Method for detecting N-methylaniline impurities in quetiapine fumarate

Abstract

The invention provides a method for simultaneously detecting the content of N-methylaniline and N, N-dimethylaniline in quetiapine fumarate, the method adopts an ultra-high performance liquid chromatography and mass spectrometry technology, a chromatographic column is a phenyl-hexyl chromatographic column, a water phase of a mobile phase is a formic acid aqueous solution, and an organic phase of the mobile phase is acetonitrile or methanol. The method is good in specificity, high in accuracy, high in sensitivity and high in durability, and the quality of quetiapine fumarate can be effectively monitored.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis, specifically relating to a method for detecting N-methylaniline impurities in quetiapine fumarate using liquid chromatography-mass spectrometry (LC-MS). Background Technology

[0002] Nitrosamines are a class of small molecule compounds in which the nitroso group is directly linked to the amine group. Under the metabolic environment in vivo, nitrosamines can be converted into alkyl diazo ions, which directly interact with the nitrogen and oxygen in molecules such as guanine, cytosine, and thymine that make up DNA, causing DNA alkylation, leading to DNA mismatches and mutations, and posing a carcinogenic risk.

[0003] In the 2017 ICH document, "Assessing and controlling DNA-reactive (mutagenic) impurities in pharmaceuticals to limit potential carcinogenic risks" (ICH M7), nitrosamines were listed as impurities in the "cohort of concern" with a high carcinogenic risk. A significant risk characteristic of these impurities is that even if the daily intake is below the toxicological concern threshold, the cumulative effect of DNA damage can still produce a quantifiable probability of carcinogenesis. Currently, drug regulatory authorities in various countries require risk assessments of nitrosamine impurities in active pharmaceutical ingredients and finished drug products, and mandate the use of effective analytical and control methods to keep their levels within safe limits.

[0004] The main pathways for the formation of nitrosamine impurities include process synthesis, degradation and transformation, and exogenous contamination. Their formation mechanism primarily involves nitrosation, where, under acidic or high-temperature conditions, secondary amine compounds undergo an electrophilic substitution reaction with nitrosating agents such as sodium nitrite, generating highly mutagenic nitrosamine compounds. Based on this reaction characteristic, if secondary amines or nitrosating agents are introduced into the materials used in the entire process of drug substance and formulation production (including starting materials, solvents, reaction reagents, catalysts, synthetic intermediates, and residual nitrites in formulation excipients), the risk of nitrosamine impurity formation will be significantly increased.

[0005] Quetiapine fumarate, an atypical antipsychotic, exerts its therapeutic effect by antagonizing 5-HT2A and D2 receptors. Clinically, it is mainly used for the acute and maintenance treatment of schizophrenia, as well as the control of manic episodes in bipolar disorder. During the synthesis of quetiapine fumarate raw material, two amine impurities, N-methylaniline and N,N-dimethylaniline, are present. These two impurities have the structural basis to react with nitrosating agents to form nitrosamines, and are a significant source of nitrosamine impurities in quetiapine fumarate raw material.

[0006] To effectively control nitrosamine impurities in quetiapine fumarate raw material, the primary prerequisite is to achieve accurate quantification of N-methylaniline and N,N-dimethylaniline. However, existing detection methods have significant technical limitations and cannot meet the stringent requirements for controlling these two impurities. For example, the detection method disclosed in the literature "Liang Jianmou, Fu Cong, Chen Yue. Determination of N,N-dimethylaniline in quetiapine fumarate by liquid chromatography-mass spectrometry [J]. Pharmaceutical Guide, 2017, 36(03):317-320" only targets N,N-dimethylaniline, has low detection sensitivity for N,N-dimethylaniline, and does not cover the detection scheme for N-methylaniline, thus failing to achieve simultaneous control of the two key amine impurities.

[0007] Because existing methods cannot simultaneously achieve highly sensitive detection of two amine impurities, trace impurities in quetiapine fumarate API may react with nitrosamine reagents to form nitrosamines, posing a potential quality risk. Therefore, establishing a highly sensitive method for the simultaneous detection of N-methylaniline and N,N-dimethylaniline is of great significance for preventing the formation of nitrosamine impurities at the source and ensuring that the API meets quality standards. Summary of the Invention

[0008] In one aspect of the present invention, the present invention provides a method for simultaneously detecting the contents of N-methylaniline and N,N-dimethylaniline in quetiapine fumarate, wherein ultra-high performance liquid chromatography coupled with mass spectrometry is employed, the chromatographic column is a phenyl-hexyl column, the aqueous phase of the mobile phase is an aqueous formic acid solution, and the organic phase of the mobile phase is acetonitrile or methanol.

[0009] In some embodiments, in the above detection method, the aqueous phase of the mobile phase is a 0.1-0.2% formic acid aqueous solution, and the organic phase of the mobile phase is methanol or acetonitrile; preferably, the aqueous phase of the mobile phase is a 0.1% formic acid aqueous solution, and the organic phase of the mobile phase is methanol; or the aqueous phase of the mobile phase is a 0.2% formic acid aqueous solution, and the organic phase of the mobile phase is acetonitrile.

[0010] In some embodiments, in any of the above detection methods, the chromatographic column is Waters ACQUITY UPLCCSH Phenyl-Hexyl.

[0011] In some embodiments, in any of the above detection methods, the sample to be tested is prepared using a 50% acetonitrile aqueous solution or a 0.1-0.2% formic acid aqueous solution (preferably 0.2% formic acid aqueous solution) as a diluent to prepare the test solution.

[0012] In some embodiments, in any of the above detection methods, the mobile phase A is a 0.1% formic acid aqueous solution, the mobile phase B is methanol, and gradient elution is used, with the gradient conditions as follows:

[0013] Time (min) Mobile phase A (%) Mobile phase B (%) 0 98 2 4 98 2 9 5 95 12 5 95 12.1 98 2 15 98 2

[0014] In some embodiments, in any of the above detection methods, the mobile phase aqueous phase A is a 0.1-0.2% formic acid aqueous solution (preferably 0.2% formic acid aqueous solution), the mobile phase organic phase B is acetonitrile, and gradient elution is used, with the gradient conditions as follows:

[0015] Time (min) Mobile phase A (%) Mobile phase B (%) 0 90 10 4 90 10 9 5 95 12 5 95 12.1 90 10 15 90 10

[0016] In some embodiments, in any of the above detection methods, the flow rate of the mobile phase is 0.35 to 0.55 mL / min, preferably 0.4 ± 0.05 mL / min.

[0017] In some embodiments, in any of the above detection methods, the column temperature of the chromatographic column is 30–45°C, preferably 35±2°C.

[0018] In some embodiments, in any of the above detection methods, the mass spectrometry conditions include: ionization mode: ESI source positive ion mode; scanning mode: multiple reaction monitoring (MRM); drying gas temperature: 350°C; drying gas flow rate: 12 L / min; nebulization pressure: 50 psi; sheath gas temperature: 400°C; sheath gas flow rate: 12 L / min; capillary voltage: 3000 V; nozzle voltage: 0 V.

[0019] In some embodiments, the present invention provides a method for simultaneously detecting the contents of N-methylaniline and N,N-dimethylaniline in quetiapine fumarate, employing ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-MS / MS). The chromatographic column is a phenyl-hexyl column, preferably a Waters ACQUITY UPLC CSH Phenyl-Hexyl column; mobile phase A is a 0.1% formic acid aqueous solution, and mobile phase B is methanol; preferably, the sample to be tested is prepared as a test solution using a 50% acetonitrile aqueous solution as a diluent; more preferably, gradient elution is used in the mobile phase, with the following gradient conditions:

[0020] Time (min) Mobile phase A (%) Mobile phase B (%) 0 98 2 4 98 2 9 5 95 12 5 95 12.1 98 2 15 98 2

[0021] In some embodiments, the present invention provides a method for simultaneously detecting the contents of N-methylaniline and N,N-dimethylaniline in quetiapine fumarate, employing ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-MS / MS). The chromatographic column is a phenyl-hexyl column, preferably a Waters ACQUITY UPLC CSH Phenyl-Hexyl column; mobile phase A is a 0.1–0.2% formic acid aqueous solution (preferably 0.2% formic acid aqueous solution), and mobile phase B is acetonitrile; preferably, the sample to be tested is prepared using a 0.1–0.2% formic acid aqueous solution (preferably 0.2% formic acid aqueous solution) as a diluent to prepare a test solution; more preferably, the mobile phase employs gradient elution, with the gradient conditions as follows:

[0022] Time (min) Mobile phase A (%) Mobile phase B (%) 0 90 10 4 90 10 9 5 95 12 5 95 12.1 90 10 15 90 10

[0023] In some embodiments, any of the above detection methods includes the preparation of one or more of the following solutions:

[0024] (1) Reference stock solution: Weigh N-methylaniline reference standard and N,N-dimethylaniline reference standard, dissolve them in ethanol and dilute them with diluent to the required concentration;

[0025] (2) Reference solution: Take the reference stock solution and dilute it with a diluent to prepare the target concentration, preferably 1.5 ng / ml;

[0026] (3) Linear solution: Take the stock solution of the reference standard and prepare it according to a linear gradient using diluent;

[0027] (4) Test solution: Take an appropriate amount of quetiapine fumarate and dilute it with a diluent to prepare the target concentration, preferably 3 mg / ml;

[0028] The diluent is a 50% acetonitrile aqueous solution or a 0.1-0.2% formic acid aqueous solution.

[0029] In some embodiments, any of the above detection methods includes the following steps: accurately measuring blank solution, reference solution or linear solution, and test solution for injection and analysis, recording the chromatogram, and calculating the content. The calculation method may be the standard curve method or the external standard single-point method.

[0030] The present invention has the following advantages and beneficial effects:

[0031] 1. High specificity; neither blank solution nor test solution interferes with the detection of N-methylaniline and N,N-dimethylaniline.

[0032] 2. High sensitivity, capable of accurately identifying and measuring trace amounts of N-methylaniline and N,N-dimethylaniline in samples. Effective detection and accurate quantification can be achieved even when the target analyte concentration in the sample is at trace or ultra-trace levels.

[0033] 3. High durability, with higher reliability and stability in practical applications.

[0034] 4. The detection method is simple to operate, quick to analyze, and highly accurate. Attached Figure Description

[0035] Figure 1-1 Example 1: The MRM chromatogram of the target compound detected using column 1 in Method 1.

[0036] Figure 1-2 Example 1: The MRM chromatogram of the target compound detected using column 2 in Method 1.

[0037] Figure 2 Example 2: Method 2 uses two chromatographic columns to detect the target compound's MRM chromatogram.

[0038] Figure 3-1 , 3-2 These are the specific property spectra of N-methylaniline and N,N-dimethylaniline detected using Method 1 described in Example 1, respectively.

[0039] Figure 3-3 , 3-4 These are the specific property spectra of N-methylaniline and N,N-dimethylaniline detected using Method 2 as described in Example 2.

[0040] Figure 4-1 Example 2: Method 2 using different diluents and an aqueous mobile phase, MRM diagram of the target compound.

[0041] Figure 4-2 Example 2: Method 2 using different mobile phase organic phases, MRM diagrams of the target compound.

[0042] Figure 4-3 Example 2: MRM diagram of the target compound when using gradient 1 in method 2.

[0043] Figure 4-4 Example 2: MRM diagram of the target compound when using gradient 2 in method 2.

[0044] Figure 5-1 MRM chromatogram of N-methylaniline detected according to literature method

[0045] Figure 5-2 MRM chromatogram of N-methylaniline was detected according to Method 2 of Example 2.

[0046] Figure 5-3 MRM chromatogram of N,N-dimethylaniline was determined according to the literature method.

[0047] Figure 5-4MRM chromatogram of N,N-dimethylaniline was detected according to Method 2 of Example 2. Detailed Implementation

[0048] Example 1, Method 1

[0049] 1.1 Experimental Instruments: Liquid Chromatography-Mass Spectrometry (LC-MS / MS): Agilent-1290 / 6470

[0050] 1.2 Solution Preparation

[0051] Diluent: 50% acetonitrile aqueous solution

[0052] Blank solution: Same as diluent

[0053] Reference stock solution (15 ng / ml): Accurately weigh 15 mg each of N-methylaniline and N,N-dimethylaniline reference standards, place them in separate 100 ml volumetric flasks, add an appropriate amount of ethanol, sonicate to dissolve and dilute to the mark, and shake well to prepare N1 and N2 solutions. Accurately measure 1 ml each of N1 and N2 solutions, place them in 50 ml volumetric flasks, dilute to the mark with ethanol, and shake well to prepare N3 solution. Accurately measure 2 ml of N3 solution, place it in a 100 ml volumetric flask, dilute to the mark with ethanol, and shake well to prepare N4 solution. Accurately measure 25 ml of N4 solution, place it in a 100 ml volumetric flask, dilute to the mark with ethanol, and shake well to prepare N5 solution.

[0054] Reference solution (1.5 ng / ml): Accurately measure 1 ml of N5 solution, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0055] System suitability solution: Same as reference solution.

[0056] Test solution: Weigh 30 mg of quetiapine fumarate accurately, place it in a 10 ml volumetric flask, add an appropriate amount of diluent, sonicate to dissolve and dilute to the mark, shake well, and the solution is ready.

[0057] 1.3 Chromatographic and Mass Spectrometric Conditions

[0058]

[0059]

[0060] 1.4 Determination Method

[0061] Accurately measure 5 μl of each of the blank solution, reference solution, and test solution for analysis, record the chromatogram, and calculate the content using the external standard one-point method according to the following formula.

[0062]

[0063] In the formula, Cs The concentration of the target compound reference solution is expressed in ng / ml.

[0064] A s The average peak area of ​​the target compound reference solution after six consecutive injections.

[0065] A u The peak area of ​​the target compound in the test solution;

[0066] V represents the dilution volume of the test sample, in ml;

[0067] M represents the sample weight of quetiapine fumarate, in mg.

[0068] System suitability requirements: For continuous injection of SST solution for 6 injections, the RSD of the peak area must not exceed 20.0%.

[0069] Two identical chromatographic columns were used to analyze the samples. The results using column 1 are as follows: Figure 1-1 As shown in the figure. The results indicate that under these assay conditions, neither the blank solution nor the test solution interferes with the detection of N-methylaniline and N,N-dimethylaniline, and the peaks of N-methylaniline and N,N-dimethylaniline are sharp and have good responses. The results using column 2 are shown in the figure. Figure 1-2 As shown, the peak of N,N-dimethylaniline showed a bifurcation phenomenon.

[0070] Example 2, Method Two

[0071] 2.1 Experimental Instruments: Liquid Chromatography-Mass Spectrometry (LC-MS / MS): Agilent-1290 / 6470

[0072] 2.2 Solution Preparation

[0073] Diluent (0.2% formic acid aqueous solution): Measure 2 ml of formic acid and place it in 1000 ml of water, shake well, and you have the solution.

[0074] Blank solution: Same as diluent.

[0075] Reference stock solution (3 ng / ml): Accurately weigh approximately 15 mg each of N-methylaniline and N,N-dimethylaniline, place them in a 50 ml volumetric flask, add an appropriate amount of ethanol, sonicate to dissolve and dilute to the mark, shake well, and this is the N1 solution; accurately measure 0.5 ml of the N1 solution, place it in a 50 ml volumetric flask, dilute to the mark with ethanol, shake well, and this is the N2 solution; accurately measure 0.5 ml of the N2 solution, place it in a 50 ml volumetric flask, dilute to the mark with ethanol, shake well, and this is the N3 solution; accurately measure 1 ml of the N3 solution, place it in a 10 ml volumetric flask, dilute to the mark with diluent, shake well, and this is the N4 solution.

[0076] Linear solutions: Accurately measure 0.1 ml, 0.5 ml, 1 ml, 1.5 ml and 2 ml of N4 solution, place them in different 10 ml volumetric flasks, dilute to the mark with diluent, and shake well to obtain L1-L5 respectively.

[0077] System suitability solution (SST solution): L3 solution.

[0078] Test solution: Weigh 30 mg of quetiapine fumarate accurately, place it in a 10 ml volumetric flask, add an appropriate amount of diluent, sonicate for 2 min to dissolve and dilute to the mark, shake well to obtain the test solution.

[0079] 2.3 Chromatographic and Mass Spectrometric Conditions

[0080]

[0081]

[0082] 2.4 Determination Methods

[0083] Accurately measure 5 μl each of blank solution, system suitability solution, linear solution and test solution for analysis, record the chromatogram, calculate the content using the standard curve method, and calculate according to the following formula.

[0084]

[0085] In the formula, C is the concentration of the target compound in the test solution, ng / ml;

[0086] V represents the dilution volume of the test sample, in ml;

[0087] m is the sample weight, in mg;

[0088] System suitability requirements: For 6 consecutive injections of SST solution, the RSD of the peak area of ​​the target compound must not exceed 20.0%; after the sequence is completed, the peak area of ​​the target compound in the SST solution after the injection is less than 20.0% of the RSD (n=7) of the 6 consecutive injections.

[0089] Two identical chromatographic columns were used to analyze the samples. The experimental results are as follows: Figure 2 As shown, using different chromatographic columns, the results for N-methylaniline and N,N-dimethylaniline were both good, with sharp peaks and good responses.

[0090] Example 3 Specificity Test

[0091] 3.1 Solution Preparation

[0092] Method 1: Prepare blank solution, reference solution and test solution according to the method in Example 1.

[0093] Method 2: Prepare blank solution, L3 solution and test solution according to the method in Example 2.

[0094] 3.2 Determination Method

[0095] Method 1: The blank solution, reference solution and test solution were determined according to the chromatographic and mass spectrometric conditions of Example 1.

[0096] Method 2: The blank solution, L3 solution and test solution were determined according to the chromatographic and mass spectrometric conditions of Example 2.

[0097] 3.3 Results and Discussion

[0098] The experimental results of Method 1 are shown in Figure 3-1 and Figure 3-2 The experimental results for Method 2 are shown in [link to experimental results]. Figure 3-3 , 3-4 The results showed that for both Method 1 and Method 2, neither the blank solution nor the test solution interfered with the detection of the target compounds N-methylaniline and N,N-dimethylaniline.

[0099] Example 4 Limit of Quantitation Test

[0100] 4.1 Solution Preparation

[0101] Method 1: Limit of Quantitation (LOQ) Solution (6 pg / ml): Accurately measure 1 ml of N4 solution (prepared according to Example 1), place it in a 100 ml volumetric flask, dilute to the mark with ethanol, and shake well to obtain N6 solution. Accurately measure 0.1 ml of N6 solution, place it in a 10 ml volumetric flask, dilute to the mark with 50% acetonitrile aqueous solution, and shake well to obtain the final solution (prepare 6 parallel portions).

[0102] Method 2: Limit of Quantitation (LOQ) solution (0.03 ng / ml): Prepare L1 solution according to Example 2.

[0103] 4.2 Determination Method

[0104] Method 1: Six solutions of reference standard at the limit of quantitation concentration were determined according to the chromatographic and mass spectrometric conditions described in Example 1.

[0105] Method 2: Six solutions of reference standard at the limit of quantitation concentration were determined according to the chromatographic and mass spectrometric conditions of Example 2.

[0106] 4.3 Acceptable Standards

[0107] The signal-to-noise ratio (S / N) should be no less than 10; the RSD of six consecutive measurements should be no greater than 20%; the limit of quantitation (LOQ) concentration, the LQ content in the test sample, and the LQ as a percentage of the limit concentration should be reported.

[0108] 4.4 Results and Discussion

[0109] The results of Method 1 are shown in Table 1. The experimental results show that the RSD values ​​of the peak areas of N-methylaniline and N,N-dimethylaniline are both less than 20%, and the S / N ratios are both greater than 10. The limit of quantitation (LOQ) concentration of N-methylaniline is 0.0058 ng / ml, equivalent to a sample content of 0.002 ppm; the LQ concentration of N,N-dimethylaniline is 0.0060 ng / ml, equivalent to a sample content of 0.002 ppm.

[0110] Table 1. Limit of Quantitation Results for Method 1

[0111]

[0112] The results of Method 2 are shown in Table 2. The experimental results show that the RSD values ​​of the peak areas of N-methylaniline and N,N-dimethylaniline are both less than 20%, and the S / N ratios are both greater than 10. The limit of quantitation (LOQ) concentration of N-methylaniline is 0.0302 ng / ml, equivalent to a sample content of 0.01 ppm; the LQ concentration of N,N-dimethylaniline is 0.0356 ng / ml, equivalent to a sample content of 0.012 ppm.

[0113] Table 2 Results of the limit of quantitation for Method 2

[0114]

[0115] Example 5 Detection Limit Test

[0116] 5.1 Solution Preparation

[0117] Method 1: Limit of Detection (LOD) solution (1.8 pg / ml): Take 3 ml of LOQ solution into a 10 mL volumetric flask, dilute to the mark with 50% acetonitrile aqueous solution, and shake well.

[0118] Method 2: Limit of Detection (LOD) solution (9 pg / ml): Take 3 ml of LOQ solution into a 10 mL volumetric flask, dilute to the mark with diluent, and shake well.

[0119] 5.2 Determination Method

[0120] Method 1: Following the chromatographic and mass spectrometric conditions of Example 1, the detection limit solution was injected twice consecutively for determination.

[0121] Method 2: Following the chromatographic and mass spectrometric conditions of Example 2, the detection limit solution was injected twice consecutively for determination.

[0122] 5.3 Acceptable Standards

[0123] The signal-to-noise ratio (S / N) is ≥3; the report includes the limit of detection concentration, the content of the test sample equivalent to the limit of detection, and the percentage of the limit of detection concentration equivalent to the limit concentration.

[0124] 5.4 Results and Discussion

[0125] The results of Method 1 are shown in Table 3. The experimental results show that, for two consecutive injections of the reference solution at the detection limit concentration, the S / N ratios for both N-methylaniline and N,N-dimethylaniline were greater than 3. The detection limit concentration for N-methylaniline was 0.0017 ng / ml, equivalent to 0.0006 ppm in the test sample; the detection limit concentration for N,N-dimethylaniline was 0.0018 ng / ml, equivalent to 0.0006 ppm in the test sample.

[0126] Table 3 shows the detection limit results for Method 1.

[0127]

[0128] The results of Method 2 are shown in Table 4. The experimental results show that, with two consecutive injections of the reference solution at the detection limit concentration, the S / N ratios for both N-methylaniline and N,N-dimethylaniline were greater than 3. The detection limit concentration for N-methylaniline was 0.0091 ng / ml, equivalent to 0.003 ppm in the test sample; the detection limit concentration for N,N-dimethylaniline was 0.0107 ng / ml, equivalent to 0.004 ppm in the test sample.

[0129] Table 4 shows the detection limits for Method 2.

[0130]

[0131] Example 6 Accuracy Test

[0132] 6.1 Solution Preparation

[0133] 6.1.1 Method 1

[0134] Diluent: 50% acetonitrile aqueous solution.

[0135] Blank solution: Same as diluent.

[0136] Test solution: Weigh 30 mg of quetiapine fumarate accurately, place it in a 10 ml volumetric flask, add an appropriate amount of diluent, sonicate to dissolve and dilute to the mark, shake well, and the solution is ready.

[0137] 50% spiked solution of test sample: Weigh 30 mg of quetiapine fumarate accurately, place it in a 10 ml volumetric flask, add an appropriate amount of diluent and sonicate to dissolve, add 0.5 ml of N5 solution (prepared according to Example 1), dilute to the mark with diluent, and shake well to obtain the solution (prepare 3 parallel portions).

[0138] 100% spiked solution of the test sample: Weigh 30 mg of quetiapine fumarate accurately, place it in a 10 ml volumetric flask, add an appropriate amount of diluent and sonicate to dissolve, add 1.0 ml of N5 solution (prepared according to Example 1), dilute to the mark with diluent, and shake well to obtain the solution (prepare 3 parallel portions).

[0139] 150% spiked solution of test sample: Weigh 30 mg of quetiapine fumarate accurately, place it in a 10 ml volumetric flask, add an appropriate amount of diluent and sonicate to dissolve, add 1.5 ml of N5 solution (prepared according to Example 1), dilute to the mark with diluent, and shake well to obtain the solution (prepare 3 parallel portions).

[0140] 6.1.2 Method Two

[0141] Blank solution, linear solution and test solution were prepared according to Example 2.

[0142] LOQ spiking solution for test sample: Weigh 30 mg of quetiapine fumarate accurately, place it in a 10 ml volumetric flask, add an appropriate amount of diluent and sonicate for 2 min to dissolve, add 0.1 ml of N4 solution (prepared according to Example 2), dilute to the mark with diluent, and shake well to obtain the solution (prepare 3 parallel portions).

[0143] 100% spiked solution of the test sample: Weigh 30 mg of quetiapine fumarate accurately, place it in a 10 ml volumetric flask, add an appropriate amount of diluent and sonicate for 2 min to dissolve it, add 1.0 ml of N4 solution (prepared according to Example 2), dilute to the mark with diluent, and shake well to obtain the solution (prepare 3 parallel portions).

[0144] 120% spiked solution of test sample: Weigh 30 mg of quetiapine fumarate accurately, place it in a 10 ml volumetric flask, add an appropriate amount of diluent and sonicate for 2 min to dissolve, add 1.2 ml of N4 solution (prepared according to Example 2), dilute to the mark with diluent, and shake well to obtain the solution (prepare 3 parallel portions).

[0145] 6.2 Determination Method

[0146] Method 1: Following the chromatographic and mass spectrometric conditions of Example 1, the blank solution, the test solution, and the spiked test solution were injected sequentially for determination. The spectra were recorded, and the measured amount and spiked recovery rate were calculated.

[0147] Method 2: Following the chromatographic and mass spectrometric conditions of Example 2, the blank solution, linear solution, test solution, and spiked test solution were injected sequentially for determination. The spectra were recorded, and the measured amounts and spiked recoveries were calculated.

[0148] Recovery rate % = (Measured amount - Background amount) / Added amount * 100%.

[0149] 6.3 Results and Discussion

[0150] The test results of Method 1 are shown in Table 5. The recoveries of N-methylaniline at the 50% limit concentration level were 101.5%–103.8%, at the 100% limit concentration level were 102.1%–102.7%, and at the 150% limit concentration level were 99.9%–103.3%; the recoveries of N,N-dimethylaniline at the 50% limit concentration level were 104.8%–106.7%, at the 100% limit concentration level were 99.2%–100.1%, and at the 150% limit concentration level were 99.8%–102.5%.

[0151] Table 5 Accuracy Results

[0152]

[0153] The test results for Method 2 are shown in Table 6. The LOQ level recoveries of N-methylaniline were 85.9%–106.0%, the 100% limit concentration level recoveries were 103.6%–111.1%, and the 120% limit concentration level recoveries were 102.3%–109.9%; the LOQ level recoveries of N,N-dimethylaniline were 106.1%–119.1%, the 100% limit concentration level recoveries were 104.7%–108.6%, and the 120% limit concentration level recoveries were 103.2%–105.4%.

[0154] Table 6. Accuracy results of Method 2

[0155]

[0156]

[0157] Example 7: Comparative Study of Method Two

[0158] 7.1 Study on diluent and mobile phase (aqueous phase)

[0159] The effect of both the diluent and the mobile phase being a 0.1% or 0.2% aqueous formic acid solution on the peak shape of the target compound was investigated. Other experimental conditions were the same as in Example 2. The results are as follows: Figure 4-1 As shown, when both the diluent and the mobile phase were 0.1% or 0.2% formic acid aqueous solution, the peak shape of the target compound did not bifurcate, and the peak shape of the target compound was better when both the diluent and the aqueous phase were 0.2% formic acid aqueous solution than when both were 0.1% formic acid aqueous solution.

[0160] 7.2 Study of the organic phase in the mobile phase

[0161] The effect of methanol or acetonitrile as the mobile phase on the peak shape of the target compound was investigated. Other experimental conditions were the same as in Example 2, and the results are as follows: Figure 4-2As shown, when the organic phase is acetonitrile, the peak shape of the target compound is better than that of methanol, and the column pressure is lower when the mobile phase is acetonitrile.

[0162] 7.3 Study on the elution gradient of the mobile phase

[0163] The effect of the mobile phase elution gradient on the specificity of the target compound was investigated. Except for the mobile phase gradient (the specific gradients are shown in Tables 7 and 8), the other experimental conditions were the same as in Example 2.

[0164] Table 7 Gradient 1

[0165] Time (min) Flow rate (ml / min) A(％) B(％) 0 0.4 98 2 4 0.4 98 2 9 0.4 5 95 12 0.4 5 95 12.1 0.4 98 2 15 0.4 98 2

[0166] Table 8 Gradient 2

[0167]

[0168]

[0169] When gradient 1 is used, i.e., the initial (0-4 min) organic phase content of the mobile phase is 2%, the results are as follows: Figure 4-3 As shown in the figure. The experimental results show that the blank solution does not interfere with N-methylaniline, but interferes with N,N-dimethylaniline. The peak area of ​​the interfering peak in the blank solution accounts for 9.2% of the peak area of ​​the limit concentration compound.

[0170] When gradient 2 conditions are used, i.e., the initial (0-4 min) organic phase content of the mobile phase is 10%, the results are as follows: Figure 4-4 As shown in the figure. The experimental results show that the blank solution has no interference with N-methylaniline and N,N-dimethylaniline.

[0171] Comparative test

[0172] The determination of N,N-dimethylaniline in quetiapine fumarate was performed according to the method described in the literature “Liang Jianmou, Fu Cong, Chen Yue. Determination of N,N-dimethylaniline in quetiapine fumarate by liquid chromatography-mass spectrometry [J]. Medical Guide, 2017, 36(03):317-320”. The solution preparation method is as follows:

[0173] Solution 1 (20 ng / ml): Accurately weigh 10 mg each of N-methylaniline and N,N-dimethylaniline, place them in a 50 mL volumetric flask, dissolve and dilute to the mark with 50% acetonitrile, and shake well to obtain STD-1; accurately measure 1 mL of STD-1, place it in a 100 mL volumetric flask, dilute to the mark with 50% acetonitrile, and shake well to obtain STD-2; accurately measure 1 mL of STD-2, place it in a 100 mL volumetric flask, dilute to the mark with 50% acetonitrile, and shake well to obtain the final solution.

[0174] Solution 2 (10 ng / ml): Accurately measure 0.5 ml of STD-2, place it in a 100 mL volumetric flask, dilute to the mark with 50% acetonitrile, and shake well.

[0175] Solution 3 (0.03 ng / ml): Accurately measure 0.3 ml of Solution 2 (10 ng / ml), place it in a 100 mL volumetric flask, dilute to the mark with 50% acetonitrile, and shake well to obtain the solution.

[0176] The test results are shown in Table 9 and Figures 5-1 to 5-4 .

[0177] Table 9 Test Results

[0178]

[0179] Based on the experimental results of Examples 4 and 5, it can be seen that the literature method not only has a wider peak shape, but also has a much lower sensitivity than the method of the present invention. The specific comparison is as follows:

[0180] (1) For N-methylaniline, when the concentration is as low as 0.03 ng / ml, the literature method can no longer detect it, while the method of the present invention can still detect it stably, and the signal-to-noise ratio (S / N) is greater than 10 (meeting the quantitative requirements); the limit of quantitation of the literature method is 10 ng / ml, while the limit of quantitation of the second method of the present invention is 0.0302 ng / ml, which is only more than 1 / 300 of it (that is, the sensitivity is improved by more than 300 times).

[0181] (2) For N,N-dimethylaniline, when the concentration is as low as 0.03 ng / ml, the literature method cannot detect it, but the method of the present invention can still detect it effectively and meet the quantitative requirements.

Claims

1. A method for simultaneously determining the content of N-methylaniline and N,N-dimethylaniline in quetiapine fumarate, wherein, Ultra-high performance liquid chromatography coupled with mass spectrometry was used. The chromatographic column was a phenyl-hexyl column, the mobile phase was an aqueous formic acid solution, and the organic phase was acetonitrile or methanol.

2. The method according to claim 1, wherein, The aqueous phase of the mobile phase is a 0.1-0.2% formic acid aqueous solution, and the organic phase of the mobile phase is methanol or acetonitrile; preferably, the aqueous phase of the mobile phase is a 0.1% formic acid aqueous solution, and the organic phase of the mobile phase is methanol; or the aqueous phase of the mobile phase is a 0.2% formic acid aqueous solution, and the organic phase of the mobile phase is acetonitrile.

3. The method according to any one of claims 1 to 2, wherein, The chromatographic column was a Waters ACQUITY UPLC CSHPhenyl-Hexyl.

4. The method according to any one of claims 1 to 3, wherein, The test sample solution is prepared using a 50% acetonitrile aqueous solution or a 0.1-0.2% formic acid aqueous solution as a diluent.

5. The method according to any one of claims 1 to 4, wherein, The mobile phase A is an aqueous solution of 0.1% formic acid, and the mobile phase B is an organic solution of methanol. Gradient elution is used, and the gradient conditions are as follows: Alternatively, the mobile phase A is a 0.2% formic acid aqueous solution, and the mobile phase B is acetonitrile, using gradient elution under the following gradient conditions.

6. The method according to any one of claims 1 to 5, wherein, The flow rate of the mobile phase is 0.35–0.55 mL / min, preferably 0.4 ± 0.05 mL / min.

7. The method according to any one of claims 1 to 6, wherein, The column temperature of the chromatographic column is 30–45℃, preferably 35±2℃.

8. The method according to any one of claims 1 to 7, wherein, Mass spectrometry conditions included: ionization mode: ESI source positive ion mode; scanning mode: multiple reaction monitoring (MRM); drying gas temperature: 350℃; drying gas flow rate: 12L / min; nebulization pressure: 50psi; sheath gas temperature: 400℃; sheath gas flow rate: 12L / min; capillary voltage: 3000V; nozzle voltage: 0V.

9. The method according to any one of claims 1 to 8, wherein, The following solutions are prepared: (1) Reference stock solution: Weigh N-methylaniline reference standard and N,N-dimethylaniline reference standard, dissolve them in ethanol and dilute them with diluent to the required concentration; (2) Reference solution: Take the reference stock solution and dilute it with a diluent to prepare the target concentration, preferably 1.5 ng / ml; (3) Linear solution: Take the stock solution of the reference standard and prepare it according to a linear gradient using diluent; (4) Test solution: Take an appropriate amount of quetiapine fumarate and dilute it with a diluent to prepare the target concentration, preferably 3 mg / ml; The diluent is a 50% acetonitrile aqueous solution or a 0.1-0.2% formic acid aqueous solution.

10. The method according to any one of claims 1 to 9, wherein, Includes the following steps: Accurately measure blank solution, reference solution or linear solution, and test solution for injection and analysis, record the chromatogram, and calculate the content.

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