General analysis and detection method for seven nitrosamines in medicine

Abstract

The invention discloses a general analysis and detection method for seven nitrosamines in a drug, which comprises sample pretreatment, liquid chromatography-triple quadrupole tandem mass spectrometry analysis and detection, and qualitative and quantitative analysis, and comprises the following steps: step 1, dissolving a sample with an organic solvent, adding water for precipitation, and centrifuging to take supernate to form a sample solution; 2, injecting the sample solution and the reference substance solution into a liquid chromatography-triple quadrupole tandem mass spectrometer for detection; and 3, qualitative and quantitative analysis. According to the method disclosed by the invention, general analysis methods of seven nitrosamines listed in the FDA guide are developed, and appropriate chromatographic conditions such as diluents, chromatographic columns and mobile phase gradients are established by investigating various sample matrixes, so that the method is really general. The sensitivity of seven nitrosamines is 10ppb level, the linear range is 100 times (10ppb-1ppm), the high and medium concentration labeling accuracy is 80%-120%, the low concentration is 70%-130% below 40ppb, the stability is more than 48h (80%-120%), and the detection requirements of most later projects can be met.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug analysis and detection, and relates to the analysis of nitrosamine genotoxic impurities in raw materials, and in particular relates to a method for simultaneously detecting nitrosamines using LC-MSMS and a high-sensitivity analysis and detection method, and specifically relates to a universal analysis and detection method involving seven nitrosamines in drugs. Background Art

[0002] The analysis and control of nitrosamine impurities have received increasing attention with the introduction of nitrosamine regulations by EMA (European Medicines Agency) and FDA (U.S. Food and Drug Administration) in 2020. The pharmaceutical industry's understanding of nitrosamine impurities has been increasingly improved, and the evaluation and control methods have become more scientific and detailed. There are 7 common nitrosamine impurities listed in the FDA guidelines, including N,N-dimethylnitrosamine (NDMA), N-methyl-4-aminobutyric acid nitrosamine (NMBA), N,N-diethylnitrosamine (NDEA), N-ethyl-N-isopropylnitrosamine (NEIPA), N,N-diisopropylnitrosamine (NDIPA), N-methyl-N-phenylnitrosamine (NMPA) and N,N-di-n-butylnitrosamine (NDBA). These nitrosamine impurities mainly come from solvents and reagents in the process. At present, there is no unified pharmacopoeia method for the analysis of nitrosamine impurities at home and abroad.

[0003] The United States Pharmacopoeia lists four methods for detecting nitrosamines, including GC-MSMS (gas chromatography-tandem mass spectrometry), HSGC-MSMS (high-sensitivity gas chromatography-tandem mass spectrometry), LC-MSMS (liquid chromatography-tandem mass spectrometry) and LC-HRMS (liquid chromatography-high-resolution mass spectrometry). GC-MSMS is suitable for detecting nitrosamines with low polarity and high volatility, but it cannot effectively detect nitrosamines with high polarity such as NMBA, and GC detection of nitrosamines is prone to false positives. The main advantage of LC-HRMS is qualitative, and its quantitative sensitivity cannot be compared with triple quadrupole mass spectrometry. The LC-MSMS method of the United States Pharmacopoeia only detects 6 nitrosamines at the same time, and only NDEA has a sensitivity of 10ppb, while the remaining nitrosamines are 20ppb; there is no special plan for sample pretreatment of the LC-MSMS method.

[0004] The company already had some internal analysis methods for nitrosamine impurities, but in actual applications, the sample solubility was insufficient and the sample matrix interfered with the detection of the target. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for detecting nitrosamine compounds in medicines, which has a sample pretreatment process step, convenient analysis and detection operation, high sensitivity and can meet the needs of low-content nitrosamine analysis and detection in medicines.

[0006] In order to solve the above technical problems, the specific technical solutions adopted by the present invention are as follows:

[0007] A general analytical detection method for seven nitrosamines in drugs, characterized by comprising sample pretreatment, liquid chromatography-triple quadrupole tandem mass spectrometry (LC-MSMS), qualitative and quantitative analysis, and the specific steps are as follows:

[0008] Step 1: dissolve the sample in an organic solvent, add water to precipitate, and centrifuge to obtain the supernatant to form a sample solution;

[0009] Step 2: injecting the sample solution and the reference solution into a liquid chromatography-triple quadrupole tandem mass spectrometer for detection;

[0010] Step 3: Qualitative and quantitative analysis.

[0011] The seven nitrosamines are N,N-nitrosodimethylamine (NDMA), N-nitrosomethyl-4-aminobutyric acid (NMBA), N,N-nitrosodiethylamine (NDEA), N-nitrosoethyl-N-isopropylamine (NEIPA), N,N-nitrosodiisopropylamine (NDIPA), N-methyl-N-phenylnitrosamine (NMPA) and N,N-nitrosodi-n-butylamine (NDBA).

[0012] In some specific embodiments, the nitrosamines detected may be some or more of the above 7 types.

[0013] In some specific embodiments, the organic solvent in step 1 is selected from one or more of methanol, acetonitrile, ethanol, isopropanol, tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc), 1,3-dimethyl-2-imidazolidinone (DMI), and N-methyl-2-pyrrolidone (NMP).

[0014] In some specific embodiments, the operation of taking the supernatant in step 1 is to take the supernatant after standing or to take the supernatant after filtering.

[0015] In some specific embodiments, the sample solution in step 1 and step 2 is selected from one or more of in-process control samples, raw material samples, intermediate samples, and product samples.

[0016] In some specific embodiments, the preparation method of the reference solution in step 2 is: weigh together the same mass of NDMA, NMBA, NDEA, NEIPA, NDIPA, NMPA, and NDBA into a volumetric flask, dissolve and adjust the volume with an organic solvent, and mix evenly; dilute gradually, and finally add water to mix; step 2 also includes a sensitivity solution, and the preparation method of the sensitivity solution is: weigh together the same mass of NDMA, NMBA, NDEA, NEIPA, NDIPA, NMPA, and NDBA into a volumetric flask, dissolve and adjust the volume with an organic solvent, and mix evenly; dilute gradually, and finally add water to mix.

[0017] In some specific embodiments, the chromatographic conditions of step 2 are as follows: chromatographic column is Agilent ZORBAX Bonus-RP 150mm×4.6mm, 3.5μm; column temperature is 40°C; injector temperature is 5°C; flow rate is 0.7mL / min; injection volume is 20μL; mobile phase A is 0.1% formic acid aqueous solution v / v; mobile phase B is methanol 0-2min, 90% A, 10% B; 2-7min, 45% A, 55% B; 7-12min, 45% A, 55% B; 12-13min, 10% A, 90% B; 13-17min in, 10% A, 90% B; 17.0-17.1min, 90% A, 10% B; 17.1-20min, 90% A, 10% B; the mass spectrometry conditions of step 2 are: the ion source is atmospheric pressure chemical ionization source APCI; the polarity is positive ion; the battery accelerator voltage is 3v; the mode is multiple reaction detection MRM; the gas temperature is 250°C; the gas flow rate is 6L / min; the nozzle pressure is 45psi; and the evaporation temperature is 350°C.

[0018] In some specific embodiments, the mass spectrometry parameters in step 2 are set as follows:

[0019]

[0020] In some specific embodiments, the qualitative and quantitative analysis in step 3 is selected from one of the following calculation formulas:

[0021] 1)

[0022] 2)

[0023] Where:

[0024] A SPL : Peak area of ​​target peak in sample solution;

[0025] W SPL : The mass of the sample, in mg;

[0026] VSPL : dilution volume of sample solution, in mL;

[0027] A STD : Peak area of ​​target peak in reference solution;

[0028] W STD : The weight of each target impurity in the reference solution, in mg;

[0029] V STD : The dilution volume of each target impurity in the reference solution, in mL.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The method of the present invention makes the sample matrix less affected and more versatile. The present invention develops a universal analytical method for the seven nitrosamines listed in the FDA guidelines. Since the regulatory limits of nitrosamines are low, the method of the present invention establishes suitable chromatographic conditions, such as diluents, chromatographic columns and mobile phase gradients, by examining a variety of sample matrices, making the method truly universal. The sensitivity of the seven nitrosamines is all at the 10ppb level, the linear range is 100 times (10ppb-1ppm), the accuracy of spiked high and medium concentrations is 80%-120%, the accuracy of low concentrations below 40ppb is 70%-130%, and the stability is more than 48h (80%-120%), which can meet the detection needs of most later projects.

[0032] The concept and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, features and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Here are the linear results and range graphs for NDMA.

[0034] Figure 2 It is the linear result and range graph of NMBA.

[0035] Figure 3 Here are the linear results and range graphs for NDEA.

[0036] Figure 4 It is the linearity result and range graph of NEIPA.

[0037] Figure 5 It is the linearity result and range graph of NDIPA.

[0038] Figure 6 It is the linearity result and range graph of NMPA.

[0039] Figure 7 The linear results and range graph for NDBA are shown below.

[0040] Figure 8 It is the LC-MSMS multiple reaction monitoring (MRM) spectrum of 7 nitrosamine reference substances.

[0041] Fig. 9 Overlay of the blank, sensitivity, control, sample, and sample spike solutions for NDMA.

[0042] Fig.10 Overlay of NMBA blank, sensitivity, control, sample, and sample spike solutions.

[0043] Fig.11 Overlay of blank, sensitivity, control, sample, and sample spike solutions for NDEA.

[0044] Fig.12 Overlay of blank, sensitivity, control, sample, and sample spike solutions of NEIPA.

[0045] Fig.13 Overlay of blank, sensitivity, control, sample, and sample spike solutions for NDIPA.

[0046] Fig.14 It is an overlay of the blank, sensitivity, control, sample, and sample spike solutions of NMPA.

[0047] Fig.15 Overlay of blank, sensitivity, control, sample, and sample spike solutions of NDBA. DETAILED DESCRIPTION

[0048] In order to make the technical means, creative features, objectives and effects of the invention easier to understand, the invention is further described below with reference to specific diagrams. However, the invention is not limited to the following implementation cases.

[0049] It should be noted that the drawings in this specification are only used to match the contents disclosed in the specification so as to facilitate understanding and reading by persons familiar with this technology, and are not used to limit the conditions under which the present invention can be implemented. Therefore, they have no substantive technical significance. Any adjustments without creative effort, without affecting the effects and purposes that can be achieved by the present invention, should still fall within the scope of the technical contents disclosed by the present invention.

[0050] All kinds of reagents, antibodies, and consumables involved in the examples are commercially available products, and all commercial products can be used.

[0051] The present invention analyzes the solubility characteristics of nitrosamine samples and reference substances, dissolves the samples, adds water to precipitate the sample matrix, and then centrifuges to take the supernatant for injection, thereby eliminating the interference of the sample matrix as a sample pretreatment method. In terms of instruments, the present invention establishes a universal analytical method for detecting seven nitrosamines through liquid chromatography-triple quadrupole tandem mass spectrometry (LC-MSMS) technology. This technology is applicable to the LC-MSMS quantitative determination of seven nitrosamine genotoxic impurities N,N-nitrosodimethylamine (NDMA, CAS#: 62-75-9), N-methyl-4-aminobutyric acid nitrosoamine (NMBA, CAS#: 61445-55-4), N,N-diethylnitrosoamine (NDEA, CAS#: 55-18-5), N-ethyl-N-isopropylnitrosoamine (NEIPA, CAS#: 16339-04-1), N,N-diisopropylnitrosoamine (NDIPA, CAS#: 601-77-4), N-methyl-N-phenylnitrosoamine (NMPA, CAS#: 614-00-6) and N,N-di-n-butylnitrosoamine (NDBA, CAS#: 924-16-3) in raw materials, in-process control, intermediates and products of clinical projects. The corresponding ranges and retention times are listed in Table 1 below:

[0052] Table 1 Detection range and retention time of 7 nitrosamines

[0053] name Working range (ng / mL) Retention time (min) NDMA 0.25-25 3.4 NMBA 0.25-25 5.7 NDEA 0.25-25 7.3 NEIPA 0.25-25 8.7 NDIPA 0.25-25 9.9 NMPA 0.25-25 10.5 NDBA 0.25-25 15.1

[0054] The instruments, equipment, reagents, etc. designed by the present invention are as follows in Table 2:

[0055] Table 2 Instruments and reagents

[0056]

[0057]

[0058] Example 1 Chromatographic and mass spectrometric conditions

[0059] Set the chromatographic and mass spectrometric conditions and parameters as shown in Tables 3, 4 and 5 below:

[0060] Table 3 Chromatographic conditions

[0061]

[0062]

[0063] Table 4 Mass spectrometry conditions

[0064]

[0065] Table 5 MS parameter settings

[0066]

[0067]

[0068] **The start time can be adjusted according to actual situation.

[0069] Note: The above conditions were established based on an Agilent 1290 UHPLC system with a G6470B MS / MS detector. Some non-critical parameters, such as re-equilibration time, may be adjusted when using other instruments.

[0070] Example 2 Solution Preparation

[0071] 1. Diluent

[0072] Diluent 1: Methanol

[0073] Diluent 2: Purified water

[0074] 2. Preparation of blank solution

[0075] Take 1.0 mL of diluent 1 (methanol) and place it in a 15 mL centrifuge tube. Transfer 3.0 mL of diluent 2 (purified water) into the tube and mix well.

[0076] 3. Preparation of control solution

[0077] The concentration of the reference solution can be adjusted according to the product's quality specifications for the limit of nitrosamine residues and the sample concentration. The selected standard concentration should be ensured to be within the validation linear range (0.25ng / mL to 25ng / mL). It is recommended to use 5ng / mL as the base concentration of the method standard. The type of impurities is determined by the quality specifications and can only include the target impurities to be tested.

[0078] Product testing: prepare 2 reference solution in parallel; raw material, in-process control and intermediate product testing: prepare 1 reference solution.

[0079] Unless otherwise specified, the reference solution is prepared as shown in Table 6:

[0080] Table 6 Preparation of reference solution

[0081]

[0082] 4. Preparation of sensitivity solution

[0083] Unless otherwise specified, the sensitivity solution is prepared as shown in Table 7:

[0084] Table 7 Sensitivity solution preparation

[0085]

[0086] 5. Sample solution preparation

[0087] The concentration and preparation of the sample solution can be determined based on the quality specification of the nitrosamine impurity, the linear range, the sensitivity solution and the solubility of the sample. The sample concentration should be selected to ensure that the quality specification limit is higher than the sensitivity solution of the method and within the linear range. 25 mg / mL is recommended.

[0088] In-process control samples: prepare 1 portion; raw material, intermediate, and product samples: prepare 2 portions in parallel.

[0089] Unless otherwise specified, the sample solution is prepared as follows:

[0090] Sample-1: Accurately weigh about 100 mg of the mixed sample into a 15 mL centrifuge tube, pipette 1.0 mL of diluent 1 (methanol) until dissolved, pipette 3.0 mL of diluent 2 (purified water) and shake well, centrifuge at 10,000 rpm for 15 min, and take the supernatant for analysis.

[0091] Sample-2 (if necessary): Repeat the above preparation process to prepare the second sample.

[0092] Note: Liquid samples are usually prepared directly in centrifuge tubes. However, if the change in volume has a significant impact on the spike recovery results, they should be prepared in a volumetric flask first, and then 1.0 ml should be transferred to a centrifuge tube.

[0093] Example 3 Calculation of test results

[0094]

[0095] Where:

[0096] A 1 : The average peak area of ​​the target peak in the first six injections (three injections for intermediate and raw materials) of the reference substance-1 solution;

[0097] A 2 : Peak area of ​​target peak in reference substance-2;

[0098] W 1 : The sample amount of each target impurity in reference substance-1 (mg);

[0099] W 2 : The sample amount of each target impurity in reference substance-2 (mg);

[0100]

[0101] Where:

[0102] A 1 : The average peak area of ​​the target peak in the reference substance-1 solution in the first 6 injections (three injections for the intermediate and raw materials);

[0103] A 2: Peak area of the target peak in spike injection.

[0104] Results of target impurities (ppm or % w / w)

[0105] 1)

[0106] 2)

[0107] Where:

[0108] A SPL : Peak area of the target peak in the sample solution;

[0109] W SPL : Mass of the sample, in mg;

[0110] V SPL : Dilution volume of the sample solution, in mL;

[0111] A STD : Peak area of the target peak in the reference solution;

[0112] W STD : Weighing amount of each target impurity in the reference solution, in mg;

[0113] V STD : Dilution volume of each target impurity in the reference solution, in mL.

[0114] Analysis and detection results of Example 4

[0115] Unless otherwise specified, when calculating the results of a single impurity, it should be compared with LOQ (ppm) first. Those ≥ LOQ (ppm) participate in the calculation, and those < LOQ (ppm) do not participate in the calculation. Finally, convert to percentage (if necessary) for reporting:

[0116] Table 8

[0117]

[0118] Method validation results of Example 5

[0119] Table 9

[0120]

[0121]

[0122]

[0123]

[0124]

[0125] Figure 1-Figure 7 They are the linear results and range diagrams of NDMA, NMBA, NDEA, NEIPA, NDIPA, NMPA, and NDBA. Figure 8 It is the LC-MSMS multiple reaction monitoring (MRM) spectrum of 7 nitrosamine reference substances. Figure 9-Figure 15 They are the overlay graphs of blank, sensitivity, reference, sample and sample spiked solution of NDMA, NMBA, NDEA, NEIPA, NDIPA, NMPA and NDBA respectively.

[0126] It can be seen that the sensitivity of the seven nitrosamines is all at the 10ppb level, the linear range is 100 times (10ppb-1ppm), the accuracy of spiked high and medium concentrations is 80%-120%, the accuracy of low concentrations below 40ppb is 70%-130%, and the stability is more than 48h (80%-120%), which can meet the detection needs of most later projects.

[0127] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A general analytical detection method for seven nitrosamines in medicines, characterized in that: It includes sample pretreatment, liquid chromatography-triple quadrupole tandem mass spectrometry, qualitative and quantitative analysis. The specific steps are as follows: Step 1: dissolve the sample in an organic solvent, add water to precipitate, and take the supernatant to form a sample solution; Step 2: injecting the sample solution and the reference solution into a liquid chromatography-triple quadrupole tandem mass spectrometer for detection; Step 3: Qualitative and quantitative analysis; The seven nitrosamines are N,N-nitrosodimethylamine (NDMA), N-nitrosomethyl-4-aminobutyric acid (NMBA), N,N-nitrosodiethylamine (NDEA), N-nitrosoethyl-N-isopropylamine (NEIPA), N,N-nitrosodiisopropylamine (NDIPA), N-methyl-N-phenylnitrosamine (NMPA) and N,N-nitrosodi-n-butylamine (NDBA).

2. The method according to claim 1, characterized in that The organic solvent in step 1 is selected from one or more of methanol, acetonitrile, ethanol, isopropanol, tetrahydrofuran, dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, 1,3-dimethyl-2-imidazolidinone, and N-methyl-2-pyrrolidone.

3. The method according to claim 1, characterized in that The operation of taking the supernatant in step 1 is to take the supernatant after standing or to take the supernatant after filtering.

4. The method according to claim 1, characterized in that: The sample solution in step 1 and step 2 is selected from one or more of in-process control samples, raw material samples, intermediate samples, and product samples.

5. The method according to claim 1, characterized in that The preparation method of the reference solution in the step 2 is: weighing the same mass of NDMA, NMBA, NDEA, NEIPA, NDIPA, NMPA, and NDBA into a volumetric flask, dissolving and fixing the volume with an organic solvent, and mixing evenly; diluting gradually, and finally adding water to mix; the step 2 also includes a sensitivity solution, and the preparation method of the sensitivity solution is: weighing the same mass of NDMA, NMBA, NDEA, NEIPA, NDIPA, NMPA, and NDBA into a volumetric flask, dissolving and fixing the volume with an organic solvent, and mixing evenly; diluting gradually, and finally adding water to mix evenly.

6. The method according to claim 1, characterized in that The chromatographic conditions of step 2 are as follows: the chromatographic column is Agilent ZORBAX Bonus-RP 150mm×4.6mm, 3.5μm; the column temperature is 40°C; the injector temperature is 5°C; the flow rate is 0.7mL / min; the injection volume is 20μL; the mobile phase A is 0.1% formic acid aqueous solution v / v; the mobile phase B is methanol 0-2min, 90% A, 10% B; 2-7min, 45% A, 55% B; 7-12min, 45% A, 55% B; 12-13min, 10% A, 90% B; 13-17min, 10% A, 90% B; 17.0~17.1min, 90%A, 10%B; 17.1~20min, 90%A, 10%B; the mass spectrometry conditions of step 2: the ion source is atmospheric pressure chemical ionization source APCI; the polarity is positive ion; the battery accelerator voltage is 3v; the mode is multiple reaction detection MRM; the gas temperature is 250℃; the gas flow rate is 6L / min; the nozzle pressure is 45psi; the evaporation temperature is 350℃.

7. The method according to claim 1, characterized in that The mass spectrometry parameters in step 2 are set as follows: * Used for quantitative calculation of ions.

8. The method according to claim 1, characterized in that: The qualitative and quantitative analysis in step 3 is selected from one of the following calculation formulas: 1) 2) Where: A SPL : Peak area of ​​target peak in sample solution; W SPL : The mass of the sample, in mg; V SPL : dilution volume of sample solution, in mL; A STD : Peak area of ​​target peak in reference solution; W STD : The weight of each target impurity in the reference solution, in mg; V STD : The dilution volume of each target impurity in the reference solution, in mL.

Images