Method for detecting genotoxic impurities in tofacitinib citrate starting material by gas chromatography-mass spectrometry
Through gas phase mass spectrometry combined with external standard method, the problem of detecting the content of genotoxic impurities bromoacetaldehyde and bromoacetaldehyde diethanol in the drug was successfully solved, achieving high sensitivity and high accuracy detection effects, and meeting the strict requirements of drug quality control.
Patent Information
- Application Number
- CN202410737866.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-06-07
- Publication Date
- 2025-02-28
AI Technical Summary
There is a lack of effective detection methods in the prior art to determine the content of the genotoxic impurities bromoacetaldehyde and bromoacetaldehyde diethanol in the drug, especially when the drug itself has a large amount of impurities and is relatively complex.
The peak area was measured by the standard limit of known concentrations of bromoacetaldehyde and bromoacetaldehyde diethanol control solution, and the correction factor between the peak area and the control concentration was determined. Then, the peak area of the test sample solution was measured under the same detection conditions, and the content of bromoacetaldehyde and bromoacetaldehyde diethanol was calculated according to the external standard method.
High sensitivity and high accuracy detection of bromine acetaldehyde and bromine acetaldehyde diethanol was achieved. The detection limit and quantification limit were significantly lower than the standard requirements, the method was strong in specificity, the sample was simple to handle, and it was not disturbed by blank solvents.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of drug analysis, and relates to a method for detecting the content of genotoxic impurities bromoacetaldehyde and bromoacetaldehyde diethanol acetal in a drug. The detection method adopts gas phase-mass spectrometry, adopts bromoacetaldehyde and bromoacetaldehyde diethanol acetal reference substance solutions with known concentrations of standard limits to respectively measure the peak areas, determine the correction factor between the peak area and the reference substance concentration, and then measure the peak area of the test solution under the same detection conditions, and calculate the content of bromoacetaldehyde and bromoacetaldehyde diethanol acetal in the test sample by the peak area according to the external standard method. Background Art
[0002] Bromoacetaldehyde is an important organic synthesis industrial raw material with a molecular formula of C2H3BrO and a molecular weight of 121.95. It can be used as a pharmaceutical chemical synthesis intermediate and a pharmaceutical chemical synthesis intermediate, and is mainly used in laboratory research and development processes and chemical production processes. Bromoacetaldehyde is a fatty aldehyde compound, which belongs to the genotoxic impurity warning structure and has potential genotoxicity.
[0003] Bromoacetaldehyde diethyl acetal is an important raw material for organic synthesis industry, with the molecular formula of C6H 13 BrO2, with a molecular weight of 197.07, is widely used in the fields of pharmaceuticals and chemicals. Bromoacetaldehyde diethanol acetal is a skin irritant and can cause poisoning if swallowed or inhaled. Moreover, bromoacetaldehyde diethanol acetal fatty halogen compounds belong to the genotoxic impurity warning structure and have potential genotoxicity.
[0004] According to the ICH M7 Draft consensus guideline assessment and control of DNAreactive (Mutagenic) impurities in pharmaceuticals to limit potentialcarcinogenic risk, quality control should be more stringent for drugs that may contain this genotoxic impurity.
[0005] During the preparation of the starting material compound B of the starting material TF101Ts, a variety of potential genotoxic impurities with warning structures are introduced, including possible bromoacetaldehyde and bromoacetaldehyde diethanol acetal. However, there is almost no detection method for the content of bromoacetaldehyde and bromoacetaldehyde diethanol acetal in drugs in the prior art. In view of the fact that the drug itself contains a large number of impurities and is relatively complex, and the content of genotoxic impurities is relatively low, it is necessary to establish a highly specific and sensitive analytical method for the detection of the content of genotoxic impurities bromoacetaldehyde and bromoacetaldehyde diethanol acetal. Summary of the invention
[0006] The invention provides a method for determining the contents of bromoacetaldehyde and bromoacetaldehyde diethyl acetal by gas phase mass spectrometry. The method is not interfered by a blank solvent, and bromoacetaldehyde and bromoacetaldehyde diethyl acetal do not interfere with each other in detection. The method has simple sample processing, strong specificity, and high sensitivity and accuracy.
[0007] In order to achieve the above-mentioned purpose, the technical solution adopted by the present invention is as follows: a method for detecting genotoxic impurities in tofacitinib citrate starting materials by gas phase mass spectrometry, wherein the impurities are bromoacetaldehyde and bromoacetaldehyde diethyl acetal, and the specific structural formula is as follows Figure Ⅰ and Ⅱ As shown: ; .
[0008] Furthermore, the method described herein uses a chromatographic column whose filler is 6% cyanopropylphenyl-94% polydimethylsiloxane (or similar polarity), and the initial column temperature is 35-45°C, maintained for 3-10 minutes, and then heated to 200°C at a rate of 20°C per minute and maintained for 3-10 minutes. Preferably, the initial column temperature is 40°C, maintained for 5 minutes, and then heated to 200°C at a rate of 20°C per minute and maintained for 3 minutes.
[0009] Furthermore, in the method described, the chromatographic column injection port temperature is 175°C to 185°C, preferably 178°C to 182°C.
[0010] Furthermore, in the method described, the initial column temperature of the chromatographic column is 38°C to 42°C, preferably 39°C to 42°C.
[0011] Furthermore, in the method described, the column flow rate temperature of the chromatographic column is 1.40 mL / min to 1.60 mL / min, preferably 1.45 mL / min to 1.55 mL / min.
[0012] Furthermore, in the method described, the split ratio is 9:1 to 11:1, preferably 9:1 to 10.5:1.
[0013] Furthermore, in the method described above, the detector ion source temperature is 200°C to 220°C, preferably 210°C.
[0014] Furthermore, when the impurities bromoacetaldehyde and bromoacetaldehyde diethyl acetal are quantitatively determined, the impurity external standard method is used for calculation.
[0015] The peak areas of bromoacetaldehyde and bromoacetaldehyde diethyl acetal reference solutions with known concentrations within the standard limits are measured respectively, and the correction factor between the peak area and the reference concentration is determined. Then, the peak area of the test solution is measured under the same detection conditions, and the content of bromoacetaldehyde and bromoacetaldehyde diethyl acetal in the test sample is calculated by the peak area according to the external standard method.
[0016] Furthermore, in the method described, the concentration of bromoacetaldehyde in the reference solution is in the range of 0.04487 to 0.67301 μg / mL (i.e., 8.97 to 134.60 ppm), and the concentration of bromoacetaldehyde diethyl acetal is in the range of 0.04726 to 0.70894 μg / mL (i.e., 9.45 to 141.79 ppm).
[0017] Furthermore, in the method described above, when quantitatively determining the impurities bromoacetaldehyde and bromoacetaldehyde diethanol acetal, the control limit is that in the quantitative limit solution, the signal-to-noise ratio of the bromoacetaldehyde and bromoacetaldehyde diethanol acetal peaks shall not be less than 10; and the RSD of the peak area shall not exceed 15.0%.
[0018] The invention has the advantages that: 1. the sample processing is simple; the blank solution has no interference on the determination of bromoacetaldehyde and bromoacetaldehyde diethanol acetal; in the reference solution, the retention time of bromoacetaldehyde is 7.400 minutes, the retention time of bromoacetaldehyde diethanol acetal is 12.233 minutes, and the retention time of bromoacetaldehyde and bromoacetaldehyde diethanol acetal is consistent with the retention time in the reference solution; the separation degree between bromoacetaldehyde and bromoacetaldehyde diethanol acetal meets the requirements, and the detection method has good specificity.
[0019] 2. The detection limit of bromoacetaldehyde in this method is 0.04375μg / mL, i.e. 8.75ppm, and the quantification limit is 0.13460μg / mL, i.e. 26.92ppm; the detection limit of bromoacetaldehyde diethanol acetal is 0.04630μg / mL, i.e. 9.26ppm, and the quantification limit is 0.14179μg / mL, i.e. 28.36ppm. The detection sensitivity is high, which is far lower than the standard requirement that the content of bromoacetaldehyde and bromoacetaldehyde diethanol acetal should not exceed 93.75ppm. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 It is the test spectrum of the spiked test solution; Figure 2 This is the quantitative limit detection spectrum of bromoacetaldehyde and bromoacetaldehyde diethyl acetal; Figure 3 It is the detection limit detection spectrum of bromoacetaldehyde and bromoacetaldehyde diethyl acetal; Figure 4 Bromoacetaldehyde linear graph; Figure 5 Linear graph of bromoacetaldehyde diethyl acetal. Specific implementation plan
[0021] The present invention is further described below through various embodiments, but these embodiments do not limit the protection scope of the present invention.
[0022] Example 1: Separation of bromoacetaldehyde, bromoacetaldehyde diethyl acetal and TF101Ts (starting material 4-chloro-7-tosyl-7H-pyrrolo[2,3-d]pyrimidine)
[0023] Chromatographic conditions: a capillary column with 6% cyanopropylphenyl-94% dimethylpolysiloxane (or similar polarity) as the stationary phase; the initial column temperature is 40°C, maintained for 5 minutes, then increased to 200°C at a rate of 20°C per minute and maintained for 3 minutes; the injection port temperature is 180°C; the split ratio is 10:1; the carrier gas is helium; the column flow rate is 1.5 mL per minute; the injection volume is 1 μl.
[0024] The detector is a single quadrupole; the mode is SIM; the ion source is an EI source; the ion source temperature is 210°C; the interface temperature is 210°C; the solvent delay is 4.0 min; the acquisition time is 4.0-16.0 min; the m / z of 4.0-11.0 min is 94; the m / z of 11.0-16.0 min is 103.
[0025] 1.1 System suitability test and specificity test
[0026] (1) Solution preparation:
[0027] Blank solution (diluent): dichloromethane.
[0028] Mixed reference substance stock solution: Take about 58.5 mg of bromoacetaldehyde reference substance, accurately weigh it, put it in a 25 mL volumetric flask, quantitatively dilute it to the mark with dichloromethane, shake well, and obtain the bromoacetaldehyde reference substance stock solution (concentration is 2.34 mg / mL); take about 117 mg of bromoacetaldehyde diethyl acetal reference substance, accurately weigh it, put it in a 50 mL volumetric flask, quantitatively dilute it to the mark with dichloromethane, shake well, and obtain the reference substance stock solution (concentration is 2.34 mg / mL). Accurately measure 1.0 mL of each of the above two reference substance stock solutions, put them in a 50 mL volumetric flask, quantitatively dilute them to the mark with dichloromethane, shake well, and obtain the mixed reference substance stock solution. (Concentration is 46.8 mg / mL).
[0029] Reference substance solution: Accurately measure 1.0 mL of each of the above mixed reference substance stock solutions, place in a 100 mL volumetric flask, quantitatively dilute to scale with dichloromethane, shake well, and obtain (concentration is 0.468 mg / mL).
[0030] Test solution: Take 0.125g of TF101Ts test sample, weigh accurately, place in a 25mL volumetric flask, add dichloromethane to dissolve and dilute to the scale, shake well, and obtain (concentration is 5mg / mL).
[0031] Spiked test solution: Take about 0.125g of the product, weigh accurately, put it in a 25mL volumetric flask, add the reference solution to dissolve and dilute to the scale, shake well, and obtain.
[0032] (2) Sampling: Analyze the blank solution, test solution, mixed reference solution, and spiked test solution once each, and analyze the reference solution 5 times. Record the chromatograms. The specific test results are shown in Tables 1, 2 and Figure 1 : ; .
[0033] Test results: The blank solution has no interference with the determination of bromoacetaldehyde and bromoacetaldehyde diethanol acetal; the theoretical plate numbers of bromoacetaldehyde and bromoacetaldehyde diethanol acetal are 150638 and 588858 respectively; in the reference solution, the retention time of bromoacetaldehyde is 7.400min, and the retention time of bromoacetaldehyde diethanol acetal is 12.233min, and the separation between bromoacetaldehyde and bromoacetaldehyde diethanol acetal meets the requirements; the retention time of bromoacetaldehyde and bromoacetaldehyde diethanol acetal is consistent with the retention time in the reference solution. The RSD of the bromoacetaldehyde peak area obtained by continuous analysis of the reference solution for 5 times is 4.2% (≤10.0%), and the RSD of the retention time is 0.02% (≤1.0%); the RSD of the bromoacetaldehyde diethanol acetal peak area is 6.3% (≤10.0%), and the RSD of the retention time is 0.02% (≤1.0%), which proves that the system applicability and specificity of this method are good.
[0034] 1.2. Sensitivity (detection limit, quantification limit) of bromoacetaldehyde and bromoacetaldehyde diethyl acetal
[0035] Detection limit solution (10%): Accurately measure 1.0 mL of the reference solution, place it in a 10 mL volumetric flask, dilute to the mark with dichloromethane, and shake well.
[0036] Quantitative limit solution (30%): Accurately measure 3.0 mL of the reference solution, place it in a 10 mL volumetric bottle, dilute to the mark with dichloromethane, and shake well. The test results are shown in Tables 3, 4, 5 and Figure 2 , Figure 3 : ; ; .
[0037] The test results show that in the detection limit solution, the signal-to-noise ratio of the bromoacetaldehyde peak is 12.4 (≥3); the signal-to-noise ratio of the bromoacetaldehyde diethanol acetal peak is 99.4 (≥3), which meets the requirements of the verification scheme. According to the test results in Table 15, in the 6 quantitative limit solutions, the signal-to-noise ratio of the bromoacetaldehyde peak is within 27.2-31.6 (≥10), and the peak area RSD is 5.3% (≤15.0%); the signal-to-noise ratio of the bromoacetaldehyde diethanol acetal peak is within 82.8-127.9 (≥10), and the peak area RSD is 4.1% (≤15.0%), which meets the requirements of the verification scheme. The results show that the detection limit of bromoacetaldehyde in this method is 0.04375 μg / mL, i.e. 8.75 ppm, and the quantification limit is 0.13460 μg / mL, i.e. 26.92 ppm; the detection limit of bromoacetaldehyde diethyl acetal is 0.04630 μg / mL, i.e. 9.26 ppm, and the quantification limit is 0.14179 μg / mL, i.e. 28.36 ppm.
[0038] Embodiment 2-9
[0039] The chromatographic condition parameters remain unchanged, and the following parameters are adjusted: (1) Chromatographic parameters Based on Example 1, the flow rates were adjusted to 1.45 mL / min and 1.55 mL / min respectively; (2) Chromatographic parameters Based on Example 1, the initial column temperatures were adjusted to 39°C and 42°C respectively; (3) Chromatographic parameters Based on Example 1, the injection port temperature was adjusted to 178°C and 182°C respectively; (4) Chromatographic parameters Based on Example 1, the split ratios were adjusted to 9:1 and 10.5:1, respectively.
[0040] Under the above chromatographic conditions, under each durability condition, the blank solution was analyzed once, the reference solution 1 was analyzed 5 times, the reference solution 2 was analyzed twice, the test solution was analyzed once, and the test spiked solution was analyzed once. The specific test results are shown in Table 6: .
[0041] The test results show that when the chromatographic conditions of this method change slightly, the recovery rates of bromoacetaldehyde and bromoacetaldehyde diethyl acetal should be between 85% and 110%. The data results show that this method has good durability.
[0042] Example 10 Quantitative determination of bromoacetaldehyde and bromoacetaldehyde diethyl acetal content
[0043] The chromatographic condition parameters remain unchanged and are the same as in Example 1.
[0044] (1) Linearity and range of bromoacetaldehyde and bromoacetaldehyde diethyl acetal
[0045] Accurately measure 5 mL of the mixed reference substance stock solution, place it in a 50 mL volumetric flask, quantitatively dilute it to the mark with dichloromethane, shake well, and obtain the mixed reference substance stock solution II (concentration is 4.68 mg / mL). Accurately measure an appropriate amount of the mixed reference substance stock solution II, quantitatively dilute it to the mark with dichloromethane, and prepare 10%, 30%, 50%, 100%, 120%, 150% linear solutions. The test results are shown in Tables 7 and 8: ; .
[0046] The experimental results show that: According to Table 7 and Figure 4 The test results show that the linear regression equation of bromoacetaldehyde is y=7,808.8689 x - 48.3004, the correlation coefficient is 0.9937 (r≥0.990), the ratio of the Y-axis intercept to the peak area of bromoacetaldehyde at 100% concentration is 0.01 (≤0.25), and the linear relationship is good in the range of bromoacetaldehyde concentration of 0.04487~0.67301μg / mL (i.e. 8.97~134.60ppm); according to Table 8 and Figure 5 The test results show that the linear regression equation of bromoacetaldehyde acetal is y=24,367.1749 x + 586.4687, the correlation coefficient is 0.9934 (r≥0.990), the ratio of the Y-axis intercept to the peak area of bromoacetaldehyde acetal at 100% concentration is 0.05 (≤0.25), and the linear relationship is good in the range of bromoacetaldehyde acetal concentration of 0.04726~0.70894μg / mL (i.e. 9.45~141.79ppm).
[0047] (2) Calculation of bromoacetaldehyde and bromoacetaldehyde diethyl acetal content
[0048] 2.1. Solution preparation
[0049] Blank solution (diluent): dichloromethane.
[0050] Mixed reference substance stock solution: Take about 58.5 mg of bromoacetaldehyde reference substance, accurately weigh it, place it in a 25 mL volumetric flask, quantitatively dilute it to the mark with dichloromethane, shake well, and you will get the bromoacetaldehyde reference substance stock solution (concentration is 2.34 mg / mL); take about 117 mg of bromoacetaldehyde diethyl acetal reference substance, accurately weigh it, place it in a 50 mL volumetric flask, quantitatively dilute it to the mark with dichloromethane, shake well, and you will get the bromoacetaldehyde diethyl acetal reference substance stock solution (concentration is 2.34 mg / mL). Accurately measure 1.0 mL of each of the above two reference substance stock solutions, place them in a 50 mL volumetric flask, quantitatively dilute them to the mark with dichloromethane, shake well, and you will get the mixed reference substance stock solution. (Concentration is 46.8 mg / mL)
[0051] Reference substance solution: Accurately measure 1.0 mL of each mixed reference substance stock solution, place in a 100 mL volumetric flask, quantitatively dilute to scale with dichloromethane, shake well, and prepare 2 portions in parallel. (Concentration is 0.468 mg / mL)
[0052] Test solution: Take 0.125g of TF101Ts test sample, weigh accurately, place in a 25mL volumetric flask, add dichloromethane to dissolve and dilute to scale, shake well, and prepare 2 portions in parallel. (Concentration is 5mg / mL)
[0053] 2.2 Calculation formula
[0054] Calculate the content of bromoacetaldehyde and bromoacetaldehyde diethyl acetal in the test sample by peak area according to the external standard method:
[0055] Where: m1—the sample weight of bromoacetaldehyde and bromoacetaldehyde diethyl acetal reference substances, g; ρ—the content of bromoacetaldehyde and bromoacetaldehyde diethyl acetal reference substance, %; m2—the weight of the test sample, g; V1—the dilution volume of the reference solution, mL; V2—the dilution volume of the test solution, mL; A1—peak area of bromoacetaldehyde and bromoacetaldehyde diethyl acetal in the reference solution; A2—Peak area of bromoacetaldehyde and bromoacetaldehyde diethyl acetal in the test solution.
[0056] The content of bromoacetaldehyde and bromoacetaldehyde diethyl acetal in the tofacitinib citrate starting material TF101Ts should not exceed 93.75ppm. The test results are shown in Table 9: .
[0057] The experimental results show that bromoacetaldehyde and bromoacetaldehyde diethyl acetal were not detected in TF101Ts (batch number: S2020030102), TF101Ts (batch number: S2016110102), TF101Ts (batch number: R2017103002) and TF101Ts (batch number: S2019010101).
[0058] (3) Recovery rate of impurities bromoacetaldehyde and bromoacetaldehyde diethyl acetal
[0059] Take an appropriate amount of impurity bromoacetaldehyde and bromoacetaldehyde diethyl acetal reference substances, add them to the tofacitinib citrate starting material TF101Ts according to the limit of 30% to 150%, and calculate the recovery rate of impurities by subtracting the amount of impurities in the sample from the measured amount and the ratio of the amount of impurities added. The test results are shown in Tables 10 and 11: ; .
[0060] The test results show that the recovery rates of bromoacetaldehyde in 9 accuracy solutions are within 100.3% to 108.7% (all within 85% to 110%), and the RSD of the recovery rate is 2.9% (≤10.0%); the recovery rates of bromoacetaldehyde diethyl acetal are within 86.4% to 103.3% (all within 85% to 110%), and the RSD of the recovery rate is 7.0% (≤10.0%). This proves that the method has good accuracy.
[0061] Embodiment 11
[0062] The chromatographic parameters were the same as those in Example 1, except that the split ratio was adjusted to 11:1.
[0063] Example 12
[0064] The chromatographic parameters were the same as those in Example 1, except that the initial column temperature was changed to 38°C.
[0065] The results are shown in Tables 12 to 15. When the split ratio was adjusted to 11:1, the recovery rate of bromoacetaldehyde was 121.7% (not within 85% to 110%); the recovery rate of bromoacetaldehyde diethanol acetal was 124.0% (not within 85% to 110%). When the split ratio was changed to 11:1, the durability of this method would be relatively poor. When the initial column temperature was adjusted to 38°C, the recovery rate of bromoacetaldehyde was 111.9% (not within 85% to 110%); the recovery rate of bromoacetaldehyde diethanol acetal was 123.7% (not within 85% to 110%). When the initial column temperature was changed to 38°C, the durability of this method would be relatively poor: ; ; ; .
Claims
1. A method for detecting genotoxic impurities in tofacitinib citrate starting materials by gas phase mass spectrometry, characterized in that: The impurities are bromoacetaldehyde and bromoacetaldehyde diethyl acetal, and the specific structural formulas are shown in Figures Ⅰ and Ⅱ: ； 。 2. The method according to claim 1, characterized in that: Use a chromatographic column filled with 6% cyanopropylphenyl-94% polydimethylsiloxane (or similar polarity), start with a column temperature of 35-45°C, maintain for 3-10 minutes, increase the temperature to 200°C at a rate of 20°C per minute, and maintain for 3-10 minutes.
3. The method according to claim 1, characterized in that The temperature of the chromatographic column injection port is 175°C to 185°C.
4. The method according to claim 1, characterized in that: The initial column temperature of the chromatographic column is 38°C to 42°C.
5. The method according to claim 1, characterized in that: The column flow rate temperature of the chromatographic column is 1.40 ml / min to 1.60 ml / min.
6. The method according to claim 1, characterized in that The split ratio is 9:1 to 11:
1.
7. The method according to claim 1, characterized in that The detector ion source temperature is 200℃~220℃.
8. The method according to claim 1, characterized in that: The concentration of bromoacetaldehyde in the reference solution is in the range of 0.04487 to 0.67301 μg / ml, and the concentration of bromoacetaldehyde diethyl acetal is in the range of 0.04726 to 0.70894 μg / ml.
9. The method according to claim 1, characterized in that: When quantitatively determining the impurities bromoacetaldehyde and bromoacetaldehyde diethyl acetal, the impurity external standard method shall be used for calculation.
10. The method according to claim 9, characterized in that, when quantitatively determining the impurities bromoacetaldehyde and bromoacetaldehyde diethanol acetal, the control limit is that in the quantitative limit solution, the signal-to-noise ratio of the bromoacetaldehyde and bromoacetaldehyde diethanol acetal peaks shall not be less than 10; and the RSD of the peak area shall not exceed 15.0%.