A Method for Detecting N-Nitrosol Ambroxol in Drugs by LC-MS / MS

Abstract

The present invention discloses a method for detecting N-nitrosoambroxol in drugs by LC-MS / MS, belonging to the technical field of pharmaceutical analysis. The method for detecting N-nitrosoambroxol in drugs by LC-MS / MS disclosed by the present invention, the drug is an ambroxol-containing preparation or ambroxol and its salt raw material drugs, and the liquid chromatography conditions include: using octadecylsilane-bonded silica gel as the filler, using 0.05-0.2% formic acid as mobile phase A, and gradient elution is carried out with acetonitrile as mobile phase B; the mass spectrometry conditions include: using a mass spectrometry detector, in the positive electrospray ionization mode, multiple reaction monitoring is carried out, and m / z377.9→279.2, m / z377.9→114.3 and m / z377.9→96.3 are selected as the detection ion pairs. The method for detecting ambroxol-containing preparations and raw materials by LC-MS / MS adopted by the present invention has high sensitivity and good accuracy, and is suitable for the quality control of trace nitrosamine impurities in preparations and raw materials.

Description

Technical Field

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

[0002] During the production and storage of ambroxol-containing preparations in raw materials and preparations, the nitrosamine impurity N-nitrosoambroxol (NDSRI) may be generated. Nitrosamine impurities are a class of compounds with a chemical structure in which a nitroso group is combined with an amine, and have high potential mutagenicity and carcinogenicity. Nitrosamine impurities belong to the "concern cohort" substances mentioned in the ICH M7 (R1) (Assessment and Control of DNA-Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk) guideline. According to the carcinogen list published by the World Health Organization, both N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) belong to Group 2A carcinogens; according to the internationally recognized database, there are already public carcinogenicity data for some nitrosamine impurities, 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 uses gas chromatography or high performance liquid chromatography. However, these two methods have limitations in terms of sensitivity, specificity, and anti-interference ability, and it is difficult to meet the detection requirements of trace nitrosamine impurities. The specific defects are as follows: (1) Insufficient sensitivity: The detection limits (LOD) and quantification limits (LOQ) of conventional methods for detecting low-concentration nitrosamine impurities are relatively high, and it is impossible to meet the trace detection requirements at the ppm or ppb level. (2) Poor specificity: In complex matrices (such as excipients in oral solutions), impurities may interfere with the identification of target peaks, resulting in false positive or false negative results. (3) Long analysis time: The elution program of traditional chromatography methods is complex and time-consuming, which affects the detection efficiency. These limitations make it difficult for the prior art to effectively meet the precise detection requirements of trace nitrosamine impurities.

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

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

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] A method for detecting N-nitroso ambroxol in drugs by LC-MS / MS according to the present invention, wherein the drug is an ambroxol-containing preparation, or ambroxol raw material drug, or a salt raw material drug of ambroxol.

[0008] The liquid chromatography conditions include: using octadecylsilane-bonded silica gel as the filler, using 0.05-0.2% formic acid as mobile phase A and acetonitrile as mobile phase B, and performing gradient elution according to the following regulations:

[0009] At 0 min, 70-80% A and 20-30% B;

[0010] At 3 min, 70-80% A and 20-30% B;

[0011] At 6 min, 25-35% A and 65-75% B;

[0012] At 8 min, 25-35% A and 65-75% B;

[0013] The mass spectrometry conditions include: using a mass spectrometry detector, in the positive electrospray ionization mode, performing multiple reaction monitoring, and selecting m / z377.9→279.2, m / z377.9→114.3, and m / z377.9→96.3 as the detected ion pairs.

[0014] 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.

[0015] In some embodiments of the present invention, the gradient elution program is as follows:

[0016] At 0 min, 75% A and 25% B;

[0017] At 3 min, 75% A and 25% B;

[0018] At 6 min, 30% A and 70% B;

[0019] At 8 min, 30% A and 70% B.

[0020] 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.

[0021] 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.

[0022] In some embodiments of the present invention, when the drug is ambroxol or its salt raw material drug, the method includes the following steps:

[0023] S1. Preparation of the test solution: Weigh an appropriate amount of ambroxol or its salt raw material accurately, dissolve it in an appropriate amount of solvent and quantitatively dilute to prepare a solution with a concentration of 0.7 - 0.9 mg / ml as the raw material - test solution.

[0024] S2. Preparation of the reference solution: Take N - nitrosambroxol reference substance, and prepare a series of linear reference solutions with mass concentration gradients of 0.6 - 5 ng / mL using the solvent; the concentration gradients of the series of linear reference solutions are at least 5.

[0025] The solvents described in S1 and S2 are both mixtures of acetonitrile and water with a volume ratio of 1:0.8 - 1.2.

[0026] S3. Testing: Accurately measure the blank solvent, the raw material - test solution and the reference solution, inject them into a high - performance liquid chromatography - mass spectrometry instrument respectively for determination, record the chromatogram, and calculate by the standard curve method to obtain the content of N - nitrosambroxol in the ambroxol or its salt raw material.

[0027] In some embodiments of the present invention, the concentrations of the linear reference solutions prepared in S2 are 0.6 ng / mL, 1.25 ng / mL, 2.5 ng / mL, 3.75 ng / mL and 5 ng / mL respectively.

[0028] The injection volume in S3 is 5 - 20 μl.

[0029] The content of N - nitrosambroxol in ambroxol hydrochloride raw material should be less than or equal to 3.3 ppm.

[0030] In some embodiments of the present invention, when the drug is an ambroxol - containing preparation, the method includes the following steps:

[0031] Step 1. Preparation of the test solution: Take an appropriate amount of the ambroxol - containing preparation, weigh it accurately, dissolve it in an appropriate amount of solvent and quantitatively dilute to prepare a solution containing 0.70 - 0.80 mg of ambroxol hydrochloride per 1 ml as the preparation - test solution.

[0032] Step 2. Preparation of the reference solution: Take N - nitrosambroxol reference substance, and prepare a series of linear reference solutions with mass concentration gradients of 0.6 - 5 ng / mL using the solvent; the concentration gradients of the series of linear reference solutions are at least 5.

[0033] The solvents described in S1 and S2 are both mixtures of acetonitrile and water with a volume ratio of 1:0.8 - 1.2.

[0034] 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.

[0035] 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;

[0036] The content of N-nitrosoambroxol in ambroxol-containing preparations should be less than or equal to 5 ng / mL.

[0037] In some embodiments of the present invention, the ambroxol-containing preparation is ambroxol oral solution, and the ambroxol salt raw material is ambroxol hydrochloride.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] 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.

[0040] 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

[0041] Attached Figure 1 is the chromatogram of blank solvent;

[0042] Attached Figure 2 is the chromatogram of blank excipient;

[0043] Attached Figure 3 is the chromatogram of the preparation;

[0044] Attached Figure 4 is the chromatogram of the API;

[0045] Attached Figure 5 is the chromatogram of the reference substance;

[0046] 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;

[0047] Attached Figure 7 is the standard curve graph;

[0048] Attached Figure 8 is the chromatogram of the 100% spiked test sample solution of the preparation;

[0049] Attached Figure 9 is the chromatogram of the 100% spiked test sample solution of the active pharmaceutical ingredient.

[0050] Figures 1 to 5 and Figures 8 to 9 The corresponding Chinese meanings of the English in are: TIC (Total Ion Chromatogram): Total Ion Chromatogram; MRM (Multiple Reaction Monitoring): Multiple Reaction Monitoring; Counts: Response; N-yaxiaoi-anxiusuo: N-nitrosol Ambroxol; weightEqual: Weight. Detailed implementation manners

[0051] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those conditions not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For the reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained by purchasing in the market.

[0052] The active pharmaceutical ingredient described in the embodiments of the present invention is Ambroxol Hydrochloride.

[0053] The instruments used in the embodiments of the present invention are as follows:

[0054] High performance liquid chromatography-tandem mass spectrometer (Agilent 1290-6470 LC / TQ), equipped with an electrospray (ESI) ionization source, the liquid chromatography separation mode is reverse phase chromatography separation, and the detector is a triple quadrupole tandem mass spectrometer.

[0055] Example 1

[0056] This example discloses the method investigation of the present invention.

[0057] 1. Chromatographic conditions

[0058] The chromatographic column is Eclipse Plus C18, 2.1*50mm, 1.8μm, 0.1% formic acid is used as mobile phase A, and acetonitrile is used as mobile phase B, and the gradient elution is carried out according to the following regulations:

[0059] 0 min, 75% A, 25% B;

[0060] 3 min, 75% A, 25% B;

[0061] 6 min, 30% A, 70% B;

[0062] 8 min, 30% A, 70% B.

[0063] The mobile phase flow rate was 0.3 ml / min, the column temperature was 30 °C, and the injection volume was 10 μL.

[0064] 2. Mass spectrometry conditions

[0065] A mass spectrometry detector was used, in the electrospray positive ion mode, for multiple reaction monitoring. The detection ion pairs were selected as m / z 377.9 → 279.2, m / z 377.9 → 114.3, and m / z 377.9 → 96.3. Valve switching: Introduce into the mass spectrometry from 6.26 - 7.30 min, and switch to the waste liquid for the rest of the time. The mass spectrometry ion parameters are shown in the following table:

[0066] Table 1 Mass spectrometry ion parameters

[0067]

[0068] 3. Solution preparation

[0069] 3.1 Solvent

[0070] Acetonitrile: water = 1:1 (volume ratio).

[0071] 3.2 Preparation of test solution

[0072] API - test solution: Weigh about 20 mg of the active pharmaceutical ingredient and place it in a 25 - ml volumetric flask. Dilute to the mark with the solvent and shake well to obtain.

[0073] Formulation - test solution: Weigh about 13.5 g of ambroxol hydrochloride oral solution and place it in a 25 - ml volumetric flask. Dilute to the mark with the solvent and shake well to obtain.

[0074] 3.3 Preparation of reference solution

[0075] Reference stock solution ①: Take the reference substance of N - nitrosambroxol, accurately weigh it to be 6.485 mg, place it in a 100 - ml volumetric flask, add an appropriate amount of the solvent to dissolve it and dilute to the mark, and shake well.

[0076] Reference stock solution ②: Accurately measure 0.2 ml of reference stock solution ① and place it in a 100 - ml volumetric flask. Dilute to the mark with the solvent and shake well.

[0077] Standard curve solution 1: Quantitation limit concentration.

[0078] Standard curve solution 2: Accurately measure 0.25 ml of reference stock solution ② and place it in a 25 - ml volumetric flask. Dilute to the mark with the solvent and shake well.

[0079] Standard curve solution 3: Accurately measure 0.50 ml of reference stock solution ②, place it in a 25-ml volumetric flask, dilute it to the mark with solvent, and shake well.

[0080] Standard curve solution 4: Accurately measure 0.75 ml of reference stock solution ②, place it in a 25-ml volumetric flask, dilute it to the mark with solvent, and shake well.

[0081] Standard curve solution 5: Accurately measure 1.00 ml of reference stock solution ②, place it in a 25-ml volumetric flask, dilute it to the mark with solvent, and shake well.

[0082] 3.4 Preparation of blank excipient solution: Accurately weigh appropriate amounts of each excipient except ambroxol hydrochloride according to the prescription, place 5 g of the blank excipient solution prepared according to the process requirements in a 10-ml volumetric flask, dilute it to the mark with solvent, and shake well to obtain.

[0083] 4. Testing

[0084] 4.1 Specificity test

[0085] Take the solvent, reference solution (standard curve solution 3), API - test solution, formulation - test solution, and blank excipient solution, inject samples respectively according to the chromatographic conditions and mass spectrometric conditions of this example, record the chromatograms, and the results are as shown in Appendix Figure 1 ~Appendix Figure 6 shown. The results show that there is no interference in the blank solution (solvent) and the blank excipient solution at the target impurity peaks, and the specificity of the method of the present invention is good.

[0086] 4.2 Detection limit and quantitation limit

[0087] Take the N-nitroso ambroxol reference solution with known concentration and dilute it step by step. When the signal-to-noise ratio (S / N) is 3:1 and 10:1 respectively, they are used as the detection limit and quantitation limit. Among them, the quantitation limit solution is injected 6 times, and the detection limit solution is injected 3 times. The detection results of the detection limit are shown in Table 2, and the detection results of the quantitation limit are shown in Table 3.

[0088] Table 2 Detection results of detection limit

[0089]

[0090] As can be seen from the above table, the detection limit concentration of the API is 0.2 ppm, and the detection limit of the formulation is 0.2 ng / ml; the signal-to-noise ratios are all greater than 3, meeting the requirements.

[0091] Table 3 Detection results of quantitation limit

[0092]

[0093] As can be seen from the above table, the quantitation limit concentration of the bulk drug is 0.75 ppm, and the quantitation limit concentration of the preparation is 0.59 ng / ml, both of which are less than 25% of the impurity limit concentration (bulk drug: 0.82 ppm, preparation: 1.25 ng / ml); the RSD of the peak area is 6.34%, less than 20.0%, and the signal-to-noise ratio is greater than 10, meeting the requirements.

[0094] 4.3 Investigation of linear range

[0095] A linear study was conducted on the N-nitrosoambroxol reference substance, and the linear relationship was investigated from the quantitation limit concentration to 200% of the limit concentration. The specific solutions were Standard Curve Solution 1, Standard Curve Solution 2, Standard Curve Solution 3, Standard Curve Solution 4, and Standard Curve Solution 5 under "3.3 Preparation of reference substance solution" in this example. Accurately measure the above solutions and inject them respectively according to the chromatographic conditions and mass spectrometry conditions in this example, and record the chromatograms. The results are shown in Table 4 and the appendix Figure 7 as follows.

[0096] Table 4 Results of linear investigation of N-nitrosoambroxol

[0097]

[0098] The linear equation is Y = 35.651064X + 2.911145 (r = 0.9980), and the correlation coefficient r is greater than 0.990; it indicates that the linear relationship of the reference substance solution concentration is good in the range of 0.60 ng / ml to 5.14 ng / ml. The intercept is 2.911145, which is much less than 25% of the peak area of the limit concentration (23), and the sum of squared residuals is 67.

[0099] 4.4 Investigation of accuracy

[0100] The accuracy of the content of N-nitrosoambroxol was investigated by recovery rate, and the verification ranges were R1 - quantitation limit, R2 - 50%, R3 - 100%, and R4 - 200%. Three parallel samples were prepared for each concentration.

[0101] 4.4.1 Investigation of the accuracy of the bulk drug

[0102] (1) Bulk drug background solution: Weigh about 20 mg of the bulk drug and place it in a 25 ml volumetric flask, dilute it to the mark with the solvent, and shake well.

[0103] (2) Bulk drug R1 - quantitation limit solution: Weigh about 20 mg of the bulk drug and place it in a 25 ml volumetric flask, add 0.12 ml of reference substance stock solution ② to make it the quantitation limit concentration, dilute it to the mark with the solvent, and shake well. Three parallel samples were prepared.

[0104] (2)Stock solution of API R2 - 50%: Weigh approximately 20 mg of the API and place it in a 25 - ml volumetric flask. Add 0.25 ml of reference stock solution ②, dilute to the mark with the solvent, and mix well. Prepare 3 parallel samples.

[0105] (3)Stock solution of API R3 - 100%: Weigh approximately 20 mg of the API and place it in a 25 - ml volumetric flask. Add 0.50 ml of reference stock solution ②, dilute to the mark with the solvent, and mix well. Prepare 3 parallel samples.

[0106] (4)Stock solution of API R4 - 200%: Weigh approximately 20 mg of the API and place it in a 25 - ml volumetric flask. Add 1.00 ml of reference stock solution ②, dilute to the mark with the solvent, and mix well. Prepare 3 parallel samples.

[0107] Precisely measure the above - mentioned solutions, inject them respectively according to the chromatographic conditions and mass - spectrometric conditions of this example, record the chromatograms, and the results are shown in Table 5.

[0108] Table 5 Results of the recovery investigation of the API

[0109]

[0110] As can be seen from the above table, the recoveries of the API at each limit concentration are 101.45% - 125.06%, all within the range of 70.0% - 130.0%; the RSD value is 8.15%, less than 20.0%. The method of the present invention has good recoveries and good accuracy.

[0111] 4.4.2 Accuracy investigation of the preparation

[0112] (1)Stock solution of the preparation: Precisely measure 12.5 ml of ambroxol hydrochloride and terbutaline sulfate oral solution and place it in a 25 - ml volumetric flask. Dilute to the mark with the solvent and mix well.

[0113] (2)Stock solution of preparation R1 - limit of quantitation: Precisely measure 12.5 ml of ambroxol hydrochloride and terbutaline sulfate oral solution and place it in a 25 - ml volumetric flask. Add 0.12 ml of reference stock solution ② to make the concentration at the limit of quantitation, dilute to the mark with the solvent, and mix well. Prepare 3 parallel samples.

[0114] (3)Stock solution of preparation R2 - 50%: Precisely measure 12.5 ml of ambroxol hydrochloride and terbutaline sulfate oral solution and place it in a 25 - ml volumetric flask. Add 0.25 ml of reference stock solution ②, dilute to the mark with the solvent, and mix well. Prepare 3 parallel samples.

[0115] (4)Stock solution of preparation R3 - 100%: Precisely measure 12.5 ml of ambroxol hydrochloride and terbutaline sulfate oral solution and place it in a 25 - ml volumetric flask. Add 0.50 ml of reference stock solution ②, dilute to the mark with the solvent, and mix well. Prepare 3 parallel samples.

[0116] (5)Formulation R4 - 200% solution: Accurately measure 12.5 ml of ambroxol hydrochloride oral solution and place it in a 25 - ml volumetric flask. Add 1.00 ml of reference substance stock solution ②, dilute to the mark with solvent, and shake well to obtain; prepare 3 portions in parallel.

[0117] Accurately measure the above - mentioned solution, inject samples separately according to the chromatographic conditions and mass spectrometric conditions of this example, record the chromatograms, and the results are shown in Table 6.

[0118] Table 6 Results of the recovery test of the formulation

[0119]

[0120] As can be seen from the above table, the recoveries of each limit concentration of the formulation are between 76.30% and 120.01%, all within the range of 70.0% - 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.

[0121] 4.5 Precision investigation

[0122] 4.5.1 Repeatability test

[0123] Prepare 6 portions of the test sample solutions of the raw material drug with 100% spiked standard and the test sample solutions of the formulation with 100% spiked standard in parallel respectively. Inject samples separately according to the chromatographic conditions and mass spectrometric conditions of this example, record the chromatograms, and calculate the RSD of the recovery rate. The results are shown in Table 7 and Appendix Figure 8 and Appendix Figure 9 as shown.

[0124] Table 7 Results of the repeatability test

[0125]

[0126] As can be seen from the above table, the repeatability recoveries of the 6 test sample solutions of the raw material drug are between 101.45% and 129.19%, all within the range of 70.0% - 130.0%; the RSD value is 8.60%, less than 20.0%. The repeatability recoveries of the 6 test sample solutions of the formulation are between 88.46% and 105.66%, all within the range of 70.0% - 130.0%; the RSD value is 6.62%, less than 20.0%. The method of the present invention has good repeatability.

[0127] 4.5.2 Intermediate precision test

[0128] Six portions of samples were prepared by different personnel according to the operation procedures of the spiked test sample solution and the reference substance described in the repeatability test, and the tests were carried out. The results are as follows:

[0129] Table 8 Results of the intermediate precision investigation

[0130]

[0131] As can be seen from the above table, the recovery rates of the test solutions for intermediate precision of 12 portions of the bulk drug were 94.55% - 129.19%, all within the range of 70.0% - 130.0%; the RSD value was 9.63%, less than 20.0%. The recovery rates of the test solutions for intermediate precision of 12 portions of the preparation were 88.46% - 124.92%, all within the range of 70.0% - 130.0%; the RSD value was 9.23%, less than 20.0%. The method of the present invention has good intermediate precision.

[0132] 4.6 Solution Stability

[0133] The reference solution, the test solution, and the 100% spiked test solution placed at room temperature for 10 h were respectively taken and injected for detection at different time points to investigate the solution stability. The results are shown in Table 9.

[0134] Table 9 Results of the investigation on solution stability

[0135]

[0136] As can be seen from the above table, within 10 h of placing the reference solution, the ratios of the concentration of N-nitrosoambroxol were all within the range of 70% - 130%, indicating that the reference solution was stable within 10 h;

[0137] Within 10 h of placing the test solution, the ratios of the concentration of N-nitrosoambroxol were all within the range of 70% - 130%, indicating that the test solution was stable within 10 h;

[0138] Within 10 h of placing the spiked test solution, the ratios of the concentration of N-nitrosoambroxol were all within the range of 70% - 130%, indicating that the spiked test solution was stable within 10 h.

[0139] Example 2

[0140] This example discloses the setting of the limit for the content of N-nitrosoambroxol in the bulk drug of ambroxol hydrochloride and ambroxol oral solution of the present invention.

[0141] According to the "Guidance for Industry on Recommended Acceptable Intake Limits for Nitrosamine Drug Substance-Related Impurities (NDSRIs)" (August 2023) issued by the FDA [Recommended Acceptable Intake Limits for Nitrosamine Drug Substance-Related Impurities (NDSRIs) Guidance for Industry (August 2023)], the activity level of ambroxol hydrochloride is determined based on its chemical structure, and thus its AI value is determined. The AI value (Acceptable Intake) refers to the daily exposure amount corresponding to an additional cancer risk of 1 case per 100,000 people under lifetime exposure (assumed to be 70 years). The specific analysis is as follows:

[0142] Table 10 Analysis Table of N-Nitroso Ambroxol Activity Index

[0143]

[0144] Activity Index = α-Hydrogen Index + Deactivation Characteristics + Activation Characteristics = 3 + 1 - 1 = 3.

[0145] The activity index of N-nitroso ambroxol is determined to be 3 through the chemical structure of NDSRI. According to the decision tree in the guideline, the activity level is confirmed to be 3, so its AI value is determined to be 400 ng / day.

[0146] The maximum daily dose of ambroxol hydrochloride oral solution is 80 ml (derived from the instructions of the reference preparation Spasmo-Mucosolvan® of ambroxol hydrochloride oral solution), which is equivalent to 120 mg of the active ingredient. Therefore, the content of N-nitroso ambroxol in ambroxol hydrochloride raw material should be less than or equal to 3.3 ppm; the content of N-nitroso ambroxol in ambroxol-containing preparations should be less than or equal to 5 ng / mL.

[0147] The embodiments described above are some, but not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the present invention claimed, but merely represents selected embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall 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 or an ambroxol raw material 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.

2. The method for detecting N-nitrosoambroxol in a drug by LC-MS / MS according to claim 1, wherein: 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 raw material, the method comprises the following steps: S1. Preparation of test solution: Take an appropriate amount of ambroxol or API, weigh accurately, add an appropriate amount of solvent to dissolve and quantitatively dilute to make a solution with a concentration of 0.7~0.9mg / ml as API-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 into HPLC-MS / MS, measure, record chromatogram, calculate according to standard curve method, and obtain the content of N-nitrosoambroxol in ambroxol 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 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.

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