Method for simultaneously determining atenolol and raw material p-hydroxyphenylacetamide thereof based on high performance liquid chromatography

Abstract

The invention discloses a method for simultaneously determining atenolol and a raw material p-hydroxyphenylacetamide thereof based on high performance liquid chromatography, and belongs to the technical field of drug analysis and detection. An instrument adopted by the method disclosed by the invention is Agilent 1260 HPLC (High Performance Liquid Chromatography Octadecylsilane chemically bonded silica is selected as a filling agent for a chromatographic column; a mobile phase is phosphoric acid water and an acetonitrile solution for gradient elution. According to the method, retention of atenolol and the raw material p-hydroxyphenylacetamide on a chromatographic column can be remarkably enhanced, the problem of co-elution caused by similar structural polarities of atenolol and p-hydroxyphenylacetamide is successfully solved, and baseline separation is realized. The method is economical and practical.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a method for the simultaneous determination of atenolol and its raw material p-hydroxyphenylacetamide based on high performance liquid chromatography. Background Technology

[0002] Atenolol, a highly cardioselective β1-receptor blocker, has shown significant efficacy in treating cardiovascular diseases such as hypertension, angina pectoris, arrhythmias, and myocardial infarction, with good safety and tolerability. It can lower blood pressure, myocardial oxygen consumption, and angina attacks, and also reduce mortality from myocardial infarction and stroke. It can also prevent and control various types of arrhythmias, especially those caused by catecholamines. Furthermore, atenolol can be used to treat glaucoma, lowering intraocular pressure. The widespread clinical application of atenolol has promoted the research and innovation of cardioselective β1-receptor blockers and made a significant contribution to the development of β1-receptor blockers.

[0003] In the synthesis of atenolol, p-hydroxyphenylacetamide is a key starting material. It undergoes an addition reaction with epichlorohydrin to form an intermediate, which is then condensed with isopropylamine to obtain the target product, atenolol. The purity of this starting material directly affects the quality, yield, and safety of the final product, and the residual amount of the starting material also directly affects the purity of the finished atenolol product. Therefore, in the production process of atenolol, accurate detection of both the main component of atenolol and the p-hydroxyphenylacetamide starting material is crucial to ensuring quality.

[0004] Currently, there are established standard methods for the individual detection of atenolol. The Pharmacopoeia of the People's Republic of China (2025 edition) uses high-performance liquid chromatography (HPLC) for content determination, with chromatographic conditions of octadecylsilane-bonded silica gel as the packing material, phosphate buffer (pH 3.0)-methanol (70:30) as the mobile phase for isocratic elution, and a detection wavelength of 226 nm. Patent CN115754102A discloses a method for detecting atenolol epoxide impurities, employing a triple quadrupole HPLC system to improve the sensitivity of impurity detection. However, the aforementioned existing technologies all have significant limitations, only detecting atenolol or specific impurities, and do not involve the simultaneous analysis of the synthetic raw material p-hydroxyphenylacetamide, failing to meet the integrated "raw material to finished product" requirements of the production process. Therefore, in order to track and control drug quality, it is essential to establish an effective detection method that can simultaneously detect atenolol and its raw material p-hydroxyphenylacetamide. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for the simultaneous determination of atenolol and its raw material, p-hydroxyphenylacetamide, using high-performance liquid chromatography (HPLC). This invention establishes a method for the simultaneous detection of two samples, enabling accurate quantification to aid in monitoring the production process and facilitating the control and improvement of product quality.

[0006] The structure of atenol is shown below:

[0007] .

[0008] The structure of p-hydroxyphenylacetamide is shown below:

[0009] .

[0010] This invention relates to a method for the simultaneous determination of atenolol and its raw material p-hydroxyphenylacetamide based on high performance liquid chromatography, comprising the following steps:

[0011] Weigh 10 mg of the sample to be tested, transfer it to a 10 mL volumetric flask with 10% acetonitrile aqueous solution, and dilute to the mark; detect the content of atenolol and its raw material p-hydroxyphenylacetamide in the sample by high performance liquid chromatography.

[0012] Chromatographic conditions were set as follows:

[0013] Detector: Diode array detector;

[0014] Column: Agilent EC-C18 (4.6×250nm, 5μm);

[0015] Mobile phase: Mobile phase A: 0.1% phosphoric acid aqueous solution; Mobile phase B: acetonitrile;

[0016] Column temperature: 30-40℃;

[0017] Injection volume: 5-10 μL;

[0018] Wavelength: 210-260 nm;

[0019] Flow rate: 0.8-1.2 mL / min;

[0020] Elution method: Gradient elution is performed using mobile phase A and mobile phase B.

[0021] The elution program is set as follows:

[0022]

[0023] Furthermore, the detection wavelength is 230 nm, which allows for the simultaneous acquisition of the optimal absorption peaks of atenolol and p-hydroxyphenylethylamine.

[0024] The beneficial effects of this invention are reflected in:

[0025] The method of this invention can effectively analyze and detect atenolol and p-hydroxyphenylacetamide. The method has good separation, with a separation degree ≥1.5 to achieve baseline separation. The theoretical plate number of both atenolol and p-hydroxyphenylacetamide is ≥5000, the retention time is controlled within 18 min, the economic cost is low, and it can be used for quality control in the production process of atenolol. Attached Figure Description

[0026] Figure 1 HPLC chromatogram under the conditions measured in Example 1 of this invention.

[0027] Figure 2 The HPLC chromatograms of Comparative Example 1 of this invention are obtained under the conditions measured.

[0028] Figure 3 The HPLC chromatograms of Comparative Example 2 of this invention are obtained under the conditions measured. Detailed Implementation

[0029] The present invention will be further illustrated below with specific embodiments. It should be understood that the embodiments of the present invention are merely for illustration and not for limiting the present invention. In the following embodiments, the acetonitrile and methanol used were of chromatographic grade from Maclean's, the water was purified water, and the high-performance liquid chromatograph used was an Agilent HPLC 1260.

[0030] Example 1:

[0031] Preparation of sample solution: Weigh 10 mg each of atenolol and p-hydroxyphenylacetamide, transfer 10 mL of 10% acetonitrile aqueous solution to a volumetric flask, and dilute to the mark and mix well.

[0032] Chromatographic determination conditions:

[0033] Column: Agilent EC-C18, 4.6×250mm, 5μm;

[0034] Mobile phase: A: 0.1% aqueous phosphoric acid solution; B: acetonitrile;

[0035] Flow rate: 1.0 mL / min;

[0036] Column temperature: 30℃;

[0037] Injection volume: 5 μL;

[0038] Wavelength: 230nm;

[0039] Gradient elution procedure:

[0040]

[0041] Under the chromatographic conditions of Example 1, the detection chromatogram of the sample solution is as follows: Figure 1 As shown, the peak shapes are good. Atenolol has a retention time of 6.013 min, a tailing factor of 1.01, and a theoretical plate number of 20533; p-hydroxyphenylacetamide has a retention time of 6.680 min, a tailing factor of 1.12, a resolution of 3.28, and a theoretical plate number of 24747, meeting the requirements for component separation. Both meet the pharmacopoeia requirements of resolution ≥2, tailing factor ≤1.2, and theoretical plate number ≥5000, indicating good column efficiency, symmetrical peak shape without tailing, and avoidance of interference from impurity peaks.

[0042] Comparative Example 1:

[0043] Preparation of solution: Weigh 10 mg each of atenolol and p-hydroxyphenylacetamide, transfer 10 ml of 10% acetonitrile aqueous solution to a volumetric flask, and dilute to the mark and mix well.

[0044] Chromatographic determination conditions:

[0045] Column: Agilent EC-C18, 4.6×250mm, 5μm;

[0046] Mobile phase: A: purified water; B: acetonitrile;

[0047] Flow rate: 1.0 mL / min;

[0048] Column temperature: 30℃;

[0049] Injection volume: 5 μL;

[0050] Gradient elution procedure:

[0051]

[0052] Under the chromatographic conditions of Comparative Example 1, the detection chromatogram of the solution is as follows: Figure 2 As shown in the figure, atenolol had a retention time of 2.411 min, a tailing factor of 3.10, and a theoretical plate number of 524, which are far below the basic requirements for plate number in the pharmacopoeia, and the peak shape showed obvious tailing. p-Hydroxyphenylacetamide had a retention time of 6.7 min, a tailing factor of 1.05, and a theoretical plate number of 24695. The figure shows that atenolol peak broadening was severe, resulting in poor separation and significant asymmetry with obvious tailing. When using a pure water-acetonitrile mobile phase for simultaneous detection of atenolol and p-hydroxyphenylacetamide, it led to poor peak shape, insufficient separation, and low quantitative accuracy for atenolol, making it difficult to meet the requirements for precise monitoring of raw material residues and product content in the synthesis process.

[0053] Comparative Example 2:

[0054] Preparation of solution: Weigh 10 mg each of atenolol and p-hydroxyphenylacetamide, transfer 10 ml of 10% acetonitrile aqueous solution to a volumetric flask, and dilute to the mark and mix well.

[0055] Chromatographic determination conditions:

[0056] Column: Agilent EC-C18 (4.6×250mm, 5μm);

[0057] Mobile phase: A: 0.1% aqueous phosphoric acid solution; B: methanol;

[0058] Flow rate: 1.0 mL / min;

[0059] Column temperature: 30℃;

[0060] Injection volume: 5 μL;

[0061] Gradient elution procedure:

[0062]

[0063] Under the chromatographic conditions of Comparative Example 2, the detection chromatogram of the sample solution is as follows: Figure 3 As shown in the figure, atenolol had a retention time of 12.338 min, a tailing factor of 3.7, and a theoretical plate number of 10427. The atenolol peak was asymmetrical and exhibited significant tailing. p-Hydroxyphenylacetamide had a retention time of 10.651 min, a tailing factor of 1.07, and a theoretical plate number of 29034. The figure shows that the change in the mobile phase altered the competitive adsorption of the stationary phase, leading to a change in the peak order of atenolol and p-hydroxyphenylacetamide. In contrast, the chromatographic results of Example 1 of this invention showed symmetrical peaks and excellent resolution, solving the problems of asymmetrical peaks and insufficient resolution in pure aqueous and methanol phase systems. This fully demonstrates that the phosphoric acid-acetonitrile mobile phase system can meet the quantitative requirements for residual raw materials and product content in the synthesis process.

[0064] Example 2: System Adaptability Test

[0065] Sample preparation: First, prepare standard stock solutions. Weigh 10 mg each of atenolol and p-hydroxyphenylacetamide, transfer 10 ml of the solution to a volumetric flask with acetonitrile aqueous solution, and dilute to the mark to obtain a 1 mg / ml atenolol and p-hydroxyphenylacetamide reference stock solution. Take 1.0 ml of each of the above stock solutions, mix them, and then dilute with 10 ml of 10% acetonitrile aqueous solution to obtain a mixed reference solution containing 100 μg / mL atenolol and p-hydroxyphenylacetamide. Filter the solution through a 0.22 μm microporous membrane before injection.

[0066] Detection: Take blank solution and system adaptability solution, inject 5 μL, and repeat the injection 6 times according to the chromatographic conditions and methods in Example 1. Record the retention time and peak area. The results are shown in Table 1.

[0067]

[0068] As shown in Table 1, the RSDs of the same mixed sample solution were 0.48% and 0.537% respectively after six repetitions, both of which meet the requirements of RSD% < 2.0% in the pharmacopoeia. This indicates that the method of the present invention has good system adaptability and high experimental reliability.

[0069] Example 3: Linearity Test

[0070] Preparation of reference solutions: Six atenolol and six p-hydroxyphenylacetamide samples were weighed at six concentrations (20%, 50%, 80%, 100%, 120%, and 150%): 5 mg, 12.5 mg, 20 mg, 25 mg, 30 mg, and 37.5 mg respectively. These were accurately weighed into 25 mL volumetric flasks, dissolved and diluted to the mark with a suitable amount of 10% acetonitrile aqueous solution, and mixed thoroughly to obtain mixed solutions of different mass concentrations. These solutions corresponded sequentially to the test solutions numbered 1 to 6. For each concentration, the samples were measured six times under the chromatographic conditions of Example 1, and the average peak area was calculated. The linearity test results are shown in Table 2.

[0071]

[0072] Based on the data in Table 2, linear regression was performed using the least squares method, with the concentrations of atenolol and p-hydroxyphenylacetamide as the x-axis and peak areas as the y-axis, respectively. For atenolol in the injection concentration range of 0.2–1.5 mg / mL, the linear regression equation was Y = 10156X + 190, and the linear regression coefficient r0 was [missing value]. 2 The linear regression coefficient for the p-hydroxyphenylacetamide intermediate within the injection concentration range of 0.2–1.5 mg / mL was 0.9958. 2 The value was 0.9961; all met the pharmacopoeia requirements. 2 A value ≥0.99 indicates that the method of the present invention has good linearity.

[0073] The above description is merely a specific embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent transformations made using the present invention, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for the simultaneous determination of atenolol and its raw material p-hydroxyphenylacetamide based on high performance liquid chromatography, characterized in that: Weigh 10 mg of the sample to be tested, transfer it to a 10 mL volumetric flask with 10% acetonitrile aqueous solution, and dilute to the mark; detect the content of atenolol and its raw material p-hydroxyphenylacetamide in the sample by high performance liquid chromatography. Chromatographic conditions were set as follows: Detector: Diode array detector; Column: Agilent EC-C18, 4.6 × 250 nm, 5 μm; Mobile phase: Mobile phase A: 0.1% phosphoric acid aqueous solution; Mobile phase B: acetonitrile; Column temperature: 30-40℃; Injection volume: 5-10 μL; Detection wavelength: 210-260 nm; Flow rate: 0.8-1.2 mL / min; Elution method: Gradient elution is performed using mobile phase A and mobile phase B.

2. The method according to claim 1, characterized in that: The elution program is set as follows: 。 3. The method according to claim 1, characterized in that: The detection wavelength is 230 nm, which can simultaneously obtain the optimal absorption peaks of atenolol and p-hydroxyphenethylamine.

4. The method according to claim 1, characterized in that: The separation between peaks is ≥1.5.

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