Method for determining and analyzing impurities in irbesartan

Abstract

The invention discloses a method for determining and analyzing impurities in irbesartan. The method comprises the following steps: (1) respectively preparing a test solution and a reference solution; (2) respectively injecting the test solution and the reference solution obtained in the step (1) into a high performance liquid chromatography-triple quadrupole tandem mass spectrometer for detection, and calculating the content of impurities in irbesartan by adopting an external standard method, the impurity is 2-butyl-1-nitroso-1, 3-diazaspiro [4.4] nonyl-2-ene-4-ketone, and the impurity is 2-butyl-1-nitroso-1, 3-diazaspiro [4.4] nonyl-2-ene-4-ketone. By optimizing the conditions of the high performance liquid chromatography and the triple quadrupole tandem mass spectrometry, the signal-to-noise ratio of the impurities is high, and the method has the beneficial effects of high sensitivity, high specificity, rapid analysis and high interference resistance.

Description

Technical Field

[0001] The present application relates to the technical field of analytical detection, and particularly relates to a method for determining and analyzing impurities in irbesartan. Background Art

[0002] Irbesartan has the chemical name of 3-((2'-(1H-tetrazol-5-yl)-[1,1'-biphenyl]-4-yl)methyl)-2-butyl-1,3-diazaspiro[4,4]non-1-en-4-one, and is a drug for treating essential hypertension and type 2 diabetic nephropathy with hypertension. In the process of irbesartan, a potential genotoxic impurity has been newly evaluated, with the chemical name of 2-butyl-1-nitroso-1,3-diazaspiro[4.4]non-2-en-4-one, whose chemical structure is shown below and is hereinafter referred to as FY-S03.

[0003]

[0004] The above-mentioned impurity FY-S03 is produced by the reaction of the irbesartan intermediate 2-butyl-1,3-diazaspiro[4,4]non-1-en-4-one (also known as irbesartan side chain) remaining in the system with nitrite ions (sodium azide reactant is in excess in the reaction, and nitrous acid is used to treat the remaining azide under acidic conditions). According to the ICH M7 guideline, the acceptable limit of impurity FY-S03 in irbesartan bulk drug should not exceed 5 ppm.

[0005] Since the acceptable limit of impurity FY-S03 in irbesartan bulk drug is only 5 ppm, the detection sensitivity of the conventional high performance liquid chromatography (HPLC) test method cannot meet the requirements, and it is technically difficult to sensitively and accurately detect trace impurity FY-S03 in irbesartan bulk drug. At present, there is no report on the detection method of 2-butyl-1-nitroso-1,3-diazaspiro[4.4]non-2-en-4-one in irbesartan.

[0006] 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-S03. Summary of the Invention

[0007] In order to solve the above technical problems, the present invention provides a method for determining and analyzing 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-S03 is high, and it has the beneficial effects of high sensitivity, strong specificity, fast analysis and strong anti-interference ability.

[0008] In order to achieve the above object of the present invention, the following technical solutions are specifically adopted: (1) Prepare the test solution and reference solution respectively; (2) Inject the test solution and 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 2-butyl-1-nitroso-1,3-diazaspiro[4.4]non-2-ene-4-one, and the structural formula of the impurity is as follows: 。

[0009] Optionally, the test conditions of the high performance liquid chromatography include: mobile phase A is 0.1% formic acid in water, 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 2-5 μL, and the column temperature is 38-42 °C.

[0010] Optionally, the chromatographic column is Welch XB C18.

[0011] Optionally, gradient elution is used in the detection of the high performance liquid chromatography, and the gradient program is as follows: Time (minutes) Mobile phase A % Mobile phase B % 0 58~62 38~42 13 58~62 38~42 15 10 90 21 10 90 21.01 58~62 38~42 23 58~62 38~42

[0012] Optionally, the test conditions of the triple quadrupole tandem mass spectrometer include: using an electrospray ionization source (AJSESI), 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.

[0013] Optionally, the limit of quantitation LOQ of the impurity is 0.10 ppm, and the limit of detection LOD is 0.05 ppm.

[0014] Optionally, the concentration of the reference solution is 0.05-5 ng / mL.

[0015] Optionally, the concentration of the test solution is 0.5-2 mg / mL, and the solvent is acetonitrile.

[0016] 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 (0.05 ppm) and limit of quantitation (0.10 ppm) are much lower than the limit (5 ppm) of impurity FY-S03; (2) The detection method of the present invention has a good linear relationship in the range of 0.05-5 ng / mL, and the linear correlation coefficient r is about 0.9996; (3) The detection method of the present invention is accurate and feasible. The average recovery rate of sample addition is 104.41%, the RSD is 5.99%, and the RSD of the peak area of FY-S03 for 5 consecutive injections of the 100% linear solution is 0.61%. (4) The present invention has developed a new detection method for potential genotoxic impurity FY-S03, and for the first time, effectively controls FY-S03 (2-butyl-1-nitroso-1,3-diazaspiro[4.4]non-2-ene-4-one) in irbesartan, ensuring the medication safety of patients. Description of the Drawings

[0018] Figure 1 It is the LC-MS / MS spectrogram of the blank solvent in the specificity verification of Example 2; Figure 2 It is the LC-MS / MS spectrogram of the test solution in the specificity verification of Example 2; Figure 3 It is the LC-MS / MS spectrogram of the reference solution in the specificity verification of Example 2; Figure 4 It is the LC-MS / MS spectrogram of the spiked test solution in the specificity verification of Example 2; Figure 5 It is the linear relationship diagram of FY-S03. Detailed Embodiments

[0019] 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 embodiments and the drawings of the specification, but it should not be construed as a limitation on the scope of implementation of the present invention.

[0020] In the embodiments of the present invention, the information of the compounds involved is shown in Table 1.

[0021] Table 1

[0022] Example 1 Detection Method (1) Prepare the test solution and the standard curve reference solution respectively: Preparation of the test solution: Take 50 mg of the sample, accurately weigh it, place it in a 100 mL volumetric flask, dissolve it with acetonitrile and dilute to the mark, and shake well; Preparation of the standard curve reference solution: Take 25 mg of the FY-S03 reference substance, dissolve it with methanol and dilute to 100 mL, and shake well; accurately measure an appropriate amount, dissolve it with acetonitrile and quantitatively dilute to prepare a solution containing about 0.05, 0.25, 1.25, 2.5, 3.75, and 5 ng of each in every 1 mL of the standard curve reference solution; (2) Inject the test sample 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-S03 in the test sample of irbesartan by the external standard method.

[0023] Chromatographic conditions: Use octadecylsilane chemically bonded silica gel as the filler (Welch XB C18, 4.6×150mm, 3μm), use 0.1% formic acid water as mobile phase A, and acetonitrile as mobile phase B, perform gradient elution according to Table 2, the flow rate is 0.4 mL / min, the column temperature is 40 °C, and the injection volume is 3 μL.

[0024] Table 2 Gradient elution conditions

[0025] Mass spectrometry conditions: Detect with a triple quadrupole tandem mass spectrometer, use an electrospray ionization source (AJS ESI), and use the positive ion scan mode for FY-S03, and the monitoring mode is multiple reaction monitoring (MRM).

[0026] The AJS ESI ion source parameters are shown in Table 3: Table 3

[0027] The specific quantitative and qualitative parent ion-daughter ion pairs are shown in Table 4: Table 4

[0028] Example 2 Methodology verification 2.1 Specificity test 2.1.1 Solution preparation

[0029] ① FY-S03 reference stock solution (50 ng / mL): Take 25 mg of FY-S03 reference substance, weigh it accurately, place it in a 100 mL volumetric flask, dissolve it with methanol and dilute to the mark, and shake well; accurately measure 1 mL, place it in a 100 mL volumetric flask, and dilute to the mark with acetonitrile, and shake well; then accurately measure 1 mL, place it in a 50 mL volumetric flask, and dilute to the mark with acetonitrile, and shake well.

[0030] ② Reference solution (2.5 ng / mL): Accurately measure 0.5 mL of the FY-S03 reference stock solution, place it in a 10 mL volumetric flask, and dilute to the mark with acetonitrile, and shake well.

[0031] ③ Test sample solution: Take 10 mg of the sample irbesartan, weigh it accurately, place it in a 20 mL volumetric flask, dissolve it by ultrasonic treatment with acetonitrile and dilute to the mark, and shake well.

[0032] ④Spiked test solution: Accurately weigh 10 mg of the sample irbesartan, place it in a 20 mL volumetric flask, dissolve it ultrasonically with acetonitrile, accurately add 1 mL of the FY-S03 reference stock solution, and then dilute it to the mark with acetonitrile and mix well.

[0033] 2.1.2 Test operation Precisely measure 3 μL each of the above 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 method of Example 1. For the reference solution, inject it continuously for 5 injections to examine the RSD of the peak area of FY-S03.

[0034] 2.1.3 Results and evaluation The test results are shown in Table 5 and Figures 1 - 4 .

[0035] Table 5 Specificity results

[0036] From Table 5 and Figures 1 - 4 it can be seen that the blank solvent peak and other peaks in the test solution should not interfere with the determination of the FY-S03 peak. The RSD of the peak area of the FY-S03 reference solution for 5 consecutive injections is 2.96%, and the spiked recovery rate is 112.61%, indicating that this method has good specificity.

[0037] 2.2 Limit of quantitation and limit of detection tests 2.2.1 Solution preparation

[0038] ① Limit of quantitation stock solution: Precisely measure 0.5 mL of the reference stock solution under the specificity item in 2.1.1, place it in a 10 mL volumetric flask, and dilute it to the mark with acetonitrile and mix well.

[0039] ② Limit of quantitation solution: Precisely measure 1 mL of the limit of quantitation stock solution, place it in a 50 mL volumetric flask, and dilute it to the mark with acetonitrile and mix well.

[0040] ③ Limit of detection solution: Precisely measure 1 mL of the limit of quantitation stock solution, place it in a 100 mL volumetric flask, and dilute it to the mark with acetonitrile and mix well.

[0041] 2.2.2 Test operation Precisely measure 3 μL each of the above-mentioned quantification limit solution and detection limit solution, inject them into the liquid chromatography-mass spectrometry instrument, and detect according to the liquid chromatography-mass spectrometry conditions and methods of Example 1. The signal measured by FY-S03 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 detection limit is determined by the response concentration at a signal-to-noise ratio of 3 / 1, and the quantification limit is determined by the response concentration at a signal-to-noise ratio of 10 / 1. After determining the quantification limit and detection limit, inject the quantification limit and detection limit into the liquid chromatograph continuously for 6 times, and examine the RSD of the peak area of FY-S03 and the signal-to-noise ratio at the quantification limit concentration, and examine the peak signal-to-noise ratio at the detection limit concentration.

[0042] 2.2.3 Results and evaluation The test results are shown in Table 6 below.

[0043] Table 6 Results of quantification limit, detection limit and precision

[0044] As can be seen from Table 6, the RSD of the peak area of FY-S03 in the quantification limit solution for 6 consecutive injections is 1.73%, and the signal-to-noise ratio is greater than 10 for all. The signal-to-noise ratio of the detection limit solution for 6 consecutive injections is greater than 3, indicating that this method is stable, sensitive and precise.

[0045] 2.3 Linear relationship and recovery test 2.3.1 Solution preparation

[0046] ① Linear stock solution: Precisely measure 2 mL of solution A under the specificity item of "2.1.1", place it in a 100 mL volumetric flask, dilute it to the mark with acetonitrile, and shake well.

[0047] ② Linear series solutions: Precisely measure 0.5, 0.5, 1.5, 1 mL of the linear stock solution respectively, place them in 20, 10, 20, 10 mL volumetric flasks in sequence, dilute them to the mark with acetonitrile, and shake well to obtain linear solutions with relative concentrations of 50%, 100%, 150%, 200%; precisely measure 1 mL of the linear solution with a relative concentration of 100%, place it in a 10 mL volumetric flask, dilute it to the mark with acetonitrile, and shake well to obtain a linear solution with a relative concentration of 10%; precisely measure 2 mL of the linear solution with a relative concentration of 10%, place it in a 10 mL volumetric flask, dilute it to the mark with acetonitrile, and shake well to obtain a linear solution with a relative concentration of 2%.

[0048] ③ Background solution: Take 50 mg of this product, accurately weigh it, place it in a 100 mL volumetric flask, add acetonitrile, dissolve it by ultrasonic treatment, and dilute it to the mark, then shake well.

[0049] ④ 10% recovery solution: Take 50 mg of this product, accurately weigh it, place it in a 100 mL volumetric flask, add acetonitrile, dissolve it by ultrasonic treatment, accurately add 0.5 mL of the linear stock solution, dilute it to the mark with acetonitrile, and shake well. Prepare 3 parallel portions.

[0050] ⑤ 50% recovery solution: Accurately weigh 50 mg of this product, place it in a 100 mL volumetric flask, dissolve it by ultrasonic treatment with acetonitrile, accurately add 2.5 mL of the linear stock solution, dilute it to the mark with acetonitrile, and shake well. Prepare 3 portions in parallel.

[0051] ⑥ 100% recovery solution: Accurately weigh 50 mg of this product, place it in a 100 mL volumetric flask, dissolve it by ultrasonic treatment with acetonitrile, accurately add 5 mL of the linear stock solution, dilute it to the mark with acetonitrile, and shake well. Prepare 3 portions in parallel.

[0052] ⑦ 150% recovery solution: Accurately weigh 50 mg of this product, place it in a 100 mL volumetric flask, dissolve it by ultrasonic treatment with acetonitrile, accurately add 7.5 mL of the linear stock solution, dilute it to the mark with acetonitrile, and shake well. Prepare 3 portions in parallel.

[0053] 2.3.2 Test operation Precisely measure 3 μL of each of the above-mentioned linear solutions and recovery solutions, inject them into the liquid chromatography-mass spectrometry instrument, and detect them according to the liquid chromatography conditions and methods of Example 1. Inject the 100% concentration linear solution 5 times, and repeat the injection of other concentration linear solutions 2 times. 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.

[0054] 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.

[0055] Table 7 Linear results

[0056] Table 8 Recovery results

[0057] From Figure 5 and Tables 7 - 8, it can be seen that the linear correlation coefficient r of FY-S03 is approximately 0.9996, the RSD of the peak area of FY-S03 for 5 consecutive injections of the 100% linear solution is 0.61%, the average recovery rate of the added sample is 104.41%, and the RSD is 5.99%, indicating that the linear relationship and precision of this method are good and the accuracy is high.

[0058] 2.4 Tolerance test Preparation of the tolerance solution: Take 50 mg of this product, accurately weigh it, place it in a 100 mL volumetric flask, dissolve it with acetonitrile, accurately add 5 mL of the linear stock solution under item "2.1.1", dilute it to the scale with acetonitrile, and shake well. Accurately measure 3 μL of the above tolerance solution and inject it into the liquid chromatography-mass spectrometry instrument to examine whether there are significant changes in the peak area of FY-S03 under different detection conditions in Table 9. The test results are shown in Table 10.

[0059] Table 9

[0060] Table 10 Tolerance Results

[0061] It can be seen from Table 10 of the test results that under different conditions, the RSD of the peak area of FY-S03 in the tolerance solution is 1.96%, and the retention time changes slightly, but there is no interference from other peaks on the determination of the FY-S03 peak, indicating that the method has good durability. Example 3 Sample Detection

[0062] Weigh 6 batches of irbesartan API samples L0102320221001, L0102320221002, L0102320221101, L0102320221102, L0102320221103 and L0102320221104, each 50 mg, place them in a 100 mL volumetric flask, dilute them to the scale with acetonitrile, and shake well; prepare 2 parallel portions for each batch of samples and perform injection detection.

[0063] The results are shown in Table 11. FY-S03 was not detected in the test samples L0102320221001, L0102320221101, L0102320221102 and L0102320221103. The detected FY-S03 content in the test samples L0102320221002 and L0102320221104 was less than 0.2 ppm, far lower than the limit requirement of the impurity FY-S03 (5 ppm).

[0064] Table 11

[0065] Of course, the above are only specific embodiments of this application and do not limit the scope of implementation of the present invention. Any equivalent changes or modifications made according to the features 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 2-butyl-1-nitroso-1,3-diazaspiro[4.4]non-2-en-4-one, 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 0.1% formic acid water, 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 2~5μL, and the column temperature is 38~42°C.

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 electrospray ionization source (AJS ESI), 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 0.10ppm and the detection limit LOD is 0.05ppm.

7. The method for determining and analyzing impurities in irbesartan according to claim 1, characterized in that: The concentration of the reference solution is 0.05-5 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 acetonitrile.

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