Method for determining and analyzing impurities in irbesartan
By optimizing the conditions of high performance liquid chromatography and triple quadratic rod tandem mass spectrometry, a method that can quickly, accurately and highly sensitive is developed to detect potential genotoxic impurity FY-S08 in irbesartan, solving the problem of difficult detection in the prior art and ensuring the safety of drug use.
Patent Information
- Application Number
- CN202510567832.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-30
- Publication Date
- 2025-06-13
AI Technical Summary
The prior art lacks methods that can quickly, accurately and highly sensitively detect potential genotoxic impurity FY-S08 in irbesartan, especially under the requirements of small sample usage and rapid analysis.
By optimizing the conditions of high performance liquid chromatography and triple quadratic rod tandem mass spectrometry, a method that can efficiently detect the impurity FY-S08 in irbesartan was developed. The method includes preparing a test sample and a reference sample solution, and testing it through a high performance liquid chromatography-triple quadratic rod tandem mass spectrometer, and calculating the content of impurities using the external standard method.
High sensitivity detection of FY-S08 in irbesartan is achieved, with small sample dosage, simple operation, and the detection limit and quantitative limit are far lower than the limit of impurities, ensuring the safety of patients' medication.
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Figure CN120142530A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the technical field of analytical detection, and particularly to a method for the determination and analysis of impurities in irbesartan. Background Art
[0002] Irbesartan, chemically named 3-((2'-(1H-tetrazol-5-yl)-[1,1'-biphenyl]-4-yl)methyl)-2-butyl-1,3-diazaspiro[4,4]non-1-en-4-one, is a drug for the treatment of essential hypertension and type 2 diabetic nephropathy with hypertension. In the irbesartan process, a potential genotoxic impurity has been newly evaluated, with the chemical name of 4'-(azidomethyl)[1,1-biphenyl]-2-carboxylic acid, whose chemical structure is shown below and is hereinafter referred to as FY-S08.
[0003]
[0004] The above-mentioned impurity FY-S08 is produced by the reaction and hydrolysis of residual 2-cyano-4'-bromomethylbiphenyl and sodium azide in the irbesartan production process system. According to the ICH M7 guideline, the acceptable limit of impurity FY-S08 in irbesartan raw material drug should not exceed 5 ppm. At present, there is no report on the detection method of 4'-(azidomethyl)[1,1-biphenyl]-2-carboxylic acid in irbesartan.
[0005] Therefore, it is an urgent problem for those skilled in the art to develop a method with high sensitivity, small sample consumption and capable of quickly and accurately detecting the genotoxic impurity FY-S08. Summary of the Invention
[0006] To solve the above technical problems, the present invention provides a method for the determination and analysis of impurities in irbesartan. By optimizing the conditions of high performance liquid chromatography and triple quadrupole tandem mass spectrometry, the signal-to-noise ratio of impurity FY-S08 is high, and it has the beneficial effects of high sensitivity, strong specificity, fast analysis and strong anti-interference ability.
[0007] To achieve the above object of the present invention, the following technical solutions are specifically adopted: (1) Prepare a test solution and a reference solution respectively; (2) Inject the test solution and the reference solution obtained in step (1) into a high performance liquid chromatography-triple quadrupole tandem mass spectrometer for detection respectively, and calculate the content of impurities in irbesartan by the external standard method; The impurity is 4'-(azidomethyl)[1,1-biphenyl]-2-carboxylic acid, and the structural formula of the impurity is as follows: .
[0008] Optionally, the test conditions of the high performance liquid chromatography include: mobile phase A is an aqueous ammonium acetate - acetonitrile solution, mobile phase B is acetonitrile, the chromatographic column uses octadecylsilane bonded silica gel as the filler, the flow rate is 0.3 - 0.5 mL / min, the injection volume is 10 - 20 μL, and the column temperature is 38 - 42 °C.
[0009] Optionally, the chromatographic column is Yuexu XB C18.
[0010] Optionally, gradient elution is used in the detection of the high performance liquid chromatography, and the gradient program is as follows: Time (min) Mobile Phase A % Mobile Phase B % 0 78~82 18~22 6 10 90 11 10 90 11.01 78~82 18~22 16 78~82 18~22
[0011] Optionally, the test conditions of the triple quadrupole tandem mass spectrometry include: using an atmospheric pressure chemical ionization ion source (APCI), the monitoring mode is multiple reaction monitoring (MRM), the drying gas temperature is 290 - 310 °C, and the gas flow rate is 3 - 6 L / min.
[0012] Optionally, the limit of quantitation LOQ of the impurity is 0.25 ppm, and the limit of detection LOD is 0.125 ppm.
[0013] Optionally, the concentration of the reference substance solution is 0.25 - 10 ng / mL.
[0014] Optionally, the concentration of the test sample solution is 0.5 - 2 mg / mL, and the solvent is 50% acetonitrile water.
[0015] In summary, the present application includes at least one of the following beneficial technical effects: (1) The detection method of the present invention has high sensitivity, small sample consumption, does not require derivatization of the sample, is easy to operate, and its limit of detection (0.125 ppm) and limit of quantitation (0.25 ppm) are much lower than the limit (5 ppm) of the impurity FY - S08; (2) The detection method of the present invention has a good linear relationship in the range of 0.25 - 10 ng / mL, and the linear correlation coefficient r is about 0.997; (3) The detection method of the present invention is accurate and feasible, the average recovery rate of sample addition is 104.39%, the RSD is 9.45%, and the RSD of the peak area of FY - S08 for 5 consecutive injections of the 100% linear solution is 6.88%; (4) The present invention has developed a new detection method for the potential genotoxic impurity FY - S08, and for the first time, effectively controls FY - S08 (4'-(azidomethyl)[1,1 - biphenyl]-2 - carboxylic acid) in irbesartan, ensuring the medication safety of patients. Description of the Drawings
[0016] Figure 1LC-MS / MS chromatogram of the blank solvent in the specificity verification of Example 2; Figure 2 LC-MS / MS chromatogram of the test solution in the specificity verification of Example 2; Figure 3 LC-MS / MS chromatogram of the reference solution in the specificity verification of Example 2; Figure 4 LC-MS / MS chromatogram of the spiked test solution in the specificity verification of Example 2; Figure 5 Linear relationship diagram of FY-S08; Figure 6 TIC diagram of detecting FY-S08 in irbesartan samples. Detailed implementation manners
[0017] For a clearer understanding of the technical features, objectives, and beneficial effects of the present application, the technical solutions of the present invention will be described in detail below in conjunction with the following specific examples and the accompanying drawings of the specification, but it should not be construed as a limitation on the scope of implementation of the present invention.
[0018] In the embodiments of the present invention, the compound information involved is shown in Table 1.
[0019] Table 1
[0020] Example 1 Detection method (1) Prepare the test solution and the standard curve reference solution respectively: Preparation of the test solution: Take 20 mg of the sample, weigh it accurately, place it in a 20 mL volumetric flask, dissolve it with 50% acetonitrile water and dilute to the mark, and shake well; Preparation of the standard curve reference solution: Take 25 mg of the FY-S08 reference substance, dissolve it with methanol and dilute to 100 mL, and shake well; accurately measure an appropriate amount, dissolve it with 50% acetonitrile water and quantitatively dilute to prepare a solution containing about 0.25, 0.5, 2.5, 5, 7.5, 10 ng per 1 mL; (2) Inject the test solution and the standard curve reference solution obtained in step (1) into an ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometer (Agilent 1290-6470B) for detection, and calculate the content of impurity FY-S08 in the irbesartan test substance by the external standard method.
[0021] Chromatographic conditions: Octadecylsilane chemically bonded silica gel was used as the filler (Welch XB C18, 4.6×150mm, 3μm). 0.01mol / L ammonium acetate aqueous solution - acetonitrile (90:10) was used as mobile phase A, and acetonitrile was used as mobile phase B. Gradient elution was carried out according to Table 2, the flow rate was 0.5mL / min, the column temperature was 40°C, and the injection volume was 15μL.
[0022] Table 2 Gradient elution conditions
[0023] Mass spectrometry conditions: Detection was performed using a triple quadrupole tandem mass spectrometer. An atmospheric pressure chemical ionization ion source (APCI) was used. FY-S08 was scanned in the positive ion mode, and the monitoring mode was multiple reaction monitoring (MRM).
[0024] The ion source parameters are shown in Table 3: Table 3
[0025] The specific quantitative and qualitative parent ion - daughter ion pairs are shown in Table 4: Table 4 Name MRM Ion Pair Use Dwell time (ms) FY-S08 m / z252.0→178.7 Quantification 250 FY-S08 m / z252.0→152.9 Qualification 250 Example 2 Methodological verification
[0026] 2.1 Specificity test 2.1.1 Solution preparation
[0027] ① FY-S08 reference stock solution (50ng / mL): Take 25mg of FY-S08 reference substance, accurately weigh it, place it in a 100mL volumetric flask, dissolve it with methanol and dilute to the mark, shake well; accurately measure 1mL, place it in a 100mL volumetric flask, dilute to the mark with 50% acetonitrile water, shake well, as Solution A; then accurately measure 1mL, place it in a 50mL volumetric flask, dilute to the mark with 50% acetonitrile water, shake well.
[0028] ② Reference solution (5ng / mL): Accurately measure 1mL of FY-S08 reference stock solution, place it in a 10mL volumetric flask, dilute to the mark with 50% acetonitrile water, shake well.
[0029] ③ Test solution: Take 20mg of the sample irbesartan, accurately weigh it, place it in a 20mL volumetric flask, dissolve it by ultrasonic treatment with 50% acetonitrile water and dilute to the mark, shake well.
[0030] ④ Spiked test solution: Take 20mg of the sample irbesartan, accurately weigh it, place it in a 20mL volumetric flask, dissolve it by ultrasonic treatment with 50% acetonitrile water, accurately add 2mL of FY-S08 reference stock solution, and then dilute to the mark with 50% acetonitrile water, shake well.
[0031] 2.1.2 Test operation Precisely measure 15 μL each of the above-mentioned reference solution, test solution, and spiked test solution, inject them into the liquid chromatography-mass spectrometry instrument, and record the chromatogram according to the liquid chromatography conditions and methods in Example 1. Among them, the reference solution is continuously injected 5 times, and the RSD of the peak area of FY-S08 is examined.
[0032] 2.1.3 Results and evaluation The test results are shown in Table 5 and Figure 1 - Figure 4 。
[0033] Table 5 Specificity results
[0034] It can be seen from Table 5 and Figure 1 - Figure 4 that the blank solvent peak and other peaks in the test solution should not interfere with the determination of the FY-S08 peak. The RSD of the peak area of the FY-S08 reference solution is 2.59% when continuously injected 5 times, and the spiked recovery rate is 100.29%, indicating that this method has good specificity.
[0035] 2.2 Limit of quantitation and limit of detection tests 2.2.1 Solution preparation
[0036] ① Limit of quantitation stock solution (5 ng / mL): Precisely measure 1 mL of the reference stock solution under the specificity item of "7.5.1", place it in a 10 mL volumetric flask, dilute it to the mark with 50% acetonitrile-water, and shake well.
[0037] ② Limit of quantitation solution: Precisely measure 1 mL of the limit of quantitation stock solution, place it in a 20 mL volumetric flask, dilute it to the mark with 50% acetonitrile-water, and shake well.
[0038] ③ Limit of detection solution: Precisely measure 0.5 mL of the limit of quantitation stock solution, place it in a 20 mL volumetric flask, dilute it to the mark with 50% acetonitrile-water, and shake well.
[0039] 2.2.2 Test operation Precisely measure 15 μL each of the above-mentioned limit of quantitation solution and limit of detection solution, inject them into the liquid chromatography-mass spectrometry instrument, and detect according to the liquid chromatography conditions and methods in Example 1. The signal of FY-S08 measured on the chromatograph is compared with the baseline at the nearby blank. The selected blank baseline segment shall not be less than 1 minute. The limit of detection is determined by the response concentration at a signal-to-noise ratio of 3 / 1, and the limit of quantitation is determined by the response concentration at a signal-to-noise ratio of 10 / 1. After determining the limit of quantitation and the limit of detection, inject the limit of quantitation and the limit of detection into the liquid chromatography instrument 6 times continuously, and examine the RSD of the peak area of FY-S08 and the signal-to-noise ratio at the limit of quantitation concentration, and examine the peak signal-to-noise ratio at the limit of detection concentration.
[0040] 2.2.3 Results and evaluation The test results are shown in Table 6 below.
[0041] Table 6 Results of Quantitation Limit, Detection Limit and Precision
[0042] As can be seen from Table 6, the RSD of the peak area of the quantitation limit solution FY-S08 for 6 consecutive injections was 6.79%, and the signal-to-noise ratio was greater than 10. The signal-to-noise ratio of the detection limit solution for 6 consecutive injections was greater than 3, indicating that this method is stable, sensitive and precise.
[0043] 2.3 Linear Relationship and Recovery Test 2.3.1 Solution Preparation
[0044] ① Linear stock solution: Accurately measure 1 mL of solution A under the specificity item of "2.1.1", place it in a 50 mL volumetric flask, dilute it to the scale with 50% acetonitrile water, and shake well.
[0045] ② Linear series solutions: Accurately measure 0.5, 0.5, 1.5, and 2 mL of the linear stock solution respectively, place them in 4 10 mL volumetric flasks in sequence, dilute them to the scale with 50% acetonitrile water, and shake well to obtain linear solutions with relative concentrations of 50%, 100%, 150%, and 200%; accurately measure 1 mL of the linear solution with a relative concentration of 100%, place it in 20 mL and 10 mL volumetric flasks respectively, dilute it to the scale with 50% acetonitrile water, and shake well to obtain linear solutions with relative concentrations of 5% and 10%.
[0046] ③ Background solution: Take 20 mg of this product, accurately weigh it, place it in a 20 mL volumetric flask, add 50% acetonitrile water, dissolve it by ultrasonic treatment, and dilute it to the scale, then shake well.
[0047] ④ 10% recovery solution: Take 20 mg of this product, accurately weigh it, place it in a 20 mL volumetric flask, dissolve it by ultrasonic treatment with the solvent, accurately add 2 mL of the 100% linear solution, dilute it to the scale with 50% acetonitrile water, and shake well. Prepare 3 parallel portions.
[0048] ⑤ 50% recovery solution: Take 20 mg of this product, accurately weigh it, place it in a 20 mL volumetric flask, add 50% acetonitrile water, dissolve it by ultrasonic treatment, accurately add 1 mL of the linear stock solution, dilute it to the scale with 50% acetonitrile water, and shake well. Prepare 3 parallel portions.
[0049] ⑥ 100% recovery solution: Take 20 mg of this product, accurately weigh it, place it in a 20 mL volumetric flask, add 50% acetonitrile water, dissolve it by ultrasonic treatment, accurately add 2 mL of the linear stock solution, dilute it to the scale with 50% acetonitrile water, and shake well. Prepare 3 parallel portions.
[0050] ⑦ 150% Recovery Solution: Take 20 mg of this product, accurately weigh it, place it in a 20 mL volumetric flask, add 50% acetonitrile water, dissolve it by ultrasonic treatment, accurately add 3 mL of the linear stock solution, dilute it to the mark with 50% acetonitrile water, and shake well. Prepare 3 portions in parallel.
[0051] 2.3.2 Test Operation Accurately measure 15 μL each of the above linear solution and recovery solution, inject them into the liquid chromatography - mass spectrometry instrument, and detect according to the liquid chromatography conditions and methods in Example 1. Inject the 100% concentration linear solution 5 times, and repeat the injection of other concentration linear solutions 2 times each. With the injection concentration C (ng / mL) as the abscissa and the peak area A as the ordinate, use the least - squares method and the regression analysis tool in Excel for data analysis.
[0052] 2.3.3 Results and Evaluation The linear results are shown in Table 7, and the linear curve is as Figure 5 shown, and the recovery results are shown in Table 8.
[0053] Table 7 Linear Results
[0054] Table 8 Recovery Results
[0055] From Figure 5 and Tables 7 - 8, it can be seen that the linear correlation coefficient r of FY - S08 is approximately 0.997. The RSD of the peak area of FY - S08 for 5 consecutive injections of the 100% linear solution is 6.88%, the average recovery rate of spiked samples is 104.39%, and the RSD is 9.45%. This indicates that this method has a good linear relationship and high accuracy.
[0056] 2.4 Tolerance Test Preparation of tolerance solution: Take 20 mg of this product, accurately weigh it, place it in a 20 mL volumetric flask, dissolve it with 50% acetonitrile water, accurately add 2 mL of the linear stock solution under item "2.1.1", dilute it to the mark with 50% acetonitrile water, and shake well. Accurately measure 15 μL of the above tolerance solution, inject it into the liquid chromatography - mass spectrometry instrument, and examine whether there are significant changes in the peak area of FY - S08 under different detection conditions in Table 9. The test results are shown in Table 10.
[0057] Table 9 Condition 1 Original Condition (Example 1) Condition 2 Drying Gas Temperature: 305°C, others remain unchanged Condition 3 Drying Gas Temperature: 295°C, others remain unchanged Condition 4 Gas Flow Rate: 6.1 L / min, others remain unchanged Condition 5 Gas Flow Rate: 5.9 L / min, others remain unchanged Condition 6 Evaporation Chamber Temperature: 305°C Condition 7 Evaporation Chamber Temperature: 295°C Condition 8 Column Temperature: 42°C, others remain unchanged Condition 9 Column Temperature: 38°C, others remain unchanged Condition 10 Flow Rate: 0.51 mL / min, others remain unchanged Condition 11 Flow Rate: 0.49 mL / min, others remain unchanged Condition 12 Initial Mobile Phase Ratio: 78:22 Condition 13 Initial Mobile Phase Ratio: 82:18 Table 10 Tolerance Results
[0058] As can be seen from Table 10 of the test results, the RSD of the peak area of FY-S08 in the tolerance solution under different conditions was 6.59%, and the retention time changed slightly, but there was no interference from other peaks in the determination of the FY-S08 peak, indicating that the method had good tolerance.
[0059] Example 3 Sample Detection Weigh 6 batches of irbesartan API samples L0102320221001, L0102320221002, L0102320221101, L0102320221102, L0102320221103 and L0102320221104, each 20 mg, place them in a 20 mL volumetric flask, dissolve them with 50% acetonitrile water and dilute to the mark, and shake well; prepare 2 parallel portions for each batch of samples, inject them for detection, and the representative recorded chromatograms are as Figure 6 shown.
[0060] The results showed that FY-S08 was not detected in any of the test samples, that is, it was less than the detection limit, and the limit report was all < 0.125 ppm. At the same time, the crude irbesartan and the sample after acid adjustment and centrifugation in the previous step of the crude product were also detected, and the results also showed that FY-S08 was not detected.
[0061] Of course, the above are only specific embodiments of the present application and do not limit the scope of implementation of the present invention. Any equivalent changes or modifications made according to the characteristics and principles described in the scope of the patent application of the present invention should be included within the scope of the patent application of the present invention.
Claims
1. A method for determining and analyzing impurities in irbesartan, characterized in that: The detection method comprises the following steps: (1) Prepare test sample solution and reference sample solution respectively; (2) injecting the test solution and the reference solution obtained in step (1) into a high performance liquid chromatography-triple quadrupole tandem mass spectrometer for detection, and calculating the content of impurities in irbesartan by an external standard method; The impurity is 4'-(azidomethyl)[1,1-biphenyl]-2-carboxylic acid, and the structural formula of the impurity is as follows: 。 2. The method for determining and analyzing impurities in irbesartan according to claim 1, characterized in that: The test conditions of the high performance liquid chromatography include: mobile phase A is ammonium acetate aqueous solution-acetonitrile, mobile phase B is acetonitrile, the chromatographic column uses octadecylsilane bonded silica gel as a filler, the flow rate is 0.3~0.5mL / min, the injection volume is 10~20μL, and the column temperature is 38~42℃.
3. The method for determining and analyzing impurities in irbesartan according to claim 2, characterized in that: The chromatographic column is Yuexu XB C18.
4. The method for determining and analyzing impurities in irbesartan according to claim 1, characterized in that: The high performance liquid chromatography detection adopts a gradient program for elution, and the gradient program is:
5. The method for determining and analyzing impurities in irbesartan according to claim 1, characterized in that: The test conditions of the triple quadrupole tandem mass spectrometer include: using an atmospheric pressure chemical ionization ion source (APCI), a monitoring mode of multiple reaction monitoring (MRM), a drying gas temperature of 290-310° C., and a gas flow rate of 3-6 L / min.
6. The method for determining and analyzing impurities in irbesartan according to claim 1, characterized in that: The quantification limit LOQ of the impurity is 0.25ppm and the detection limit LOD is 0.125ppm.
7. The method for determining and analyzing impurities in irbesartan according to claim 1, characterized in that: The concentration of the reference solution is 0.25-10 ng / mL.
8. The method for determining and analyzing impurities in irbesartan according to claim 1, characterized in that: The concentration of the test solution is 0.5-2 mg / mL, and the solvent is 50% acetonitrile water.