Analytical Method for Genotoxic Impurity Content of N-Nitrosodiclofenac in Chemical Drugs

Abstract

The present invention belongs to the technical field of analysis and detection, and specifically relates to an analysis method for the content of N-nitroso diclofenac genotoxic impurities in chemical drugs. The analysis method uses a triple quadruple liquid-mass spectrometer to determine the content of N-nitroso diclofenac impurities in chemical drugs, using a 50 vol% acetonitrile aqueous solution (containing 0.1 vol% formic acid) as a solvent and dichloromethane as an extractant, and uses a standard curve method to analyze the content of N-nitroso diclofenac impurities. By dissolving the test sample with a solvent first and then extracting it with an extractant, the problems of low impurity limit, complex and easy interference of the test sample matrix, and easy false positive caused by the impurity structure being similar to the main component of the test sample are overcome, and at the same time, the sensitivity of N-nitroso diclofenac impurities is improved by high performance liquid chromatography-mass spectrometry, and the detection method is efficient and accurate.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis and detection, and particularly relates to a method for analyzing the content of N-nitrosodiclofenac genotoxic impurities in chemical drugs. Background Art

[0002] N-nitrosodiclofenac is a macromolecular nitroso impurity closely related to the main component structure and is highly carcinogenic to humans. In view of its harmfulness, countries currently have extremely strict control standards for the residual amount of nitrosamine impurities in drugs. Every year, a large number of drugs are recalled from the market due to excessive nitrosamine impurity content, and this issue has attracted widespread attention.

[0003] Secondary and tertiary amines, as chemical precursors of nitrosamines, are ubiquitous in many drugs and play an important role. According to drug databases, 41.4% of active pharmaceutical ingredients (APIs) and 33% of impurities are secondary or tertiary amines. These amine compounds are crucial in chemical reactions. They effectively improve the solubility of drugs by acting as hydrogen bond donors and acceptors, and their unique structures help promote pharmacological interactions. Given the wide application of secondary and tertiary amines in drugs, it is not realistic to completely avoid their use, which leads to an extremely high risk of generating nitrosamine impurities in different drugs.

[0004] Nitrosamine impurities not only have strict limit requirements, but also the matrix of the test sample is complex, which can easily interfere with the detection of impurities. Traditional chromatography is difficult to meet the needs of nitrosamine impurity detection due to its insufficient sensitivity and specificity. If you try to increase the detection sensitivity by increasing the concentration of the test solution, it may cause overload of the instrument, chromatographic column, etc., and then cause contamination or even damage to the chromatographic column and other serious consequences. Therefore, it is particularly important to develop an efficient, accurate and reliable analysis method for the content of N-nitrosodiclofenac genotoxic impurities in chemical drugs. Summary of the invention

[0005] In view of the deficiencies in the prior art, the object of the present invention is to provide a method for analyzing the content of N-nitrosodiclofenac genotoxic impurities in chemical drugs. The method overcomes the problems of low impurity limit, complex matrix of the test sample, easy interference, and false positive caused by impurity structure similar to the main component of the test sample by dissolving the test sample with a solvent and then extracting it with an extractant. At the same time, the sensitivity of N-nitrosodiclofenac impurities is improved by high performance liquid chromatography-mass spectrometry, and the detection method is efficient and accurate.

[0006] The technical solution adopted by the present invention is as follows:

[0007] The method for analyzing the content of N-nitrosodiclofenac genotoxic impurities in the chemical medicine comprises the following steps:

[0008] (1) Add the test sample to a 50 vol% acetonitrile aqueous solution (containing 0.1 vol% formic acid) as solvent, mix well to obtain a test sample solution with a concentration of 90 mg / mL, then centrifuge the test sample solution, take the supernatant after centrifugation, extract the supernatant with dichloromethane (the volume ratio of supernatant to dichloromethane is 1:3), collect the dichloromethane layer after stratification, and obtain a test solution with a concentration of 30 mg / mL;

[0009] (2) Setting the parameters of liquid chromatography and mass spectrometry of triple quadrupole LC-MS instrument;

[0010] (3) Inject the prepared test solution into the injection bottle, directly inject the sample through the instrument, collect data, and then perform data analysis;

[0011] (4) After the analysis is completed, the peak area of ​​N-nitrosodiclofenac measured in the test solution is compared with the standard curve, and the content of N-nitrosodiclofenac in the test sample is calculated.

[0012] The parameters of the liquid chromatography are as follows:

[0013] The chromatographic column is a C18 column with octadecylsilane bonded silica as filler, with specifications of 2.1 mm × 100 mm, 2.7 μm;

[0014] Mobile phase A was pure water, and mobile phase B was methanol;

[0015] The flow rate was 0.2 mL / min.

[0016] The column temperature was 35°C; the injection volume was 2 μL;

[0017] The gradient program is as follows:

[0018] 0.01min, the volume percentage of mobile phase A was 45%, and the volume percentage of mobile phase B was 55%;

[0019] 4.5 min, the volume percentage of mobile phase A was 45%, and the volume percentage of mobile phase B was 55%;

[0020] 5 min, the volume percentage of mobile phase A is 0%, and the volume percentage of mobile phase B is 100%;

[0021] 10 min, the volume percentage of mobile phase A is 0%, and the volume percentage of mobile phase B is 100%;

[0022] 10.1 min, the volume percentage of mobile phase A was 45%, and the volume percentage of mobile phase B was 55%;

[0023] 15min, the volume percentage of mobile phase A is 45%, and the volume percentage of mobile phase B is 55%.

[0024] The mass spectrometry parameters are as follows:

[0025] The MRM positive ion mode was used;

[0026] The ion source was ESI source, and the interface temperature was 300 °C;

[0027] Atomizing gas flow rate 3L / min, heater flow rate 10L / min;

[0028] DL tube temperature was 250°C, heating block temperature was 350°C;

[0029] The ion pair of N-nitrosodiclofenac is 295>242, and the collision voltage is -19V.

[0030] The method for determining the standard curve comprises the following steps:

[0031] (1) Prepare a stock solution using 50 vol% acetonitrile aqueous solution (containing 0.1 vol% formic acid) as solvent and N-nitrosodiclofenac reference substance as solute;

[0032] (2) Take an appropriate amount of the stock solution and dilute it with a solvent to obtain reference solutions of different concentrations. Extract the reference solution with dichloromethane at a volume ratio of 1:3. After the solution is allowed to stand and separate into layers, separate and collect the dichloromethane layer as the standard solution;

[0033] (3) Setting the parameters of the liquid chromatography and mass spectrometry of the triple quadrupole LC / MS instrument, injecting standard solutions of different concentrations into injection bottles respectively, and measuring the peak areas corresponding to the standard solutions of different concentrations by the instrument;

[0034] (4) Based on the peak area data corresponding to each concentration of the standard solution, a standard curve between N-nitrosodiclofenac concentration and peak area was drawn.

[0035] The concentration of the stock solution is 70 ng / mL.

[0036] In the step (2), the concentration of the reference solution is 2-14 ng / mL.

[0037] The equation of the standard curve is: Y=22138X+528.11, and the linear coefficient r=0.9997.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] (1) The present invention adopts high performance liquid chromatography-mass spectrometry, which greatly improves the detection sensitivity of N-nitrosodiclofenac content. It can not only accurately distinguish the target compound from the interfering components in the complex matrix, but also effectively solve the false positive problem caused by the similarity between the impurity structure and the main component of the test sample, thereby ensuring the accuracy and reliability of the test results;

[0040] (2) The present invention optimizes the sample processing steps. The sample is first dissolved in an acetonitrile aqueous solution (containing 0.1 vol% formic acid) in a specific ratio as a solvent, and then extracted with dichloromethane. This not only simplifies the operation, but also significantly reduces the matrix effect, effectively eliminates the interference of complex components in the sample on the test results, and improves the specificity of the analysis; at the same time, it effectively avoids the contamination of the chromatographic column and ion source by the sample, thereby protecting the analytical instrument, extending its service life, and reducing long-term operating costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is the standard curve of N-nitrosodiclofenac;

[0042] Figure 2 This is the quantitative graph of spiked solution 1 in Example 2. DETAILED DESCRIPTION

[0043] The present invention is further described below with reference to the embodiments, but they do not limit the implementation of the present invention.

[0044] Unless otherwise specified, the raw materials used in the examples and comparative examples are conventional commercially available raw materials, and the process methods used in the examples and comparative examples are conventional methods in the art unless otherwise specified.

[0045] The instruments and equipment used in the examples are as follows:

[0046] Triple quadruple liquid spectrometer-mass spectrometer: model is Shimadzu LCMS-8050;

[0047] The parameters of the liquid chromatography and mass spectrometry used in the examples are as follows:

[0048] The parameters of liquid chromatography are:

[0049] The chromatographic column is a C18 column with octadecylsilane bonded silica as filler, with specifications of 2.1 mm × 100 mm, 2.7 μm;

[0050] Mobile phase A was pure water, and mobile phase B was methanol;

[0051] The flow rate was 0.2 mL / min.

[0052] The column temperature was 35°C; the injection volume was 2 μL;

[0053] The gradient program is as follows:

[0054] 0.01min, the volume percentage of mobile phase A was 45%, and the volume percentage of mobile phase B was 55%;

[0055] 4.5 min, the volume percentage of mobile phase A was 45%, and the volume percentage of mobile phase B was 55%;

[0056] 5 min, the volume percentage of mobile phase A is 0%, and the volume percentage of mobile phase B is 100%;

[0057] 10 min, the volume percentage of mobile phase A is 0%, and the volume percentage of mobile phase B is 100%;

[0058] 10.1 min, the volume percentage of mobile phase A was 45%, and the volume percentage of mobile phase B was 55%;

[0059] 15min, the volume percentage of mobile phase A is 45%, and the volume percentage of mobile phase B is 55%.

[0060] The parameters of mass spectrometry are:

[0061] The MRM positive ion mode was used;

[0062] The ion source was ESI source, and the interface temperature was 300 °C;

[0063] Atomizing gas flow rate 3L / min, heater flow rate 10L / min;

[0064] DL tube temperature was 250°C, heating block temperature was 350°C;

[0065] The ion pair of N-nitrosodiclofenac is 295>242, and the collision voltage is -19V.

[0066] Example 1

[0067] Linearity and Range Analysis:

[0068] Here are the steps:

[0069] (1) Using 50 vol% acetonitrile aqueous solution (containing 0.1 vol% formic acid) as solvent and N-nitrosodiclofenac reference substance as solute, prepare a stock solution with a concentration of 70 ng / mL;

[0070] (2) Accurately measure an appropriate amount of the 70 ng / mL stock solution and dilute it with a 50 vol% acetonitrile aqueous solution (containing 0.1 vol% formic acid) to obtain reference solutions with concentrations of 2.1 ng / mL, 3.5 ng / mL, 7.0 ng / mL, 10.5 ng / mL and 14.0 ng / mL, respectively. Accurately measure the reference solution of each concentration and extract it with dichloromethane at a volume ratio of 1:3. After the solution is allowed to stand and separate, separate and collect the dichloromethane layer to obtain a standard solution with a concentration of 0.7-4.7 ng / mL;

[0071] (3) Setting the parameters of the liquid chromatography and mass spectrometry of the triple quadrupole LC / MS instrument, injecting standard solutions of different concentrations into injection bottles respectively, and measuring the peak areas corresponding to the standard solutions of different concentrations by the instrument;

[0072] (4) Based on the peak area data corresponding to each concentration of the standard solution, a standard curve between N-nitrosodiclofenac concentration and peak area was drawn.

[0073] The standard curve of N-nitrosodiclofenac is as follows Figure 1 As shown, the equation of the standard curve is Y=22138X+528.11, and the linear coefficient r=0.9997, indicating that N-nitrosodiclofenac has a good linear relationship between concentration and peak area in the concentration range of 0.7~4.7ng / mL.

[0074] Example 2

[0075] Repeatability analysis:

[0076] Here are the steps:

[0077] (1) Add 90 mg of the test sample to a 50 vol% acetonitrile aqueous solution (containing 0.1 vol% formic acid) as solvent, then add 1 mL of a 7.0 ng / mL reference solution, mix well to obtain a 90 mg / mL test sample solution, then centrifuge the test sample solution for 3 min, take the supernatant after centrifugation, extract it with dichloromethane at a volume ratio of 1:3, collect the dichloromethane layer after stratification as the test solution, prepare 6 portions in parallel, and record them as spiked solution 1 to spiked solution 6;

[0078] (2) Setting the parameters of liquid chromatography and mass spectrometry of triple quadrupole LC-MS instrument;

[0079] (3) Inject the prepared test solution into the injection bottle, directly inject the sample through the instrument, collect data, and then perform data analysis;

[0080] (4) After the analysis is completed, the peak area of ​​N-nitrosodiclofenac measured in the test solution is compared with the standard curve, and the content of N-nitrosodiclofenac in the test sample is calculated.

[0081] The results of the precision investigation of N-nitrosodiclofenac are shown in Table 1.

[0082] Table 1 Results of precision study of N-nitrosodiclofenac

[0083]

[0084] It can be concluded from Table 1 that the RSD of the retention time of N-nitrosodiclofenac in the 6 test sample spiked solutions is 0.3%, less than 2%; the RSD of the content is 5.0%, less than 15.0%, indicating that the detection method has good repeatability. Figure 2 shown.

[0085] Example 3

[0086] Accuracy analysis:

[0087] Here are the steps:

[0088] (1) Blank test solution: Take the test sample and add 50 vol% acetonitrile aqueous solution (containing 0.1 vol% formic acid) to make a 90 mg / mL test solution. Vortex and mix well, centrifuge for 3 min, and take the supernatant. Accurately pipette a certain volume and extract with dichloromethane in a ratio of 1:3. Let the solution stand for stratification, then take the dichloromethane layer for testing. Prepare 3 copies in parallel.

[0089] (2) LOQ spiked test solution: Take 90 mg of the test sample, weigh accurately, add 1 mL of a 2.1 ng / mL reference solution to make a 90 mg / mL test solution, oscillate and mix, centrifuge for 3 min, and take the supernatant; accurately pipette a certain volume and extract with dichloromethane at a ratio of 1:3. Let the solution stand for separation, then take the dichloromethane layer for testing, and prepare 3 copies in parallel.

[0090] (3) 100% test sample spiked solution: Take 90 mg of the test sample, weigh accurately, add 1 mL of 7.0 ng / mL reference solution to make a 90 mg / mL test sample solution, oscillate and mix, centrifuge for 3 min, and take the supernatant; accurately pipette a certain volume and extract with dichloromethane at a ratio of 1:3. Let the solution stand for separation, then take the dichloromethane layer for testing. Prepare 3 copies in parallel.

[0091] (4) 150 vol% test sample spiked solution: Take 90 mg of the test sample, weigh accurately, add 1 mL of a 10.5 ng / mL reference solution to make a 90 mg / mL test sample solution, oscillate and mix, centrifuge for 3 min, and take the supernatant; accurately pipette a certain volume and extract with dichloromethane at a ratio of 1:3. Let the solution stand for separation, then take the dichloromethane layer for testing. Prepare 3 portions in parallel.

[0092] The accuracy analysis results of N-nitrosodiclofenac are shown in Table 2.

[0093] Table 2 Results of accuracy analysis of N-nitrosodiclofenac

[0094]

[0095] It can be seen from Table 2 that the average recoveries of LOQ spiked test solution, 100% test spiked solution and 150 vol% test spiked solution were 82%, 90% and 88%, respectively. The average recovery rate of 9 injections was 87%, all within the range of 70%-130%. The RSD of the recovery rate of 9 injections was 7.5%, which was less than 15.0%, and its accuracy met the requirements.

[0096] Example 4

[0097] Detection limit analysis:

[0098] Here are the steps:

[0099] Accurately measure an appropriate amount of 70 ng / mL stock solution, dilute it with a 50 vol% acetonitrile aqueous solution (containing 0.1 vol% formic acid) to prepare a sensitivity solution with a concentration of 1.4 ng / mL, then accurately measure a certain volume, extract it with dichloromethane in a ratio of 1:3, let the solution stand for stratification, then take the dichloromethane layer for testing, collect 3 injections continuously, and the signal-to-noise ratios are 7, 16 and 25, respectively. The detection limit concentration of N-nitrosodiclofenac is 0.4 ng / mL, which is equivalent to the content of 0.014 ng / mg in the test sample; the minimum signal-to-noise ratio is 7 (greater than 3), which meets the requirements.

[0100] Example 5

[0101] Test product analysis:

[0102] Here are the steps:

[0103] (1) Add the test sample to a 50 vol% acetonitrile aqueous solution (containing 0.1 vol% formic acid) as the solvent, mix well to obtain a test sample solution with a concentration of 90 mg / mL, oscillate and mix well, centrifuge for 3 min, take the supernatant, extract with dichloromethane at a volume ratio of 1:3, collect the dichloromethane layer after stratification, and obtain a test solution with a concentration of 30 mg / mL;

[0104] (2) Setting the parameters of liquid chromatography and mass spectrometry of triple quadrupole LC-MS instrument;

[0105] (3) Inject the prepared test solution into the injection bottle, directly inject the sample through the instrument, collect data, and then perform data analysis;

[0106] (4) After the analysis is completed, the peak area of ​​N-nitrosodiclofenac measured in the test solution is compared with the standard curve, and the content of N-nitrosodiclofenac in the test sample is calculated.

[0107] The calculation formula is:

[0108] ;

[0109] The unit of impurity content is ng / mg or ppm, C is the concentration of the solution to be tested (ng / mL), V is the volume of the solution to be tested (mL), and m is the mass of the test sample (mg).

[0110] The contents of N-nitrosodiclofenac in three different batches of test samples were analyzed, and the results are shown in Table 3.

[0111] Table 3 Analysis results of impurity content in the test samples

[0112]

[0113] It can be seen from the above examples that the analytical method of the present invention is suitable for detecting the content of N-nitrosodiclofenac, overcomes the problems of low impurity limit, complex and easy interference of the test sample matrix, and false positive caused by the impurity structure being similar to the main component of the test sample. The method has good precision, high accuracy, low detection limit, saves detection time, and the detection method is efficient and accurate.

Claims

1. A method for analyzing the content of genotoxic impurities of N-nitrosodiclofenac in chemical drugs, characterized in that: The following steps are involved: (1) Using acetonitrile aqueous solution as solvent, adding the test sample thereto, mixing evenly to obtain a test sample solution, then centrifuging the test sample solution, taking the supernatant after centrifugation, extracting the supernatant with dichloromethane, and collecting the dichloromethane layer after stratification to obtain a test solution; the acetonitrile aqueous solution is a 50 vol% acetonitrile aqueous solution, and the acetonitrile aqueous solution also contains 0.1 vol% formic acid; the volume ratio of the supernatant to dichloromethane is 1:3, and the concentration of the test solution is 30 mg / mL; (2) Setting the parameters of liquid chromatography and mass spectrometry of triple quadrupole LC-MS instrument; The parameters of the liquid chromatography are as follows: The chromatographic column is a C18 column with octadecylsilane bonded silica as filler, with specifications of 2.1 mm × 100 mm, 2.7 μm; Mobile phase A was pure water, and mobile phase B was methanol; The flow rate was 0.2 mL / min. The column temperature was 35°C; the injection volume was 2 μL; The gradient program is as follows: 0.01min, the volume percentage of mobile phase A was 45%, and the volume percentage of mobile phase B was 55%; 4.5 min, the volume percentage of mobile phase A was 45%, and the volume percentage of mobile phase B was 55%; 5 min, the volume percentage of mobile phase A is 0%, and the volume percentage of mobile phase B is 100%; 10 min, the volume percentage of mobile phase A is 0%, and the volume percentage of mobile phase B is 100%; 10.1 min, the volume percentage of mobile phase A was 45%, and the volume percentage of mobile phase B was 55%; 15 min, the volume percentage of mobile phase A was 45%, and the volume percentage of mobile phase B was 55%; The mass spectrometry parameters are as follows: The MRM positive ion mode was used; The ion source was ESI source, and the interface temperature was 300 °C; Atomizing gas flow rate 3L / min, heater flow rate 10L / min; DL tube temperature was 250°C, heating block temperature was 350°C; The ion pair of N-nitrosodiclofenac is 295>242, and the collision voltage is -19V; (3) Inject the prepared test solution into the injection bottle, directly inject the sample through the instrument, collect data, and then perform data analysis; (4) After the analysis is completed, the peak area of ​​N-nitroso-diclofenac measured in the test solution is compared with the standard curve, and the content of N-nitroso-diclofenac in the test sample is calculated; the detection limit concentration of N-nitroso-diclofenac is 0.4 ng / mL.

2. The method for analyzing the content of genotoxic impurities of N-nitrosodiclofenac in the chemical medicine according to claim 1, characterized in that: The concentration of the test solution is 90 mg / mL.

3. The method for analyzing the content of genotoxic impurities of N-nitrosodiclofenac in the chemical medicine according to claim 1, characterized in that: The method for determining the standard curve comprises the following steps: 1) Prepare a stock solution using 50 vol% acetonitrile aqueous solution as solvent, the acetonitrile aqueous solution also contains 0.1 vol% formic acid, and N-nitrosodiclofenac reference substance as solute; 2) Take the stock solution, dilute it with a solvent to obtain reference solutions of different concentrations, extract the reference solution with dichloromethane at a volume ratio of 1:3, and after the solution is allowed to stand and separate, separate and collect the dichloromethane layer as the standard solution; 3) Set the parameters of the liquid chromatography and mass spectrometry of the triple quadrupole-LC-MS instrument, inject standard solutions of different concentrations into the injection bottles respectively, and measure the peak area corresponding to the standard solutions of each concentration through the instrument; 4) Based on the peak area data corresponding to each concentration of the standard solution measured, draw a standard curve between the concentration and peak area of ​​N-nitrosodiclofenac.

4. The method for analyzing the content of genotoxic impurities of N-nitrosodiclofenac in the chemical medicine according to claim 3, characterized in that: The concentration of the stock solution is 70 ng / mL.

5. The method for analyzing the content of genotoxic impurities of N-nitrosodiclofenac in chemical medicine according to claim 3, characterized in that: In the step 2), the concentration of the reference solution is 2-14 ng / mL.

6. The method for analyzing the content of genotoxic impurities of N-nitrosodiclofenac in chemical medicine according to claim 3, characterized in that: The equation of the standard curve is: Y=22138X+528.11.

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