to obtain linear stock solutions for the three impurities;

[0100] (3) Preparation of linear and range solutions: Accurately measure 200 μl, 1000 μl, 2000 μl, 3000 μl and 4000 μl of the linear stock solution of the three impurities respectively, put them into different 20 ml volumetric flasks, add methanol to dilute to the mark, shake well, and use them as linear and range solutions 1 to 5 for the three impurities.

[0101] (4) Preparation of solutions with quantitative limits: i.e. linear and range solutions 1.

[0102] (5) Preparation of detection limit solutions: Accurately measure 5 ml of linear and range solutions 1 for the three impurities respectively, place them in a 10 ml volumetric flask, dilute with methanol to the mark, shake well, and obtain detection limit solutions for the three impurities.

[0103] (6) Detection and analysis: Accurately measure 2 μl of the limit of quantitation solution and limit of detection solution of the three impurities, inject them into the liquid chromatograph-mass spectrometer respectively, and record the mass spectrum.

[0104] The results of the investigation of the limit of detection and limit of quantitation are shown in Tables 6 and 7:

[0105] Table 6 Results of Limit of Quantitation Test

[0106]

[0107] Table 7 Results of the detection limit test

[0108]

[0109]

[0110] According to the results of the investigation in Tables 6 and 7:

[0111] (1) The limit of quantitation (LOQ) of N2-nitrosopyroxycaine is 66 ng / ml, which is equivalent to 6.6 ppm of the test sample, accounting for 10% of the limit. The minimum signal-to-noise ratio (SNR) is 54.6, which is greater than 10 and meets the standard requirements. The limit of quantitation (LOQ) of impurity D in N2-nitrosopyroxycaine is 17 ng / ml, which is equivalent to 1.7 ppm of the test sample, accounting for 10% of the limit. The minimum SNR is 26.5, which is greater than 10 and meets the standard requirements. The limit of quantitation (LOQ) of impurity E in N2-nitrosopyroxycaine is 17 ng / ml, which is equivalent to 1.7 ppm of the test sample, accounting for 10% of the limit. The minimum SNR is 26.1, which is greater than 10 and meets the standard requirements.

[0112] (2) The detection limits of the three impurities are all no higher than 5% of the limit and the quantitation limits are all no higher than 10% of the limit. This indicates that the three impurities in the active pharmaceutical ingredient prilocaine can be detected when the concentration reaches 5% of the limit concentration. Moreover, the concentration of the three impurities in this invention is much lower than the control threshold of the impurities, indicating that the detection method of this method has high sensitivity.

[0113] The limits here refer to the quality control limits for nitrosamine impurities in the prilocaine raw material used in lidocaine prilocaine aerosols. Those skilled in the art will readily understand that the detection method of this invention is also applicable to the detection of the three nitrosamine impurities in the prilocaine raw material used in other formulations with a maximum daily dose of prilocaine that is lower than the maximum daily dose of prilocaine in the above-mentioned aerosols.

[0114] Example 4 Recovery rate investigation

[0115] (1) Preparation of reference stock solutions: Accurately weigh appropriate amounts of N2-nitrosopyracocaine reference standard, N2-nitrosopyracocaine impurity D reference standard and N2-nitrosopyracocaine impurity E reference standard, dissolve and dilute with methanol, and prepare N2-nitrosopyracocaine reference stock solution with N2-nitrosopyracocaine content of 100 μg / ml; N2-nitrosopyracocaine impurity D reference stock solution with N2-nitrosopyracocaine impurity D content of 100 μg / ml; N2-nitrosopyracocaine impurity E reference stock solution with N2-nitrosopyracocaine impurity E content of 100 μg / ml;

[0116] (2) Preparation of linear stock solutions: Accurately measure 1320 μl of N2-nitrosopyracocaine reference stock solution, 340 μl of N2-nitrosopyracocaine impurity D reference stock solution and 340 μl of N2-nitrosopyracocaine impurity E reference stock solution, place them in 20 ml volumetric flasks, dilute with methanol to the mark, and shake well to obtain linear stock solutions for the three impurities;

[0117] (3) Preparation of linear and range solutions: Accurately measure 200 μl, 1000 μl, 2000 μl, 3000 μl and 4000 μl of the linear stock solution of the three impurities respectively, put them into different 20 ml volumetric flasks, add methanol to dilute to the mark, shake well, and use them as linear and range solutions 1 to 5 for the three impurities.

[0118] (4) Preparation of N2-nitrosopyracocaine recovery spiking solution:

[0119] Preparation of the quantitation limit spiked test solution: Weigh approximately 20 mg of the active pharmaceutical ingredient prilocaine accurately, add 2 ml of N2-nitrosopyprilocaine linear and range solution 1, and vortex to dissolve.

[0120] Preparation of 100% spiked test solution: Weigh approximately 20 mg of the active pharmaceutical ingredient prilocaine accurately, add 2 ml of N2-nitrosopyprilocaine linear and range solution 3, and vortex to dissolve.

[0121] Preparation of 150% spiked test solution: Accurately weigh approximately 20 mg of the active pharmaceutical ingredient prilocaine, add 2 ml of N2-nitrosopyprilocaine linear and range solution 4, and vortex to dissolve. Prepare 3 parallel solutions using the same method.

[0122] (5) Preparation of spiked solution for the recovery of N2-nitrosopyracocaine impurity D:

[0123] Preparation of 100% spiked test solution: Weigh approximately 20 mg of the active pharmaceutical ingredient prilocaine accurately, add 2 ml of N2-nitrosopylocaine impurity D linearity and range solution 1, and vortex to dissolve. Prepare 6 parallel solutions using the same method.

[0124] (6) Preparation of spiked solution for the recovery of N2-nitrosopyracocaine impurity E:

[0125] Preparation of 100% spiked test solution: Weigh approximately 20 mg of the active pharmaceutical ingredient prilocaine accurately, add 2 ml of N2-nitrosopylocaine impurity E linearity and range solution 1, and vortex to dissolve. Prepare 6 parallel solutions using the same method.

[0126] (7) Test solution: Weigh approximately 20 mg of the test sample accurately, add 2 ml of methanol, and vortex to dissolve.

[0127] (8) Detection and analysis: Accurately measure 2 μl of the recovery rate spiked solution and the test solution of the three impurities respectively, inject them into the liquid chromatograph-mass spectrometer, and record the mass spectrum.

[0128] The recovery rate results are shown in Tables 8 and 9.

[0129] Table 8 Recovery Rate Test Results - N2-Nitropropylcaine

[0130]

[0131] Table 9. Recovery Test Results - N2-nitrosopyracocaine Impurity D, N2-nitrosopyracocaine Impurity E

[0132]

[0133]

[0134] According to the recovery rate results in Tables 8 and 9:

[0135] The recoveries of N2-nitrosopyroxycaine at various concentrations were within the range of 94.7% to 98.9%, which is within the standard range of 70% to 130%, and the RSD of the recoveries was 1.5%, less than 20.0%, which meets the standard requirements. The recoveries of N2-nitrosopyroxycaine impurity D in the 100% spiked test solution were within the range of 100.15% to 102.08%, which is within the standard range of 70% to 130%, and the RSD of the recoveries was 0.9%, less than 20.0%, which meets the standard requirements. The recoveries of N2-nitrosopyroxycaine impurity E in the 100% spiked test solution were within the range of 98.22% to 101.07%, which is within the standard range of 70% to 130%, and the RSD of the recoveries was 1.1%, less than 20.0%, which meets the standard requirements. This indicates that the detection method of the present invention complies with the recovery rate requirements of the Chinese Pharmacopoeia.

[0136] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for detecting three isomeric nitrosamine impurities in a raw drug propylcaine, comprising the following steps: (1) Preparation of reference substance stock solution: precisely weigh N2-nitrosopropylcaine reference substance, N2-nitrosopropylcaine impurity D reference substance and N2-nitrosopropylcaine impurity E reference substance, respectively, dissolve them in solvent to obtain N2-nitrosopropylcaine reference substance stock solution, N2-nitrosopropylcaine impurity D reference substance stock solution and N2-nitrosopropylcaine impurity E reference substance stock solution; (2) Preparation of standard curve solution: precisely measure the reference substance stock solution of step (1), dilute them in solvent to obtain N2-nitrosopropylcaine standard curve solution, N2-nitrosopropylcaine impurity D standard curve solution and N2-nitrosopropylcaine impurity E standard curve solution; (3) Preparation of test sample solution: take a proper amount of test sample, precisely weigh and dissolve it in solvent; (4) Detection and analysis: precisely measure N2-nitrosopropylcaine standard curve solution, N2-nitrosopropylcaine impurity D standard curve solution, N2-nitrosopropylcaine impurity E standard curve solution and test sample solution, inject them into liquid chromatography-mass spectrometry respectively, and record mass spectrum; wherein the liquid chromatography conditions are as follows: octadecylsilane bonded silica gel as filler; mobile phase A is 0.05% formic acid solution, and mobile phase B is acetonitrile. In the liquid chromatography conditions, the volume ratio of mobile phase A to mobile phase B is 18:82-22:78; preferably, the volume ratio of mobile phase A to mobile phase B is 20:

80. In the liquid chromatography conditions, the flow rate of mobile phase is 0.3-0.5 ml / min; preferably, the flow rate of mobile phase is 0.4 ml / min. The liquid chromatography adopts isocratic elution. The solvent in step (1), step (2) and step (3) is selected from one or more of methanol, acetonitrile, acetone; preferably, the solvent is methanol. In step (4), the mass spectrometry conditions of liquid chromatography-mass spectrometry are as follows: ESI+ as ion source; cone gas pressure is 25 psi; atomization gas pressure is 50 psi; transmission tube temperature is 500℃; mass spectrum collection time is 6.5-11 min; scanning mode is MRM; ion mode is negative ion.

2. The detection method according to claim 1, characterized in that, The detection method further comprises drawing a standard curve.

3. The method of claim 1, wherein, The method for obtaining the standard curve comprises:

4. The method of claim 1, wherein, (1) Preparation of standard sample: dilute the three isomeric nitrosamine impurities into standard samples with different concentrations using the solvent; the solvent is selected from one or more of methanol, acetonitrile, acetone; 5. The method of claim 1, wherein (2) Detection and analysis: precisely measure the standard samples with different concentrations of the three isomeric nitrosamine impurities of step (1), inject them into liquid chromatography-mass spectrometry respectively, and record mass spectrum; calculate the regression equation with concentration and corresponding peak area to obtain the standard curve; 6. The method of claim 1, wherein wherein the chromatography / mass spectrometry conditions of liquid chromatography-mass spectrometry are as follows: ​ 7. The method of claim 1, wherein, ​ 8. The detection method according to claim 7, characterized in that, ​ ​ ​ ​ Chromatography conditions: octadecylsilane-bonded silica gel as the filler; flow rate of 0.3-0.5 ml / min; mobile phase A is 0.05% formic acid solution, mobile phase B is acetonitrile; isocratic elution; the volume ratio of the mobile phase A to the mobile phase B is 18:82-22:78; Mass spectrometry conditions: ESI+as the ion source; cone hole gas pressure is 25 psi; atomization gas pressure is 50 psi; transmission tube temperature is 500 DEG C; mass spectrometry collection time is 6.5-11 min; scanning mode is MRM; ion mode is negative ion.

9. The method of claim 1, wherein, The content calculation formula of nitrosamine impurities in the raw drug prilocaine is as follows: Impurity content (ng / mg) = C x V / M 粉末 Wherein, C represents the corresponding impurity concentration (ng / ml) calculated according to the standard curve of claim 7; V represents the dilution volume (ml) of the test sample; M 粉末 represents the sample weight (mg) of the test sample.

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