Method for detecting N-nitrosodiethylamine in diclofenac epolamine

Abstract

The invention discloses a method for detecting N-nitrosodiethylamine in diclofenac epolamine, which comprises the following steps: dissolving a diclofenac epolamine sample in a methanol solution, adding a formic acid aqueous solution, mixing, and precipitating out diclofenac epolamine in the sample; centrifugally separating, and taking supernate; and carrying out quantitative detection on the N-nitrosodiethylamine in the supernate by adopting a liquid chromatography-tandem mass spectrometry method. Based on the principle that diclofenac is converted into free acid to be precipitated under the acidic condition, 2% formic acid is creatively selected as a precipitator, matrix interference of main components is effectively removed, the problem that the main components are separated out in a chromatographic system is solved, and a mass spectrometer is protected. According to methodology verification, the method disclosed by the invention is extremely high in sensitivity, good in accuracy, strong in reproducibility, rapid in analysis and good in durability. The method provides a reliable and efficient detection means for quality control of trace N-nitrosodiethylamine in the diclofenac epolamine bulk drug, and has extremely high practical value.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a method for detecting N-nitrosodiethylamine in diclofenac epoisen. Background Technology

[0002] N-nitrosodiethylamine (NDEA) is an impurity that may be generated during the production of diclofenac epoise. This impurity may remain in trace amounts in the finished diclofenac epoise product. Since N-nitrosodiethylamine has a potentially genotoxic structure, its acceptable intake (AI) is 26.5 ng / day. Referring to the maximum daily dose (MDD) of diclofenac epoise, which is 360 mg / day, the residue limit for N-nitrosodiethylamine, determined using the AI / MDD method, is not more than 0.000007% (0.07 ppm).

[0003] Existing detection technologies face two major challenges: First, diclofenac epoisene itself easily precipitates in the conventional liquid chromatography mobile phase, clogging the system and interfering with detection; second, the high background of the main component severely masks the signal of trace N-nitrosodiethylamine, leading to insufficient sensitivity. Therefore, there is an urgent need to develop a method that can effectively remove interference from the main component matrix while meeting the requirements for sensitivity, accuracy, and reproducibility in trace detection. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a method for detecting N-nitrosodiethylamine in diclofenac epoise. The method utilizes formic acid to precipitate the diclofenac epoise sample, and combines this with liquid chromatography-mass spectrometry (LC-MS) tandem to quantitatively analyze the N-nitrosodiethylamine in the supernatant. This reduces background interference and minimizes its influence on trace N-nitrosodiethylamine.

[0005] This invention provides a method for detecting N-nitrosodiethylamine in diclofenac epoisen, comprising the following steps: S1, Test solution: Accurately weigh the diclofenac epoise sample, add D6-N-nitrosodimethylamine internal standard solution, add solvent and shake to dissolve, add volatile acid aqueous solution and shake well, let stand, centrifuge and filter to prepare the test solution. S2, Reference Solution: Accurately weigh diclofenac epoisen free of N-nitrosodiethylamine, add D6-N-nitrosodimethylamine internal standard solution and N-nitrosodiethylamine reference stock solution a, add solvent and shake to dissolve, add volatile acid aqueous solution and shake well, let stand, centrifuge and filter to prepare reference solution, wherein the concentration of N-nitrosodiethylamine is 7 ng / mL and the concentration of D6-N-nitrosodimethylamine is 7 ng / mL; S3, accurately transfer the test solution and the reference solution, inject them into the liquid chromatography-tandem mass spectrometer, and record the chromatogram; S4. If there is a chromatographic peak in the chromatogram of the test solution that has the same retention time as N-nitrosodiethylamine, the content of N-nitrosodiethylamine is calculated by peak area using the internal standard method.

[0006] In some embodiments, the liquid chromatography conditions are as follows: column: Poroshell 120 EC-C18 2.7 μm, 3.0 mm × 100 mm; mobile phase: A: water containing 0.1% formic acid; B: methanol containing 0.1% formic acid; mobile phase gradient: 0 min: Mobile phase A 95%, Mobile phase B 5%; 3 min: Mobile phase A 95%, mobile phase B 5%; 7 min: Mobile phase A 5%, Mobile phase B 95%; 9 min: Mobile phase A 5%, Mobile phase B 95%; 9.1 min: Mobile phase A 95%, Mobile phase B 5%; 10 min: Mobile phase A 95%, Mobile phase B 5%.

[0007] In some embodiments, the liquid chromatography conditions also include column temperature: 40°C; flow rate: 0.50 mL / min; injection volume: 15 μL.

[0008] In some implementations, the mass spectrometry parameters are as follows: ion source: APCI, positive ion mode; scanning mode: MRM multiple reaction monitoring, quantitative ion pair for N-nitrosodiethylamine is m / z 103→75.1, qualitative ion pair is m / z 103→47.1.

[0009] In some embodiments, the solvent is methanol.

[0010] In some embodiments, the volatile acid aqueous solution is a formic acid aqueous solution.

[0011] The detection principle of this invention is as follows: Diclofenac epoise is soluble in methanol-water solution, but when it encounters a mobile phase containing formic acid, it gradually precipitates, causing blockage of the chromatographic system and rendering it undetectable. To reduce matrix interference and equipment contamination, it is necessary to extract trace amounts of N-nitrosodiethylamine from diclofenac epoise while simultaneously reducing the concentration interference of diclofenac epoise. Since diclofenac epoise contains an amino group, similar in properties to the target analyte, acid precipitation of diclofenac is chosen.

[0012] The precipitation of diclofenac epoise under acidic conditions is essentially due to the transformation of its active ingredient, diclofenac (a weak acid with a pKa of approximately 4.0), from its salt form (epoiset) to its free acid form. Free acid has extremely low solubility in water, thus crystallizing out. For complete precipitation to occur, the required pH value generally needs to be at least two units lower than the pKa of diclofenac. Theoretically, when pH < 2.0, the diclofenac free acid is almost completely precipitated (i.e., over 99% exists in molecular form).

[0013] Commonly used acids in chromatographic detection include phosphoric acid, sulfuric acid, hydrochloric acid, formic acid, acetic acid, and trifluoroacetic acid. However, phosphoric acid, sulfuric acid, and hydrochloric acid are all non-volatile acids, which can deposit on the ion source and mass analyzer, leading to a sharp decrease in sensitivity and making them difficult to clean. Sulfonic acids are ion-pairing reagents that severely suppress signals and contaminate the instrument. Commonly used volatile acids are formic acid, acetic acid, and trifluoroacetic acid. Among these, formic acid is the most versatile and mild, suitable for the analysis of most basic compounds in positive ion mode, and can promote the [M+H] reaction. + Peak formation. Therefore, formic acid is preferred as the volatile acid.

[0014] Formic acid is a commonly used acidifying agent. Its pH relationship with concentration is not linear and depends on other components in the solution (such as organic solvents and buffer salts). In a pure aqueous system, if the goal is simply to create an acidic environment (pH < 2), a 0.5% to 1% (v / v) formic acid aqueous solution is usually sufficient, resulting in a pH of approximately 2.0-2.3. In systems containing organic solvents, i.e., methanol in the mobile phase, methanol increases the solubility of diclofenac. Therefore, a higher concentration of formic acid is needed to provide a stronger acidic environment to overcome the solubilizing effect of the organic solvent, requiring the formic acid concentration to be increased to 1%-5% or even higher. To ensure a pH below 2 and complete precipitation of diclofenac, the added formic acid should not be too concentrated to avoid localized over-acidification, which could cause the target analyte to be encapsulated within crystals, leading to lower results. Furthermore, excessively high acid concentrations can not only suppress the signal and reduce sensitivity but also corrode stainless steel components of the equipment. Therefore, a 2% (v / v) formic acid concentration is preferred.

[0015] In some embodiments, the internal standard solution is a methanol solution of 140 μg / L D6-N-nitrosodimethylamine.

[0016] In some embodiments, the preparation method of the N-nitrosodiethylamine reference stock solution a includes: transferring the N-nitrosodiethylamine standard solution into a volumetric flask, making up to volume with methanol, mixing evenly, and obtaining the N-nitrosodiethylamine reference stock solution a with a concentration of 140 μg / L.

[0017] In some embodiments, in step S1, the mixing ratio of the diclofenac epoise sample to the formic acid aqueous solution is 1.8 mL of 2% formic acid aqueous solution per 0.2 g sample; and / or, in step S2, the mixing ratio of the diclofenac epoise sample without N-nitrosodiethylamine to the formic acid aqueous solution is 1.8 mL of 2% formic acid aqueous solution per 0.2 g sample.

[0018] In some embodiments, after adding the volatile acid aqueous solution in steps S1 and S2, the standing time is 1 to 3 hours, preferably 2 hours, to allow diclofenac epoise to fully precipitate.

[0019] In some implementations, in steps S1 and S2, the centrifugation step is performed at 4000 r / min for 5 minutes.

[0020] In some embodiments, the method has a limit of quantitation of 0.7 ng / mL, a limit of detection of 0.35 ng / mL, a linear range of 0.7–10.5 ng / mL, and a correlation coefficient R ≥ 0.999.

[0021] This invention also provides the application of the above detection method in the quality control of trace N-nitrosodiethylamine in diclofenac epollide raw material.

[0022] Compared with the prior art, the present invention has the following technical effects: This invention provides a method for detecting the content of N-nitrosodiethylamine in diclofenac epollide using LC-MS / MS. The method is highly sensitive, accurate, and reproducible, and is suitable for the quality control of trace N-nitrosodiethylamine in diclofenac epollide technical grade.

[0023] This invention utilizes formic acid to precipitate diclofenac epoise in solution, thereby reducing background and minimizing its interference with trace N-nitrosodiethylamine.

[0024] The method of this invention has high sensitivity, with a limit of quantitation of 0.7 ng / mL and a limit of detection of 0.35 ng / mL, meeting the requirements for trace detection.

[0025] The method of this invention not only precipitates the sample and reduces the influence of the sample matrix, but also effectively resists interference by eliminating matrix interference through gradient elution and valve switching technology; the total analysis time is 10 minutes, improving detection efficiency. This method has undergone systematic validation, covering key parameters such as specificity, linearity, precision, limit of quantitation, limit of detection, accuracy, stability, and robustness, ensuring the reliability of the results. Attached Figure Description

[0026] Figure 1 The above are the qualitative and quantitative ion chromatograms of the N-nitrosodiethylamine reference solution in Example 2 of this invention. Figure 2 The above are the qualitative and quantitative ion chromatograms of N-nitrosodiethylamine in the blank solution in Example 2 of this invention. Figure 3 The above are the qualitative and quantitative ion chromatograms of N-nitrosodiethylamine in the mixed sample solution in Example 2 of this invention. Figure 4 The present invention provides qualitative and quantitative ion chromatograms of N-nitrosodiethylamine in the diclofenac epoise solution of Example 2 of this invention. Figure 5 This is a linear graph of N-nitrosodiethylamine in Example 4 of the present invention. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

[0028] The following examples illustrate the preparation of sample and standard solutions: D6-N-nitrosodimethylamine internal standard solution: Transfer 1 mL of 1000 mg / L D6-N-nitrosodimethylamine solution to a 10 mL volumetric flask, dilute to volume with methanol, and mix thoroughly to obtain an intermediate standard solution of D6-N-nitrosodimethylamine with a concentration of 100 mg / L; Transfer 140 μL of 100 mg / L D6-N-nitrosodimethylamine intermediate standard solution to a 100 mL volumetric flask, dilute to volume with methanol, and mix thoroughly to obtain an internal standard solution of D6-N-nitrosodimethylamine with a concentration of 140 μg / L.

[0029] D6-N-nitrosodimethylamine internal standard solution a: Transfer 0.1 mL of 1000 mg / L D6-N-nitrosodimethylamine solution to a 10 mL volumetric flask, dilute to volume with methanol, and mix thoroughly to obtain D6-N-nitrosodimethylamine intermediate standard solution with a concentration of 10 mg / L; Transfer 280 μL of 10 mg / L D6-N-nitrosodimethylamine intermediate standard solution to a 10 mL volumetric flask, dilute to volume with methanol, and mix thoroughly to obtain D6-N-nitrosodimethylamine internal standard solution a with a concentration of 280 μg / L.

[0030] N-nitrosodiethylamine reference standard stock solution a: Transfer 140 μL of 100 mg / L N-nitrosodiethylamine standard solution into a 100 mL volumetric flask, dilute to volume with methanol, mix well, and obtain N-nitrosodiethylamine reference standard stock solution a with a concentration of 140 μg / L.

[0031] N-nitrosodiethylamine reference standard stock solution b: Transfer 1 mL of N-nitrosodiethylamine reference standard stock solution a into a 10 mL volumetric flask, dilute to volume with methanol, mix well, and obtain N-nitrosodiethylamine reference standard stock solution b with a concentration of 14 μg / L.

[0032] N-N-nitrosodiethylamine reference solution: Take 0.2 g of diclofenac epoisetine (without the analyte) and place it in a 15 mL stoppered centrifuge tube. Add 0.1 mL of 140 μg / L D6-N-nitrosodimethylamine internal standard solution and 0.1 mL of N-nitrosodiethylamine reference standard stock solution a. After shaking to dissolve, add 1.8 mL of 2% formic acid aqueous solution and shake well. After standing for 2 h, centrifuge and filter through a 0.22 μm organic filter membrane to obtain the supernatant. The concentration of N-nitrosodiethylamine and the concentration of D6-N-nitrosodimethylamine in the supernatant are 7 ng / mL and 7 ng / mL, respectively.

[0033] Diclofenac epollamine test solution: Weigh 0.2 g of diclofenac epollamine test sample and place it in a 15 mL stoppered centrifuge tube. Add 0.10 mL of D6-N-nitrosodimethylamine internal standard solution, then add 0.1 mL of methanol and shake to dissolve. Add 1.8 mL of 2% formic acid aqueous solution and shake well. Let stand for 2 h until precipitation is complete. Centrifuge at 4000 r / min for 5 min. Filter the supernatant through a 0.22 μm needle organic filter membrane to prepare the diclofenac epollamine test solution. The concentration of diclofenac epollamine is 0.1 g / mL.

[0034] Blank solution: Add 0.10 mL of D6-N-nitrosodimethylamine internal standard solution, 0.1 mL of methanol, and 1.8 mL of 2% formic acid aqueous dilution to a 15 mL stoppered centrifuge tube. Shake well and let stand for 2 hours until precipitation is complete. Centrifuge at 4000 r / min for 5 min. Filter the supernatant through a 0.22 μm needle organic filter membrane to obtain the blank solution.

[0035] Mixed sample solution: Weigh 0.2 g of diclofenac epoise as a test sample and place it in a 15 mL stoppered centrifuge tube. Add 0.10 mL of D6-N-nitrosodimethylamine internal standard solution, then add 0.1 mL of N-nitrosodiethylamine reference standard stock solution a and shake to dissolve. Add 1.8 mL of 2% formic acid aqueous dilution and shake well. Let stand for 2 h until precipitation is complete. Centrifuge at 4000 r / min for 5 min. Filter the supernatant through a 0.22 μm needle organic filter membrane to prepare a mixed sample solution, wherein the concentration of diclofenac epoise is 0.1 g / mL and the concentration of N-nitrosodiethylamine is 7 μg / L.

[0036] Quantitative limit level mixed sample solution: Weigh 0.2 g of diclofenac epoise and place it in a 15 mL stoppered centrifuge tube. Add 0.10 mL of D6-N-nitrosodimethylamine internal standard solution, then add 0.1 mL of N-nitrosodiethylamine reference stock solution b. Shake to dissolve, add 1.8 mL of 2% formic acid aqueous dilution and shake well. Let stand for 2 h until precipitation is complete. Centrifuge at 4000 r / min for 5 min. Filter the supernatant through a 0.22 μm needle organic filter membrane to prepare a quantitative limit level mixed sample solution, wherein the concentration of diclofenac epoise is 0.1 g / mL and the concentration of N-nitrosodiethylamine is 0.7 μg / L.

[0037] Instrument: LC-MS / MS AB SCIEX Triple Quad TM 5500+; Column: Poroshell 120 EC-C18 2.7μm, 3.0 mm × 100 mm Chromatographic conditions: Column temperature: 40℃; Flow rate: 0.50 mL / min; Injection volume: 15 μL; Running time: 10 min; Mobile phase: A: water containing 0.1% formic acid; B: methanol containing 0.1% formic acid; liquid phase gradient is shown in Table 1; Table 1. Liquid phase gradient determination of N-nitrosodiethylamine

[0038] Mass spectrometry parameters: Ion source: APCI, positive ion mode; Scanning method: MRM (Multiple Reaction Monitoring); Air curtain gas CUR: 25 psi; NC current: 3.0 μA; source temperature: 300℃; atomizing gas Gas1: 40 psi, auxiliary gas Gas2: 55 Psi; MRM parameters are shown in Table 2: Table 2 Mass Spectrometry Parameters

[0039] Note: N-nitrosodiethylamine (NDEA1) is the quantitative ion of N-nitrosodiethylamine.

[0040] Example 1 System Suitability Test Test method: After the chromatographic system is stabilized, the N-nitrosodiethylamine reference solution is injected into the instrument for detection. Six consecutive injections are performed, and the chromatograms are recorded. The results are shown in Table 3.

[0041] Table 3. Suitability of the N-nitrosodiethylamine System

[0042] The N-nitrosodiethylamine reference solution was injected six times consecutively. The RSD of the ratio of the peak area of ​​N-nitrosodiethylamine to that of D6-N-nitrosodimethylamine was 3.33% (acceptable standard: not more than 4%), and the RSD of the retention time was 0.46% (acceptable standard: not more than 1%), which met the method requirements.

[0043] Example 2: Specificity Test Test method: After the chromatographic system stabilized, the blank solution, N-nitrosodiethylamine reference solution, diclofenac epoise solution, and mixed sample solution were tested on the instrument, and the chromatograms were recorded. The results are shown in Table 4. Figures 1-4 .

[0044] Table 4. Specificity of N-nitrosodiethylamine

[0045] The results showed that the blank sample had no interference, the diclofenac epoise solution had no target peak, and the retention time of the N-nitrosodiethylamine peak in the mixed sample solution was consistent with that in the N-nitrosodiethylamine reference solution. Because multiple reaction monitoring (MRM) was used, only the ion pair peaks of N-nitrosodiethylamine were observed. Furthermore, the quantitative and qualitative ion pairs of N-nitrosodiethylamine in the mixed solution (103→75.1 and 103→47.1) were free from interference from other peaks within 0.3 min around the target peak position. Therefore, the resolution was considered to be greater than 1.5, which met the method requirements.

[0046] Example 3: Test for Limit of Detection and Limit of Quantification Limit of detection and limit of quantitation: Take the N-nitrosodiethylamine reference standard stock solution and dilute it stepwise. The solution concentration with a signal-to-noise ratio of 10:1 or greater is taken as the limit of quantitation, and the solution concentration with a signal-to-noise ratio of 3:1 or greater is taken as the limit of detection. Detect the solution and record the chromatogram.

[0047] Quantitative limit repeatability: After determining the quantitative limit concentration, prepare 6 mixed sample solutions at the quantitative limit level as quantitative limit repeatability solutions, perform instrument detection, and record the chromatograms. The results are shown in Tables 5-6.

[0048] Table 5. Limits of Detection and Limits of Quantification for N-Nitrosaminodiethylamine

[0049] Table 6 Repeatability of the Limit of Quantitation for N-Nitrosaminodiethylamine

[0050] The limit of detection (LOD) for N-nitrosodiethylamine was 0.35 ng / mL, equivalent to a sample volume of 0.0035 ppm, and the limit of quantitation (LOQ) was 0.7 ng / mL, equivalent to a sample volume of 0.007 ppm. The LOQ repeatability solution was injected in parallel six times. The RSD of the ratio of the N-nitrosodiethylamine peak area to the internal standard peak area was 3.94% (acceptable standard: not exceeding 10%), meeting the method requirements.

[0051] Example 4: Linear Range Test Test method: Weigh 0.2 g of diclofenac epoise as a test sample and place it in a 15 mL stoppered centrifuge tube. Add 0.10 mL of D6-N-nitrosodimethylamine internal standard solution, then add an appropriate amount of N-nitrosodiethylamine reference standard stock solution a or N-nitrosodiethylamine reference standard stock solution b. Add 2% formic acid aqueous dilution and shake well. Let stand for 2 h until precipitation is complete. Centrifuge at 4000 r / min for 5 min. Filter the supernatant through a 0.22 μm needle organic filter membrane to prepare linear L1 (50%) with concentrations of 0.7 ng / mL (limit of quantitation), 3.5 ng / mL, 5.6 ng / mL (80%), 7 ng / mL (100%), 8.4 ng / mL (120%), and 10.5 ng / mL, respectively. The linear L5 (150%) of ng / mL was determined by instrumental analysis, and chromatograms were recorded. A linear regression curve was plotted using the concentration and the corresponding peak area ratio of the target analyte to the internal standard to obtain the linear equation and correlation coefficient R. Chromatograms were recorded, and the results are shown in Table 7. Figure 5 .

[0052] Table 7. Linearity results for N-nitrosodiethylamine

[0053] Within the LOQ (limit of quantitation) to 150% of the limit concentration, the concentration of N-nitrosodiethylamine showed a good linear relationship with the ratio of the peak area of ​​the corresponding target peak and the internal standard peak. The linear equation was: y = 0.04199x + 0.00457, r = 0.99944. The method showed good linearity and met the method requirements.

[0054] Example 5 Accuracy Test Quantitative limit level accuracy solution: Weigh 0.2 g of diclofenac epoise as a test sample and place it in a 15 mL stoppered centrifuge tube. Add 0.10 mL of D6-N-nitrosodimethylamine internal standard solution, then add 0.1 mL of N-nitrosodiethylamine reference standard stock solution b and shake to dissolve. Add 1.8 mL of 2% formic acid aqueous dilution and shake well. Let stand for 2 h until precipitation is complete. Centrifuge at 4000 r / min for 5 min. Filter the supernatant through a 0.22 μm needle organic filter membrane. Prepare 3 aliquots using the same method.

[0055] 50% limit level accuracy solution: Weigh 0.2 g of diclofenac epoise as a test sample and place it in a 15 mL stoppered centrifuge tube. Add 0.10 mL of D6-N-nitrosodimethylamine internal standard solution, then add 0.05 mL of N-nitrosodiethylamine reference standard stock solution a. Add 0.05 mL of methanol and shake to dissolve. Add 1.8 mL of 2% formic acid aqueous dilution and shake well. Let stand for 2 h until precipitation is complete. Centrifuge at 4000 r / min for 5 min. Filter the supernatant through a 0.22 μm needle organic filter membrane. Prepare 3 aliquots using the same method.

[0056] 100% limit level accuracy solution: Weigh 0.2 g of diclofenac epoise as a test sample and place it in a 15 mL stoppered centrifuge tube. Add 0.10 mL of D6-N-nitrosodimethylamine internal standard solution, then add 0.10 mL of N-nitrosodiethylamine reference standard stock solution a and shake to dissolve. Add 1.8 mL of 2% formic acid aqueous dilution and shake well. Let stand for 2 hours until precipitation is complete. Centrifuge at 4000 r / min for 5 min. Filter the supernatant through a 0.22 μm needle organic filter membrane. Prepare 3 aliquots using the same method.

[0057] 150% limit level accuracy solution: Weigh 0.2 g of diclofenac epoise as a test sample and place it in a 15 mL stoppered centrifuge tube. Add 0.05 mL of D6-N-nitrosodimethylamine internal standard solution a, then add 0.15 mL of N-nitrosodiethylamine reference standard stock solution a and shake to dissolve. Add 1.8 mL of 2% formic acid aqueous dilution and shake well. Let stand for 2 hours until precipitation is complete. Centrifuge at 4000 r / min for 5 min. Filter the supernatant through a 0.22 μm needle organic filter membrane. Prepare 3 aliquots using the same method.

[0058] The test method involved testing solutions at each accuracy limit level using an instrument and recording the chromatograms. The results are shown in Table 8.

[0059] Table 8. Accuracy Test Results of N-nitrosodiethylamine

[0060] The results showed that the average recovery rate of N-nitrosodiethylamine at the limit of quantitation was 83.8%, with an RSD of 3.94%; the average recovery rate at the 50% limit was 83.7%, with an RSD of 4.92%; the average recovery rate at the 100% limit was 87.0%, with an RSD of 5.67%; and the average recovery rate at the 150% limit was 90.0%, with an RSD of 6.08% (acceptable standard: not exceeding 8%). The RSD of the 12 recoveries at the limit of quantitation to 150% limit level was 5.48% (acceptable standard: not exceeding 10%), meeting the method requirements.

[0061] Example 6 Precision Test The average recovery rate of N-nitrosodiethylamine should be between 75% and 120%, and the RSD of three parallel tests at each concentration level should not exceed 8%, and the RSD of 12 recovery tests should not exceed 10%.

[0062] Repeatable reference solution: Weigh 0.2 g of diclofenac epoise as a test sample and place it in a 15 mL stoppered centrifuge tube. Add 0.10 mL of D6-N-nitrosodimethylamine internal standard solution, then add 0.10 mL of N-nitrosodiethylamine reference standard stock solution a and shake to dissolve. Add 1.8 mL of 2% formic acid aqueous dilution and shake well. Let stand for 2 h until precipitation is complete. Centrifuge at 4000 r / min for 5 min. Filter the supernatant through a 0.22 μm needle organic filter membrane. Prepare 6 aliquots using the same method.

[0063] Repeatability solution: Weigh 0.2 g of diclofenac epoise as a test sample and place it in a 15 mL stoppered centrifuge tube. Add 0.10 mL of D6-N-nitrosodimethylamine internal standard solution, then add 0.10 mL of N-nitrosodiethylamine reference standard stock solution a and shake to dissolve. Add 1.8 mL of 2% formic acid aqueous solution and shake well. Let stand for 2 h until precipitation is complete. Centrifuge at 4000 r / min for 5 min. Filter the supernatant through a 0.22 μm needle organic filter membrane. Prepare 6 aliquots using the same method.

[0064] Test method: Intermediate precision solution: The intermediate precision solution was prepared by different experimenters at different times according to the method in the repeatability solution. Six mixed sample solutions were prepared by weighing the same batch of diclofenac epollamine test sample according to the preparation method in the repeatability solution as intermediate precision solutions.

[0065] Repeatability test method: The reference solution and 6 repeatability solutions were tested by instrument, and the chromatograms were recorded. The results are shown in Table 9.

[0066] Intermediate precision test method: The intermediate precision reference solution and 6 intermediate precision solutions were tested by the instrument, and the chromatograms were recorded. The results are shown in Table 9.

[0067] Table 9 Precision of N-nitrosodiethylamine

[0068] The results showed that under these chromatographic conditions, the RSD of N-nitrosodiethylamine concentration in the six reproducible reference solutions was 3.36%, and the RSD of N-nitrosodiethylamine content in the six reproducible solutions was 4.54%, indicating good repeatability of the method. Six intermediate precision solutions were measured in parallel by different experimenters at different times. The RSD of N-nitrosodiethylamine concentration in the six intermediate precision reference solutions was 2.04%, and the RSD of N-nitrosodiethylamine content in the intermediate precision solutions was 4.64%, indicating good intermediate precision of the method. The RSD of N-nitrosodiethylamine concentration in the 12 reproducible reference solutions prepared by different experimenters at different times was 2.93%, and the RSD of N-nitrosodiethylamine content in the 12 reproducible solutions was 4.41%, indicating good precision of the method and meeting the method requirements.

[0069] Example 7 Solution Stability Test For the determination of 6 mixed sample solutions placed for a fixed time, the RSD values ​​of the peak areas of N-nitrosodiethylamine and internal standard should not exceed 8%.

[0070] Test method: Weigh 0.2 g of diclofenac epoise as a test sample and place it in a 15 mL stoppered centrifuge tube. Add 0.10 mL of D6-N-nitrosodimethylamine internal standard solution, then add 0.10 mL of N-nitrosodiethylamine reference standard stock solution a and shake to dissolve. Add 1.8 mL of 2% formic acid aqueous solution for dilution and shake well. Let stand for 2 h until precipitation is complete. Centrifuge at 4000 r / min for 5 min. Filter the supernatant through a 0.22 μm needle organic filter membrane. Prepare 6 aliquots using the same method. Inject the above solutions at time intervals of approximately 0 h, 1 h, 2 h, 3 h, 10 h, and 11 h for analysis and record the chromatograms. The results are shown in Table 10.

[0071] Table 10 Stability of N-nitrosodiethylamine solution

[0072] The results showed that the RSD of the peak area ratio of N-nitrosodiethylamine to internal standard was 5.12% for six mixed sample solutions that were left to stand for 0 h, 1 h, 2 h, 3 h, 10 h, and 11 h, respectively, which met the method requirements.

[0073] Example 8 Durability Test After minor adjustments to the chromatographic conditions, the resolution between each chromatographic peak should be greater than 1.5.

[0074] Test method: N-nitrosodiethylamine reference solution was used as the robustness test solution to investigate the effect of changes in chromatographic conditions on resolution. The mobile phase gradient and mobile phase flow rate were changed, and the results are shown in Tables 11-12.

[0075] Table 11 Changing the mobile phase gradient

[0076] Table 12 Changing the flow rate

[0077] The results show that by making minor adjustments to the mobile phase gradient and flow rate, and by using the multiple reaction monitoring mode, there are no other impurity peaks interfering with the quantitative and qualitative ion peaks corresponding to N-nitrosodiethylamine within 0.3 min to the left and right. Therefore, the resolution is considered to be greater than 1.5, with good robustness, and it meets the method requirements.

[0078] Example 9 Sample Testing Using this analytical method, a test solution of diclofenac epoise was prepared, injected, and the chromatogram was recorded. If a target peak with the same position as N-nitrosodiethylamine was found in the chromatogram, the N-nitrosodiethylamine content was calculated by peak area using the internal standard method. The content of N-nitrosodiethylamine should not exceed its limit of 0.07 ppm. The test results showed no N-nitrosodiethylamine target peak in the sample, and all results were negative. The N-nitrosodiethylamine detection results are shown in Table 13.

[0079] Table 13 Test Results of the Samples

Claims

1. A method for detecting N-nitrosodiethylamine in diclofenac epoisen, comprising the following steps: S1, Test solution: Accurately weigh the diclofenac epoise sample, add D6-N-nitrosodimethylamine internal standard solution, add solvent and shake to dissolve, add volatile acid aqueous solution and shake well, let stand, centrifuge and filter to prepare the test solution. S2, Reference Solution: Accurately weigh diclofenac epoisen free of N-nitrosodiethylamine, add D6-N-nitrosodimethylamine internal standard solution and N-nitrosodiethylamine reference stock solution a, add solvent and shake to dissolve, add volatile acid aqueous solution and shake well, let stand, centrifuge and filter to prepare reference solution, wherein the concentration of N-nitrosodiethylamine is 7 ng / mL and the concentration of D6-N-nitrosodimethylamine is 7 ng / mL; S3, accurately transfer the test solution and the reference solution, inject them into the liquid chromatography-tandem mass spectrometer, and record the chromatogram; S4. If there is a chromatographic peak in the chromatogram of the test solution that has the same retention time as N-nitrosodiethylamine, the content of N-nitrosodiethylamine is calculated by peak area using the internal standard method.

2. The detection method according to claim 1, wherein, The liquid chromatography conditions were as follows: column: Poroshell 120EC-C18 2.7μm, 3.0 mm × 100 mm; mobile phase: A: water containing 0.1% formic acid. B: Methanol containing 0.1% formic acid, with the following mobile phase gradient: 0 min: Mobile phase A 95%, Mobile phase B 5%; 3 min: Mobile phase A 95%, mobile phase B 5%; 7 min: Mobile phase A 5%, Mobile phase B 95%; 9 min: Mobile phase A 5%, Mobile phase B 95%; 9.1 min: Mobile phase A 95%, Mobile phase B 5%; 10 min: Mobile phase A 95%, Mobile phase B 5%.

3. The detection method according to claim 2, wherein, The liquid chromatography conditions also included column temperature: 40℃; flow rate: 0.50 mL / min; injection volume: 15 μL.

4. The detection method according to claim 1, wherein, The mass spectrometry parameters were as follows: ion source: APCI, positive ion mode; scanning mode: MRM multiple reaction monitoring; quantitative ion pair for N-nitrosodiethylamine was m / z 103→75.1, and qualitative ion pair was m / z 103→47.

1.

5. The detection method according to claim 1, wherein, The solvent is methanol, and / or the volatile acid aqueous solution is formic acid aqueous solution.

6. The detection method according to claim 1, wherein, The internal standard solution was a methanol solution of 140 μg / L D6-N-nitrosodimethylamine.

7. The detection method according to claim 1, wherein, The preparation method of the N-nitrosodiethylamine reference stock solution a includes: transferring the N-nitrosodiethylamine standard solution into a volumetric flask, making up to volume with methanol, mixing evenly, and obtaining the N-nitrosodiethylamine reference stock solution a with a concentration of 140 μg / L.

8. The detection method according to claim 1, wherein, In step S1, the mixing ratio of the diclofenac epoise sample to the formic acid aqueous solution is 1.8 mL of 2% formic acid aqueous solution for every 0.2 g of sample; and / or, in step S2, the mixing ratio of the diclofenac epoise sample without N-nitrosodiethylamine to the formic acid aqueous solution is 1.8 mL of 2% formic acid aqueous solution for every 0.2 g of sample.

9. The detection method according to claim 1, wherein, The method has a limit of quantitation of 0.7 ng / mL, a limit of detection of 0.35 ng / mL, a linear range of 0.7–10.5 ng / mL, and a correlation coefficient R ≥ 0.

999.

10. The application of the detection method according to claims 1-9 in the quality control of trace N-nitrosodiethylamine in diclofenac epollide raw material.

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