Method for detecting nitrosamine impurities in terbinafine hydrochloride spray

Abstract

The application belongs to the technical field of drug detection, and particularly relates to a detection method of nitrosamine impurities in terbinafine hydrochloride spray. The application adopts liquid chromatography-mass spectrometry to detect test sample solution and control sample solution; chromatographic conditions of the liquid chromatography are as follows: a phenyl-hexyl silane bonded silica gel chromatographic column is used, a 0.1% formic acid aqueous solution is used as mobile phase A, a 0.1% formic acid methanol solution is used as mobile phase B, and gradient elution is adopted. Mass spectrometry conditions are as follows: an APCI source is used, positive ion mode detection is adopted, the acquisition mode is MRM, the fragmentation voltage is 60-80 V, the collision energy is 10-20 eV, the EMV voltage is 0-100 V, and suitable parent ions and daughter ions are selected according to nitrosamine impurities. The application solves the problems of poor specificity, low sensitivity and unqualified recovery rate of the detection of nitrosamine impurities in terbinafine hydrochloride spray by systematically optimizing pretreatment methods and chromatographic and mass spectrometry parameters, and provides a reliable technical means for product quality control.

Description

Technical Field

[0001] This invention belongs to the field of drug detection technology, specifically relating to a method for detecting nitrosamine impurities in terbinafine hydrochloride spray. Background Technology

[0002] Terbinafine hydrochloride spray is a topical antifungal medication for dermatology. Its main ingredient is terbinafine hydrochloride, and excipients include ethanol, 1,2-propanediol, and polycetol 1000. It works by inhibiting fungal squalene epoxidase, disrupting fungal cell membrane synthesis, and is used to treat fungal infections such as tinea manuum, tinea pedis, and tinea corporis.

[0003] Nitrosamine impurities are generally classified into two categories: small-molecule nitrosamines, represented by N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA), and nitrosamines introduced due to the presence of secondary amine structures in drug molecules (or impurities related to drug molecules). The European Medicines Agency (EMA) list of nitrosamine impurities includes three types related to terbinafine hydrochloride: N-nitrosodemethylterbinafine (NHNA), N-nitrosoterbinafine impurity A (NMNA), and N-nitrosoterbinafine degradation product (NHMA). The maximum daily intake (AI) for these three nitrosamine impurities is 18 ng / day. Based on a maximum daily dose of 5 ml for terbinafine hydrochloride spray, the limit for each of the three nitrosamine impurities is 0.36 ppm.

[0004] However, due to the unique formulation and excipient composition of terbinafine hydrochloride spray, a series of problems arise when detecting the above three nitrosamine impurities with extremely low limits:

[0005] (1) Difficult to separate by chromatography: All three nitrosamine impurities are impurities introduced by the secondary amine structure in the terbinafine molecule. They are similar in structure to terbinafine and are difficult to separate by conventional chromatographic methods.

[0006] (2) Low sensitivity: The limits for the three nitrosamine impurities are extremely low, and the sensitivity cannot meet the requirements by using conventional operations such as diluting the sample to prepare the test solution.

[0007] (3) Low recovery rate: Polycetol 1000 in the excipients is a surfactant, which significantly reduces the ionization efficiency of nitrosamine impurities, resulting in a low recovery rate.

[0008] Currently, there are no reports on methods for detecting nitrosamine impurities in terbinafine hydrochloride spray. Therefore, developing a method for detecting nitrosamine impurities in terbinafine hydrochloride spray to achieve accurate quantification of nitrosamine impurities is of great significance for the quality control of terbinafine hydrochloride spray. Summary of the Invention

[0009] The purpose of this invention is to provide a method for accurately quantifying nitrosamine impurities in terbinafine hydrochloride spray. The method has high sensitivity, strong specificity, good reproducibility, and good recovery rate.

[0010] The method for detecting nitrosamine impurities in terbinafine hydrochloride spray described in this application uses liquid chromatography-mass spectrometry to detect the test solution and the reference solution.

[0011] The chromatographic conditions for the liquid chromatography are as follows: a phenyl-hexylsilane bonded silica column is used, with a mobile phase A of 0.1% formic acid aqueous solution and a mobile phase B of 0.1% formic acid methanol solution, and gradient elution is performed.

[0012] The mass spectrometry conditions are as follows: APCI source, positive ion mode detection, MRM acquisition mode, fragmentation voltage 60-80V, collision energy 10-20eV, EMV voltage 0-100V, and appropriate parent and daughter ions are selected according to the nitrosamine impurities.

[0013] The nitrosamine impurity is N-nitrosodemethylterbinafine or N-nitrosoterbinafine impurity A or N-nitrosoterbinafine degradation product.

[0014] The precursor ion of N-nitrosodemethylterbinafine is 307.2, and the daughter ion is 141.1; the precursor ion of N-nitrosoterbinafine impurity A is 201.1, and the daughter ion is 141.1; the precursor ion of N-nitrosoterbinafine degradation product is 181.1, and the daughter ion is 93.1.

[0015] The preparation steps of the test solution are as follows:

[0016] (1) Take terbinafine hydrochloride spray, add diluent, freeze, centrifuge, and obtain supernatant;

[0017] (2) Take the supernatant, add dichloromethane, concentrate by nitrogen blowing to obtain a concentrated solution; use a diluent to redissolve the concentrated solution to obtain the test solution.

[0018] The diluent is a mixed solution of methanol and water, with a volume ratio of methanol:water = 1:1.

[0019] In step (1), the volume ratio of terbinafine hydrochloride spray to diluent is 1:1.

[0020] In step (1), the freezing temperature is -20℃ and the freezing time is 30min.

[0021] In step (2), the supernatant and dichloromethane are in a volume ratio of 1:1, and the volume after nitrogen blowing concentration is 1 ml.

[0022] The preparation steps of the reference solution are as follows: take nitrosamine impurity reference standard and prepare a reference solution with diluent.

[0023] Compared with existing technologies, this invention has the following beneficial effects: By using low-temperature precipitation followed by centrifugation, polycetrol 1000 in terbinafine hydrochloride spray can be separated, reducing the inhibitory effect of surfactants on mass spectrometry ionization, improving the ionization efficiency of nitrosamine impurities, and increasing the recovery rate. By using nitrogen blowing concentration and resolution, this invention effectively enriches the sample, increasing the sample concentration. By optimizing the gradient program, column temperature, and EMV voltage for each nitrosamine impurity using a preferred phenyl-hexylsilane-bonded silica gel column and APCI source, this invention improves the separation of the impurity from the main component and enhances the compound response. In summary, this invention, through systematic optimization of pretreatment methods and chromatographic and mass spectrometric parameters, solves the problems of poor specificity, low sensitivity, and unsatisfactory recovery rate in the detection of nitrosamine impurities in terbinafine hydrochloride spray, providing a reliable technical means for product quality control. Attached Figure Description

[0024] Figure 1 This is the methodological specificity result for the detection of N-nitrosodemethylterbinafine (NHNA) impurities in Example 1;

[0025] Figure 2 The graph shows the linearity of the N-nitrosodemethylterbinafine (NHNA) impurity detection methodology in Example 1.

[0026] Figure 3 This is the methodological specificity result for the detection of N-nitrosoterbinafine impurity A (NMNA) in Example 2;

[0027] Figure 4 The graph shows the linearity of the method for detecting N-nitrosoterbinafine impurity A (NMNA) in Example 2.

[0028] Figure 5 This is the methodological specificity result for the detection of N-nitrosoterbinafine degradation products (NHMA) impurities in Example 3;

[0029] Figure 6 The graph shows the linearity of the methodology for detecting N-nitrosoterbinafine degradation products (NHMA) impurities in Example 3. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is described with reference to the following specific embodiments, but these are not intended to limit the invention. The following descriptions are preferred embodiments of the invention and are used only to describe the invention; they should not be construed as limiting the invention. It should be noted that any modifications, equivalent substitutions, and improvements made within the spirit and principles of this invention should be included within the scope of protection of this invention.

[0031] Because the simultaneous determination of three nitrosamine impurities involves cyclically collecting fragment ions of all three impurities by mass spectrometry, compared to collecting fragment ions of only one impurity, the collection time for the same ion per unit time becomes shorter, resulting in increased baseline noise and reduced sensitivity. Therefore, in the following examples, one of the three nitrosamine impurities is measured separately to improve sensitivity.

[0032] Example 1: Validation of the method for detecting N-nitrosodemethylterbinafine (NHNA) impurities

[0033] Liquid chromatography conditions:

[0034] Column: Phenyl-Hexyl (4.6 mm × 50 mm, 2.6 µm);

[0035] Mobile phase A: 0.1% formic acid aqueous solution;

[0036] Mobile phase B: 0.1% formic acid in methanol solution;

[0037] Flow rate: 0.3 ml / min;

[0038] Column temperature: 40°C;

[0039] Injection volume: 20 µl;

[0040] Gradient procedure:

[0041] .

[0042] Mass spectrometry conditions:

[0043] Ion source: APCI; Polarity: Positive ion; Drying gas temperature: 325°C; Drying gas flow rate: 4L / min; Nebulizing gas pressure: 20psi; Capillary voltage: 4500V; Corona needle current: 4μA; Evaporation chamber temperature: 350°C; Acquisition mode: MRM; Mother ion: 307.2; Daughter ion: 141.1; Fragmentation voltage: 80V; Acceleration voltage: 3V; Collision energy: 20eV; EMV voltage: 0V.

[0044] Reference stock solution: Accurately weigh approximately 5 mg of the impurity NHNA reference standard, place it in a 10 ml volumetric flask, dissolve and dilute to the mark with methanol, and shake well; accurately measure 450 μl, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well; accurately measure 50 μl, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0045] Reference solution: Accurately measure 400 μl of the reference stock solution, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0046] Sensitivity solution: Accurately measure 40 μl of the reference stock solution, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0047] Test solution: Accurately measure 10 ml of terbinafine hydrochloride spray test sample, place it in a 50 ml centrifuge tube, add 10 ml of diluent, freeze in a -20℃ freezer for 30 min, remove and centrifuge to obtain the supernatant; accurately measure 12.5 ml of the supernatant into the concentration cup of a nitrogen blower, add 12.5 ml of dichloromethane, concentrate to 1 ml by nitrogen blower, transfer to a 5 ml volumetric flask, rinse the concentration cup 3 times with 1 ml of diluent, combine the washings into the volumetric flask, dilute to the mark with diluent, shake well, and the solution is ready.

[0048] Blank excipient solution: Accurately measure 10 ml of blank excipient and place it in a 50 ml centrifuge tube. Add 10 ml of diluent and freeze in a -20°C freezer for 30 min. After freezing, centrifuge to obtain the supernatant. Accurately measure 12.5 ml of the supernatant into the concentration cup of a nitrogen blower. Add 12.5 ml of dichloromethane and concentrate to 1 ml by nitrogen blowing. Transfer to a 5 ml volumetric flask. Rinse the concentration cup three times with 1 ml of diluent. Combine the washings in the volumetric flask and dilute to the mark with diluent. Shake well to obtain the final solution.

[0049] Mixed solution: Accurately measure 10 ml of terbinafine hydrochloride spray test sample and 320 μl of reference stock solution into a 50 ml centrifuge tube, add 9.68 ml of diluent, freeze in a -20℃ freezer for 30 min, remove and centrifuge to obtain the supernatant; accurately measure 12.5 ml of the supernatant into the concentration cup of a nitrogen blower, add 12.5 ml of dichloromethane, concentrate to 1 ml by nitrogen blower, transfer to a 5 ml volumetric flask, rinse the concentration cup 3 times with 1 ml of diluent, combine the washings into the volumetric flask, dilute to the mark with diluent, shake well, and the solution is ready.

[0050] Methodological investigation

[0051] 1.1 System Applicability

[0052] After the system reached equilibrium, inject one injection of the sensitivity solution and six injections of the reference solution, and record the extracted ion chromatogram. The results are shown in Table 1.

[0053] Table 1 System Applicability Results

[0054] .

[0055] Conclusion: The sensitivity solution S / N was 43.25. With six consecutive injections of the reference solution, the NHNA peak area RSD was 3.8%, less than 20%, meeting the standard. This indicates that the method is well-suited for NHNA detection of impurities in test samples.

[0056] 1.2 Specificity

[0057] After the system has reached equilibrium, inject one syringe each of the diluent, reference solution, blank excipient solution, test solution, and mixed solution, and record the extracted ion chromatogram. Results are shown below. Figure 1 and Table 2 below:

[0058] Table 2 Specificity Results

[0059] .

[0060] From Table 2 and Figure 1 It can be seen that the diluent, blank excipient and test sample do not interfere with the NHNA detection of impurities. The resolution between peak 1 in the mixed solution and the unknown peak is 1.8, indicating that this method has good specificity for the NHNA detection of impurities in the test sample.

[0061] 1.3 Limit of detection and limit of quantitation

[0062] Limit of Quantitation Solution: Accurately measure 40 μl of the reference standard stock solution, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0063] Detection limit solution: Accurately measure 3 ml of the quantitation limit solution, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0064] After the system reached equilibrium, inject one injection of the limit of detection solution and six injections of the limit of quantitation solution, and record the extracted ion chromatogram. The results are shown in Table 3.

[0065] Table 3 Results of Limit of Detection and Limit of Quantification

[0066] .

[0067] The above test results show that the NHNA impurity in the detection limit solution is equivalent to 3% of the limit concentration, which is equivalent to 10.6 ppb of terbinafine hydrochloride; the S / N ratio in the detection limit solution is 4.43, which is greater than 3. This meets the acceptable standard, indicating that this method has a good detection limit for the inspection of NHNA impurities in the test sample.

[0068] The NHNA impurity in the limit of quantitation solution was equivalent to 10% of the limit concentration, which is equivalent to 35.4 ppb of terbinafine hydrochloride. The RSD of the peak area in the 6-needle limit of quantitation solution was 9.0%, less than 20%, and the S / N ranged from 11.63 to 23.56, all greater than 10. These results meet the acceptable criteria, indicating that this method is effective for determining the limit of quantitation of NHNA impurities in the test sample.

[0069] 1.4 Linearity and Range

[0070] 10% linear solution: Accurately measure 40 μl of the reference stock solution, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0071] 50% linear solution: Accurately measure 200 μl of the reference stock solution, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0072] 100% linear solution: Accurately measure 400 μl of the reference stock solution, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0073] 120% linear solution: Accurately measure 480 μl of the reference stock solution, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0074] 150% linear solution: Accurately measure 600 μl of the reference stock solution, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0075] After the system reached equilibrium, one injection of each linear solution was administered, and the extracted ion chromatogram was recorded. The results are shown in Table 4.

[0076] Table 4 Linearity and Range Results

[0077] .

[0078] From Table 4 and Figure 2 It can be seen that the linear correlation coefficient r of the impurity NHNA in the range of 0.443 ng / ml to 6.640 ng / ml (10% to 150% limit concentration) is 0.9992, which meets the acceptable standard, indicating that the linearity and range of this method are good for the detection of impurity NHNA in the test sample.

[0079] 1.5 accuracy

[0080] 50% Recovery Sample Solution: Accurately measure 10 ml of terbinafine hydrochloride spray test sample and 160 μl of reference stock solution into a 50 ml centrifuge tube. Add 9.84 ml of diluent and freeze at -20°C for 30 min. After freezing, centrifuge to obtain the supernatant. Accurately measure 12.5 ml of the supernatant into the concentration cup of a nitrogen evaporator, add 12.5 ml of dichloromethane, concentrate to 1 ml under nitrogen evaporation, transfer to a 5 ml volumetric flask, rinse the concentration cup three times with 1 ml of diluent, combine the washings into the volumetric flask, dilute to the mark with diluent, and mix well. Prepare 3 aliquots using the same method.

[0081] 100% Recovery Sample Solution: Accurately measure 10 ml of terbinafine hydrochloride spray test sample and 320 μl of reference stock solution into a 50 ml centrifuge tube, add 9.68 ml of diluent, freeze at -20℃ for 30 min, and centrifuge to obtain the supernatant. Accurately measure 12.5 ml of the supernatant into the concentration cup of a nitrogen evaporator, add 12.5 ml of dichloromethane, concentrate to 1 ml under nitrogen evaporation, transfer to a 5 ml volumetric flask, rinse the concentration cup three times with 1 ml of diluent, combine the washings into the volumetric flask, dilute to the mark with diluent, and shake well. Prepare 3 aliquots using the same method.

[0082] 120% Recovery Sample Solution: Accurately measure 10 ml of terbinafine hydrochloride spray test sample and 384 μl of reference stock solution into a 50 ml centrifuge tube, add 9.62 ml of diluent, freeze at -20℃ for 30 min, and centrifuge to obtain the supernatant. Accurately measure 12.5 ml of the supernatant into the concentration cup of a nitrogen evaporator, add 12.5 ml of dichloromethane, concentrate to 1 ml under nitrogen evaporation, transfer to a 5 ml volumetric flask, rinse the concentration cup three times with 1 ml of diluent, combine the washings into the volumetric flask, dilute to the mark with diluent, and shake well. Prepare 3 aliquots using the same method.

[0083] After the system reached equilibrium, inject one sample of the test solution and one sample solution of each recovery rate, and record the extracted ion chromatogram. The results are shown in Table 5.

[0084] Table 5 Accuracy Results

[0085]

[0086] Conclusion: The recovery rate of NHNA impurities was 89.4%–98.3%, all within the range of 70%–125%. The RSD of the recovery rate was 3.2%, which is less than 20% and meets the acceptable standard, indicating that the method has good accuracy for the detection of NHNA impurities in the test sample.

[0087] 1.6 Precision

[0088] Repeatability: After the system has reached equilibrium, inject one sample of the mixed solution into each sample and record the extracted ion chromatogram. Calculate the impurity content and RSD value of the six mixed solutions (n=6).

[0089] Intermediate precision: In the same laboratory, at different times, different analysts performed the above repeatability test to calculate the NHNA impurity content and RSD value in 12 mixed solutions (n=12). The results are shown in Table 6.

[0090] Table 6 Precision Results

[0091]

[0092] The above test results show that the RSD value of the NHNA impurity content in the 6 mixed solutions is 3.4%, which is less than 20%, indicating that the method has good repeatability for the test of NHNA impurities in the test samples; the RSD value of the NHNA impurity content in the 12 mixed solutions is 9.3%, which is less than 25%, indicating that the method has good precision for the test of NHNA impurities in the test samples.

[0093] Example 2: Validation of the method for detecting N-nitrosoterbinafine impurity A (NMNA)

[0094] Liquid chromatography conditions:

[0095] Column: Phenyl-Hexyl (4.6 mm × 50 mm, 2.6 µm);

[0096] Mobile phase A: 0.1% formic acid aqueous solution;

[0097] Mobile phase B: 0.1% formic acid in methanol solution;

[0098] Flow rate: 0.3 ml / min;

[0099] Column temperature: 20°C;

[0100] Injection volume: 20 µl;

[0101] Gradient procedure:

[0102]

[0103] Mass spectrometry conditions:

[0104] Ion source: APCI; Polarity: Positive ion; Drying gas temperature: 325°C; Drying gas flow rate: 4L / min; Nebulizing gas pressure: 20psi; Capillary voltage: 4500V; Corona needle current: 4μA; Evaporation chamber temperature: 350°C; Acquisition mode: MRM; Mother ion: 201.1; Daughter ion: 141.1; Fragmentation voltage: 60V; Acceleration voltage: 3V; Collision energy: 16eV; EMV voltage: 100V.

[0105] Reference stock solution: Accurately weigh approximately 5 mg of impurity NMNA reference standard, place it in a 10 ml volumetric flask, dissolve and dilute to the mark with methanol, and shake well; accurately measure 450 μl, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well; accurately measure 50 μl, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0106] Reference solution: Accurately measure 400 μl of the reference stock solution, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0107] Sensitivity solution: Accurately measure 40 μl of the reference stock solution, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0108] Test solution: Accurately measure 10 ml of terbinafine hydrochloride spray test sample, place it in a 50 ml centrifuge tube, add 10 ml of diluent, freeze in a -20℃ freezer for 30 min, remove and centrifuge to obtain the supernatant; accurately measure 12.5 ml of the supernatant into the concentration cup of a nitrogen blower, add 12.5 ml of dichloromethane, concentrate to 1 ml by nitrogen blower, transfer to a 5 ml volumetric flask, rinse the concentration cup 3 times with 1 ml of diluent, combine the washings into the volumetric flask, dilute to the mark with diluent, shake well, and the solution is ready.

[0109] Blank excipient solution: Accurately measure 10 ml of blank excipient and place it in a 50 ml centrifuge tube. Add 10 ml of diluent and freeze in a -20°C freezer for 30 min. After freezing, centrifuge to obtain the supernatant. Accurately measure 12.5 ml of the supernatant into the concentration cup of a nitrogen blower. Add 12.5 ml of dichloromethane and concentrate to 1 ml by nitrogen blowing. Transfer to a 5 ml volumetric flask. Rinse the concentration cup three times with 1 ml of diluent. Combine the washings in the volumetric flask and dilute to the mark with diluent. Shake well to obtain the final solution.

[0110] Mixed solution: Accurately measure 10 ml of terbinafine hydrochloride spray test sample and 320 μl of reference stock solution into a 50 ml centrifuge tube, add 9.68 ml of diluent, freeze in a -20℃ freezer for 30 min, remove and centrifuge to obtain the supernatant; accurately measure 12.5 ml of the supernatant into the concentration cup of a nitrogen blower, add 12.5 ml of dichloromethane, concentrate to 1 ml by nitrogen blower, transfer to a 5 ml volumetric flask, rinse the concentration cup 3 times with 1 ml of diluent, combine the washings into the volumetric flask, dilute to the mark with diluent, shake well, and the solution is ready.

[0111] Methodological investigation

[0112] 2.1 System Applicability

[0113] After the system reached equilibrium, 6 syringes of reference solution and 1 syringe of sensitivity solution were injected, and the extracted ion chromatograms were recorded. The results are shown in Table 7.

[0114] Table 7 System Applicability Results

[0115]

[0116] The sensitivity solution S / N was 12.78. With six consecutive injections of the reference solution, the NMNA peak area RSD was 5.0%, less than 20%, meeting the standard. This indicates that the method is well-suited for the NMNA detection of impurities in the test sample.

[0117] 2.2 Specificity

[0118] After the system has reached equilibrium, inject one syringe each of the diluent, reference solution, blank excipient solution, test solution, and mixed solution, and record the extracted ion chromatogram. The results are shown in Table 8. Figure 3 .

[0119] Table 8 Specificity Results

[0120]

[0121] From Table 8 and Figure 3 The results show that the diluent, blank excipient, and test sample do not interfere with the NMNA detection of impurities, indicating that this method has good specificity for the detection of NMNA impurities in test samples.

[0122] 2.3 Limit of detection and limit of quantitation

[0123] Limit of Quantitation Solution: Accurately measure 40 μl of the reference standard stock solution, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0124] Detection limit solution: Accurately measure 3 ml of the quantitation limit solution, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0125] After the system reached equilibrium, one injection of the limit of detection solution and six injections of the limit of quantitation solution were administered, and the extracted ion chromatogram was recorded. The results are shown in Table 9.

[0126] Table 9 Results of Limit of Detection and Limit of Quantification

[0127]

[0128] Conclusion: The above experimental results show that the NMNA impurity in the detection limit solution is equivalent to 3% of the limit concentration, which is equivalent to 11.3 ppb of terbinafine hydrochloride; the S / N ratio in the detection limit solution is 5.48, which is greater than 3. This meets the acceptable standard, indicating that this method has a good detection limit for the detection of NMNA impurities in the test sample.

[0129] The NMNA impurity in the limit of quantitation solution was equivalent to 10% of the limit concentration, which is equivalent to 37.8 ppb of terbinafine hydrochloride. The RSD of the peak area in the 6-needle limit of quantitation solution was 4.9%, less than 20%, and the S / N ranged from 22.88 to 34.33, all greater than 10. These results meet the acceptable criteria, indicating that this method is effective for determining the limit of quantitation of NMNA impurities in the test sample.

[0130] 2.4 Linearity and Range

[0131] 10% linear solution: Accurately measure 40 μl of the reference stock solution, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0132] 50% linear solution: Accurately measure 200 μl of the reference stock solution, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0133] 100% linear solution: Accurately measure 400 μl of the reference stock solution, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0134] 120% linear solution: Accurately measure 480 μl of the reference stock solution, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0135] 150% linear solution: Accurately measure 600 μl of the reference stock solution, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0136] After the system reached equilibrium, one injection of each linear solution was performed, and the extracted ion chromatogram was recorded. The results are shown in Table 10.

[0137] Table 10 Linearity and Range Results

[0138]

[0139] From Table 10 and Figure 4It can be seen that the linear correlation coefficient r of impurity NMNA in the range of 0.472 ng / ml ~ 7.087 ng / ml (10% ~ 150% limit concentration) is 0.9993, which meets the acceptable standard, indicating that the linearity and range of this method are good for the detection of impurity NMNA in the test sample.

[0140] 2.5 accuracy

[0141] 50% Recovery Sample Solution: Accurately measure 10 ml of terbinafine hydrochloride spray test sample and 160 μl of reference stock solution into a 50 ml centrifuge tube. Add 9.84 ml of diluent and freeze at -20°C for 30 min. After freezing, centrifuge to obtain the supernatant. Accurately measure 12.5 ml of the supernatant into the concentration cup of a nitrogen evaporator, add 12.5 ml of dichloromethane, concentrate to 1 ml under nitrogen evaporation, transfer to a 5 ml volumetric flask, rinse the concentration cup three times with 1 ml of diluent, combine the washings into the volumetric flask, dilute to the mark with diluent, and mix well. Prepare 3 aliquots using the same method.

[0142] 100% Recovery Sample Solution: Accurately measure 10 ml of terbinafine hydrochloride spray test sample and 320 μl of reference stock solution into a 50 ml centrifuge tube, add 9.68 ml of diluent, freeze at -20℃ for 30 min, and centrifuge to obtain the supernatant. Accurately measure 12.5 ml of the supernatant into the concentration cup of a nitrogen evaporator, add 12.5 ml of dichloromethane, concentrate to 1 ml under nitrogen evaporation, transfer to a 5 ml volumetric flask, rinse the concentration cup three times with 1 ml of diluent, combine the washings into the volumetric flask, dilute to the mark with diluent, and shake well. Prepare 3 aliquots using the same method.

[0143] 120% Recovery Sample Solution: Accurately measure 10 ml of terbinafine hydrochloride spray test sample and 384 μl of reference stock solution into a 50 ml centrifuge tube, add 9.62 ml of diluent, freeze at -20℃ for 30 min, and centrifuge to obtain the supernatant. Accurately measure 12.5 ml of the supernatant into the concentration cup of a nitrogen evaporator, add 12.5 ml of dichloromethane, concentrate to 1 ml under nitrogen evaporation, transfer to a 5 ml volumetric flask, rinse the concentration cup three times with 1 ml of diluent, combine the washings into the volumetric flask, dilute to the mark with diluent, and shake well. Prepare 3 aliquots using the same method.

[0144] After the system reached equilibrium, inject one sample of the test solution and one sample solution of each recovery rate, and record the extracted ion chromatogram. The results are shown in Table 11.

[0145] Table 11 Accuracy Results

[0146]

[0147] Conclusion: The recovery rate of impurity NMNA was 94.4%–105.0%, all within the range of 70%–125%. The RSD of the recovery rate was 3.1%, which is less than 20% and meets the acceptable standard, indicating that the method is accurate for the detection of impurity NMNA in the test sample.

[0148] 2.6 Precision

[0149] Repeatability: After the system has reached equilibrium, inject one sample of the mixed solution into each sample and record the extracted ion chromatogram. Calculate the impurity content and RSD value of the six mixed solutions (n=6).

[0150] Intermediate precision: In the same laboratory, at different times, different analysts performed the above repeatability test to calculate the NMNA impurity content and RSD value in 12 mixed solutions (n=12). The results are shown in Table 12.

[0151] Table 12 Precision Results

[0152]

[0153] The above test results show that the RSD value of the NMNA impurity content in the 6 mixed solutions is 2.2%, which is less than 20%, indicating that the method has good repeatability for the test of NMNA impurity in the test samples; the RSD value of the NMNA impurity content in the 12 mixed solutions is 2.2%, which is less than 25%, indicating that the method has good precision for the test of NMNA impurity in the test samples.

[0154] Example 3: Validation of the method for detecting impurities in N-nitrosoterbinafine degradation products (NHMA)

[0155] Chromatographic conditions:

[0156] Column: Phenyl-Hexyl (4.6 mm × 50 mm, 2.6 µm);

[0157] Mobile phase A: 0.1% formic acid aqueous solution;

[0158] Mobile phase B: 0.1% formic acid in methanol solution;

[0159] Flow rate: 0.3 ml / min;

[0160] Column temperature: 40°C;

[0161] Injection volume: 20 µl;

[0162] Gradient procedure:

[0163]

[0164] Mass spectrometry conditions:

[0165] Ion source: APCI; Polarity: Positive ion; Drying gas temperature: 325°C; Drying gas flow rate: 4L / min; Nebulizing gas pressure: 20psi; Capillary voltage: 4500V; Corona needle current: 4μA; Evaporation chamber temperature: 350°C; Acquisition mode: MRM; Mother ion: 181.1; Daughter ion: 93.1; Fragmentation voltage: 60V; Acceleration voltage: 3V; Collision energy: 10eV; EMV voltage: 0V.

[0166] Reference stock solution: Accurately weigh approximately 5 mg of NHMA reference standard, place it in a 10 ml volumetric flask, dissolve and dilute to the mark with methanol, and shake well; accurately measure 450 μl, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well; accurately measure 50 μl, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0167] Reference solution: Accurately measure 400 μl of the reference stock solution, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0168] Sensitivity solution: Accurately measure 40 μl of the reference stock solution, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0169] Test solution: Accurately measure 10 ml of terbinafine hydrochloride spray test sample, place it in a 50 ml centrifuge tube, add 10 ml of diluent, freeze in a -20℃ freezer for 30 min, remove and centrifuge to obtain the supernatant; accurately measure 12.5 ml of the supernatant into the concentration cup of a nitrogen blower, add 12.5 ml of dichloromethane, concentrate to 1 ml by nitrogen blower, transfer to a 5 ml volumetric flask, rinse the concentration cup 3 times with 1 ml of diluent, combine the washings into the volumetric flask, dilute to the mark with diluent, shake well, and the solution is ready.

[0170] Blank excipient solution: Accurately measure 10 ml of blank excipient and place it in a 50 ml centrifuge tube. Add 10 ml of diluent and freeze in a -20°C freezer for 30 min. After freezing, centrifuge to obtain the supernatant. Accurately measure 12.5 ml of the supernatant into the concentration cup of a nitrogen blower. Add 12.5 ml of dichloromethane and concentrate to 1 ml by nitrogen blowing. Transfer to a 5 ml volumetric flask. Rinse the concentration cup three times with 1 ml of diluent. Combine the washings in the volumetric flask and dilute to the mark with diluent. Shake well to obtain the final solution.

[0171] Mixed solution: Accurately measure 10 ml of terbinafine hydrochloride spray test sample and 320 μl of reference stock solution into a 50 ml centrifuge tube, add 9.68 ml of diluent, freeze in a -20℃ freezer for 30 min, remove and centrifuge to obtain the supernatant; accurately measure 12.5 ml of the supernatant into the concentration cup of a nitrogen blower, add 12.5 ml of dichloromethane, concentrate to 1 ml by nitrogen blower, transfer to a 5 ml volumetric flask, rinse the concentration cup 3 times with 1 ml of diluent, combine the washings into the volumetric flask, dilute to the mark with diluent, shake well, and the solution is ready.

[0172] 3.1 System Applicability

[0173] After the system reached equilibrium, inject 6 syringes of the reference solution and 1 syringe of the sensitivity solution, and record the extracted ion chromatogram. The results are shown in Table 13.

[0174] Table 13 System Applicability Results

[0175]

[0176] As shown in Table 13, the sensitivity solution S / N is 144.56, and the reference solution was injected continuously for 6 injections. The NHMA peak area RSD value was 3.7%, which is less than 20%, meeting the standard. This indicates that the method is well-suited for the NHMA detection system for impurities in test samples.

[0177] 3.2 Specificity

[0178] After the system has reached equilibrium, inject one syringe each of the diluent, reference solution, blank excipient solution, test solution, and mixed solution, and record the extracted ion chromatogram. The results are shown in Table 14. Figure 5 :

[0179] Table 14 Specificity Results

[0180]

[0181] From Table 14 and Figure 5 The results show that the diluent, blank excipient, and test sample do not interfere with the NHMA test for impurities, indicating that this method has good specificity for the NHMA test of impurities in test samples.

[0182] 3.3 Limit of detection and limit of quantitation

[0183] Limit of Quantitation Solution: Accurately measure 40 μl of the reference standard stock solution, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0184] Detection limit solution: Accurately measure 3 ml of the quantitation limit solution, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0185] After the system reached equilibrium, one injection of the limit of detection solution and six injections of the limit of quantitation solution were administered, and the extracted ion chromatogram was recorded. The results are shown in Table 15.

[0186] Table 15 Results of Limit of Detection and Limit of Quantitation

[0187]

[0188] As shown in Table 15, the NHMA impurity in the detection limit solution is equivalent to 3% of the limit concentration, which is equivalent to 10.9 ppb of terbinafine hydrochloride; the S / N ratio in the detection limit solution is 94.30, which is greater than 3. This meets the acceptable standard, indicating that the detection limit of this method for checking the NHMA impurity in the test sample is good.

[0189] The NHMA impurity in the limit of quantitation solution was equivalent to 10% of the limit concentration, which is equivalent to 36.4 ppb of terbinafine hydrochloride. The RSD of the peak area in the 6-needle limit of quantitation solution was 3.9%, less than 20%, and the S / N ranged from 172.73 to 309.31, all greater than 10. These results meet the acceptable criteria, indicating that this method is effective for detecting NHMA impurities in the test sample.

[0190] 3.4 Linearity and Range

[0191] 10% linear solution: Accurately measure 40 μl of the reference stock solution, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0192] 50% linear solution: Accurately measure 200 μl of the reference stock solution, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0193] 100% linear solution: Accurately measure 400 μl of the reference stock solution, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0194] 120% linear solution: Accurately measure 480 μl of the reference stock solution, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0195] 150% linear solution: Accurately measure 600 μl of the reference stock solution, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0196] After the system reached equilibrium, one injection of each linear solution was performed, and the extracted ion chromatogram was recorded. The results are shown in Table 16.

[0197] Table 16 Linearity and Range Results

[0198]

[0199] From Table 16 and Figure 6It can be seen that the linear correlation coefficient r of the impurity NHMA in the range of 0.456 ng / ml ~ 6.833 ng / ml (10%~150% limit concentration) is 0.9967, which meets the acceptable standard, indicating that the linearity and range of this method for the detection of impurity NHMA in the test sample are good.

[0200] 3.5 accuracy

[0201] 50% Recovery Sample Solution: Accurately measure 10 ml of terbinafine hydrochloride spray test sample and 160 μl of reference stock solution into a 50 ml centrifuge tube. Add 9.84 ml of diluent and freeze at -20°C for 30 min. After freezing, centrifuge to obtain the supernatant. Accurately measure 12.5 ml of the supernatant into the concentration cup of a nitrogen evaporator, add 12.5 ml of dichloromethane, concentrate to 1 ml under nitrogen evaporation, transfer to a 5 ml volumetric flask, rinse the concentration cup three times with 1 ml of diluent, combine the washings into the volumetric flask, dilute to the mark with diluent, and mix well. Prepare 3 aliquots using the same method.

[0202] 100% Recovery Sample Solution: Accurately measure 10 ml of terbinafine hydrochloride spray test sample and 320 μl of reference stock solution into a 50 ml centrifuge tube, add 9.68 ml of diluent, freeze at -20℃ for 30 min, and centrifuge to obtain the supernatant. Accurately measure 12.5 ml of the supernatant into the concentration cup of a nitrogen evaporator, add 12.5 ml of dichloromethane, concentrate to 1 ml under nitrogen evaporation, transfer to a 5 ml volumetric flask, rinse the concentration cup three times with 1 ml of diluent, combine the washings into the volumetric flask, dilute to the mark with diluent, and shake well. Prepare 3 aliquots using the same method.

[0203] 120% Recovery Sample Solution: Accurately measure 10 ml of terbinafine hydrochloride spray test sample and 384 μl of reference stock solution into a 50 ml centrifuge tube, add 9.62 ml of diluent, freeze at -20℃ for 30 min, and centrifuge to obtain the supernatant. Accurately measure 12.5 ml of the supernatant into the concentration cup of a nitrogen evaporator, add 12.5 ml of dichloromethane, concentrate to 1 ml under nitrogen evaporation, transfer to a 5 ml volumetric flask, rinse the concentration cup three times with 1 ml of diluent, combine the washings into the volumetric flask, dilute to the mark with diluent, and shake well. Prepare 3 aliquots using the same method.

[0204] After the system reached equilibrium, one injection of the test solution and one injection of each recovery rate sample solution were performed, and the extracted ion chromatogram was recorded. The results are shown in Table 17.

[0205] Table 17 Accuracy Results

[0206]

[0207] As shown in Table 17, the recovery rate of NHMA impurity was 75.4% to 86.8%, all within the range of 70% to 125%. The RSD of the recovery rate was 5.1%, which is less than 20% and meets the acceptable standard, indicating that the method is accurate for the detection of NHMA impurity in the test sample.

[0208] 3.6 Precision

[0209] Repeatability: After the system has reached equilibrium, inject one sample of the mixed solution into each sample and record the extracted ion chromatogram. Calculate the impurity content and RSD value of the six mixed solutions (n=6).

[0210] Intermediate precision: In the same laboratory, at different times, different analysts performed the above repeatability test to calculate the NHMA impurity content and RSD value in 12 mixed solutions (n=12). The results are shown in Table 18.

[0211] Table 18 Precision Results

[0212]

[0213] As shown in Table 18, the RSD of the NHMA impurity content in the 6 mixed solutions was 5.7%, which is less than 20%, indicating that the method has good repeatability for the test of NHMA impurities in the test samples; the RSD of the NHMA impurity content in the 12 mixed solutions was 4.6%, which is less than 25%, indicating that the method has good precision for the test of NHMA impurities in the test samples.

[0214] Comparative Example 1: Screening of Chromatographic Column Types

[0215] Three nitrosamine impurities were attempted to be separated chromatographically using C18, C8, HILIC, phenyl, biphenyl, and phenyl-hexyl columns, respectively. After system equilibration, one injection each of the diluent, reference solution, test solution, and mixed solution was administered, and the extracted ion chromatograms were recorded. The results are shown in Table 19.

[0216] Table 19 Column Screening Results

[0217] .

[0218] As shown in Table 19, using a HILIC column, the three nitrosamine impurities either had poor peak shapes or did not produce any peaks, making it unsuitable as a column. Using C18 and C8 columns, the NHNA and NHMA peaks overlapped with the terbinafine peak, and optimized gradient programs still could not separate them; moreover, the NMNA peak had poor shape and low sensitivity. Using phenyl and biphenyl columns, the NHNA or NHMA peaks overlapped with the terbinafine peak, and optimized gradient programs could not remove the interfering peaks, although the NMNA peak shape improved. Using a phenyl-hexyl column, both the NHNA and NHMA peaks were separated from terbinafine; optimized gradient programs could remove the unknown peak interference, and the NMNA peak shape and sensitivity improved. Setting the EMV voltage to 100V in the mass spectrometry parameters significantly enhanced the response, and the sensitivity met the requirements.

[0219] Comparative Example 2: Three types of nitrosamine impurities were determined to investigate the effect of different ion sources on the recovery rate.

[0220] Using ESI and APCI sources respectively, after the system equilibrated, diluent, reference solution, test solution, and mixed solution were injected, and the extracted ion chromatograms were recorded. The effect of different ion sources on the recovery rate of three nitrosamine impurities was investigated. The results are shown in Table 20.

[0221] Table 20 Recovery Rate Study

[0222] .

[0223] As shown in Table 20, the recovery rates of the three nitrosamine impurities were low when using the ESI source, which did not meet the requirements for method validation. When using the APCI source, the recovery rates of the three nitrosamine impurities could meet the requirements of 70%-130%, and the RSD was less than 15%. The APCI source is more suitable as the ion source for this method.

[0224] Comparative Example 3: Investigation of the preparation method of the test solution

[0225] Test solution preparation method 1: Direct injection of stock solution

[0226] Accurately measure 10 ml of terbinafine hydrochloride spray test sample, place it in a 10 ml volumetric flask, and shake well to obtain the solution.

[0227] Test solution preparation method 2: Dilute by one time before injection

[0228] Accurately measure 5 ml of terbinafine hydrochloride spray test sample, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the final product.

[0229] Test solution preparation method 3: After low-temperature precipitation and centrifugation, take the supernatant and dilute it.

[0230] Accurately measure 10 ml of terbinafine hydrochloride spray test sample, place it in a 50 ml centrifuge tube, add 10 ml of diluent, freeze in a -20℃ freezer for 30 min, remove and centrifuge to obtain the supernatant; accurately measure 10 ml of the supernatant into a volumetric flask, shake well, and the test result is obtained.

[0231] Preparation method 4 for test solution: direct nitrogen blowing concentration

[0232] Accurately measure 12.5 ml of terbinafine hydrochloride spray into the concentration cup of a nitrogen evaporator, concentrate it to 1 ml with nitrogen, transfer it to a 5 ml volumetric flask, rinse the concentration cup three times with 1 ml of diluent, combine the washings into the volumetric flask, dilute to the mark with diluent, and shake well to obtain the final product.

[0233] Method 5 for preparing the test solution: concentration by adding dichloromethane followed by nitrogen blowing.

[0234] Accurately measure 12.5 ml of terbinafine hydrochloride spray into the concentration cup of a nitrogen evaporator, add 12.5 ml of dichloromethane, concentrate to 1 ml by nitrogen evaporation, transfer to a 5 ml volumetric flask, rinse the concentration cup three times with 1 ml of diluent, combine the washings into the volumetric flask, dilute to the mark with diluent, and shake well to obtain the final product.

[0235] Preparation method 6 for the test solution: After low-temperature precipitation and centrifugation, take the supernatant, add dichloromethane, and concentrate by nitrogen blowing.

[0236] Accurately measure 10 ml of terbinafine hydrochloride spray test sample and place it in a 50 ml centrifuge tube. Add 10 ml of diluent and freeze in a -20°C freezer for 30 min. After freezing, centrifuge to obtain the supernatant. Accurately measure 12.5 ml of the supernatant into the concentration cup of a nitrogen blower, add 12.5 ml of dichloromethane, concentrate under nitrogen to 1 ml, transfer to a 5 ml volumetric flask, rinse the concentration cup three times with 1 ml of diluent, combine the washings into the volumetric flask, dilute to the mark with diluent, and shake well to obtain the final product.

[0237] The test solution and mixed solution were prepared using the methods described in steps 1-6 above. After system equilibration, the diluent, reference solution, test solution, and mixed solution were injected, and the extracted ion chromatograms were recorded to investigate the effect of different test solution preparation methods on the method recovery and sensitivity. The results are shown in Table 22.

[0238] Table 22 Investigation of the preparation method of the test sample

[0239] .

[0240] As shown in Table 22, the methods of directly injecting the original sample solution and preparing the sample solution by dilution of one time resulted in severe ionization inhibition, and the sample solution concentration was too low, failing to meet the sensitivity requirements. The method of preparing the sample solution by low-temperature precipitation and centrifugation alone could eliminate ionization inhibition, but the sample solution concentration was too low, failing to meet the sensitivity requirements. The method of preparing the sample solution by nitrogen blowing concentration alone could increase the sample solution concentration, and the addition of dichloromethane could significantly shorten the nitrogen blowing concentration time, but ionization inhibition was severe, failing to meet the sensitivity requirements. The method of preparing the sample solution by taking the supernatant after low-temperature precipitation and centrifugation, adding dichloromethane, and nitrogen blowing concentration could significantly shorten the nitrogen blowing concentration time, increase the sample solution concentration, and basically eliminate ionization inhibition, achieving a higher sensitivity.

[0241] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting nitrosamine impurities in terbinafine hydrochloride spray, characterized in that, The test solution and the reference solution were detected by liquid chromatography-mass spectrometry. The chromatographic conditions for the liquid chromatography are as follows: a phenyl-hexylsilane bonded silica gel column is used, with a mobile phase A of 0.1% formic acid aqueous solution and a mobile phase B of 0.1% formic acid methanol solution, and the column temperature is 20-40℃, with gradient elution. The mass spectrometry conditions are as follows: APCI source, positive ion mode detection, MRM acquisition mode, fragmentation voltage 60-80V, collision energy 10-20eV, EMV voltage 0-100V, and appropriate parent and daughter ions are selected according to the nitrosamine impurities.

2. The method for detecting nitrosamine impurities in terbinafine hydrochloride spray according to claim 1, characterized in that, The nitrosamine impurity is N-nitrosodemethylterbinafine or N-nitrosoterbinafine impurity A or N-nitrosoterbinafine degradation product.

3. The method for detecting nitrosamine impurities in terbinafine hydrochloride spray according to claim 2, characterized in that, The precursor ion of the N-nitrosodemethylterbinafine is 307.2, and the daughter ion is 141.1; the precursor ion of N-nitrosoterbinafine impurity A is 201.1, and the daughter ion is 141.

1. The parent ion of the N-nitrosoterbinafine degradation product is 181.1, and the daughter ion is 93.

1.

4. The method for detecting nitrosamine impurities in terbinafine hydrochloride spray according to claim 1, characterized in that, The preparation steps for the test solution are as follows: (1) Take terbinafine hydrochloride spray, add diluent, freeze, centrifuge, and obtain supernatant; (2) Take the supernatant, add dichloromethane, and concentrate by nitrogen blowing to obtain the concentrate; The concentrate was reconstituted with a diluent to obtain the test solution.

5. The method for detecting nitrosamine impurities in terbinafine hydrochloride spray according to claim 4, characterized in that, The diluent is a mixed solution of methanol and water, with a volume ratio of methanol:water = 1:

1.

6. The method for detecting nitrosamine impurities in terbinafine hydrochloride spray according to claim 4, characterized in that, In step (1), the volume ratio of terbinafine hydrochloride spray to diluent is 1:

1.

7. The method for detecting nitrosamine impurities in terbinafine hydrochloride spray according to claim 4, characterized in that, In step (1), the freezing temperature is -20℃ and the freezing time is 30min.

8. The method for detecting nitrosamine impurities in terbinafine hydrochloride spray according to claim 4, characterized in that, In step (2), the supernatant and dichloromethane are in a volume ratio of 1:1, and the volume after nitrogen blowing concentration is 1 ml.

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