A method for detecting the content of a nitrosamine impurity N-nitroso-N-decyclohexyl bromohexane

Abstract

The application discloses a detection method of a nitrosamine impurity N-nitroso-N-decyclohexane bromohexane hexene content, relates to the technical field of drug analysis, and comprises the following steps: dissolving a bromhexine hydrochloride sample with a diluent to prepare a test sample solution; and adopting liquid chromatography-mass spectrometry to detect the test sample solution; the diluent comprises one or more of methanol, ethanol, acetonitrile or an acetonitrile aqueous solution, and the detection method has the characteristics of high accuracy, precision, sensitivity, linearity, specificity and system adaptability.

Description

Technical Field

[0001] This invention relates to the technical field of pharmaceutical analysis, specifically to a method for detecting the content of N-nitroso-N-decyclohexane bromide, a potential nitrosamine impurity, in bromhexine hydrochloride. Background Technology

[0002] Bromhexine hydrochloride, chemically named N-(2-amino-3,5-dibromobenzyl)-N-methylcyclohexylamine hydrochloride, has the molecular formula C1. 14 H 20 Br2N2·HCl molecular weight: 412.60, CAS number: 611-75-6, its structural formula is shown in the following compound formula:

[0003]

[0004] Bromhexine hydrochloride is used for acute and chronic bronchitis, asthma, bronchiectasis, and emphysema. It acts directly on bronchial glands, stimulating the release of lysosomes from mucus-secreting cells, causing the differentiation and breakdown of mucosal glycofibrils in sputum. It also inhibits the synthesis of acidic glycoproteins in mucus glands and goblet cells, leading to the secretion of smaller, less viscous glycoproteins, thus reducing sputum viscosity and making it easier to cough up. During the synthesis of bromhexine hydrochloride, N-methylcyclohexylamine may introduce impurities such as cyclohexylamine and methylamine, potentially resulting in the formation of the nitrosamine impurity N-nitroso-N-decyclohexanebromhexine. The structural formula of this impurity is as follows: . Summary of the Invention

[0005] To control the content of the aforementioned nitrosamine impurities in the active pharmaceutical ingredient bromhexine hydrochloride, ensure the quality of bromhexine hydrochloride, and guarantee the safety of patients, this invention provides a novel method for detecting potential nitrosamine impurities in bromhexine hydrochloride.

[0006] The main objective of this invention is to overcome the problems existing in the prior art and provide a method for determining the content of nitrosamine impurities in bromhexine hydrochloride. The method uses liquid chromatography-mass spectrometry (LC-MS) to determine the content of N-nitroso-N-decyclohexane bromhexine, which has high accuracy, precision, and sensitivity, and good linearity, specificity, and system adaptability.

[0007] The technical solution provided by this invention is as follows:

[0008] A method for determining the content of N-nitroso-N-decyclohexane bromide, a nitrosamine impurity, includes the following steps:

[0009] Step 1: Weigh approximately bromhexine hydrochloride raw material into a volumetric flask, add diluent to dissolve and dilute to the mark to obtain the test solution; wherein, the diluent includes any one or more of methanol, ethanol and acetonitrile.

[0010] The second step is to perform liquid chromatography-mass spectrometry (LC-MS) analysis on the test solution, and calculate the content of N-nitroso-N-decyclohexane bromhexine in the bromhexine hydrochloride raw material based on the test results. The detection conditions for LC-MS analysis include high performance liquid chromatography (HPLC) parameters. In the HPLC parameters, a CS-C18 HPLC column is used, and the mobile phase consists of an aqueous solution of formic acid and an acetonitrile solution.

[0011] This method can effectively extract N-nitroso-N-decyclohexane bromide, a low-content nitrosamine impurity, from bromide hydrochloride and perform quantitative detection using liquid chromatography-mass spectrometry (LC-MS), thereby achieving the determination of the content of this nitrosamine impurity. It has the advantages of high accuracy and high separation.

[0012] Preferably, in the first step, the diluent is methanol, ethanol, and acetonitrile.

[0013] More preferably, the diluent is methanol.

[0014] By adopting the above preferred scheme, the diluent used in the first step can be further optimized, thereby more effectively extracting the nitrosamine impurity N-nitroso-N-decyclohexanebromohexene, reducing interfering components, and improving detection accuracy.

[0015] Preferably, in the first step, during dissolution, the ratio of the sample to the diluent, calculated as bromhexine hydrochloride, is 2 mg: 5-20 mL.

[0016] Preferably, the specific process of the first step is as follows:

[0017] Accurately weigh bromhexine hydrochloride raw material and place it in a volumetric flask. Accurately measure the predetermined amount of diluent and add it to the volumetric flask, then dilute to the mark. Shake well to obtain the test solution.

[0018] By adopting the above preferred scheme, the specific technical details of the first step can be further optimized to achieve better extraction results.

[0019] Preferably, in the second step, the high-performance liquid chromatography (HPLC) parameters are as follows: the HPLC instrument is used; the column has an inner diameter of 4.6 mm, a length of 100 mm or more, and a particle size of 2.7 μm; the mobile phase consists of an aqueous solution of formic acid with a volume fraction of 0.5 ± 0.2% and an acetonitrile solution in a volume ratio of 70–60:30–40.

[0020] More preferably, in the high performance liquid chromatography parameters, the column type is Agilent Poroshell 120CS-C18, 4.6×100mm, 2.7μm; the flow rate is 0.4~0.6ml / min, the column temperature is 30℃~45℃, and the injection volume is 4~10μl.

[0021] By adopting the above preferred scheme, the high-performance liquid chromatography parameters in the second step can be further optimized, the detection resolution and accuracy can be further improved, and better detection results can be achieved.

[0022] Preferably, in the second step, the detection conditions for liquid chromatography-mass spectrometry (LC-MS) detection also include mass spectrometry parameters; in the mass spectrometry parameters, the ion source is an ESI ion source with positive polarity, the ion source temperature is 250℃, the transfer line temperature is 280℃, the quadrupole temperature 1 is 150℃, the quadrupole temperature 2 is 150℃, and the time segment is 6-7 minutes.

[0023] By adopting the above optimized scheme, the mass spectrometry parameters in the second step can be further optimized, the detection separation and accuracy can be further improved, and better detection results can be achieved.

[0024] Preferably, the specific process of the second step is as follows:

[0025] First, N-nitroso-N-decyclohexane bromhexine reference standard was dissolved in methanol. Different volumes of reference standard solutions with a predetermined series of concentrations were prepared using 50% acetonitrile, and each solution was analyzed by liquid chromatography-mass spectrometry (LC-MS). Based on the detection results, a standard curve of concentration-peak area for N-nitroso-N-decyclohexane bromhexine was established. Then, the test solution was analyzed by LC-MS, and the content of nitrosamine impurity N-nitroso-N-decyclohexane bromhexine in bromhexine hydrochloride was calculated based on the detection results and the standard curve.

[0026] More preferably, in the second step, the preset series concentrations are in the concentration range of 0.01 to 1000 ng / ml, and the number of preset series concentrations is at least 3; the limit of quantitation is first determined based on the detection results of the reference solution of the preset series concentrations, and then a standard curve is established with the concentration of the limit of quantitation as the lower limit.

[0027] By adopting the above preferred scheme, the specific technical details of the second step can be further optimized to achieve better detection results.

[0028] This invention extracts N-nitroso-N-decyclohexane bromhexene from bromhexine hydrochloride raw material and uses liquid chromatography-mass spectrometry (LC-MS) to quantitatively detect this nitrosamine impurity, thus successfully overcoming the problems of low content, difficulty in detection, and susceptibility to interference associated with the N-nitroso-N-decyclohexane bromhexene impurity. The method of this invention has high accuracy, precision, and sensitivity, and also exhibits good linearity, specificity, and system adaptability. Attached Figure Description

[0029] Figure 1 This is a route diagram for the synthesis of bromhexine hydrochloride according to the present invention.

[0030] Figure 2 This is a chromatogram of the blank solution in an embodiment of the present invention.

[0031] Figure 3 This is a chromatogram of the spiked test solution in an embodiment of the present invention.

[0032] Figure 4 This is a chromatogram of the test solution in an embodiment of the present invention.

[0033] Figure 5 This is a chromatogram of the reference solution in an embodiment of the present invention. Detailed Implementation

[0034] A method for determining the content of N-nitroso-N-decyclohexane bromide, a nitrosamine impurity, includes the following steps:

[0035] The first step is to dissolve the bromhexine hydrochloride raw material to obtain the test solution.

[0036] The diluent is one of methanol, ethanol, acetonitrile, or aqueous solutions of acetonitrile in different proportions;

[0037] Methanol is preferred as the diluent.

[0038] For dilution, weigh approximately 20 mg (20-22 mg) of bromhexine hydrochloride raw material into a 10 mL volumetric flask, add diluent to dissolve and dilute to the mark, mix well, and the product is obtained. Label it as S-1.

[0039] Prepare S-2 using the same method.

[0040] The second step is to perform liquid chromatography-mass spectrometry (LC-MS) analysis on the test solution, and calculate the content of N-nitroso-N-decyclohexane bromide, a nitrosamine impurity, in bromide hydrochloride based on the test results.

[0041] The detection conditions for liquid chromatography-mass spectrometry (LC-MS) include high-performance liquid chromatography (HPLC) parameters.

[0042] In the high-performance liquid chromatography (HPLC) parameters, a high-performance liquid chromatograph (HPLC) is used; the chromatographic column is a CS-C18 HPLC column with an inner diameter of 4.6 mm, a length of 100 mm or more, and a particle size of 2.7 μm (e.g., Agilent Poroshell 120CS-C18 column, 4.6 × 100 mm, 2.7 μm); the mobile phase consists of an aqueous solution of 0.2% formic acid and an acetonitrile solution in a volume ratio of 70–60:30–40.

[0043] In addition, the high performance liquid chromatography parameters were: flow rate 0.4–0.6 ml / min, column temperature 30℃–45℃, and injection volume 4–10 μl.

[0044] The detection conditions for liquid chromatography-mass spectrometry (LC-MS) also include mass spectrometry parameters. Among these parameters, the LC-MS parameters include: an ESI ion source with positive polarity, an ion source temperature of 250°C, a transfer line temperature of 280°C; quadrupole temperature 1 of 150°C; quadrupole temperature 2 of 150°C; and a time interval of 6-7 minutes.

[0045] Specifically, the second step involves the following process:

[0046] First, N-nitroso-N-decyclohexane bromide reference standard was used. Methanol was used as the diluent, and the solution was diluted to volume and shaken well. Different volumes were measured and diluted with 50% acetonitrile to prepare reference standard solutions of a preset series of concentrations. Liquid chromatography-mass spectrometry was used to detect them separately. Based on the detection results, a standard curve of concentration-peak area for N-nitroso-N-decyclohexane bromide was established.

[0047] The test solution was then subjected to liquid chromatography-mass spectrometry (LC-MS) analysis. Based on the test results and the standard curve, the content of N-nitroso-N-decyclohexane bromide, an nitrosamine impurity, in bromide hydrochloride was calculated.

[0048] The preset series concentrations are in the range of 0.01 to 1000 ng / ml, and the number of preset series concentrations is at least 3. The limit of quantitation is first determined based on the detection results of the reference solution of the preset series concentrations, and then a standard curve is established with the concentration of the limit of quantitation as the lower limit.

[0049] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. However, the present invention is not limited to the examples given. Unless otherwise specified, experimental procedures described below shall be performed under conventional conditions or as recommended by the manufacturer. Unless otherwise specified, all experimental materials and reagents described below are commercially available products.

[0050] Example 1

[0051] This embodiment examines the methodology of the technical solution of the present invention as described above, and determines the content of the nitrosamine impurity N-nitroso-N-decyclohexane bromide in bromhexine hydrochloride.

[0052] The specific details of this embodiment are as follows.

[0053] (1) Preparation of mobile phase

[0054] Mobile phase A is an aqueous solution of 0.2% formic acid. The specific preparation process is as follows: measure 1000 mL of ultrapure water into a reagent bottle, add 2 mL of formic acid, mix well, and degas by sonication to obtain the mobile phase A.

[0055] Mobile phase B is an acetonitrile solution. Measure an appropriate amount of acetonitrile into a reagent bottle and degas it by sonication to obtain the solution.

[0056] Mobile phase: Measure mobile phase A and mobile phase B according to the set ratio, and mix them to obtain the mobile phase.

[0057] (2) Preparation of the test solution

[0058] Weigh approximately 20 mg (20-22 mg) of bromhexine hydrochloride raw material into a 10 mL volumetric flask, add diluent to dissolve and dilute to the mark, mix well, and the product is obtained. Label it S-1. Prepare S-2 using the same method.

[0059] (3) Preparation of reference solution

[0060] Weigh approximately 2 mg of N-nitroso-N-decyclohexanebromohexine reference standard and place it in a 20 ml volumetric flask. Dilute to the mark with methanol and mix well. Accurately measure 0.75 ml of the above solution and place it in a 10 ml volumetric flask. Dilute to the mark with 50% acetonitrile and mix well. Accurately measure 0.1 ml of the above solution and place it in a 10 ml volumetric flask. Dilute to the mark with 50% acetonitrile and mix well.

[0061] Accurately measure 2 ml of the above solution and place it in a 20 ml volumetric flask. Dilute to the mark with 50% acetonitrile and shake well. Accurately measure 1 ml of the above solution and place it in a 10 ml volumetric flask. Dilute to the mark with 50% acetonitrile and shake well.

[0062] (4) LC-MS detection conditions

[0063] i) High-performance liquid chromatography (HPLC) parameters

[0064] High performance liquid chromatography was used;

[0065] Column: Agilent Poroshell 120CS-C18, 4.6×100mm, 2.7μm;

[0066] Flow rate: 0.5 ml / min;

[0067] Column temperature: 40℃;

[0068] Injection volume: 5 μl;

[0069] Running time: 20 minutes;

[0070] Mobile phase: Mobile phase A: Mobile phase B = 60:40, v / v;

[0071] ii) Mass spectrometer parameters

[0072] A liquid chromatography-mass spectrometry system, model Agilent 1290-6495, was used.

[0073] Ion source: ESI;

[0074] Polarity: Positive;

[0075] Ion source temperature: 250℃;

[0076] Transmission line temperature: 280℃;

[0077] Quadrupole temperature 1:150℃;

[0078] Quadrupole temperature 2: 150℃;

[0079] Injection mode: Split;

[0080] Flow split ratio: 10:1;

[0081] The mass spectrometry detection parameters are shown in Table 1 below.

[0082] Table 1. Mass spectrometry detection parameters for N-nitroso-N-decyclohexanebromohexene

[0083]

[0084] (5) Methodological investigation

[0085] The methodology of the detection method using the above-mentioned liquid chromatography-mass spectrometry (LC-MS) detection conditions was investigated, and the specific contents are as follows.

[0086] i) Sample reproducibility:

[0087] The reference solution was injected six times consecutively. In the results, the peak areas of the N-nitroso-N-decyclohexane bromide peak were 26872, 26977, 26157, 26577, 26716, and 26713, respectively. The calculated RSD was 1%, which is less than 20.0%, indicating that the above detection method has good precision.

[0088] ii) Exclusivity:

[0089] Inject the reference solution, blank solvent (i.e., 50% acetonitrile), and test sample separately. For example... Figure 5 As shown, the retention time of N-nitroso-N-decyclohexanebromohexene in the chromatogram of the reference solution is 4.204 minutes, and the retention time of the blank solvent is... Figure 2 The retention time was interference-free. This result indicates that the above detection method has good specificity.

[0090] iii) Linearity, limit of quantitation, and limit of detection:

[0091] An appropriate amount of N-nitroso-N-decyclohexanebromohexine reference standard was accurately weighed, dissolved and diluted with methanol, and then different volumes of the solution were diluted with 50% acetonitrile to obtain a series of reference standard solutions of different concentrations. These solutions were then injected separately. The experimental results showed that the linear concentration range was 0.0636~1.4127 ng / mL, the linear regression equation was y = 35813.1043 x - 23.6306, and the correlation coefficient r was 0.9996, which met the acceptance criteria.

[0092] Therefore, when the limit of quantitation concentration is 0.0636 ng / mL (0.0318 ppm), the S / N of the six-shot limit of quantitation is greater than 10.0, and the RSD of the peak area of ​​the target impurity in the six-shot limit of quantitation solution is 2%. This result shows that the above detection method has good precision.

[0093] Limit of Detection Solution: Measure 2.5 mL of the limit of quantitation solution into a 5 mL volumetric flask, dilute to the mark with diluent, and mix well. Prepare one aliquot and inject for detection. The results show that when the limit of detection concentration of N-nitroso-N-decyclohexanebromohexene is 0.0318 ng / mL (0.0159 ppm), the limit of detection solution S / N is 25.9. This result shows that the above detection method has good precision.

[0094] iv) Recovery rate:

[0095] Experimental results show that in the accuracy experiment, the recovery rates of the LOQ, 100%, and 150% spiked test solutions of N-nitroso-N-decyclohexanebromohexene were between 70% and 130%, and the average recovery rate of the nine spiked test solutions was 107%, with a recovery RSD of 3%, all of which met the requirements.

[0096] Recovery rate calculation formula:

[0097]

[0098] Table 2. Recovery rate data for this embodiment

[0099]

[0100] Nine solutions containing 100% N-nitroso-N-decyclohexane bromide were prepared for determination. The recoveries of N-nitroso-N-decyclohexane bromide were 101%, 101%, 107%, 110%, 110%, 107%, 109%, 109%, and 110%, respectively.

[0101] The RSD was 3% (limit: not exceeding 20.0%). This result shows that the above detection method has good precision.

[0102] v) Repeatability:

[0103] Six solutions containing 100% N-nitroso-N-decyclohexane bromide were prepared for determination. The recoveries of N-nitroso-N-decyclohexane bromide were 110%, 110%, 107%, 109%, 109%, and 109%, respectively, with an RSD of 1% (limit: not exceeding 20.0%). These results indicate that the above detection method has good precision.

[0104] Table 3. Recovery rate data of this embodiment

[0105] (6) Testing of test samples

[0106] Table 4. Test results of the test samples in this embodiment

[0107]

[0108] Take the test solution and inject it for testing according to the above-described liquid chromatography-mass spectrometry (LC-MS) detection conditions. The results are as follows. Figure 4 As shown, the calculated test results are as follows: N-nitroso-N-decyclohexane bromide was not detected in the test solution.

[0109] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art. In addition to the specific methods, devices, and materials used in the embodiments, based on the knowledge of those skilled in the art and the description of the present invention, any prior art methods, devices, and materials similar to or equivalent to those described, used, and materials in the embodiments of the present invention can be used to implement the present invention. Any person skilled in the art can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A method for detecting the content of N-nitroso-N-decyclohexanebromohexene, a nitrosamine impurity, characterized in that: The method includes the following steps: Dissolve the bromhexine hydrochloride sample in a diluent to prepare a test solution; The test solution was detected using liquid chromatography-mass spectrometry. The diluent includes one or more of methanol, ethanol, acetonitrile, or an aqueous solution of acetonitrile.

2. The method according to claim 1, characterized in that, The diluent is methanol.

3. The method according to claim 1, characterized in that, The ratio of bromhexine hydrochloride sample to diluent is 2 mg: 5-20 mL.

4. The method according to claim 1, characterized in that, The conditions for the liquid chromatography-mass spectrometry (LC-MS) instrument include: The chromatographic column is a CS C18 high-performance liquid chromatography column; The mobile phase consists of an aqueous solution of formic acid and an acetonitrile solution.

5. The method according to claim 4, characterized in that, The chromatographic column was an Agilent Poroshell 120CS-C18 with an inner diameter of 4.6 mm, a length of ≥100 mm, and a particle size of 2.7 μm. The volume fraction of the formic acid aqueous solution in the mobile phase is 0.2%; The volume ratio of formic acid aqueous solution to acetonitrile solution is 70-60:30-40; The flow rate is 0.4–0.6 mL / min; The column temperature is 30℃~45℃; The injection volume is 4–10 μl.

6. The method according to claim 1, characterized in that, The conditions for the liquid chromatography-mass spectrometry (LC-MS) instrument include: The ion source is an ESI source; The polarity is positive; The ion source temperature is 250℃; The quadrupole temperature 1 is 150℃; The transmission line temperature is 280℃; The quadrupole temperature 2 is 150℃; The data collection time is 6-7 minutes.

7. The method according to claim 6, characterized in that, The mass spectrometry conditions also include the following ion pair conditions for N-nitroso-N-decyclohexanebromohexene: The ion pair has a m / z of 181 / 150 and a collision energy of 5 eV. The ion pair has a m / z of 151 / 51 and a collision energy of 20 eV. The ion pair has a m / z of 150 / 78 and a collision energy of 10 eV.

8. The method according to claim 1, characterized in that, The method further includes the step of preparing a reference solution containing N-nitroso-N-decyclohexane bromide, and using the reference solution as an external standard to calculate the content of N-nitroso-N-decyclohexane bromide in the test solution.

9. The method according to claim 1, characterized in that, The method has a detection limit of 0.0159 ppm and a quantitation limit of 0.0318 ppm for N-nitroso-N-decyclohexanebromohexene.

10. The method according to claim 1, characterized in that, The method is used for the quality control of N-nitroso-N-decyclohexane bromide in bromide hydrochloride raw material or bromide hydrochloride preparation.

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