A method for detecting residual n-nitrosoduloxetine in duloxetine hydrochloride enteric-coated tablets

Abstract

The application belongs to the technical field of chemical component detection, and particularly relates to a detection method of N-nitrosoldoxetin residual in doxepin hydrochloride enteric-coated tablets; the detection method comprises the following steps: S1, diluting doxepin hydrochloride enteric-coated tablets with a dilution solvent to prepare a test sample solution; S2, diluting N-nitrosoldoxetin with a dilution solvent to prepare a control sample solution; S3, determining the test sample solution and the control sample solution by using a liquid chromatography triple quadrupole tandem mass spectrometer, and calculating the content of N-nitrosoldoxetin according to a detection spectrum; the detection method of N-nitrosoldoxetin residual in doxepin hydrochloride enteric-coated tablets provided by the application has lower detection limit and quantitative limit, and has higher accuracy, repeatability and intermediate precision, and can quickly detect the content of N-nitrosoldoxetin in doxepin hydrochloride enteric-coated tablets.

Description

Technical Field

[0001] This invention belongs to the field of chemical component detection technology, specifically relating to a method for detecting N-nitrosoduloxetine residues in duloxetine hydrochloride enteric-coated tablets. Background Technology

[0002] Duloxetine hydrochloride enteric-coated tablets are serotonin and norepinephrine reuptake inhibitors used to treat certain mood disorders such as depression and anxiety, as well as to relieve central pain such as diabetic peripheral neuropathy and fibromyalgia in women. They can also be used to treat stress urinary incontinence in women.

[0003] Duloxetine hydrochloride enteric-coated tablets may generate nitrosamine impurities, specifically N-nitrosoduloxetine, during production and storage. The formation mechanism is as follows:

[0004] .

[0005] N-nitrosoduloxetine is classified as a genotoxic impurity and carries a genotoxic impurity warning structure. According to relevant studies, N-nitrosoduloxetine in duloxetine hydrochloride is considered to have potential genotoxicity and may have adverse effects on human health. According to the European Medicines Agency's (EMA) regulatory policy on nitrosamine impurities, the ADI (Acceptable Daily Intake) limit for N-nitrosoduloxetine is 100 ng / day, which translates to 0.83 ppm. Therefore, it is necessary to control the residual amount of N-nitrosoduloxetine in duloxetine hydrochloride enteric-coated tablets.

[0006] Compared to duloxetine hydrochloride raw material, enteric-coated duloxetine hydrochloride tablets contain excipients, which can interfere with the detection accuracy of N-nitrosoduloxetine. In existing technologies, Shohei Fukuda et al. published "Simple and Practical Method for the Quantitative High-Sensitivity Analysis of N-Nitroso Duloxetine in Duloxetine Drug Products Utilizing LC-MS / MS," which successfully determined N-nitrosoduloxetine enteric-coated tablets using liquid chromatography-tandem mass spectrometry. However, this method has a relatively long analysis time. Summary of the Invention

[0007] The present invention aims to solve one or more technical problems existing in the prior art, and at least provides a beneficial solution. Specifically, the present invention provides a method for detecting N-nitrosoduloxetine residues in duloxetine hydrochloride enteric-coated tablets. The method provided by the present invention has a lower limit of detection and limit of quantitation, while having a wider detection range, higher accuracy, repeatability, and intermediate precision, and can rapidly detect the N-nitrosoduloxetine content in duloxetine hydrochloride enteric-coated tablets.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A method for detecting N-nitrosoduloxetine residues in duloxetine hydrochloride enteric-coated tablets, comprising the following steps:

[0010] S1. Mix duloxetine hydrochloride enteric-coated tablets and extraction solvent, vortex, shake, filter, take the supernatant and filter through a 0.22μm filter membrane, add diluent to dilute and prepare the test solution;

[0011] S2, N-nitrosoduloxetine was diluted with a diluent to prepare a reference solution;

[0012] S3. The test solution and reference solution were determined by liquid chromatography-triple quadrupole tandem mass spectrometry, and the content of N-nitrosoduloxetine was calculated based on the detection chromatograms.

[0013] The extraction solvent is a methanol-water solution containing ammonium formate;

[0014] The chromatographic conditions for the liquid chromatography triple quadrupole tandem mass spectrometer include:

[0015] Column: packed with pentafluorophenyl bonded silica gel;

[0016] 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.

[0017] Furthermore, the extraction solvent is a methanol-water solution containing 0.01-0.02 mol / L ammonium formate.

[0018] Furthermore, the extraction solvent is a methanol-water solution containing 0.02 mol / L ammonium formate.

[0019] Furthermore, the methanol-water solution is methanol and water in a volume ratio of 40-60:40-60.

[0020] Furthermore, the methanol-water solution is methanol and water in a volume ratio of 50:50.

[0021] Furthermore, the diluent is methanol and water in a volume ratio of 40-60:40-60.

[0022] Furthermore, the diluent is methanol and water in a volume ratio of 50:50.

[0023] Furthermore, the solid-liquid ratio of the duloxetine hydrochloride enteric-coated tablets to the extraction solvent is 0.1-0.3 g: 10 mL.

[0024] Furthermore, the vortex duration is 1-3 minutes, and the oscillation duration is 8-12 minutes.

[0025] Further, 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.

[0026] Furthermore, the gradient elution procedure is as follows:

[0027] 0 min, 60-70% A, 30-40% B;

[0028] 9 min, 5-15% A, 85-95% B;

[0029] 11 min, 5-15% A, 85-95% B;

[0030] 12 min, 60-70% A, 30-40% B;

[0031] 14 min, 60-70% A, 30-40% B.

[0032] Furthermore, the gradient elution procedure is as follows:

[0033] 0 min, 65% A, 35% B;

[0034] 9 min, 10%A, 90%B;

[0035] 11 min, 10%A, 90%B;

[0036] 12 min, 65% A, 35% B;

[0037] 14 min, 65% A, 35% B.

[0038] Furthermore, the chromatographic column of the liquid chromatography triple quadrupole tandem mass spectrometer is an Agilent Infinity LabPoroshell 120 PFP 3.0*150mm 2.7μm.

[0039] Furthermore, the chromatographic conditions also include:

[0040] Column temperature: 38-42℃;

[0041] Injection volume: 4-6 μL;

[0042] Sample tray temperature: 8-12℃;

[0043] Flow rate: 0.4-0.6 mL / min;

[0044] Needle washing solution: methanol and water in a 1:1 volume ratio.

[0045] Furthermore, the mass spectrometry conditions of the liquid chromatography triple quadrupole tandem mass spectrometer include:

[0046] Ion source: ESI; Scan mode: positive ion mode; Multiple reaction monitoring; Ion source temperature: 500℃.

[0047] Furthermore, the mass spectrometry conditions of the liquid chromatography triple quadrupole tandem mass spectrometer also include:

[0048] Air curtain gas: 30 psi;

[0049] Collision gas: 9 psi;

[0050] Ionization voltage: 5500V;

[0051] Spray gas: 50 psi;

[0052] Auxiliary heating gas: 50 psi.

[0053] Furthermore, the mass spectrometry conditions of the liquid chromatography triple quadrupole tandem mass spectrometer also include monitoring ion pairs and related parameters, as detailed in Table 1 below:

[0054] Table 1

[0055] .

[0056] Compared with the prior art, the present invention has the following beneficial effects:

[0057] 1. This invention provides a detection method for the rapid detection of N-nitrosoduloxetine in duloxetine hydrochloride enteric-coated tablets by optimizing the chromatographic column, extraction solvent, mobile phase, and elution procedure. It has good specificity, high sensitivity, small sample volume, and simple operation. Its detection limit (0.0415 ppm) and quantitation limit (0.083 ppm) are far lower than the limit of N-nitrosoduloxetine (0.83 ppm).

[0058] 2. The detection method provided by this invention exhibits good linearity in the range of 0.083ppm-1.66ppm, with a linear correlation coefficient greater than 0.9999;

[0059] 3. The detection method provided by this invention has high accuracy, with average spiked recoveries of 84.5%, 84.3%, and 85.1% at 50%, 100%, and 150% levels, respectively; the RSD of the average spiked recoveries is 0.6%.

[0060] 4. The detection method provided by this invention has good repeatability, with an RSD of 0.8% for the recovery rate of 6 100% spiked solutions; it also has high intermediate precision, with an RSD of 1.2% for the recovery rate of 6 100% spiked solutions.

[0061] 5. The detection method provided by this invention also has good durability and solution stability, and the selected instruments are low in cost. Attached Figure Description

[0062] Figure 1 The chromatogram of the test solution in Example 1;

[0063] Figure 2 The chromatogram is for a blank solution;

[0064] Figure 3 The chromatogram is of the reference solution;

[0065] Figure 4 A linear relationship diagram;

[0066] Figure 5 This is the chromatogram of the spiked sample solution at the 100% limit level for Comparative Example 1;

[0067] Figure 6 This is the chromatogram of the spiked sample solution at the 100% limit level for Comparative Example 2;

[0068] Figure 7 The chromatogram of the spiked sample solution at the 100% limit level for Comparative Example 3;

[0069] Figure 8 The chromatogram of the spiked sample solution at the 100% limit level for Comparative Example 4;

[0070] Figure 9 Chromatogram of the spiked sample solution at the 100% limit level for Comparative Example 5;

[0071] Figure 10 This is the chromatogram of the spiked sample solution at the 100% limit level for Comparative Example 6. Detailed Implementation

[0072] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0073] In the embodiments and comparative examples of this invention:

[0074] Duloxetine hydrochloride enteric-coated tablets, brand name: Ausmin;

[0075] N-nitrosoduloxetine source: CPRD (Shenzhen Zhuoyue Biotechnology Co., Ltd.)

[0076] Example 1: A method for detecting N-nitrosoduloxetine residues in duloxetine hydrochloride enteric-coated tablets, comprising the following steps:

[0077] 1. Solution preparation:

[0078] Diluent: Methanol and water in a volume ratio of 50:50;

[0079] Extraction solvent: methanol-water solution containing 0.02 mol / L ammonium formate; methanol-water solution: methanol and water in a volume ratio of 40-60:40-60;

[0080] Blank solution: methanol and water in a volume ratio of 50:50.

[0081] 1.1 Test solution: Weigh 2g of duloxetine hydrochloride enteric-coated tablets, add 10mL of extraction solvent and mix, vortex for 1min, shake for 10min, filter, take the supernatant and filter through a 0.22μm filter membrane, add dilution solvent to prepare a solution containing 2mg / mL duloxetine hydrochloride;

[0082] 1.2 Stock solution: Weigh 10 mg of N-nitrosoduloxetine, dilute it with 10 mL of methanol, and then dilute it with a diluent to 17.3 ng / mL;

[0083] 1.3 Reference solution: Transfer 1 mL of the stock solution and dilute it with diluent to 1.73 ng / mL.

[0084] 2. Inject the test sample and reference solution separately into a triple quadrupole tandem mass spectrometer for detection; the detection conditions for the triple quadrupole tandem mass spectrometer are shown in Table 2:

[0085] Table 2

[0086] .

[0087] Test results as follows Figure 1 As shown: Duloxetine hydrochloride enteric-coated tablets contain 0.11 ppm of N-nitrosoduloxetine.

[0088] Example 2: Methodological Validation

[0089] 1. Specificity: The test solution, blank solution, and reference solution were tested according to the detection method in Example 1. The detection chromatograms are shown in [reference needed]. Figures 2-3 As shown.

[0090] The results showed that the blank solution, test solution, and reference solution did not interfere with the target peak, indicating that the method of this invention has good specificity.

[0091] 2. Limit of Detection: Take 1 mL of the stock solution and dilute it with diluent to 5% of the control solution (the theoretical relative content in the sample is: N-nitrosoduloxetine 0.0415 ppm) as the LOD solution. According to the detection method in Example 1, inject the LOD solution into the liquid chromatography triple quadrupole tandem mass spectrometer. The injection was performed 3 times, and the test results are shown in Table 3 below.

[0092] Table 3

[0093] .

[0094] As can be seen from Table 3, the detection limit of the method of the present invention is 0.0415 ppm, and the signal-to-noise ratio is greater than 3.

[0095] 3. Limit of Quantitation: Transfer 1 mL of the stock solution and dilute it with diluent to prepare a 10% LOQ solution of the control solution (the theoretical relative content in the sample is: N-nitrosoduloxetine 0.083 ppm). According to the detection method in Example 1, inject the LOQ solution into the liquid chromatography triple quadrupole tandem mass spectrometer. The injection was performed six times, and the test results are shown in Table 4 below.

[0096] Table 4

[0097] .

[0098] As can be seen from Table 4, the limit of quantitation of the method of the present invention is 0.083 ppm, and the signal-to-noise ratio is greater than 10.

[0099] 4. Linear Range: Transfer 1 mL of the stock solution and dilute it with a diluent to prepare a series of linearity test solutions (10%, 50%, 100%, 150%, and 200% of the reference solution). Analyze each solution once according to the detection method in Example 1. The results are shown in Table 5 below, and the linear relationship graph is shown below. Figure 4 As shown.

[0100] Table 5

[0101] .

[0102] As shown in Table 5, the linear equation is y = 397724x - 4283 (r = 1.0000), indicating a good linear relationship within the concentration range of 0.17 ng / mL to 3.45 ng / mL.

[0103] 5. Accuracy: (1) Test solution: Weigh 2g of duloxetine hydrochloride enteric-coated tablets, add 10mL of extraction solvent and mix, vortex for 1min, shake for 10min, filter, take the supernatant and filter through a 0.22μm filter membrane, add dilution solvent to prepare a solution containing 2mg / mL duloxetine hydrochloride, and prepare 2 parallel samples;

[0104] (2) 50% limit level spiked sample solution: The test solution is diluted with 50% limit level stock solution and three parallel preparations are made.

[0105] (3) 100% limit level spiked sample solution: The test solution is diluted with 100% limit level stock solution and three parallel preparations are made.

[0106] (4) 150% limit level spiked sample solution: The test solution is mixed with 150% limit level stock solution, diluted with diluent, and three parallel preparations are made.

[0107] Following the detection method of Example 1, each solution was analyzed once, and the results are shown in Table 6 below.

[0108] Table 6

[0109] .

[0110] As shown in Table 6, the average recovery rate of the nine samples was 84.6%, and the RSD was 0.6%, indicating that the method of the present invention has high accuracy.

[0111] 6. Repeatability: Prepare 100% limit level spiked sample solutions (using the same preparation method as the accuracy test), and prepare 6 parallel solutions; analyze each solution once according to the detection method in Example 1. The results are shown in Table 7 below.

[0112] Table 7

[0113] .

[0114] As shown in Table 7, the reproducibility recovery rate of the 6 samples was 83.9%-85.7%, and the RSD was 0.8%, indicating that the method of the present invention has high reproducibility.

[0115] 7. Intermediate precision: Six spiked sample solutions at 100% limit level were prepared at different times by another researcher (using the same preparation method as the accuracy test), and the results are shown in Table 8 below.

[0116] Table 8

[0117] .

[0118] As shown in Table 8, the recovery rate of the 12 samples was 83.9%-86.8%, and the RSD was 1.2%, indicating that the intermediate precision of the method of the present invention is high.

[0119] 8. Robustness: Prepare spiked sample solutions at 100% limit level (using the same preparation method as the accuracy test), and inject the sample once under each condition by changing the initial organic phase ratio, column temperature, and flow rate. The RSD of the peak area of ​​the six test samples under a single condition is no greater than 20.0%, which meets the requirements.

[0120] 9. Solution stability: Prepare one portion each of the reference solution and the spiked sample solution at 100% limit level. Analyze these solutions at 24 hours or other time intervals and compare them with freshly prepared solutions. If the N-nitrosoduloxetine content of the stable solution is between 70% and 130% of the initial N-nitrosoduloxetine content, it indicates that the solution has good stability.

[0121] 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; all other aspects are the same. The sample solution spiked at the 100% limit level was tested under the above conditions, and the results are as follows... Figure 5 As shown, extraneous peaks appear, the peak shape becomes wider, and the response becomes lower.

[0122] Comparative Example 2: The only difference from Example 1 is that mobile phase A is 0.1% formic acid aqueous solution; mobile phase B is acetonitrile; all other aspects are the same. The sample solution spiked at the 100% limit level was tested under the above conditions, and the results are as follows: Figure 6 As shown, a tailing effect and poor peak shape are observed.

[0123] Comparative Example 3: The only difference from Example 1 is that mobile phase A is an aqueous solution of 2 mmol / L ammonium acetate + 0.1% formic acid; all other conditions are the same. The sample solution spiked at the 100% limit level was tested under the above conditions, and the results are as follows: Figure 7 As shown, a tailing effect and poor peak shape are observed.

[0124] Comparative Example 4: The only difference from Example 1 is that isocratic elution was performed using mobile phase A (5 mM ammonium formate + 0.1% aqueous formic acid) - mobile phase B (0.1% methanolic formic acid) (11:9, v / v), with a run time of 14 min; all other conditions were the same. The sample solution spiked at the 100% limit level was tested under the above conditions, and the results are as follows... Figure 8 As shown, no peak was observed within 13 minutes.

[0125] Comparative Example 5: The only difference from Example 1 is that the gradient elution procedure is as follows:

[0126] 0-2 min, 50% A, 50% B;

[0127] 2-5 min, 50-32% A, 50-68% B;

[0128] 5-7 min, 32%A, 68%B;

[0129] 7-8 min, 32-10% A, 68-90% B;

[0130] 8-9 min, 10%A, 90%B;

[0131] 9-10min, 10-50%A, 90-50%B;

[0132] 10-14 min, 50% A, 50% B; all other conditions are the same; using the above conditions to test the 100% limit level spiked sample solution, the results are as follows. Figure 9 As shown, a new impurity peak appears after eluting N-nitrosoduloxetine.

[0133] Comparative Example 6: The only difference from Example 1 is that the gradient elution procedure is as follows:

[0134] 0 min, 75% A, 25% B;

[0135] 9 min, 20% A, 80% B;

[0136] 11 min, 20% A, 80% B;

[0137] 12 min, 75% A, 25% B;

[0138] 14 min, 75% A, 25% B; all other conditions were the same. The above conditions were used to test the 100% limit level spiked sample solution, and the results are as follows: Figure 10 As shown, the peak shape is poor, and new impurity peaks appear after eluting N-nitrosoduloxetine.

[0139] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for detecting N-nitrosoduloxetine residues in duloxetine hydrochloride enteric-coated tablets, characterized in that, Includes the following steps: S1. Mix duloxetine hydrochloride enteric-coated tablets and extraction solvent, vortex, shake, filter, take the supernatant and filter through a 0.22μm filter membrane, add diluent to dilute and prepare the test solution; S2, N-nitrosoduloxetine was diluted with a diluent to prepare a reference solution; S3. The test solution and reference solution were determined by liquid chromatography-triple quadrupole tandem mass spectrometry, and the content of N-nitrosoduloxetine was calculated based on the detection chromatograms. The extraction solvent is a methanol-water solution containing 0.01-0.02 mol / L ammonium formate; the methanol-water solution is methanol and water in a volume ratio of 40-60:40-60. The diluent is methanol and water in a volume ratio of 40-60:40-60; The chromatographic conditions for the liquid chromatography triple quadrupole tandem mass spectrometer include: Column: packed with pentafluorophenyl bonded silica gel; 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. The gradient elution procedure is as follows: 0min, 60-70%A, 30-40%B; 9min, 5-15%A, 85-95%B; 11min, 5-15%A, 85-95%B; 12min, 60-70%A, 30-40%B; 14min, 60-70%A, 30-40%B.

2. The method for detecting N-nitrosoduloxetine residue in duloxetine hydrochloride enteric-coated tablets according to claim 1, 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.

3. The method for detecting N-nitrosoduloxetine residue in duloxetine hydrochloride enteric-coated tablets according to claim 2, characterized in that, The gradient elution procedure 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.

4. The method for detecting N-nitrosoduloxetine residue in duloxetine hydrochloride enteric-coated tablets according to claim 3, characterized in that, The chromatographic column of the liquid chromatography triple quadrupole tandem mass spectrometer is an Agilent Infinity LabPoroshell 120PFP 3.0*150mm 2.7μm.

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

6. The method for detecting N-nitrosoduloxetine residue in duloxetine hydrochloride enteric-coated tablets according to claim 5, 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℃.

7. The method for detecting N-nitrosoduloxetine residue in duloxetine hydrochloride enteric-coated tablets according to claim 6, characterized in that, The mass spectrometry conditions for the liquid chromatography triple quadrupole tandem mass spectrometer also include: Air curtain gas: 30 psi; Collision gas: 9 psi; Ionization voltage: 5500V; Spray gas: 50 psi; Auxiliary heating gas: 50 psi.

8. The method for detecting N-nitrosoduloxetine residue in duloxetine hydrochloride enteric-coated tablets according to claim 7, characterized in that, The mass spectrometry conditions for the liquid chromatography triple quadrupole tandem mass spectrometer also include monitoring ion pairs and parameters:

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