Method for detecting N-nitroso ambroxol in medicine by LC-MS / MS (liquid chromatography-mass spectrometry / mass spectrometry)

Abstract

The invention discloses a method for detecting N-nitroso ambroxol in a medicine through LC-MS / MS, and belongs to the technical field of medicine analysis. According to the method for detecting N-nitroso ambroxol in the medicine through LC-MS / MS, the medicine is an ambroxol-containing preparation or ambroxol and a salt bulk drug thereof, and liquid chromatography conditions comprise that octadecylsilane chemically bonded silica is used as a filler, 0.05-0.2% formic acid is used as a mobile phase A, and acetonitrile is used as a mobile phase B for gradient elution; the mass spectrum conditions comprise that a mass spectrum detector and an electrospray positive ion mode are adopted, multi-reaction detection is carried out, and m / z 377.9 to 279.2, m / z 377.9 to 114.3 and m / z 377.9 to 96.3 are selected as detection ion pairs. The method for detecting the ambroxol-containing preparation and the raw materials by adopting LC-MS / MS is high in sensitivity, good in accuracy and suitable for quality control of trace nitrosamine impurities in the preparation and the raw materials.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to a method for detecting N-nitrosoambroxol in drugs by LC-MS / MS. Background Art

[0002] Preparations containing ambroxol may produce nitrosamine impurities N-nitrosoambroxol (NDSRI) during the production and storage of raw materials and preparations. Nitrosamine impurities are a class of compounds with a chemical structure of nitroso combined with amines, which have high potential mutagenicity and carcinogenicity. Nitrosamine impurities belong to the "concern queue" substances mentioned in the ICH M7 (R1) (Evaluation and Control of DNA Reactive (Mutagenic) Impurities in Drugs to Limit Potential Carcinogenic Risk) guidelines. According to the list of carcinogens published by the World Health Organization, N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) are both Class 2A carcinogens; according to internationally recognized databases, some nitrosamine impurities have public carcinogenicity data, such as NDMA, NDEA, N-nitroso-N-methyl-4-aminobutyric acid (NMBA), N-nitrosodibutylamine (NDBA), etc.

[0003] In the prior art, the detection of nitrosamine impurities generally adopts gas chromatography or high performance liquid chromatography. However, these two methods have limitations in sensitivity, specificity and anti-interference ability, and it is difficult to meet the detection needs of trace nitrosamine impurities. The specific defects are as follows: (1) Insufficient sensitivity: The detection limit (LOD) and quantification limit (LOQ) of conventional methods for low-concentration nitrosamine impurities are high, and cannot meet the trace detection requirements at the ppm or ppb level. (2) Poor specificity: In complex matrices (such as oral solution excipients), impurities may interfere with the identification of target peaks, resulting in false positive or false negative results. (3) Long analysis time: The elution procedure of traditional chromatographic methods is complicated and time-consuming, which affects the detection efficiency. These limitations make it difficult for existing technologies to effectively meet the needs of accurate detection of trace nitrosamine impurities.

[0004] Therefore, it is necessary to develop a method for detecting N-nitrosoambroxol with high sensitivity and good accuracy for quality control of ambroxol preparations, ambroxol raw materials, or ambroxol salt raw materials, which has become an urgent problem to be solved by those skilled in the art. Summary of the invention

[0005] The object of the present invention is to provide a method for detecting N-nitrosoambroxol in medicine by LC-MS / MS, which has high sensitivity and good accuracy and can be used for quality control of ambroxol-containing preparations and raw materials thereof.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows: The present invention discloses a method for detecting N-nitrosoambroxol in a drug by LC-MS / MS, wherein the drug is an ambroxol preparation, an ambroxol bulk drug, or an ambroxol salt bulk drug. The liquid chromatography conditions include: using octadecylsilane bonded silica gel as a filler, 0.05-0.2% formic acid as a mobile phase A, acetonitrile as a mobile phase B, and gradient elution as follows: 0min, 70~80%A, 20~30%B; 3min, 70~80%A, 20~30%B; 6min, 25~35%A, 65~75%B; 8min, 25~35%A, 65~75%B; The mass spectrometry conditions include: using a mass spectrometer detector in electrospray positive ion mode, performing multiple reaction detection, and selecting m / z 377.9→279.2, m / z 377.9→114.3, and m / z 377.9→96.3 as detection ion pairs.

[0007] In some embodiments of the present invention, 0.1% formic acid is used as mobile phase A and acetonitrile is used as mobile phase B for gradient elution.

[0008] In some embodiments of the present invention, the gradient elution procedure is as follows: 0 min, 75% A, 25% B; 3min, 75% A, 25% B; 6min, 30% A, 70% B; 8min, 30%A, 70%B.

[0009] In some embodiments of the present invention, the column temperature of the chromatographic column is 28-32° C., and the flow rate of the mobile phase is 0.25-0.35 ml / min.

[0010] In some embodiments of the present invention, the column temperature of the chromatographic column is 30° C. and the flow rate of the mobile phase is 0.3 ml / min.

[0011] In some embodiments of the present invention, when the drug is ambroxol or its salt raw material, the method comprises the following steps: S1. Preparation of test solution: Take an appropriate amount of ambroxol or its salt raw material drug, accurately weigh it, add an appropriate amount of solvent to dissolve it and quantitatively dilute it to make a solution with a concentration of 0.7~0.9mg / ml as the raw material drug-test solution; S2. Preparation of reference solution: Take N-nitrosoambroxol reference substance and add solvent to prepare a series of linear reference solutions with a mass concentration gradient of 0.6~5 ng / mL; the concentration gradient of the series of linear reference solutions should be at least 5; The solvents described in S1 and S2 are both mixtures of acetonitrile and water, with a volume ratio of 1:0.8~1.2; S3. Test: Accurately measure blank solvent, API-test solution and reference solution, respectively inject them into HPLC-MS / MS instrument, measure, record chromatogram, calculate according to standard curve method, and obtain the content of N-nitrosoambroxol in ambroxol or its salt API.

[0012] In some embodiments of the present invention, the concentrations of the linear control solution prepared by S2 are 0.6 ng / mL, 1.25 ng / mL, 2.5 ng / mL, 3.75 ng / mL and 5 ng / mL; The injection volume of S3 is 5~20μl; The content of N-nitrosoambroxol in ambroxol hydrochloride raw material should be less than or equal to 3.3ppm.

[0013] In some embodiments of the present invention, when the drug is an ambroxol preparation, the method comprises the following steps: Step 1. Preparation of the test solution: Take an appropriate amount of the preparation containing ambroxol, accurately weigh it, add an appropriate amount of solvent to dissolve it, and quantitatively dilute it to make a solution containing 0.70-0.80 mg of ambroxol hydrochloride per 1 ml, as the preparation-test solution; Step 2. Preparation of reference solution: Take N-nitrosoambroxol reference substance and add solvent to prepare a series of linear reference solutions with a mass concentration gradient of 0.6-5 ng / mL; the concentration gradient of the series of linear reference solutions is at least 5; The solvents described in S1 and S2 are both mixtures of acetonitrile and water, with a volume ratio of 1:0.8~1.2; Step 3. Test: Accurately measure the blank solvent, preparation-test solution and reference solution, respectively inject them into the HPLC-MS / MS instrument, measure, and calculate according to the standard curve method to obtain the N-nitrosoambroxol content in the preparation containing ambroxol.

[0014] In some embodiments of the present invention, the concentrations of the linear reference solution prepared in step 2 are 0.6 ng / mL, 1.25 ng / mL, 2.5 ng / mL, 3.75 ng / mL and 5 ng / mL respectively; The content of N-nitrosoambroxol in ambroxol-containing preparations should be less than or equal to 5 ng / mL.

[0015] In some embodiments of the present invention, the ambroxol-containing preparation is ambroxol oral solution, and the ambroxol salt raw material is ambroxol hydrochloride.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The method of the invention adopts LC-MS / MS to detect the preparations and raw materials containing ambroxol, has high sensitivity, good accuracy and strong reproducibility, and is suitable for quality control of trace nitrosamine impurities in the preparations and raw materials.

[0017] The method of the present invention has high sensitivity, with a quantitative limit of 0.59ng / ml and a detection limit of 0.2ng / ml, which meets the needs of trace detection. The method of the present invention is highly resistant to interference and eliminates matrix interference through gradient elution and valve switching technology; the total analysis time is 10 minutes, which improves detection efficiency. The method of the present invention has been systematically verified, covering key parameters such as specificity, linearity, precision, quantitative limit, detection limit, accuracy, and durability to ensure the reliability of the results. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Attached Figure 1 is the chromatogram of blank solvent; Attached Figure 2 is the chromatogram of blank excipient; Attached Figure 3 is the chromatogram of the preparation; Attached Figure 4 is the chromatogram of the API; Attached Figure 5 is the chromatogram of the reference substance; Attached Figure 6 is the control mass spectrum; Figure 6 Counts vs. Mass-to-Charge (m / z) in the table represents the response and mass-to-charge ratio; Attached Figure 7 is the standard curve graph; Attached Figure 8 This is the chromatogram of the test solution spiked with 100% of the preparation; Attached Fig. 9 This is the chromatogram of the 100% spiked test solution of the API.

[0019] Figure 1~Figure 5 , Figure 8~Figure 9 The corresponding Chinese meanings of the English words are: TIC (Total Ion Chromatogram): total ion current chromatogram; MRM (Multiple Reaction Monitoring): multiple reaction monitoring; Counts: responses; N-yaxiaoi-anxiusuo: N-nitrosoambroxol; weightEqual: weight. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0021] The bulk drug described in the embodiment of the present invention is ambroxol hydrochloride.

[0022] The instruments used in the embodiments of the present invention are as follows: A high performance liquid chromatography-tandem mass spectrometer (Agilent 1290-6470 LC / TQ) was used, equipped with an electrospray ionization (ESI) source, a reversed-phase chromatography separation mode, and a triple quadrupole tandem mass spectrometer as the detector.

[0023] Example 1 This example discloses a methodological investigation of the present invention.

[0024] 1. Chromatographic conditions The chromatographic column was Eclipse Plus C18, 2.1*50mm, 1.8μm, with 0.1% formic acid as mobile phase A and acetonitrile as mobile phase B, and the gradient elution was as follows: 0 min, 75% A, 25% B; 3min, 75% A, 25% B; 6min, 30% A, 70% B; 8min, 30%A, 70%B.

[0025] The mobile phase flow rate was 0.3 ml / min, the column temperature was 30 °C, and the injection volume was 10 μL.

[0026] 2. Mass spectrometry conditions The mass spectrometer was used in electrospray positive ion mode for multiple reaction detection, and m / z377.9→279.2, m / z377.9→114.3 and m / z377.9→96.3 were selected as the detection ion pairs. Valve switching: 6.26~7.30 min to introduce into the mass spectrometer, and the rest of the time was switched to waste liquid. The mass spectrometer ion parameters are shown in the following table: Table 1 Mass spectrometry ion parameters

[0027] 3. Solution Preparation 3.1 Solvent Acetonitrile: water = 1:1 (volume ratio).

[0028] 3.2 Preparation of test solution API-test solution: weigh about 20 mg of API into a 25 ml volumetric flask, dilute to the mark with solvent, shake well, and obtain.

[0029] Preparation - Test solution: Weigh about 13.5 g of ambroterol oral solution into a 25 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0030] 3.3 Preparation of reference solution Reference substance stock solution ①: Take N-nitrosoambroxol reference substance, accurately weigh 6.485 mg, place in a 100 ml volumetric flask, add appropriate amount of solvent to dissolve and dilute to the scale, and shake well.

[0031] Reference substance stock solution ②: Accurately measure 0.2 ml of reference substance stock solution ① and place it in a 100 ml volumetric flask, add solvent to dilute to the scale, and shake well.

[0032] Standard curve solution 1: quantitation limit concentration.

[0033] Standard curve solution 2: Accurately measure 0.25 ml of reference stock solution ② and place it in a 25 ml volumetric flask, add solvent to dilute to the scale, and shake well.

[0034] Standard curve solution 3: Accurately measure 0.50 ml of reference stock solution ② and place it in a 25 ml volumetric flask, add solvent to dilute to the scale, and shake well.

[0035] Standard curve solution 4: Accurately measure 0.75 ml of reference stock solution ② and place it in a 25 ml volumetric flask, add solvent to dilute to the scale, and shake well.

[0036] Standard curve solution 5: Accurately measure 1.00 ml of reference stock solution ② and place it in a 25 ml volumetric flask, add solvent to dilute to the scale, and shake well.

[0037] 3.4 Preparation of blank excipient solution: Accurately weigh appropriate amounts of excipients except ambroxol hydrochloride according to the prescription, put 5 g of blank excipient solution prepared according to the process requirements into a 10 ml volumetric flask, dilute to the scale with solvent, shake well, and obtain.

[0038] 4. Testing 4.1 Specificity test Take the solvent, reference solution (standard curve solution 3), raw material drug-test solution, preparation-test solution, and blank excipient solution, and inject them according to the chromatographic conditions and mass spectrometry conditions of this example, record the chromatogram, and the results are as shown in the attached Figure 1 ~Attached Figure 6 The results show that the blank solution (solvent) and the blank excipient solution have no interference at the target impurity peak, and the method of the present invention has good specificity.

[0039] 4.2 Limit of detection and limit of quantification Take the N-nitrosoambroxol reference solution of known concentration and dilute it step by step. The detection limit and quantification limit are respectively taken when the signal-to-noise ratio (S / N) is 3:1 and 10:1. The quantification limit solution is injected 6 times, and the detection limit solution is injected 3 times. The detection limit test results are shown in Table 2, and the quantification limit test results are shown in Table 3.

[0040] Table 2 Detection limit test results

[0041] It can be seen from the above table that the detection limit concentration of the raw material drug is 0.2ppm, and the detection limit of the preparation is 0.2ng / ml; the signal-to-noise ratio is greater than 3, which meets the requirements.

[0042] Table 3 Quantitation limit detection results

[0043] It can be seen from the above table that the quantitative limit concentration of the raw material is 0.75ppm, and the quantitative limit concentration of the preparation is 0.59ng / ml, both of which are less than the impurity limit concentration of 25% (raw material: 0.82ppm, preparation: 1.25ng / ml); the RSD of the peak area is 6.34%, less than 20.0%, and the signal-to-noise ratio is greater than 10, which meets the requirements.

[0044] 4.3 Linear range investigation The N-nitrosoambroxol reference substance was subjected to a linearity study, covering the quantitative limit concentration to 200% of the limit concentration for linear relationship investigation. The specific solutions are standard curve solution 1, standard curve solution 2, standard curve solution 3, standard curve solution 4 and standard curve solution 5 under the item "3.3 Preparation of reference substance solution" of this embodiment. The above solutions were accurately measured, and samples were injected according to the chromatographic conditions and mass spectrometry conditions of this embodiment, and the chromatograms were recorded. The results are shown in Table 4 and Attached Figure 7 shown.

[0045] Table 4 Results of linearity test of N-nitrosoambroxol

[0046] The linear equation is Y=35.651064X+2.911145 (r =0.9980), and the correlation coefficient r is greater than 0.990, indicating that the concentration of the reference solution has a good linear relationship in the range of 0.60ng / ml to 5.14ng / ml, the intercept is 2.911145, which is much smaller than the 25% peak area of ​​the limit concentration (23), and the residual sum of squares is 67.

[0047] 4.4 Accuracy test The accuracy of N-nitrosoambroxol content was examined by recovery rate, and the verification range was limit concentration R1-quantification limit, R2-50%, R3-100%, R4-200%. Three replicates were prepared for each concentration.

[0048] 4.4.1 Accuracy assessment of API (1) API background solution: Weigh approximately 20 mg of API into a 25 ml volumetric flask, dilute to volume with solvent, and shake well.

[0049] (2) API R1 - quantitation limit solution: Weigh about 20 mg of API into a 25 ml volumetric flask, add 0.12 ml of reference substance stock solution ② to make the quantitation limit concentration, add solvent to dilute to the scale, shake well, and the solution is ready; prepare 3 copies in parallel.

[0050] (2) API R2-50% solution: weigh about 20 mg of API into a 25 ml volumetric flask, add 0.25 ml of reference substance stock solution ②, dilute to the mark with solvent, shake well, and prepare 3 portions in parallel.

[0051] (3) API R3-100% solution: weigh about 20 mg of API into a 25 ml volumetric flask, add 0.50 ml of reference substance stock solution ②, dilute to the mark with solvent, shake well, and prepare 3 portions in parallel.

[0052] (4) API R4-200% solution: weigh about 20 mg of API into a 25 ml volumetric flask, add 1.00 ml of reference substance stock solution ②, dilute to the mark with solvent, shake well, and prepare 3 portions in parallel.

[0053] The above solution was accurately measured and injected according to the chromatographic conditions and mass spectrometry conditions of this embodiment, and the chromatogram was recorded. The results are shown in Table 5.

[0054] Table 5 Results of investigation on recovery rate of raw materials

[0055] As can be seen from the above table, the recovery rates of each limit concentration of the raw material drug are 101.45% to 125.06%, all between 70.0% and 130.0%; the RSD value is 8.15%, less than 20.0%. The method of the present invention has a good recovery rate and good accuracy.

[0056] 4.4.2 Accuracy of preparations (1) Preparation background solution: Accurately measure 12.5 ml of ambroterol oral solution and place it in a 25 ml volumetric flask. Add solvent to dilute to the mark and shake well.

[0057] (2) Preparation R1 - Quantitative limit solution: Accurately measure 12.5 ml of ambroterol oral solution and place it in a 25 ml volumetric flask. Add 0.12 ml of reference substance stock solution ② to make the quantitative limit concentration. Add solvent to dilute to the scale and shake well. Prepare 3 copies in parallel.

[0058] (3) Preparation R2-50% solution: Accurately measure 12.5 ml of ambroterol oral solution and place it in a 25 ml volumetric flask. Add 0.25 ml of reference substance stock solution ②, dilute to the mark with solvent, shake well, and prepare 3 portions in parallel.

[0059] (4) Preparation R3-100% solution: Accurately measure 12.5 ml of ambroterol oral solution and place it in a 25 ml volumetric bottle. Add 0.50 ml of reference substance stock solution ②, dilute to the mark with solvent, shake well, and prepare 3 copies in parallel.

[0060] (5) Preparation R4-200% solution: Accurately measure 12.5 ml of ambroterol oral solution and place it in a 25 ml volumetric bottle. Add 1.00 ml of reference substance stock solution ②, dilute to the mark with solvent, shake well, and prepare 3 copies in parallel.

[0061] The above solution was accurately measured and injected according to the chromatographic conditions and mass spectrometry conditions of this embodiment, and the chromatogram was recorded. The results are shown in Table 6.

[0062] Table 6 Results of the recovery rate study of the preparations

[0063] As can be seen from the above table, the recovery rates of each limit concentration of the preparation are 76.30% to 120.01%, all between 70.0% and 130.0%; the RSD value is 12.08%, less than 20.0%. The method of the present invention has a good recovery rate and good accuracy.

[0064] 4.5 Precision investigation 4.5.1 Repeatability test Six portions of 100% spiked test solution of the bulk drug and 100% spiked test solution of the preparation were prepared in parallel, and the samples were injected according to the chromatographic conditions and mass spectrometry conditions of this embodiment, the chromatograms were recorded, and the recovery RSD was calculated. The results are shown in Table 7 and Attached Figure 8 , Attachment Fig. 9 shown.

[0065] Table 7 Results of repeatability test

[0066] As can be seen from the above table, the reproducibility of the 6 test solutions of the raw material drug is 101.45% to 129.19%, all between 70.0% and 130.0%; the RSD value is 8.60%, less than 20.0%. The reproducibility of the 6 test solutions of the preparation is 88.46% to 105.66%, all between 70.0% and 130.0%; the RSD value is 6.62%, less than 20.0%. The method of the present invention has good reproducibility.

[0067] 4.5.2 Intermediate precision test Different personnel prepared 6 samples according to the operation procedures of spiked test solution and reference substance described under repeatability test and conducted the test. The results are as follows: Table 8 Intermediate precision inspection results

[0068] As can be seen from the above table, the recovery rates of the intermediate precision test solutions of 12 portions of the raw material drug were 94.55% to 129.19%, all between 70.0% and 130.0%; the RSD value was 9.63%, less than 20.0%. The recovery rates of the intermediate precision test solutions of 12 portions of the preparation were 88.46% to 124.92%, all between 70.0% and 130.0%; the RSD value was 9.23%, less than 20.0%. The intermediate precision of the method of the present invention is good.

[0069] 4.6 Solution stability The reference solution, the test solution, and the 100% spiked test solution that were placed at room temperature for 10 hours were sampled and tested at different time points to investigate the stability of the solution. The results are shown in Table 9.

[0070] Table 9 Solution stability test results

[0071] It can be seen from the table above that within 10 hours of the reference solution, the ratio of N-nitrosoambroxol concentration is in the range of 70% to 130%, indicating that the reference solution is stable within 10 hours; Within 10 hours of the test solution being placed, the ratio of N-nitrosoambroxol concentrations was in the range of 70% to 130%, indicating that the test solution was stable within 10 hours; Within 10 hours of the spiked test solution, the ratio of N-nitrosoambroxol concentration was in the range of 70%~130%, indicating that the spiked test solution was stable within 10 hours.

[0072] Example 2 This embodiment discloses the setting of the limit of the content of N-nitrosoambroxol in the ambroxol hydrochloride bulk drug and the ambroxol oral solution of the present invention.

[0073] According to the "Recommended Acceptable Intake Limits for Nitrosamine Drug Substance-Related Impurities (NDSRIs) Guidance for Industry (August 2023)" issued by the FDA, the activity level of ambroxol hydrochloride is determined in combination with its chemical structure, thereby determining its AI value. The AI ​​value (Acceptable Intake) refers to the daily exposure corresponding to an additional cancer risk of 1 case per 100,000 people under lifetime exposure (assuming 70 years). The specific analysis is as follows: Table 10 N-nitrosoambroxol activity index analysis table

[0074] Activity index = α-hydrogen index + deactivation characteristics + activation characteristics = 3 + 1-1 = 3.

[0075] The activity index of N-nitrosoambroxol was determined to be 3 through the chemical structure of NDSRI. According to the decision tree in the guidelines, the activity level was confirmed to be 3, so its AI value was determined to be 400 ng / day.

[0076] The maximum daily dose of ambroxol oral solution is 80 ml (derived from the instructions for use of the reference preparation of ambroxol oral solution, Spasmo-Mucosolvan®), which is equivalent to 120 mg of the raw material. Therefore, the content of N-nitrosoambroxol in the ambroxol hydrochloride raw material should be less than or equal to 3.3 ppm; the content of N-nitrosoambroxol in ambroxol-containing preparations should be less than or equal to 5 ng / mL.

[0077] The embodiments described above are part of the embodiments of the present invention, rather than all of the embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Claims

1. A method for detecting N-nitrosoambroxol in medicine by LC-MS / MS, characterized in that, The drug is an ambroxol preparation, an ambroxol raw material, or an ambroxol salt raw material. The liquid chromatography conditions include: using octadecylsilane bonded silica gel as filler, 0.05-0.2% formic acid as mobile phase A, acetonitrile as mobile phase B, and gradient elution as follows: 0min, 70~80%A, 20~30%B; 3min, 70~80%A, 20~30%B; 6min, 25~35%A, 65~75%B; 8min, 25~35%A, 65~75%B; The mass spectrometry conditions include: using a mass spectrometer detector in electrospray positive ion mode, performing multiple reaction detection, and selecting m / z 377.9→279.2, m / z 377.9→114.3, and m / z 377.9→96.3 as detection ion pairs.

2. The method for detecting N-nitrosoambroxol in a drug by LC-MS / MS according to claim 1, characterized in that: Gradient elution was performed with 0.1% formic acid as mobile phase A and acetonitrile as mobile phase B.

3. The method for detecting N-nitrosoambroxol in a drug by LC-MS / MS according to claim 1, characterized in that: The gradient elution program is as follows: 0 min, 75% A, 25% B; 3min, 75% A, 25% B; 6min, 30% A, 70% B; 8min, 30%A, 70%B.

4. The method for detecting N-nitrosoambroxol in medicine by LC-MS / MS according to claim 1, characterized in that: The column temperature was 28-32°C, and the mobile phase flow rate was 0.25-0.35 ml / min.

5. The method for detecting N-nitrosoambroxol in medicine by LC-MS / MS according to claim 4, characterized in that: The column temperature was 30°C and the mobile phase flow rate was 0.3 ml / min.

6. The method for detecting N-nitrosoambroxol in medicine by LC-MS / MS according to claim 1, characterized in that: The mass spectrometry acquisition time was 6.0~7.5min.

7. A method for detecting N-nitrosoambroxol in a drug by LC-MS / MS according to any one of claims 1 to 6, characterized in that: When the drug is ambroxol or its salt raw material drug, the method comprises the following steps: S1. Preparation of test solution: Take an appropriate amount of ambroxol or its salt raw material drug, accurately weigh it, add an appropriate amount of solvent to dissolve it and quantitatively dilute it to make a solution with a concentration of 0.7~0.9mg / ml as the raw material drug-test solution; S2. Preparation of reference solution: Take N-nitrosoambroxol reference substance and add solvent to prepare a series of linear reference solutions with a mass concentration gradient of 0.6~5ng / mL; the concentration gradient of the series of linear reference solutions should be at least 5; The solvents described in S1 and S2 are both mixtures of acetonitrile and water, with a volume ratio of 1:0.8~1.2; S3. Test: Accurately measure blank solvent, API-test solution and reference solution, respectively inject them into HPLC-MS / MS instrument, measure, record chromatogram, calculate according to standard curve method, and obtain the content of N-nitrosoambroxol in ambroxol or its salt API.

8. The method for detecting N-nitrosoambroxol in a drug by LC-MS / MS according to claim 7, characterized in that: The concentrations of the linear reference solutions prepared in S2 were 0.6 ng / mL, 1.25 ng / mL, 2.5 ng / mL, 3.75 ng / mL, and 5 ng / mL; The injection volume of S3 is 5~20μl; The content of N-nitrosoambroxol in ambroxol or its salt raw materials should be less than or equal to 3.3ppm.

9. A method for detecting N-nitrosoambroxol in a drug by LC-MS / MS according to any one of claims 1 to 6, characterized in that: When the drug is an ambroxol-containing preparation, the method comprises the following steps: Step 1. Preparation of the test solution: Take an appropriate amount of the preparation containing ambroxol, accurately weigh it, add an appropriate amount of solvent to dissolve it, and quantitatively dilute it to make a solution containing 0.70-0.80 mg of ambroxol hydrochloride per 1 ml, as the preparation-test solution; Step 2. Preparation of reference solution: Take N-nitrosoambroxol reference substance and add solvent to prepare a series of linear reference solutions with a mass concentration gradient of 0.6-5 ng / mL; the concentration gradient of the series of linear reference solutions is at least 5; The solvents described in S1 and S2 are both mixtures of acetonitrile and water, with a volume ratio of 1:0.8~1.2; Step 3. Test: Accurately measure the blank solvent, preparation-test solution and reference solution, respectively inject them into the HPLC-MS / MS instrument, measure, and calculate according to the standard curve method to obtain the N-nitrosoambroxol content in the preparation containing ambroxol.

10. The method for detecting N-nitrosoambroxol in drugs by LC-MS / MS according to claim 9, characterized in that: The concentrations of the linear reference solutions prepared in step 2 were 0.6 ng / mL, 1.25 ng / mL, 2.5 ng / mL, 3.75 ng / mL, and 5 ng / mL, respectively; The content of N-nitrosoambroxol in ambroxol-containing preparations should be less than or equal to 5 ng / mL.

Images