Method for detecting N-nitrosoduloxetine residue in duloxetine hydrochloride enteric-coated tablet

Abstract

The invention belongs to the technical field of chemical component detection, and particularly relates to a method for detecting N-nitrosoduloxetine residues in duloxetine hydrochloride enteric-coated tablets. The detection method comprises the following steps: S1, diluting duloxetine hydrochloride enteric-coated tablets with a diluting solvent to prepare a test solution; s2, diluting N-nitrosoduloxetine with a diluting solvent to prepare a reference solution; s3, determining the test solution and the reference solution by adopting a liquid chromatography triple quadrupole tandem mass spectrometer, and calculating the content of N-nitrosoduloxetine according to a detection map; the method for detecting the residual N-nitrosoduloxetine in the duloxetine hydrochloride enteric-coated tablet, provided by the invention, has relatively low detection limit and quantitation limit, also has relatively high accuracy, repeatability and intermediate precision, and can be used for rapidly detecting the content of the N-nitrosoduloxetine in the duloxetine hydrochloride enteric-coated tablet.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of chemical component detection, and particularly relates to a detection method for N-nitrosoduloxetine residue in duloxetine hydrochloride enteric-coated tablets. BACKGROUND

[0002] Duloxetine hydrochloride enteric-coated tablets are 5-hydroxytryptamine and norepinephrine reuptake inhibitors, and are used for treating certain mood disorders such as depression and anxiety and relieving central pain such as diabetic peripheral neuropathic pain and women's fibromyalgia, and can also be used for treating women's stress incontinence.

[0003] Duloxetine hydrochloride enteric-coated tablets may produce nitrosamine impurity N-nitrosoduloxetine during production and storage, and the production mechanism is as follows: .

[0004] N-nitrosoduloxetine belongs to a genotoxic impurity and has a genotoxic impurity warning structure. According to relevant research, N-nitrosoduloxetine in duloxetine hydrochloride is considered to have potential genotoxicity, which may have adverse effects on human health. According to the nitrosamine impurity supervision policy of the European Medicines Agency (EMA), the ADI (Acceptable Daily Intake) limit of N-nitrosoduloxetine is 100 ng / day, which is equivalent to 0.83 ppm after conversion. Therefore, the residual amount of N-nitrosoduloxetine in duloxetine hydrochloride enteric-coated tablets needs to be controlled.

[0005] Compared with duloxetine hydrochloride raw materials, duloxetine hydrochloride enteric-coated tablets contain excipients, which will interfere with N-nitrosoduloxetine and affect the detection accuracy of N-nitrosoduloxetine. Shohei Fukuda et al. published a Simple and Practical Method for the Quantitative High-Sensitivity Analysis of N-Nitroso Duloxetine in Duloxetine Drug Products Utilizing LC-MS / MS, which successfully realized the determination of N-nitrosoduloxetine enteric-coated tablets by using liquid chromatography-tandem mass spectrometry method. However, the analysis time of the technical scheme is relatively long. SUMMARY

[0006] The present application aims to solve one or more technical problems existing in the prior art described above, and at least provide a beneficial alternative. Specifically, the present application provides a detection method for N-nitroso-duloxetine residues in duloxetine hydrochloride enteric-coated tablets. The detection method for N-nitroso-duloxetine residues in duloxetine hydrochloride enteric-coated tablets provided by the present application has a lower detection limit and a quantitative limit, and at the same time has a wider detection range, higher accuracy, repeatability and intermediate precision, and can quickly detect the content of N-nitroso-duloxetine in duloxetine hydrochloride enteric-coated tablets.

[0007] To achieve the above-mentioned purpose, the present application adopts the following technical solutions: The present application provides a detection method for N-nitroso-duloxetine residues in duloxetine hydrochloride enteric-coated tablets, comprising the following steps: S1, mixing duloxetine hydrochloride enteric-coated tablets and an extraction solvent, vortexing, oscillating, filtering, taking the supernatant through a 0.22 mu m filter membrane, and adding a dilution solvent to dilute and prepare a test sample solution; S2, diluting N-nitroso-duloxetine with a dilution solvent to prepare a control sample solution; S3, using a liquid chromatography triple quadrupole tandem mass spectrometer to determine the test sample solution and the control sample solution, and calculating the content of N-nitroso-duloxetine according to the detection spectrum; The extraction solvent is a methanol-water solution containing ammonium formate; The chromatographic conditions of the liquid chromatography triple quadrupole tandem mass spectrometer include: The chromatographic column is filled with five fluorophenyl bonded silica gel; A water solution containing 4-6 mmol / L ammonium formate and 0.05-0.15% formic acid is used as mobile phase A, and a methanol solution containing 0.05-0.15% formic acid is used as mobile phase B, and gradient elution is performed.

[0008] Further, the extraction solvent is a methanol-water solution containing 0.01-0.02 mol / L ammonium formate.

[0009] Further, the extraction solvent is a methanol-water solution containing 0.02 mol / L ammonium formate.

[0010] Further, the methanol-water solution is a mixture of methanol and water in a volume ratio of 40-60:40-60.

[0011] Further, the methanol-water solution is a mixture of methanol and water in a volume ratio of 50:50.

[0012] Further, the dilution solvent is a mixture of methanol and water in a volume ratio of 40-60:40-60.

[0013] Further, the dilution solvent is a mixture of methanol and water in a volume ratio of 50:50.

[0014] Further, the solid-liquid ratio of the duloxetine hydrochloride enteric-coated tablets and the extraction solvent is 0.1-0.3 g:10 mL.

[0015] Further, the vortexing time is 1-3 min, and the shaking time is 8-12 min.

[0016] Further, the mobile phase A is a water solution containing 5 mmol / L ammonium formate and 0.1% formic acid; and the mobile phase B is a methanol solution containing 0.1% formic acid.

[0017] Further, the gradient elution program is as follows: 0 min, 60-70% A, 30-40% B; 9 min, 5-15% A, 85-95% B; 11 min, 5-15% A, 85-95% B; 12 min, 60-70% A, 30-40% B; 14 min, 60-70% A, 30-40% B.

[0018] Further, the gradient elution program is as follows: 0 min, 65% A, 35% B; 9 min, 10% A, 90% B; 11 min, 10% A, 90% B; 12 min, 65% A, 35% B; 14 min, 65% A, 35% B.

[0019] Further, the chromatographic column of the liquid chromatography triple quadrupole mass spectrometer is Agilent InfinityLab Poroshell 120 PFP 3.0*150mm 2.7μm.

[0020] Further, the chromatographic conditions further include: Chromatographic column temperature: 38-42℃; Injection volume: 4-6μL; Sample disc temperature: 8-12℃; Flow rate: 0.4-0.6mL / min; Needle washing liquid: methanol and water in a volume ratio of 1:1.

[0021] Further, the mass spectrometry conditions of the liquid chromatography triple quadrupole mass spectrometer include: Ion source: ESI; scanning mode: positive ion mode; multiple reaction monitoring; ion source temperature: 500℃.

[0022] Further, the mass spectrometry conditions of the liquid chromatography triple quadrupole mass spectrometer further include: Gas curtain: 30 psi; Collision gas: 9 psi; Ionization voltage: 5500 V; Spray gas: 50 psi; Auxiliary heating gas: 50 psi.

[0023] Further, the mass spectrometry conditions of the liquid chromatography triple quadrupole mass spectrometer further include monitoring ion pairs and related parameters, specifically as shown in Table 1 below: Table 1 .

[0024] Compared with the prior art, the present application has the following beneficial effects: 1. The detection method provided by the present application can quickly detect N-nitrosoduloxetine in duloxetine hydrochloride enteric-coated tablets by optimizing the chromatographic column, extraction solvent, mobile phase and elution program, and has good specificity, high sensitivity, small sample dosage, simple operation, and a detection limit (0.0415 ppm) and a quantitative limit (0.083 ppm) far lower than the limit (0.83 ppm) of N-nitrosoduloxetine; 2. The detection method provided by the present application has a good linear relationship in the range of 0.083 ppm-1.66 ppm, and the linear correlation coefficient is greater than 0.9999; 3. The detection method provided by the present application has high accuracy, and the average spiked recovery rates at 50%, 100% and 150% levels are 84.5%, 84.3% and 85.1% respectively; the RSD of the average spiked recovery rate is 0.6%; 4. The detection method provided by the present application has good repeatability, and the RSD of the recovery rate of 6 100% spiked solutions is 0.8%; the method has high intermediate precision, and the RSD of the recovery rate of 6 100% spiked solutions is 1.2%; 5. The detection method provided by the present application also has good durability and solution stability, and the selected instrument has low cost. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 A chromatogram of the test sample solution for Example 1; Figure 2 A chromatogram of the blank solution; Figure 3 A chromatogram of the reference solution; Figure 4 A linear relationship diagram; Figure 5 A chromatogram of the 100% limit level spiked sample solution of Comparative Example 1; Figure 6 Chromatogram of the sample solution spiked at the 100% limit level for Comparative Example 2; Figure 7 Chromatogram of the sample solution spiked at the 100% limit level for Comparative Example 3; Figure 8 Chromatogram of the sample solution spiked at the 100% limit level for Comparative Example 4; Figure 9 Chromatogram of the sample solution spiked at the 100% limit level for Comparative Example 5; Figure 10 Chromatogram of the sample solution spiked at the 100% limit level for Comparative Example 6. DETAILED DESCRIPTION

[0026] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. If specific conditions are not indicated in the embodiments, conventional conditions or conditions suggested by the manufacturers are adopted. If the reagents or instruments used are not indicated by the manufacturers, they are all conventional products that can be purchased in the market.

[0027] In the embodiments and comparative examples of the present application: Duloxetine hydrochloride enteric-coated tablets, trade name: Oxsilan; N-nitrosoduloxetine source: CPRD (Shenzhen Zhuo Rui Biological Technology Co., Ltd.).

[0028] Example 1: A method for detecting N-nitrosoduloxetine residues in duloxetine hydrochloride enteric-coated tablets, comprising the following steps: 1. Preparation of solution: Dilution solvent: methanol and water at a volume ratio of 50:50; Extraction solvent: methanol-water solution containing 0.02 mol / L ammonium formate; methanol-water solution: methanol and water at a volume ratio of 40-60:40-60; Blank solution: methanol and water at a volume ratio of 50:50.

[0029] 1.1 Test solution: 2 g of duloxetine hydrochloride enteric-coated tablets was weighed, mixed with 10 mL of extraction solvent, vortexed for 1 min, oscillated for 10 min, filtered, and the supernatant was passed through a 0.22 μm filter membrane, and diluted with dilution solvent to a duloxetine hydrochloride concentration of 2 mg / mL; 1.2 Stock solution: 10 mg of N-nitrosoduloxetine was weighed, diluted with 10 mL of methanol, and then diluted with dilution solvent to a concentration of 17.3 ng / mL; 1.3 Control solution: 1 mL of the stock solution was transferred, and diluted with dilution solvent to a concentration of 1.73 ng / mL.

[0030] 2, the test sample and control product solution are injected into liquid chromatography triple quadrupole mass spectrometer for detection; the detection conditions of liquid chromatography triple quadrupole mass spectrometer are shown in table 2: Table 2 .

[0031] The detection results are shown in table 3: Figure 1 The detection results are shown in table 3:

[0032] Example 2: method validation 1, specificity: the test sample solution, blank solution and control product solution are detected according to the detection method of example 1, and the detection spectrum is shown in table 1: Figures 2-3

[0033] The results show that the blank solution, test sample solution and control product solution have no interference at the target peak, and the specificity of the method is good.

[0034] 2, detection limit: 1 mL of the stock solution is taken, and the dilution solvent is diluted to 5% of the control solution (the theoretical relative content in the sample is: N-nitroso duloxetine 0.0415 ppm) as the LOD solution, the LOD solution is injected into the liquid chromatography triple quadrupole mass spectrometer according to the detection method of example 1, and the test results are shown in table 3.

[0035] Table 3 .

[0036] As shown in table 3, the detection limit of the method is 0.0415 ppm, and the signal-to-noise ratio is greater than 3.

[0037] 3, limit of quantification: 1 mL of the stock solution is taken, and the dilution solvent is diluted to 10% of the control solution (the theoretical relative content in the sample is: N-nitroso duloxetine 0.083 ppm) as the LOQ solution, the LOQ solution is injected into the liquid chromatography triple quadrupole mass spectrometer according to the detection method of example 1, and the test results are shown in table 4.

[0038] Table 4 .

[0039] As shown in table 4, the limit of quantification of the method is 0.083 ppm, and the signal-to-noise ratio is greater than 10.

[0040] ​4. Linear range: 1 mL of the stock solution was taken and diluted with dilution solvent to prepare a series of linear test solutions (10%, 50%, 100%, 150%, 200% of the control solution). Each solution was analyzed once by the detection method of Example 1. The results are shown in Table 5, and the linear relationship is shown in Figure Figure 4 .

[0041] Table 5 .

[0042] As shown in Table 5, the linear equation is y = 397724x - 4283 (r = 1.0000), which indicates that the linear relationship is good in the concentration range of 0.17 ng / mL to 3.45 ng / mL.

[0043] 5. Accuracy: (1) Test sample solution: 2 g of duloxetine hydrochloride enteric-coated tablets was weighed and mixed with 10 mL of extraction solvent, vortexed for 1 min, oscillated for 10 min, filtered, and the supernatant was filtered through a 0.22 μm filter membrane. The dilution solvent was added to prepare a solution containing 2 mg / mL of duloxetine hydrochloride. Two solutions were prepared in parallel; (2) 50% limit level spiked sample solution: the test sample solution was added with a 50% limit level stock solution and diluted with dilution solvent. Three solutions were prepared in parallel; (3) 100% limit level spiked sample solution: the test sample solution was added with a 100% limit level stock solution and diluted with dilution solvent. Three solutions were prepared in parallel; (4) 150% limit level spiked sample solution: the test sample solution was added with a 150% limit level stock solution and diluted with dilution solvent. Three solutions were prepared in parallel.

[0044] Each solution was analyzed once by the detection method of Example 1. The results are shown in Table 6.

[0045] Table 6 .

[0046] As shown in Table 6, the average recovery rate of the 9 samples was 84.6%, and the RSD was 0.6%, which indicates that the accuracy of the method is high.

[0047] 6. Reproducibility: a 100% limit level spiked sample solution was prepared (in the same preparation method as the accuracy test), and six solutions were prepared in parallel. Each solution was analyzed once by the detection method of Example 1. The results are shown in Table 7.

[0048] Table 7 .

[0049] As shown in Table 7, the repeatability recovery rate of 6 samples is 83.9%-85.7%, and the RSD is 0.8%, indicating that the repeatability of the method is high.

[0050] 7. Intermediate precision: another researcher prepared 6 spiked sample solutions of 100% limit level at different times (same preparation method as the accuracy test), and the results are shown in Table 8.

[0051] Table 8 .

[0052] As shown in Table 8, the recovery rate of 12 samples is 83.9%-86.8%, and the RSD is 1.2%, indicating that the intermediate precision of the method is high.

[0053] 8. Robustness: prepare spiked sample solutions of 100% limit level (same preparation method as the accuracy test), and change the initial organic phase ratio, column temperature, and flow rate, respectively. Each condition is injected once, and the RSD of the peak area of 6 tests under a single condition is not greater than 20.0%, which meets the requirements.

[0054] 9. Solution stability: prepare one control solution and one spiked sample solution of 100% limit level, respectively, and analyze these solutions at 24 hours or other time periods, and compare them with freshly prepared solutions. The N-nitrosoduloxetine content of the stability solution is between 70% and 130% of the initial N-nitrosoduloxetine content, indicating that the solution stability is good.

[0055] Comparative Example 1: The only difference from Example 1 is that the extraction solvent is methanol and water in a volume ratio of 50:50; the rest is the same; and the 100% limit level spiked sample solution is detected using the above conditions, and the results are shown in Figure 5 , showing that the peak shape is broadened and the response is lowered.

[0056] Comparative Example 2: The only difference from Example 1 is that the mobile phase A is 0.1% formic acid aqueous solution; the mobile phase B is acetonitrile; the rest is the same; and the 100% limit level spiked sample solution is detected using the above conditions, and the results are shown in Figure 6 , showing that the peak shape is poor.

[0057] Comparative Example 3: The only difference from Example 1 is that the mobile phase A is 2 mmol / L ammonium acetate + 0.1% formic acid aqueous solution; the rest is the same; and the 100% limit level spiked sample solution is detected using the above conditions, and the results are shown in Figure 7 , showing that the peak shape is poor.

[0058] Comparative Example 4: The only difference from Example 1 is that the gradient elution program is isocratic elution with mobile phase A (5 mM ammonium formate + 0.1% formic acid in water) - mobile phase B (0.1% formic acid in methanol) (11 :9, v / v) with a run time of 14 min; the rest is the same; the 100% limit level spiked sample solution is detected using the above conditions, and the results are shown in Table 1, no new impurity peak eluted within 13 min. Figure 8

[0059] Comparative Example 5: The only difference from Example 1 is that the gradient elution program is: 0-2 min, 50% A, 50% B; 2-5 min, 50-32% A, 50-68% B; 5-7 min, 32% A, 68% B; 7-8 min, 32-10% A, 68-90% B; 8-9 min, 10% A, 90% B; 9-10 min, 10-50% A, 90-50% B; 10-14 min, 50% A, 50% B; the rest is the same; the 100% limit level spiked sample solution is detected using the above conditions, and the results are shown in Table 1, a new impurity peak eluted after N-nitroso-duloxetine. Figure 9

[0060] Comparative Example 6: The only difference from Example 1 is that the gradient elution program is: 0 min, 75% A, 25% B; 9 min, 20% A, 80% B; 11 min, 20% A, 80% B; 12 min, 75% A, 25% B; 14 min, 75% A, 25% B; the rest is the same, the 100% limit level spiked sample solution is detected using the above conditions, and the results are shown in Table 1, the peak shape is poor, and a new impurity peak eluted after N-nitroso-duloxetine. Figure 10

[0061] Finally, it should be noted that the above content is only used to illustrate the technical solutions of the present application, and is not a limitation on the protection scope of the present application. Simple modifications or equivalent replacements of the technical solutions of the present application made by those skilled in the art do not deviate from the essence and scope of the technical solutions of the present application.​​​

Claims

1. A method for detecting N-nitroso-duloxetine residues in duloxetine hydrochloride enteric-coated tablets, characterized in that: The following steps are involved: S1. Mix duloxetine hydrochloride enteric-coated tablets and the extraction solvent, vortex, shake, and filter. Pass the supernatant through a 0.22 μm filter membrane and dilute with the dilution solvent to prepare the test solution. S2, N-nitroso-duloxetine is diluted with diluent to prepare reference solution; S3. Determine the test solution and the reference solution using a liquid chromatography triple quadrupole tandem mass spectrometer, and calculate the content of N-nitrosoduloxetine based on the detection spectrum; The extraction solvent is a methanol-water solution containing ammonium formate; The chromatographic conditions of the liquid chromatography triple quadrupole tandem mass spectrometer include: Chromatographic column: Pentafluorophenyl bonded silica gel as filler; Gradient elution was performed using an aqueous solution containing 4-6 mmol / L ammonium formate and 0.05-0.15% formic acid as mobile phase A and a methanol solution containing 0.05-0.15% formic acid as mobile phase B.

2. The method for detecting N-nitroso-duloxetine residue in duloxetine hydrochloride enteric-coated tablets according to claim 1, characterized in that: The procedure of the gradient elution is as follows: 0 min, 60-70% A, 30-40% B; 9min, 5-15%A, 85-95%B; 11min, 5-15%A, 85-95%B; 12 min, 60-70% A, 30-40% B; 14min, 60-70%A, 30-40%B.

3. The method for detecting N-nitroso-duloxetine residue in duloxetine hydrochloride enteric-coated tablets according to claim 2, characterized in that: The extraction solvent is a methanol-water solution containing 0.01-0.02 mol. / L ammonium formate.

4. The method for detecting N-nitroso-duloxetine residue in duloxetine hydrochloride enteric-coated tablets according to claim 3, characterized in that: The mobile phase A is an aqueous solution containing 5 mmol / L ammonium formate and 0.1% formic acid; the mobile phase B is a methanol solution containing 0.1% formic acid.

5. The method for detecting N-nitroso-duloxetine residue in duloxetine hydrochloride enteric-coated tablets according to claim 4, characterized in that: The procedure of the gradient elution is: 0 min, 65% A, 35% B; 9 min, 10% A, 90% B; 11 min, 10% A, 90% B; 12 min, 65% A, 35% B; 14min, 65%A, 35%B.

6. The method for detecting N-nitroso-duloxetine residue in duloxetine hydrochloride enteric-coated tablets according to claim 5, characterized in that: The chromatographic column of the liquid chromatography triple quadrupole tandem mass spectrometer is Agilent InfinityLab Poroshell 120 PFP 3.0*150mm 2.7μm.

7. The method for detecting N-nitroso-duloxetine residue in duloxetine hydrochloride enteric-coated tablets according to claim 6, characterized in that: The chromatographic conditions also include: Column temperature: 38-42°C; Injection volume: 4-6 μL; Sample tray temperature: 8-12°C; Flow rate: 0.4-0.6 mL / min; Needle wash solution: methanol and water in a volume ratio of 1:

1.

8. The method for detecting N-nitroso-duloxetine residue in duloxetine hydrochloride enteric-coated tablets according to claim 7, characterized in that: The mass spectrometry conditions of the liquid chromatography triple quadrupole tandem mass spectrometer include: ion source: ESI; scanning mode: positive ion mode; multiple reaction monitoring; ion source temperature: 500°C.

9. The method for detecting N-nitroso-duloxetine residue in duloxetine hydrochloride enteric-coated tablets according to claim 8, characterized in that: The mass spectrometry conditions of the liquid chromatography triple quadrupole tandem mass spectrometer also include: Curtain gas: 30psi; Collision gas: 9 psi; Ionization voltage: 5500V; Spray gas: 50psi; Auxiliary heating gas: 50psi.

10. The method for detecting N-nitroso-duloxetine residue in duloxetine hydrochloride enteric-coated tablets according to claim 9, characterized in that: The mass spectrometry conditions of the liquid chromatography triple quadrupole tandem mass spectrometer also include monitoring ion pairs and parameters: 。

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