Detection method of genotoxic impurities and application thereof

Abstract

The invention discloses a genotoxic impurity detection method, which can simultaneously detect genotoxic impurities NDMA, NDPA and dimethyl sulfate, adopts a gas chromatography-mass spectrometry (GC-MS) technology, dissolves a sample through a formic acid-containing anhydrous / ultra-dry organic solvent, and combines chromatography-mass spectrometry conditions to realize efficient and synchronous detection of the three impurities. The chromatographic conditions are simple, the mass spectrum detection limit is excellent, the impurity stability is remarkably improved by the method, and the recovery rate RSD value is 1t; the method is good in detection specificity and high in precision, so that the genotoxic impurities in the medicine are effectively controlled, the quality of the medicine is guaranteed, the safety of clinical medication is further guaranteed, and the method has remarkable technical advantages and industrial application value.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to a method for detecting trace genotoxic impurities, specifically a method for detecting three genotoxic impurities, NDMA, NDPA and dimethyl sulfate, in a pharmaceutical composition. Background Technology

[0002] Rotigotine is a selective dopamine D2 analogue agonist. Its mechanism of action primarily involves stimulating dopamine receptors in the brain to help alleviate symptoms in Parkinson's disease patients. Compared to other treatments, rotigotine not only improves motor function but may also provide significant pain relief. In clinical applications, rotigotine is typically administered via transdermal patches, offering a single-dose, sustained-release formulation. The starting materials for its active pharmaceutical ingredient, rotigotine, may contain DMF and dipropylamine, and the intermediates may contain imine structures or imine-containing byproducts. Therefore, rotigotine may contain mutagenic impurities such as N-nitrosodimethylamine (NDMA) and N-nitrosodi-n-propylamine (NDPA). Both NDMA and NDPA are classified as Group 2A carcinogens, representing highly mutagenic and carcinogenic genotoxic impurities that pose a serious threat to human health. According to the technical requirements for nitrosamine impurities and the continuously updated list of nitrosamine impurities published by international drug regulatory agencies (FDA, EMA, WHO, etc.), the acceptable daily intake (AI) for NDMA and NDPA is 96 ng / day and 26.5 ng / day, respectively. Based on the maximum daily dose of rotigotine (16 mg), the limits for NDMA and NDPA are 6 ppm and 1.6 ppm, respectively. Furthermore, based on the synthetic route, it is speculated that dimethyl sulfate may be introduced into the starting materials of rotigotine. Since its sulfate group is a warning structure, it should be studied and controlled as a genotoxic impurity. While there is carcinogenicity data for dimethyl sulfate, it is insufficient to deduce the AI. Therefore, based on the ICH M7 genotoxic impurities guideline's threshold for toxicological concern (TTC) of 1.5 μg / day, and calculated based on the maximum daily dose of 16 mg, the limit for dimethyl sulfate is 93.75 ppm, with a tightened limit of 90 ppm.

[0003] In the field of genotoxic impurity detection, existing technologies have developed various detection methods for single impurities (such as NDMA, NDPA, or dimethyl sulfate). Currently, the main methods for detecting NDMA and NDPA are HPLC-MS and GC-MS, while the main methods for detecting dimethyl sulfate residues are GC, HPLC, and GC-MS. For example, CN112730663A discloses a method for detecting N-nitrosamines based on gas chromatography-mass spectrometry and proposes a GC-MS detection scheme for NDMA. CN111693631A discloses a liquid chromatography-mass spectrometry method for detecting N-nitrosopiperazine compounds in wine, using the GC-MS / MS method. CN107966507A discloses a gas chromatography-mass spectrometry detection technique for dimethyl sulfate, involving the GC-MS detection of dimethyl sulfate. However, the above technologies are all limited to the detection of single impurities or single types of impurities and have not solved the technical problem of simultaneous analysis of multiple different types of components. Furthermore, existing methods generally use aqueous solvents (such as acetonitrile and methanol), which makes impurities prone to degradation during detection (e.g., the peak area of ​​dimethyl sulfate decreases by 22.46% within 7.5 hours in conventional solvents). However, the pharmaceutical industry has not yet established a multi-component simultaneous analysis technology capable of detecting NDMA, NDPA, and dimethyl sulfate. Therefore, to ensure the quality control of subsequent products, it is essential to develop a method that can simultaneously detect trace amounts of NDMA, NDPA, and dimethyl sulfate in rotigotine and guarantee the stable and accurate detection of NDMA, NDPA, and dimethyl sulfate. Moreover, developing a genotoxic impurity analysis method that combines high sensitivity, high stability, and multi-component simultaneous detection capability is a technical bottleneck that urgently needs to be addressed in the field of drug quality control. Summary of the Invention

[0004] This invention provides a method for detecting NDMA, NDPA, and dimethyl sulfate, and its applications. The method involves dissolving the sample in an anhydrous or ultra-dry organic solvent containing formic acid (water content ≤ 50 ppm), and then detecting the sample using gas chromatography-mass spectrometry (GC-MS). The chromatographic conditions are: capillary column, helium as carrier gas, nitrogen as collision gas, and temperature-programmed detection; the mass spectrometry conditions are: mass spectrometer detector, EI source, and MRM mode selected.

[0005]

[0006] Specifically, the present invention provides a method for detecting genotoxic impurities NDMA, NDPA, and dimethyl sulfate in rotigotine, characterized in that the method comprises the following steps:

[0007] The rotigotine sample was dissolved in an anhydrous or ultra-dry organic solvent containing formic acid, wherein the water content of the organic solvent was ≤50ppm;

[0008] The detection was performed using gas chromatography-mass spectrometry (GC-MS), and the chromatographic conditions were as follows:

[0009] Separation is performed using a capillary column, with either cyanopropylphenyl-dimethylpolysiloxane or diphenyl-dimethylpolysiloxane as the stationary phase.

[0010] The temperature program is as follows: start at 50-70℃ and hold for 1-3 minutes, then increase to 250-280℃ at a rate of 20-40℃ / min and hold for 1-3 minutes.

[0011] Injector temperature 250-280℃, split ratio 5:1-15:1, column flow rate 1.2-1.8 ml / min;

[0012] The mass spectrometry conditions are as follows:

[0013] The ion source is an EI source, and the MRM mode is selected.

[0014] In a preferred embodiment of the present invention, the anhydrous or ultra-dry organic solvent is acetonitrile, and the concentration of formic acid is 0.05%-0.5%.

[0015] In a preferred embodiment of the present invention, the concentration of the test sample is 5-20 mg / ml.

[0016] In a preferred embodiment of the present invention, the stationary phase of the capillary column is selected from any of the following:

[0017] 6% Cyanopropylphenyl-94% Dimethylpolysiloxane;

[0018] 35% diphenyl-65% dimethylpolysiloxane;

[0019] 5% diphenyl-95% dimethylpolysiloxane;

[0020] 50% diphenyl-50% dimethyl polysiloxane.

[0021] In a preferred embodiment of the present invention, the chromatographic column conditions are: injection port temperature 250-280℃, split ratio 5:1, column flow rate 1.5 ml / min, initial column temperature 60℃, and programmed column temperature rise conditions as follows: start at 60℃ and hold for 1 min, then rise to 260℃ at a rate of 30℃ / min and hold for 1 min.

[0022] Alternatively, the column conditions are: injection port temperature 260℃, split ratio 5:1-15:1, column flow rate 1.5 ml / min, initial column temperature 60℃, and column temperature program conditions: start at 60℃ and hold for 1 min, increase to 260℃ at a rate of 30℃ / min, and hold for 3 min.

[0023] Alternatively, the column conditions are: injection port temperature 260℃, split ratio 5:1, column flow rate 1.2-1.8, initial column temperature 60℃, and column temperature program conditions: start at 60℃ and hold for 1 min, then increase to 260℃ at a rate of 30℃ / min and hold for 1 min.

[0024] Alternatively, the column conditions are: injection port temperature 260℃, split ratio 5:1, column flow rate 1.5 ml / min, initial column temperature 50-70℃, and programmed column temperature as follows: start at 60℃ and hold for 1 min, then increase to 260℃ at a rate of 30℃ / min and hold for 1 min.

[0025] In a preferred embodiment of the present invention, the mass spectrometry conditions further include the following MRM ion pairs:

[0026] The quantitative ion pair for NDMA was m / z 74→44 with a collision energy of 5 eV; the qualitative ion pair was m / z 74→28 with a collision energy of 10 eV. The quantitative ion pair for NDPA was m / z 130→113 with a collision energy of 5 eV; the qualitative ion pair was m / z 130→88 with a collision energy of 5 eV. The quantitative ion pair for dimethyl sulfate was m / z 125→95 with a collision energy of 5 eV; the qualitative ion pair was m / z 125→79 with a collision energy of 10 eV. The MRM residence time was 150 ms, the solvent delay time was 3 min, and the mass spectrometry mode was low resolution mode.

[0027] In a preferred embodiment of the present invention, the concentration of formic acid is 0.1%-0.2%, more preferably 0.1%.

[0028] In a preferred embodiment of the present invention, the concentration of the test sample is 8-12 mg / ml, more preferably 10 mg / ml.

[0029] In a preferred embodiment of the present invention, the method further includes: dissolving the reference solution and the test solution in 0.1% formic acid anhydrous acetonitrile to maintain the detection system in a weakly acidic environment at all times.

[0030] Another object of the present invention is to provide the method for detecting NDMA, NDPA and dimethyl sulfate residues for use in the quality control of small molecule drugs or peptide drugs.

[0031] In a preferred embodiment of the present invention, the chemical drug is selected from active pharmaceutical ingredients or preparations thereof that are presumed to contain nitrosamines or sulfate esters.

[0032] This invention provides a method for the detection of NDMA, NDPA, and dimethyl sulfate. The method employs gas chromatography-mass spectrometry (GC-MS) with a capillary column and an EI source for mass spectrometry. Reference standards and samples are dissolved in 0.1% anhydrous or ultra-dry organic solvent (anhydrous formic acid) and then detected using GC-MS, overcoming the problem of poor stability of sulfate ester compounds in GC-MS detection. Attached Figure Description

[0033] Figure 1 This is a typical mass spectrum of a blank solvent.

[0034] Figure 2 Typical mass spectra of NDMA, NDPA, and dimethyl sulfate reference solutions.

[0035] Figure 3 This is a typical mass spectrum of the test sample solution.

[0036] Figure 4 Typical mass spectra of solutions with detection limits for NDMA, NDPA, and dimethyl sulfate.

[0037] Figure 5 This is a linear graph of NDMA.

[0038] Figure 6 The graph shows the linearity of dimethyl sulfate.

[0039] Figure 7 This is a linear graph of NDPA. Detailed Implementation

[0040] The following describes specific embodiments of the present invention, providing a clear and complete description of the technical solution of the present invention.

[0041] As the technical principle of this invention, the inventors, through in-depth research, discovered that NDMA, NDPA, and dimethyl sulfate are all unstable under both acidic and alkaline conditions. Dimethyl sulfate is easily degraded in aqueous solutions, and rotigotine solution is weakly alkaline. Therefore, in order to neutralize the weak alkalinity of rotigotine without making the system too acidic or too alkaline and to strictly control the water content of the experimental reagents, this invention specifically selects anhydrous acetonitrile formic acid as a diluent, so that both the reference solution and the test solution always maintain a weakly acidic state, ensuring the stable and accurate detection of the target impurities.

[0042] The anhydrous or ultra-dry organic solvents used in this invention are selected from one or more of methanol, ethanol, acetonitrile, N-methylpyrrolidone, dimethyl sulfoxide, N,N-dimethylformamide, or N,N-dimethylacetamide. In some embodiments, anhydrous acetonitrile is preferred as it provides a better balance between the signal-to-noise ratio of the three target analytes in terms of chromatographic separation.

[0043] The formic acid solution described in this invention has a concentration of 0.05%-0.5%, preferably 0.1%. This concentration maintains a weakly acidic state and is beneficial for sample stability and separation. It should be noted that the formic acid solution concentration in this invention is a volume-based concentration.

[0044] The concentration of the test sample described in this invention is 5-20 mg / ml, and in some embodiments the concentration of the test sample is 10 mg / ml.

[0045] The capillary column described in this invention is selected from one of the following: a capillary column with 6% cyanopropylphenyl-94% dimethylpolysiloxane as the stationary phase, a capillary column with 35% diphenyl-65% dimethylpolysiloxane as the stationary phase, a capillary column with 5% diphenyl-95% dimethylpolysiloxane as the stationary phase, or a capillary column with 50% diphenyl-50% dimethylpolysiloxane as the stationary phase. In some embodiments, the capillary column is Aglient VF-624ms with specifications of 30m*0.32mm*1.8μm.

[0046] The chromatographic conditions described in this invention are: injection port temperature: 250-280℃, preferably 260℃; auxiliary heater temperature: 260℃; split ratio: 5:1-15:1, preferably 5:1; column flow rate: 1.2-1.8, preferably...

[0047] The flow rate is 1.5 ml / min, and the initial column temperature is 50-70℃, preferably 60℃. In some embodiments, the chromatographic conditions described in this invention are: injection port temperature 260℃, split ratio 5:1, column flow rate 1.5 ml / min, and initial column temperature 60℃.

[0048] The chromatographic-detection condition combination of this invention is not a simple superposition, but a synergistic effect achieved through creative effort. The synergistic effect of the anhydrous / ultra-dry solvent system (water content ≤ 50 ppm) and the acidic environment (0.1% formic acid) solves the stability problem of genotoxic impurities (such as dimethyl sulfate) during detection (29-hour change < 9%). Furthermore, it forms a mutually supportive technical system with the programmed temperature rise rate (30℃ / min), split ratio (5:1), and MRM ion pair selection, jointly achieving high sensitivity (NDMA detection limit 1.92 ng / ml). Existing technologies only focus on single impurities or do not address solvent stability issues, and cannot derive the multi-component simultaneous detection scheme of this invention through conventional logic or limited experiments.

[0049] As a preferred embodiment of the chromatographic column of the present invention using programmed temperature rise, the temperature program is to start at 50-70°C and hold for 1-3 min, then rise to 250-280°C at a rate of 20-40°C / min and hold for 1-3 min. In some embodiments, the temperature program is to start at 60°C and hold for 1 min, then rise to 260°C at a rate of 30°C / min and hold for 1 min.

[0050] As an example of condition optimization for the detection method described in this invention, the following parameters are included: the sample containing rotigotine is dissolved in 0.1% formic acid anhydrous acetonitrile, and the residual amounts of NDMA, NDPA and dimethyl sulfate in the sample are detected by gas chromatography-mass spectrometry. The chromatographic column conditions are: injection port temperature 250-280℃, split ratio 5:1, column flow rate 1.5 ml / min, initial column temperature 60℃, and the programmed temperature conditions are: initial temperature of 60℃ for 1 min, increased to 260℃ at a rate of 30℃ / min, and held for 1 min.

[0051] Or include the following parameters:

[0052] The sample containing rotigotine was dissolved in 0.1% formic acid anhydrous acetonitrile. The residual amounts of NDMA, NDPA and dimethyl sulfate in the sample were detected by gas chromatography-mass spectrometry. The chromatographic column conditions were: injection port temperature 260℃, split ratio 5:1-15:1, column flow rate 1.5 ml / min, initial column temperature 60℃, and temperature program conditions: initial temperature of 60℃ for 1 min, increased to 260℃ at a rate of 30℃ / min, and held for 3 min.

[0053] Or include the following parameters:

[0054] The sample containing rotigotine was dissolved in 0.1% formic acid anhydrous acetonitrile. The residual amounts of NDMA, NDPA and dimethyl sulfate in the sample were detected by gas chromatography-mass spectrometry. The chromatographic column conditions were: injection port temperature 260℃, split ratio 5:1, column flow rate 1.2-1.8, column initial temperature 60℃, and column temperature program conditions: start at 60℃ and hold for 1 min, increase to 260℃ at a rate of 30℃ / min and hold for 1 min.

[0055] Or include the following parameters:

[0056] The sample containing rotigotine was dissolved in 0.1% formic acid anhydrous acetonitrile. The residual amounts of NDMA, NDPA and dimethyl sulfate in the sample were detected by gas chromatography-mass spectrometry. The column conditions were as follows: injection port temperature 260℃, split ratio 5:1, column flow rate 1.5 ml / min, initial column temperature 50-70℃, and temperature program conditions: initial temperature of 60℃ for 1 min, increased to 260℃ at a rate of 30℃ / min, and held for 1 min.

[0057] The mass spectrometry detection conditions of this invention can be any known technical solution. For example, existing mass spectrometry detection methods may employ an ionization source (such as electron bombardment, electrospray ionization, or matrix-assisted laser desorption / ionization) to convert sample molecules into charged ions, followed by separation of ions according to mass-to-charge ratio by a mass analyzer (such as a time-of-flight analyzer, quadrupole analyzer, or ion trap), and recording of the signal by a detector (such as an electron multiplier) to generate a mass spectrum. However, considering cost and equipment availability, the preferred mass spectrometry conditions of this invention are: the detector is a mass spectrometer detector. The ion source is an EI source, and the MRM mode is selected. For NDMA, the quantitative ion energy is 74→44 (5 eV) and the qualitative ion energy is 74→28 (10 eV). For NDPA, the quantitative ion energy is 130→113 (5 eV) and the qualitative ion energy is 130→88 (5 eV). For dimethyl sulfate, the quantitative ion energy is 125→95 (5 eV) and the qualitative ion energy is 125→79 (10 eV). The residence time is 150 ms, the solvent delay time is 3 min, and the low-resolution mode is used. The transmission line temperature is 250–280°C, the ion source temperature is 220–240°C, and the quadrupole temperature is 140–160°C. In a preferred embodiment of the invention, the transmission line temperature is 260°C, the ion source temperature is 230°C, and the quadrupole temperature is 150°C.

[0058] After optimizing the conditions and exploring the methods described above, under ideal conditions, the method of this invention can simultaneously detect nitrosamines and sulfate esters, with detection limits of 1.92 ng / ml for NDMA, 0.55 ng / ml for NDPA, and 27.10 ng / ml for dimethyl sulfate, equivalent to 0.19 ppm, 0.055 ppm, and 2.71 ppm of the test samples, respectively. The method exhibits good specificity and high precision, and can be used for trace detection of NDMA, NDPA, and dimethyl sulfate in rotigotine, which helps ensure drug quality and thus the safety of clinical use. More specific methodological practices and the technical effects of this invention can be found in the following examples.

[0059] Example

[0060] The following examples further illustrate typical embodiments of the present invention, including methodological verification. The experimental schemes described below are merely examples and not intended to limit the scope of the invention. Those skilled in the art can make arbitrary modifications based on an understanding of the principles and spirit of the embodiments. The described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0061] Example 1: Specificity Investigation of the Detection Method

[0062] 1. Experimental conditions

[0063]

[0064]

[0065] 2. Solution preparation

[0066] Blank solvent: 0.1% formic acid and anhydrous acetonitrile (0.1% by volume)

[0067] Reference stock solution:

[0068] Accurately weigh approximately 12 mg of NDMA reference standard, place it in a 20 ml volumetric flask, add solvent to dissolve and dilute to the mark, shake well to obtain NDMA reference standard stock solution (0.6 mg / ml), and prepare two parallel solutions.

[0069] Accurately weigh approximately 18 mg of dimethyl sulfate reference standard, place it in a 20 ml volumetric flask, add solvent to dissolve and dilute to the mark, shake well to obtain dimethyl sulfate reference standard stock solution (0.9 mg / ml), and prepare two parallel solutions.

[0070] Accurately weigh approximately 16 mg of NDPA reference standard, place it in a 100 ml volumetric flask, add solvent to dissolve and dilute to the mark, shake well to obtain the NDPA reference standard stock solution (0.16 mg / ml), and prepare two parallel solutions.

[0071] Mixed reference standard stock solution:

[0072] Take 1 ml each of NDMA and NDPA reference stock solutions and place them in a 10 ml volumetric flask. Dilute to the mark with solvent and shake well. Accurately measure 1 ml of the solution and place it in a 100 ml volumetric flask. Add 1 ml of the above dimethyl sulfate reference stock solution and dilute to the mark with solvent. Shake well to obtain the mixed reference stock solution (NDMA 600 ng / ml, dimethyl sulfate 9 ug / ml, NDPA 160 ng / ml). Prepare two parallel aliquots.

[0073] Reference solution: Accurately measure 1 ml of the mixed reference stock solution, place it in a 10 ml volumetric flask, dilute to the mark with solvent, and shake well to obtain the solution.

[0074] Test solution:

[0075] Accurately weigh approximately 100 mg of rotigotine sample, place it in a 10 ml volumetric flask, dissolve and dilute to the mark with solvent, and shake well to obtain (10 mg / ml).

[0076] 3. Recommended testing process

[0077] One or more needles can be used to inject blank solvent;

[0078] Inject one syringe of the reference solution;

[0079] One injection of the test solution;

[0080] According to the mass spectrometry method in General Chapter 0431 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, under the experimental conditions described, blank solvent, reference solution, and test solution were injected into the gas chromatograph-mass spectrometer, and the mass spectra were recorded. Figure 1 , 2 As shown in Figure 3, the blank solvent acetonitrile showed no significant peak interference at the elution positions of NDMA, dimethyl sulfate, and NDPA.

[0081] The detection method provided by this invention can be used for trace detection of genotoxic impurities NDMA, dimethyl sulfate and NDPA. The method has high specificity, good precision, high sensitivity and is easy to operate.

[0082] Example 2: Examination of the linearity and system suitability of the detection method

[0083] 1. Solution preparation

[0084] Blank solvent: 0.1% formic acid and anhydrous acetonitrile

[0085] Linear stock solution: Take the mixed reference stock solution (Example 1) as the linear stock solution and prepare each linear solution according to Table 1.

[0086] Table 1. Preparation of linear solutions

[0087]

[0088] Note: The QC point is the solution prepared for control solution 2, and its purpose is to verify the accuracy of control solution 1. The QC point is not included in the impurity calculation.

[0089] 2. Measurement Method

[0090] One injection was performed on each linear solution of each concentration. The results were plotted with concentration on the x-axis and peak area on the y-axis, where r > 0.990. The peak areas at each QC point were substituted into the equation for calculation. The standard deviation of the calculated concentration from the actual concentration was no greater than 10%. The calculation formula is as follows:

[0091]

[0092] In the formula:

[0093] A represents the purified concentration of each impurity based on its weight.

[0094] B represents the correction concentration for each impurity standard curve.

[0095] 3. Recommended testing process

[0096] One or more needles can be used to inject blank solvent;

[0097] One injection of each concentration of the linear solution was administered;

[0098] One injection of each of the two QC point concentrations of the reference standard;

[0099] 1 μl of blank solvent and linear solution were injected into the gas chromatograph-mass spectrometer, and the mass spectra were recorded. The results are shown in Table 2-7.

[0100] Table 2 NDMA linearity results

[0101]

[0102] Table 3 NDMA QC Point Results

[0103]

[0104] Table 4. Linearity results for dimethyl sulfate

[0105]

[0106] Table 5. Results of QC points for dimethyl sulfate.

[0107]

[0108]

[0109] Table 6. NDPA Linearity Results

[0110]

[0111] Table 7 NDPA QC Point Results

[0112]

[0113] The table above shows the linear equation for NDMA: y = 327.209909*x - 749.828303, with a linear coefficient r = 0.9987 and an intercept percentage of 3.8%. The RD% value at the 75% QC point at 0 hours is 4.12%, and the RD% value at the 125% QC point is 0.57%.

[0114] The linear equation for dimethyl sulfate is: y = 16.261531*x - 0.748924, with a linear coefficient r = 0.9999 and an intercept percentage of 0.005%. The RD% value at the 75% QC point at 0 hours is 8.51%, and the RD% value at the 125% QC point is 5.56%.

[0115] NDPA linear equation: y = 73.543576*x + 95.675103, linear coefficient r = 0.9970, intercept percentage is 6.6%; RD% value at 75% QC point at 0 hour is 8.32%, and RD% value at 125% QC point is 2.89%;

[0116] The linear equations and linear coefficients r for NDMA, dimethyl sulfate, and NDPA, as well as the QC points, all meet the verification requirements.

[0117] Example 3: Limit of Detection Study of Detection Method

[0118] 1. Solution preparation

[0119] Blank solvent: 0.1% formic acid and anhydrous acetonitrile

[0120] Limit of quantitation solution: Linear STD-L1 was used as the limit of quantitation solution.

[0121] Limit of detection solution: Take an appropriate amount of limit of quantitation solution and dilute it stepwise with solvent until the S / N ratio is approximately 3 to obtain the limit of detection solution.

[0122] Inject 1 μl each of the above limit of quantitation and limit of detection solutions into the gas chromatography-mass spectrometry (GC-MS) instrument and record the mass spectra. The results are shown in the table below.

[0123] Table 8. Limit of Quantitation / Limit of Detection for NDMA. Detection Limit Results

[0124]

[0125]

[0126] Table 9. Limit of Quantification / Limit of Detection for Dimethyl Sulfate: Results

[0127]

[0128] Table 10 NDPA Detection Limit Results

[0129]

[0130] The experimental results showed that the limit of quantitation (LOQ) concentration of NDMA was 12.84 ng / ml, the peak area RSD was 3.84%, and the LQ / N result was satisfactory. The limit of detection (LOD) concentration was 1.92 ng / ml, and the LQ / N result was 21.0. All results met the validation requirements.

[0131] The limit of quantitation (LOQ) concentration of dimethyl sulfate was 180.68 ng / ml, the peak area RSD was 5.25%, and the S / N of the LOQ was acceptable. The limit of detection (LOD) concentration was 27.10 ng / ml, and the S / N of the LOD was 187.7. All results met the validation requirements.

[0132] The limit of quantitation (LOQ) concentration of NDPA was 3.70 ng / ml, the peak area RSD was 19.38%, and the LQ / N result was acceptable. The limit of detection (LOD) concentration was 0.55 ng / ml, and the LQ / N result was 24.0. All results met the validation requirements.

[0133] Example 4: Selection of Sample Concentration

[0134] 1. Experimental conditions are the same as in Example 1.

[0135] 2. The test procedure adopted the above detection conditions and tested the samples at different concentrations, using sample solubility and sensitivity as evaluation indicators. The results are shown in Table 11:

[0136] Table 11 Test results of sample concentration

[0137] Concentration of test sample C (mg / ml) Dissolution Sensitivity C <5 mg / ml Completely dissolved Sensitivity does not meet the requirements of the genotoxic limit 5 mg / ml < C < 20 mg / ml Completely dissolved Sensitivity meets the requirements C > 20 mg / ml Cannot be completely dissolved /

[0138] Conclusion: As shown in Table 11, the test sample concentration in the range of 5 mg / ml to 20 mg / ml can be fully dissolved and can meet the sensitivity requirements of the sample determination.

[0139] Example 5: Accuracy Assessment of the Detection Method

[0140] The experimental conditions were the same as in Example 1.

[0141] Solution preparation:

[0142] The mixed reference standard stock solution prepared in the same manner as in Example 1 was used as the accuracy stock solution.

[0143] The test solution was prepared according to the method described in Example 1.

[0144] Preparation of R1-50% solution:

[0145] Weigh approximately 100 mg of this product accurately and place it in a 10 ml volumetric flask. Dissolve the product in an appropriate amount of solvent, then accurately measure 0.5 ml of the mixed control stock solution and place it in the flask. Dilute the solution with solvent and bring it to volume to obtain a 50% spiked recovery sample solution (prepare three parallel aliquots).

[0146] Preparation of R2-100% solution:

[0147] Weigh approximately 100 mg of this product accurately and place it in a 10 ml volumetric flask. Dissolve the product in an appropriate amount of solvent, then accurately measure 1.0 ml of the mixed control stock solution and place it in the flask. Dilute the solution with solvent and bring it to volume to obtain a 50% spiked recovery sample solution (prepare three parallel aliquots).

[0148] Preparation of R3-150% solution:

[0149] Weigh approximately 100 mg of this product accurately and place it in a 10 ml volumetric flask. Dissolve the product in an appropriate amount of solvent, then accurately measure 1.5 ml of the mixed control stock solution and place it in the flask. Dilute the solution with solvent and bring it to volume to obtain a 50% spiked recovery sample solution (prepare three parallel aliquots).

[0150] Inject 1 μl of blank solvent and accuracy solution into the gas chromatograph-mass spectrometer and record the mass spectra. The results are shown in the table below.

[0151] Table 12 NDMA Accuracy Results

[0152]

[0153] Table 13 Accuracy Results of Dimethyl Sulfate

[0154]

[0155] Table 14 NDPA Accuracy Results

[0156]

[0157] As shown in the table above, the recoveries of NDMA, dimethyl sulfate, and NDPA, all genotoxic impurities, are in the range of 70% to 130%, and the RSD is less than 15%, indicating that the method has good accuracy.

[0158] Example 6: Selection of formic acid concentration

[0159] 1. Experimental conditions are the same as in Example 1.

[0160] 2. The experimental procedure adopted the above detection conditions to investigate formic acid at different concentrations, using peak shape and recovery rate RSD as evaluation indicators. The results are shown in Table 15:

[0161] Table 15 Test results of formic acid concentration

[0162]

[0163] Conclusion: As shown in Table 12, formic acid in the range of 0.05%-0.5% exhibits good peak shapes for each genotoxic impurity, NDMA, NDPA, and dimethyl sulfate, and the recovery rates of each impurity meet the requirements with low RSD. Therefore, the method of this application is the most effective.

[0164] Example 7: Repeatability Test of the Detection Method

[0165] Repeatability solutions: Six parallel preparations of the 100% recovery solution from Example 4 were prepared as repeatability solutions. 1 μl of each repeatability solution was injected into a gas chromatography-mass spectrometry (GC-MS) instrument, and the chromatograms were recorded. Repeatability was calculated based on the recovery rate. The experimental results are shown in Tables 16-18.

[0166] Table 16 NDMA repeatability results

[0167]

[0168] Table 17 Repeatability Results of Dimethyl Sulfate

[0169]

[0170] Table 18 NDPA Repeatability Results

[0171]

[0172] Results and Conclusions: For NDMA, 6 parallel sample solutions were prepared, and the measured content showed a recovery rate RSD of 3.50%; for dimethyl sulfate, 6 parallel sample solutions were prepared, and the measured content showed a recovery rate RSD of 7.45%; for NDPA, 6 parallel sample solutions were prepared, and the measured content showed a recovery rate RSD of 4.63%. All results met the validation requirements.

[0173] Example 8: Stability Study of the Detection Method for the Solution

[0174] 1. Experimental conditions are the same as in Example 1.

[0175] 2. Solution preparation:

[0176] The reference solution was prepared using the same method as in Example 1.

[0177] The spiked test solution was prepared according to the method described in Example 4R2-100% solution preparation.

[0178] Determination: Mass spectrometry was performed on the reference solution and the spiked test solution at different time points. The changes in the results obtained at different time points were compared with the initial results to examine the stability of the reference solution and the spiked test solution. The results are shown in the table below.

[0179] Table 19 Stability of NDMA Reference Standard

[0180]

[0181] Table 20 Stability of NDMA-Spiked Test Samples

[0182]

[0183] Table 21 Stability of Dimethyl Sulfate Reference Standard

[0184]

[0185] Table 22 Stability of Dimethyl Sulfate Spiked Test Samples

[0186]

[0187] Table 23 Stability of NDPA Reference Standard

[0188]

[0189] Table 24 Stability of NDPA-Spiked Test Samples

[0190]

[0191] Results and Conclusions: At room temperature, the NDMA reference standard was stable for 28.5 hours, with a maximum change of 5.60%; the spiked test sample was stable for 29 hours, with a maximum change of 9.07%. The dimethyl sulfate reference standard was stable for 28.5 hours, with a maximum change of 8.65%; the spiked test sample was stable for 29 hours, with a maximum change of 7.79%.

[0192] The NDPA reference standard was stable for 28.5 hours, with a maximum change of 7.98%; the spiked test sample was stable for 29 hours, with a maximum change of 7.48%. Both results met the validation requirements.

[0193] Comparative Example 1: Existing conventional detection methods

[0194] The experimental conditions are as described in Example 6 above. The diluents used were methanol and acetonitrile (moisture content 100-1000 ppm).

[0195] According to the mass spectrometry method in General Chapter 0431 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, methanol and acetonitrile were used as solvents to prepare the reference solution and the spiked test solution in the same way as in Example 6. The stability of the solution was investigated by injection at different times. The results showed that the dimethyl sulfate reference standard had poor stability in methanol and acetonitrile, and the peak area decreased significantly after 7.5 hours. The results of using methanol and acetonitrile as solvents are shown in the table below.

[0196] Table 25 Stability of methanol as a solvent reference standard

[0197] Time (h) Concentration ng / ml Change rate (%) 0h 724.2626 \ 2h 717.9580 0.87 5h 657.8916 9.16 7.5h 561.6128 22.46 8.5h 530.1981 26.79

[0198] Table 26 Stability of Acetonitrile as a Solvent Reference Standard

[0199] Time (h) Concentration ng / ml Change rate (%) 0h 782.1557 \ 2h 731.2223 6.51 5h 684.9732 12.42 7.5h 625.7669 19.99 8.5h 612.3789 21.71

[0200] As shown in the table above, when methanol and acetonitrile were used as solvents, the peak area of ​​dimethyl sulfate gradually decreased with continuous injection of the reference standard. At 7.5 hours, the changes in dimethyl sulfate concentration were 22.46% and 19.99%, respectively, far exceeding 10%, indicating poor solution stability when using ordinary methanol and acetonitrile as solvents. Furthermore, the pharmacopoeia detection method has poor specificity, and its precision and sensitivity are insufficient for the simultaneous detection of genotoxic impurities NDMA, NDPA, and dimethyl sulfate in rotigotine.

[0201] In summary, the above embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention. The technical features disclosed above are not limited to the combinations of the disclosed features with other features. Those skilled in the art can also make other combinations between the technical features according to the purpose of the invention to achieve the purpose of the present invention. Various improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit of the present invention should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for detecting genotoxic impurities, characterized in that, The method includes the following steps: The sample is dissolved in an anhydrous or ultra-dry organic solvent containing formic acid, wherein the water content of the organic solvent is ≤50ppm; The detection was performed using gas chromatography-mass spectrometry (GC-MS), and the chromatographic conditions were as follows: Separation is performed using a capillary column, with either cyanopropylphenyl-dimethylpolysiloxane or diphenyl-dimethylpolysiloxane as the stationary phase. The temperature program is as follows: start at 50-70℃ and hold for 1-3 minutes, then increase to 250-280℃ at a rate of 20-40℃ / min and hold for 1-3 minutes. Injector temperature 250-280℃, split ratio 5:1-15:1, column flow rate 1.2-1.8 ml / min; The mass spectrometry conditions are as follows: EI source, MRM mode selected. The genotoxic impurities mentioned are NDMA, NDPA, and dimethyl sulfate. The sample mentioned therein is a pharmaceutical composition containing a nitrosamine or sulfate ester structure.

2. The method according to claim 1, characterized in that, The anhydrous or ultra-dry organic solvent is acetonitrile, and the concentration of formic acid is 0.05%-0.5%.

3. The method according to claim 1, characterized in that, The concentration of the test sample is 5-20 mg / ml.

4. The method according to claim 1, characterized in that, The stationary phase of the capillary column is selected from any of the following: 6% Cyanopropylphenyl-94% Dimethylpolysiloxane; 35% diphenyl-65% dimethylpolysiloxane; 5% diphenyl-95% dimethylpolysiloxane; 50% diphenyl-50% dimethyl polysiloxane.

5. The method according to claim 1, characterized in that, The column conditions were: injection port temperature 250-280℃, split ratio 5:1, column flow rate 1.5 ml / min, initial column temperature 60℃, and programmed temperature rise conditions: start at 60℃ and hold for 1 min, then increase to 260℃ at a rate of 30℃ / min and hold for 1 min. Alternatively, the column conditions are: injection port temperature 260℃, split ratio 5:1-15:1, column flow rate 1.5 ml / min, initial column temperature 60℃, and column temperature program conditions: start at 60℃ and hold for 1 min, increase to 260℃ at a rate of 30℃ / min, and hold for 3 min. Alternatively, the column conditions are: injection port temperature 260℃, split ratio 5:1, column flow rate 1.2-1.8, initial column temperature 60℃, and column temperature program conditions: start at 60℃ and hold for 1 min, then increase to 260℃ at a rate of 30℃ / min and hold for 1 min. Alternatively, the column conditions are: injection port temperature 260℃, split ratio 5:1, column flow rate 1.5 ml / min, initial column temperature 50-70℃, and programmed column temperature as follows: start at 60℃ and hold for 1 min, then increase to 260℃ at a rate of 30℃ / min and hold for 1 min.

6. The method according to claim 1, characterized in that, The mass spectrometry conditions also include the following MRM ion pairs: The quantitative ion pair for NDMA was m / z 74→44 with a collision energy of 5 eV; the qualitative ion pair was m / z 74→28 with a collision energy of 10 eV. The quantitative ion pair for NDPA was m / z 130→113 with a collision energy of 5 eV; the qualitative ion pair was m / z 130→88 with a collision energy of 5 eV. The quantitative ion pair for dimethyl sulfate was m / z 125→95 with a collision energy of 5 eV; the qualitative ion pair was m / z 125→79 with a collision energy of 10 eV. The MRM residence time was 150 ms, the solvent delay time was 3 min, and the mass spectrometry mode was low resolution mode.

7. The method according to claim 2, characterized in that, The concentration of formic acid is 0.1%-0.2%.

8. The method according to claim 3, characterized in that, The concentration of the test sample is 8-12 mg / ml.

9. The method according to claim 1, characterized in that, The method further includes dissolving the reference solution and the test solution in 0.1% formic acid anhydrous acetonitrile to maintain the detection system in a weakly acidic environment.

10. The method according to claim 1, characterized in that, The method is used for the quality control of genotoxic impurities in rotigotine raw materials or preparations.

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