Method for detecting N-nitroso bisoprolol by liquid chromatography-mass spectrometry

Abstract

This invention discloses a method for detecting N-nitrosobisoprolol impurities in bisoprolol / amlodipine tablets, belonging to the field of pharmaceutical impurity detection. The method involves preparing a reference solution and a test sample aqueous solution (ultrasonic dispersion, volume adjustment, and centrifugation), followed by HPLC-MS / MS detection. The chromatographic column is a C18 column (particle size 2-3 μm, inner diameter 2.1-4.6 mm, length 100-250 mm). The mobile phase contains 0.1-0.5% formic acid, 5-20 mM ammonium acetate aqueous solution, and methanol / acetonitrile, with gradient elution. The mass spectrometry is performed in ESI positive ion mode with MRM scanning, monitoring characteristic ion pairs (mother ion 372±1 amu, daughter ion 145±1 amu, etc.). This method features simple pretreatment, environmentally friendly solvents, high sensitivity, good durability, and accurate detection of trace impurities, providing reliable support for the quality control of bisoprolol / amlodipine tablets.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis and detection technology, and more specifically, to a method for determining the impurity N-nitrosobisoprolol in bisoprolol-amlodipine combination preparations using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). Background Technology

[0002] Bisoprolol-amlodipine tablets are a commonly used combination antihypertensive drug in clinical practice, composed of the beta-blocker bisoprolol and the calcium channel blocker amlodipine. The two work synergistically to lower blood pressure—bisoprolol inhibits myocardial contractility, slows heart rate, and reduces cardiac output; amlodipine dilates peripheral blood vessels and reduces vascular resistance. The combined use of these two drugs significantly improves antihypertensive efficacy and reduces adverse reactions associated with high doses of single-drug therapy. It is suitable for patients with hypertension complicated by coronary heart disease, heart failure, and other complications, and plays an important role in the clinical treatment of cardiovascular diseases. With the widespread use of this combination preparation, its quality and safety evaluation has become a core aspect of drug research and development and production, especially the control of potential impurities in the preparation, which directly relates to medication safety.

[0003] N-nitrosopyrosol is a genotoxic impurity that may be generated during the production or storage of bisopyrosol / amlodipine combination formulations. Its formation pathway is related to the synthetic process of the drug raw materials, interactions with excipients in the formulation, or oxidation and degradation reactions during storage. N-nitroso compounds are recognized as potent genotoxic substances with a clear carcinogenic risk. Their mechanism of action involves damaging cellular DNA, inducing gene mutations or chromosomal aberrations, and long-term intake significantly increases the probability of cancer in humans. According to the M7(R1) guidance issued by the International Council for Harmonisation of Pharmaceutical Regulatory Authorities (ICH), genotoxic impurities require a "risk assessment-control" management model. For impurities with clear evidence of carcinogenicity, their residual levels must be controlled within extremely low acceptable limits (usually at the nanogram level). Therefore, establishing a highly sensitive and selective detection method for this impurity is crucial to ensuring the safety of bisopyrosol / amlodipine tablets.

[0004] However, currently there is no effective detection method for N-nitrosobisoprolol in bisoprolol-amlodipine combination preparations, mainly due to the following technical gaps and difficulties:

[0005] First, the complexity of the matrix in compound preparations significantly interferes with impurity detection. In addition to the two active pharmaceutical ingredients, bisoprolol-amlodipine tablets also contain various excipients such as lactose, microcrystalline cellulose, and magnesium stearate. The concentrations of the active pharmaceutical ingredients and excipients are much higher than the potential N-nitrosobisoprolol, easily leading to a strong matrix effect during detection. This can mask impurity peaks or result in false positives or false negatives, making it difficult to accurately identify and quantify low-content impurities.

[0006] Secondly, the physicochemical properties and detection requirements of N-nitrosopysoprolol place stringent demands on analytical methods. This impurity exists in trace amounts, and its structure is somewhat similar to that of the active pharmaceutical ingredient, bisoprolol. Conventional detection methods, such as high-performance liquid chromatography-ultraviolet (HPLC-UV), lack sufficient sensitivity and cannot reach the nanogram-level detection limit. Gas chromatography (GC), on the other hand, is limited by the polarity and thermal stability of this impurity, making it difficult to achieve effective separation and detection.

[0007] Furthermore, existing methods for detecting impurities in bisoprolol or amlodipine single-component formulations do not cover the detection of N-nitrosobisoprolol. Since the matrix interference in combination formulations differs fundamentally from that in single-component formulations, existing detection methods for single-component formulations cannot be directly applied to combination formulations. Simultaneously, domestic and international drug standards have not yet established relevant detection standards and methods for N-nitrosobisoprolol in this combination formulation, resulting in a lack of effective quality control measures during drug research and development and production. This makes it impossible to accurately assess the residual level of this impurity, posing a potential risk to medication safety.

[0008] Therefore, developing a detection method for N-nitrosobisoprolol that can effectively eliminate matrix interference in compound preparations and has high sensitivity and selectivity, filling the existing technological gap, is of great significance for improving the quality control system of bisoprolol amlodipine tablets and ensuring the safety of clinical medication. Summary of the Invention

[0009] This invention provides a method for determining nitrosamine-related impurities in bisoprolol amlodipine tablets using high performance liquid chromatography-tandem triple quadrupole mass spectrometry (HPLC-MS / MS), providing a basis for improving the quality standards of the formulation.

[0010] In a first aspect, the present invention provides a method for detecting N-nitrosobisoprolol impurities in bisoprolol amlodipine tablets, comprising the following steps:

[0011] (1) Preparation of impurity reference solution: Take an appropriate amount of N-nitrosobisoprolol reference standard, accurately weigh it, dissolve it in methanol and dilute it to prepare a stock solution; accurately measure an appropriate amount of the stock solution and dilute it quantitatively with water to prepare a reference solution;

[0012] (2) Preparation of test solution: Take 1 tablet of bisoprolol amlodipine (equivalent to 5 mg of bisoprolol fumarate), place it in a 50 mL volumetric flask, add an appropriate amount of water and sonicate until dispersed, dilute to the mark with water, shake well, centrifuge, and take the supernatant to obtain the test solution.

[0013] (3) Detection was performed using high performance liquid chromatography-tandem mass spectrometry, wherein:

[0014] a. Chromatographic conditions: C18 column, particle size 2-3 μm, inner diameter 2.1-4.6 mm, length 100-250 mm; mobile phase A is an aqueous solution of 0.1-0.5% formic acid and 5-20 mM ammonium acetate, mobile phase B is methanol or acetonitrile; gradient elution, initial stage 80-95% mobile phase A, intermediate stage 5-45% mobile phase A, final stage return to equilibrium with 80-95% mobile phase A, mobile phase A and mobile phase B are both expressed as volume percentages, and their sum is 100%; flow rate 0.3-0.5 mL / min, column temperature 35-45℃, injection volume 1-5 μL;

[0015] b. Mass spectrometry conditions: ESI source, positive ion mode, MRM scan; the target analyte characteristic ion pairs include the parent ion 372±1 amu and daughter ions 145±1 amu, 251±1 amu, and 102±1 amu.

[0016] (4) Data processing: Plot a standard curve with the concentration of the reference solution as the abscissa and the peak area as the ordinate. Substitute the peak area of ​​the impurities in the test solution into the curve to calculate the content of N-nitrosobisoprolol impurities in the test sample.

[0017] In some embodiments, in step (1), the stock solution contains 0.2 mg of N-nitrosobisoprolol per 1 mL, and the reference solution contains 15 ng of N-nitrosobisoprolol per 1 mL.

[0018] In some implementations, in step (3), the chromatographic column is a core-shell C18 chromatographic column with a particle size of 2.7 μm, an inner diameter of 3.0 mm, and a length of 150 mm.

[0019] In some embodiments, in step (3), the mobile phase A is a 0.2% formic acid 10mM ammonium acetate aqueous solution, and the mobile phase B is methanol.

[0020] In some implementations, the gradient elution program in step (3) is as follows: 0-1 min 85-95% mobile phase A, 1-3 min 35-45% mobile phase A, 3-15 min 5-15% mobile phase A, 15-17 min 5-15% mobile phase A, 17-17.1 min 85-95% mobile phase A, 17.1-20 min 85-95% mobile phase A.

[0021] In some implementations, the gradient elution program in step (3) is as follows: 0-1 min 90% mobile phase A, 1-3 min 40% mobile phase A, 3-15 min 10% mobile phase A, 15-17 min 10% mobile phase A, 17-17.1 min 90% mobile phase A, and 17.1-20 min 90% mobile phase A.

[0022] In some implementations, in step (3), the flow rate is 0.4 mL / min, the column temperature is 40 °C, and the injection volume is 2 μL.

[0023] In some implementations, the gradient elution process is characterized by a runtime of 20 minutes, wherein the eluent is diverted to waste liquid from 0 to 8.6 minutes and introduced into the mass spectrometer for detection from 8.6 to 20 minutes.

[0024] Secondly, the present invention provides the application of the detection method described in the first aspect in the detection of N-nitrosobisoprolol impurities in bisoprolol amlodipine tablets.

[0025] Beneficial Effects: This invention discloses a specific detection method for N-nitrosobisoprolol impurities in bisoprolol amlodipine tablets, filling the gap in detection methods for this type of genotoxic impurity in this formulation, and providing reliable technical support for the quality control and safety evaluation of bisoprolol amlodipine tablets. Specific advantages are as follows:

[0026] 1. High detection sensitivity, meeting the requirements for trace impurity control.

[0027] This method exhibits excellent detection sensitivity. Under the condition of a test sample concentration of 0.1 mg / mL, the detection limit of N-nitrosobisoprolol can be as low as 0.008 ng / mL, equivalent to 0.08 ppm, which is far below the impurity limit requirement of 150 ppm. It can accurately capture trace amounts of N-nitrosobisoprolol impurities in the formulation and effectively avoid the medication safety risks caused by impurity residues.

[0028] 2. Green and environmentally friendly, simple pretreatment and minimal matrix interference.

[0029] This method uses water as the solvent to dissolve the test sample, which is more environmentally friendly than traditional organic solvent systems, while reducing experimental costs and environmental burden. The pretreatment process is simplified, eliminating the need for complex extraction or purification steps, and effectively reducing matrix interference to ensure the accuracy of the test results.

[0030] 3. The method has excellent accuracy and strong data reliability.

[0031] Spiked recovery tests were conducted at four levels equivalent to the impurity limit concentrations of 10%, 50%, 100%, and 150%. The recoveries of the 12 test solutions ranged from 94.52% to 111.42%, with an overall average recovery of 106.0% and an RSD of only 5.7%, which meets the accuracy requirements for drug impurity detection and proves that the quantitative results of this method are accurate and reliable.

[0032] 4. Good durability and wide range of applications

[0033] This method exhibits good tolerance to fluctuations in chromatographic conditions. When parameters are adjusted within the range of 0.35-0.45 mL / min, column temperature 30-40℃, and initial organic phase ratio ±2%, the deviation of the content determination results is less than 2%. It can be stably carried out without strict condition control and is suitable for the routine testing needs of different laboratories, thus having strong promotion and application value. Detailed Implementation

[0034] The present invention will be further described below with reference to specific embodiments, but these embodiments should not be construed as limiting the present invention.

[0035] Example 1: A preliminary exploration of a method for determining N-nitrosobisoprolol impurities in bisoprolol / amlodipine tablets using high performance liquid chromatography-tandem triple quadrupole mass spectrometry (HPLC-MS / MS).

[0036] 1. Instruments and Reagents

[0037] Shimadzu LC-20A-8045 high performance liquid chromatography-tandem triple quadrupole mass spectrometer (Shimadzu Corporation, Japan); XPR2 electronic balance (Mettler AG, Switzerland); BCE12i-1CCN electronic balance (Sartorius Corporation).

[0038] N-Nitrosaminobisoprolol (batch number: 83870; content: 97.70%; manufacturer: QCS); Bisoprolol Amlodipine Tablets (Jiangsu Wango Pharmaceutical Co., Ltd., batch numbers: 2403194, 2403211, 2403212, specifications: bisoprolol fumarate 5mg and amlodipine besylate (calculated as amlodipine) 5mg); Methanol was HPLC grade (Merck, Germany); Formic acid was HPLC grade (Maclean); Ammonium acetate was chromatographic grade (Aladdin); Water was (Watsons).

[0039] 2. Methods and Results

[0040] 2.1 Instrument Conditions

[0041] Chromatographic conditions

[0042] Column: Agilent Infinity Lab Poroshell 120EC-C18 (3.0×150mm, 2.7µm); Mobile phase A was 0.2% formic acid in 10mM ammonium acetate aqueous solution, and mobile phase B was methanol; Gradient elution: 0-1 min, 90% A, 1-7 min, 10% A, 7-12 min, 10% A, 12-12.1 min, 90% A, 12.1-15 min, 90% A; Run time 15 min (0-8.5 min: To Waste; 8.6-10.1 min: To MS; 10.1-15 min: To Waste); Flow rate: 0.4 mL / min; Column temperature: 40℃; Injection volume: 2 µl.

[0043] Mass spectrometry conditions

[0044] Ion source: ESI source, positive ion mode; Scanning mode: Multiple reaction monitoring (MRM); Ion pairs: 372.35>145.15, CE value 27 (quantitative), 372.35>251.20, CE value 28 (qualitative), 372.35>102.95, CE value 27 (qualitative); Nebulizing gas flow rate: 3L / min; Heating gas flow rate: 10L / min; Drying gas flow rate: 10L / min; Interface temperature: 300℃; DL tube temperature: 250℃; Heating block temperature: 400℃.

[0045] 2.2 Solution Preparation

[0046] 2.2.1 Preparation of impurity reference solution

[0047] Take an appropriate amount of N-nitrosobisoprolol reference standard, accurately weigh it, dissolve it in methanol and dilute it to prepare a solution containing 0.2 mg of N-nitrosobisoprolol per 1 mL as a stock solution; accurately measure an appropriate amount of the stock solution and dilute it quantitatively with water to prepare a solution containing approximately 15 ng of N-nitrosobisoprolol per 1 mL as a reference solution.

[0048] 2.2.2 Preparation of test solution

[0049] Take one tablet of this product (equivalent to 5 mg of bisoprolol fumarate), place it in a 50 mL volumetric flask, add an appropriate amount of water, sonicate until dispersed, dilute to the mark with water, shake well, centrifuge, and take the supernatant.

[0050] 2.3 Recovery Rate Test

[0051] Three different levels of N-nitrosobisoprolol spiked recovery tests were conducted.

[0052] Take an appropriate amount of N-nitrosobisoprolol reference standard, accurately weigh it, dissolve it in methanol and dilute it to prepare a solution containing approximately 0.2 mg of N-nitrosobisoprolol per 1 mL, as the stock solution; accurately measure an appropriate amount of the stock solution and dilute it quantitatively with water to prepare a solution containing approximately 750 ng of N-nitrosobisoprolol per 1 mL, as the linear stock solution.

[0053] Accurately measure 0.1 mL, 0.5 mL, and 1 mL of the linear stock solution into 50 mL volumetric flasks, add one tablet of this product, disperse with water by sonication, and dilute to the mark with solvent to obtain different concentrations of spiked test solutions; calculate the recovery rate using the external standard method.

[0054] The recoveries of N-nitrosobisoprolol ranged from 108.54% to 129.40%, with an average recovery of 120.4% and an RSD of 6.3%. The recovery rates were not ideal, as shown in Table 1.

[0055] Table 1 Results of recovery rate test

[0056]

[0057] Recovery rate is a core indicator for evaluating the reliability of a detection method, and its deviation directly determines whether the method can be used for accurate quantification of the target analyte. Given the systematic errors and stability defects in the above results, this invention further explores optimization of detection conditions, focusing on adjusting the gradient elution procedure parameters (which may lead to incomplete separation of the target analyte and matrix, causing a matrix enhancement effect and thus resulting in a higher recovery rate) to obtain an analytical method that meets quantitative requirements.

[0058] Example 2: Establishment and validation of a method for determining N-nitrosobisoprolol impurities in bisoprolol / amlodipine tablets using high performance liquid chromatography-tandem triple quadrupole mass spectrometry (HPLC-MS / MS).

[0059] 1. Instruments and Reagents

[0060] Shimadzu LC-20A-8045 high performance liquid chromatography-tandem triple quadrupole mass spectrometer (Shimadzu Corporation, Japan); XPR2 electronic balance (Mettler AG, Switzerland); BCE12i-1CCN electronic balance (Sartorius Corporation).

[0061] N-Nitrosaminobisoprolol (batch number: 83870; content: 97.70%; manufacturer: QCS); Bisoprolol Amlodipine Tablets (Jiangsu Wango Pharmaceutical Co., Ltd., batch numbers: 2403194, 2403211, 2403212, specifications: bisoprolol fumarate 5mg and amlodipine besylate (calculated as amlodipine) 5mg); Methanol was HPLC grade (Merck, Germany); Formic acid was HPLC grade (Maclean); Ammonium acetate was chromatographic grade (Aladdin); Water was (Watsons).

[0062] 2. Methods and Results

[0063] 2.1 Instrument Conditions

[0064] Chromatographic conditions

[0065] Column: Agilent Infinity Lab Poroshell 120EC-C18 (3.0×150mm, 2.7µm); Mobile phase A was 0.2% formic acid in 10mM ammonium acetate aqueous solution, and mobile phase B was methanol; Gradient elution: 0-1 min, 90% A, 1-3 min, 40% A, 3-15 min, 10% A, 15-17 min, 10% A, 17-17.1 min, 90% A, 17.1-20 min, 90% A; Run time: 20 min (0-8.6 min: To Waste; 8.6-20 min: To MS); Flow rate: 0.4 mL / min; Column temperature: 40℃; Injection volume: 2 µl.

[0066] Mass spectrometry conditions

[0067] Ion source: ESI source, positive ion mode; Scanning mode: Multiple reaction monitoring (MRM); Ion pairs: 372.35>145.15, CE value 27 (quantitative), 372.35>251.20, CE value 28 (qualitative), 372.35>102.95, CE value 27 (qualitative); Nebulizing gas flow rate: 3L / min; Heating gas flow rate: 10L / min; Drying gas flow rate: 10L / min; Interface temperature: 300℃; DL tube temperature: 250℃; Heating block temperature: 400℃.

[0068] 2.2 Solution Preparation

[0069] 2.2.1 Preparation of impurity reference solution

[0070] Take an appropriate amount of N-nitrosobisoprolol reference standard, accurately weigh it, dissolve it in methanol and dilute it to prepare a solution containing 0.2 mg of N-nitrosobisoprolol per 1 mL as a stock solution; accurately measure an appropriate amount of the stock solution and dilute it quantitatively with water to prepare a solution containing approximately 15 ng of N-nitrosobisoprolol per 1 mL as a reference solution.

[0071] 2.2.2 Preparation of test solution

[0072] Take one tablet of this product (equivalent to 5 mg of bisoprolol fumarate), place it in a 50 mL volumetric flask, add an appropriate amount of water, sonicate until dispersed, dilute to the mark with water, shake well, centrifuge, and take the supernatant.

[0073] 2.3 Limit of Detection and Limit of Quantification

[0074] Accurately weigh an appropriate amount of N-nitrosobisoprolol reference standard, dissolve and dilute it in methanol to prepare a solution containing approximately 0.2 mg of N-nitrosobisoprolol per mL, as the stock solution. Accurately measure an appropriate amount of the stock solution and quantitatively dilute it with water to prepare a solution containing approximately 1.5 ng of N-nitrosobisoprolol per mL, as the limit of quantitation (LOQ) solution. Dilute the LQ solution to prepare the limit of detection (LOD) solution. A signal-to-noise ratio (S / N) > 10 is required for the LQ, and an S / N > 3 is required for the LOD. The LQ of N-nitrosobisoprolol is 1.485 ng / mL, and the LOD is 0.7425 ng / mL, which effectively meets the high sensitivity requirements for the detection of N-nitrosobisoprolol.

[0075] 2.4 Linear Relationship and Range

[0076] Accurately weigh an appropriate amount of N-nitrosobisoprolol reference standard, dissolve and dilute it in methanol to prepare a solution containing approximately 0.2 mg of N-nitrosobisoprolol per mL, as a stock solution. Accurately measure an appropriate amount of the stock solution and quantitatively dilute it with water to prepare a solution containing approximately 750 ng of N-nitrosobisoprolol per mL, as a linear stock solution. Accurately measure the linear stock solution and dilute it with solvent to prepare reference standard solutions of different concentrations. Inject the solutions, record the chromatograms, and perform linear regression with concentration (x, ng / mL) on the x-axis and peak area (y) on the y-axis. Within the concentration range of 1.485 ng / mL to 29.70 ng / mL, the linear equation for N-nitrosobisoprolol is y = 167335.8299*x - 14636.9250 (n = 6), with a correlation coefficient r of 1.0000, indicating high quantitative accuracy.

[0077] 2.5 Instrument precision test

[0078] A precise 2 µl volume of the reference solution was injected into the high-performance liquid chromatography-tandem triple quadrupole mass spectrometer (HPLC-MS / MS) six times. The peak area RSD of N-nitrosobisoprolol was measured to be 0.6%. The results indicate that the instrument has good precision.

[0079] 2.6 Recovery Rate Test

[0080] Recovery tests were conducted at four levels of N-nitrosobisoprolol: minimum, low, medium, and high.

[0081] Take an appropriate amount of N-nitrosobisoprolol reference standard, accurately weigh it, dissolve it in methanol and dilute it to prepare a solution containing approximately 0.2 mg of N-nitrosobisoprolol per 1 mL, as the stock solution; accurately measure an appropriate amount of the stock solution and dilute it quantitatively with water to prepare a solution containing approximately 750 ng of N-nitrosobisoprolol per 1 mL, as the linear stock solution.

[0082] Accurately measure 0.1 mL, 0.5 mL, 1 mL, and 1.5 mL of the linear stock solution into 50 mL volumetric flasks, add one tablet of this product, disperse by sonication with water, and dilute to the mark with solvent to obtain different concentrations of spiked test solutions; calculate the recovery rate using the external standard method.

[0083] The recoveries of N-nitrosobisoprolol ranged from 94.52% to 111.42%, with an average recovery of 106.0% and an RSD of 5.7%. The recovery was good, and the results are shown in Table 2.

[0084] Table 2 Results of Recovery Test

[0085]

[0086] 2.7 Durability Test

[0087] Using impurity reference solutions and medium-concentration spiked recovery solutions as test samples, the following robustness tests were conducted on the method: column temperature ±2℃, flow rate ±0.01mL / min, and initial mobile phase ratio ±1%. The results showed that parameter changes had no effect on the results.

[0088] 2.8 Stability Test

[0089] The impurity reference solution and the medium-concentration spiked recovery solution were used as test samples. The stability at room temperature was investigated at 0, 7, 14.5 h and 20 h, respectively. The results showed that the reference solution and the test solution were stable at room temperature for 11 hours. The results are shown in Table 3.

[0090] Table 3 Solution stability results

[0091]

[0092] 2.9 Repeatability and intermediate precision tests

[0093] Six medium-concentration spiking solutions were prepared by different personnel on different days, and repeatability and intermediate precision were calculated. The results showed that N-nitrosobisoprolol had good repeatability and intermediate precision, as shown in Table 4.

[0094] Table 4 Repeatability and Intermediate Precision Results

[0095]

[0096] 2.10 Sample Analysis

[0097] This method was used to analyze three batches of bisoprolol amlodipine tablets, and the detection amount of N-nitrosobisoprolol was less than the LOQ in all cases.

[0098] Finally, it should be noted that the above examples are merely a few specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A method for detecting N-nitroso bisoprolol impurity in bisoprolol amlodipine tablet, characterized by, The detection method includes the following steps: (1) Preparation of impurity reference solution: Take an appropriate amount of N-nitrosobisoprolol reference standard, accurately weigh it, dissolve it in methanol and dilute it to prepare a stock solution; accurately measure an appropriate amount of the stock solution and dilute it quantitatively with water to prepare a reference solution; (2) Preparation of test solution: Take 1 tablet of bisoprolol amlodipine, which is equivalent to 5 mg of bisoprolol fumarate, place it in a 50 mL volumetric flask, add an appropriate amount of water, sonicate until dispersed, dilute to the mark with water, shake well, centrifuge, and take the supernatant to obtain the test solution. (3) Detection was performed using high performance liquid chromatography-tandem mass spectrometry, wherein: a. Chromatographic conditions: C18 column, particle size 2-3 μm, inner diameter 2.1-4.6 mm, length 100-250 mm; mobile phase A is 0.2% formic acid in 10 mM ammonium acetate aqueous solution, mobile phase B is methanol; gradient elution, 0-1 min 90% mobile phase A, 1-3 min 40% mobile phase A, 3-15 min 10% mobile phase A, 15-17 min 10% mobile phase A, 17-17.1 min 90% mobile phase A, 17.1-20 min 90% mobile phase A, mobile phases A and B are both by volume percentage, and their sum is 100%; flow rate 0.4 mL / min, column temperature 40℃, injection volume 2 μL; b. Mass spectrometry conditions: ESI source, positive ion mode, MRM scan; the target analyte characteristic ion pairs include the parent ion 372±1 amu and daughter ions 145±1 amu, 251±1 amu, and 102±1 amu. (4) Data processing: Plot a standard curve with the concentration of the reference solution as the abscissa and the peak area as the ordinate. Substitute the peak area of ​​the impurities in the test solution into the curve to calculate the content of N-nitrosobisoprolol impurities in the test sample. In step (1), each 1 mL of the stock solution contains 0.2 mg of N-nitrosobisoprolol, and each 1 mL of the reference solution contains 15 ng of N-nitrosobisoprolol. In step (3), the chromatographic column is a core-shell C18 chromatographic column with a particle size of 2.7 μm, an inner diameter of 3.0 mm, and a length of 150 mm. The gradient elution program runs for 20 minutes, with the eluent being diverted to waste liquid from 0 to 8.6 minutes and introduced into the mass spectrometer for detection from 8.6 to 20 minutes.

2. The application of the detection method according to claim 1 in the detection of N-nitrosobisoprolol impurities in bisoprolol amlodipine tablets.

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