Method for determining genotoxic impurities in crisaborole bulk drug

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and relates to a method for detecting genotoxic impurities in a crisaborole raw material by gas chromatography-mass spectrometry, the detection method adopts the gas chromatography-mass spectrometry, the detected genotoxic impurities are 2-bromo-5-(4-cyanophenoxy) benzyl acetate, the sample treatment is simple, the specificity is strong, the detection sensitivity is high, and the method is suitable for industrial production. And the detection quantity in a sample is far lower than the requirement of the limit (not greater than 0.0625 ppm) of 2-bromo-5-(4-cyanophenoxy) benzyl acetate.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical chemistry and analysis, and particularly relates to a method for determining genotoxic impurities in a crisaborole raw material drug. Background Art

[0002] Atopic dermatitis is a common, recurring, chronic inflammatory skin disease characterized by a chronic rash characterized by inflammation and itching, often occurring in skin folds. Symptoms typically persist for more than 14 days. An estimated 25 million people in the United States suffer from atopic dermatitis (eczema), with infants and children accounting for 8% to 18%. However, a significant number of people with mild to moderate atopic dermatitis lack safe and effective topical treatments.

[0003] Crisaborole is a phosphodiesterase 4 (PDE-4) inhibitor, primarily used for the topical treatment of mild to moderate atopic dermatitis in patients 3 months of age and older. Its primary mechanism of action is phosphodiesterase 4 (PDE-4). Inhibitors prevent the conversion of cyclic adenosine monophosphate (cAMP) to adenosine monophosphate (AMP), thereby increasing intracellular cAMP levels and suppressing inflammation.

[0004] Benzyl 2-bromo-5-(4-cyanophenoxy)acetate is an important intermediate and residual impurity in the production process of crisaborole. Its structural formula is as follows:

[0005]

[0006] Analysis of this structure revealed a genotoxicity warning structure. According to the ICH M7 Draft consensus guideline assessment and control of DNA-reactive (Mutagenic) impurities in pharmaceuticals to limit potential carcinogenic risk, drugs containing this genotoxic impurity must be subject to strict quality control. Based on the maximum daily dose of the crisaborole API, the limit of this impurity is as low as 0.0625 ppm. Conventional liquid phase-related detection methods cannot meet the detection limit and sensitivity requirements. Currently, there are no reports on related genotoxic impurities and their control methods. Therefore, there is an urgent need to develop relevant technologies to strictly control crisaborole genotoxic impurities and achieve the purpose of quality control. Summary of the Invention

[0007] The purpose of the present invention is to establish a method for analyzing genotoxic impurities in crisaborole raw materials. The method has higher sensitivity and stronger specificity, can control the genotoxic impurities in crisaborole raw materials, and ensure the quality of the final product.

[0008] The complete technical solution of the present invention is:

[0009] A method for detecting genotoxic impurity I in crisaborole raw materials by gas chromatography-mass spectrometry, wherein impurity I is 2-bromo-5-(4-cyanophenoxy)benzyl acetate, and the specific structural formula is shown in Formula I:

[0010]

[0011] Furthermore, the method described above uses a chromatographic column filled with (5%-phenyl)-methylpolysiloxane, with an initial column temperature of 40°C to 45°C, maintained for 0 to 2 minutes, and then heated to 220 to 260°C at a rate of 5 to 15°C per minute and maintained for 1 to 4 minutes.

[0012] Benzyl 2-bromo-5-(4-cyanophenoxy)acetate is a polar compound. During the research process, it was unexpectedly discovered that the use of a chromatographic column filled with (5%-phenyl)-methylpolysiloxane has a significant separation advantage for this impurity, and the ideal peak elution effect can be achieved through programmed temperature increase.

[0013] Moreover, the limit of this impurity is as low as 0.0625 ppm, calculated based on the maximum daily dose of the crisaborole raw material. Conventional liquid phase-related detection methods cannot meet the requirements of detection limit and sensitivity. By using gas chromatography-mass spectrometry and relevant method research, on the one hand, the sensitivity of the method was improved, and the detection limit was increased from 0.1590 ppm to 0.0117 ppm; on the other hand, the operation was simplified, and there was no need for the preparation of mobile phase and diluent, and the analysis time was shortened to 25 minutes, which saved time in sample configuration, instrument balance and method duration, and reduced detection costs.

[0014] Furthermore, in the method described, the temperature of the chromatographic column injection port is 270-290°C.

[0015] Furthermore, in the method described, the flow rate temperature of the chromatographic column is 1.40 ml / min to 1.60 ml / min.

[0016] Furthermore, in the method described, the split ratio is 9:1 to 11:1.

[0017] Furthermore, in the method described above, the detector ion source temperature is 220°C to 240°C.

[0018] Furthermore, the method described above is used to quantitatively determine genotoxic impurities by calculating using the peak area external standard method.

[0019] Furthermore, when the method is used to quantitatively determine genotoxic impurities, the control limit is that the signal-to-noise ratio in the quantitative limit solution must not be less than 10:1.

[0020] The beneficial effects brought about by the technical solution of the present invention are:

[0021] Due to the use of the above-mentioned analytical method, the present invention has good separation, strong specificity, high sensitivity and good accuracy, and can control the genotoxic impurity 2-bromo-5-(4-cyanophenoxy)acetic acid benzyl ester in the crisaborole raw material to ensure the quality of the final product. At the same time, it can also provide a useful reference and basis for the detection of other impurities with extremely low limits in crisaborole. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The quantitative limit solution chromatogram in Example 2;

[0023] Figure 2 This is the chromatogram of the quantitative limit solution in Comparative Example 1; DETAILED DESCRIPTION

[0024] The present invention will be further described by the following examples, however, the present invention is not limited to the following examples, and these examples do not limit the scope of the present invention in any way. Certain changes and adjustments made by those skilled in the art within the scope of the claims should also be considered to fall within the scope of the present invention.

[0025] The following examples all use the following reagents, instruments and detection methods:

[0026] Acetonitrile: mass spectrometry grade

[0027] Instrument: Agilent 8890GC+5977MS+7697HS Gas Chromatography / Mass Spectrometry

[0028] Chromatographic conditions:

[0029] Chromatographic column: (5% phenyl)-methyl polysiloxane as filler (HP-5MS 30m×0.25mm, 0.25μm or equivalent performance column);

[0030] Column flow rate: 1.0 ml per minute;

[0031] Inlet temperature: 280°C;

[0032] Transmission line temperature: 280°C;

[0033] The split ratio is 10:1;

[0034] Program temperature rise: start column temperature 40℃, hold for 0 min; increase to 240℃ at 10℃ / min, hold for 2 min.

[0035] Collection time: 25 min;

[0036] Detector: mass spectrometer;

[0037] Carrier gas: nitrogen;

[0038] Scan mode: Scan;

[0039] Scan range: 35-500;

[0040] Ion source: EI;

[0041] Ion source temperature: 230°C;

[0042] Quadrupole temperature: 150°C;

[0043] Injection volume: 1 μl.

[0044] Determination method: Accurately measure the test solution and reference solution, inject them into gas chromatography-mass spectrometry, and record the mass spectrum. Calculate the impurity I content by the peak area external standard method. The calculation formula is as follows

[0045]

[0046] Where: A 供试品 : impurity peak area in the test solution;

[0047] A 对照品 : main peak area in reference solution;

[0048] C 对照品 : The concentration of the reference solution, in mg / ml;

[0049] C 供试品 : The concentration of the test solution, in mg / ml.

[0050] Example 1 System suitability and specificity test

[0051] (1) Solution preparation:

[0052] Blank solution: acetonitrile

[0053] Reference substance stock solution: Accurately weigh 4.856 mg of impurity I reference substance, place it in a 100 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and obtain.

[0054] Reference substance mother solution: Accurately measure 2.5 ml of reference substance stock solution, place it in a 200 ml volumetric flask, add acetonitrile to dilute to the scale, shake well, and obtain.

[0055] Reference substance solution: Accurately measure 1.0 ml of the reference substance mother solution, place it in a 10 ml volumetric flask, dilute to the scale with acetonitrile, and shake well.

[0056] Test solution: Accurately weigh 497.43 mg of the test sample (crisiborol API) into a 10 ml volumetric flask, dissolve it in acetonitrile and dilute to the mark, shake well, and obtain the solution.

[0057] Spiked test solution: Accurately weigh 509.80 mg of the test sample and place it in a 10 ml volumetric flask. Accurately add 1.0 ml of the reference sample stock solution, dissolve it in acetonitrile and dilute to the scale, shake well, and obtain the solution.

[0058] (2) Injection: blank solution, test solution, and reference solution were analyzed 7 times and the chromatograms were recorded. The specific test results are shown in Tables 1, 2, and Figure 1 .

[0059] Table 1 System suitability test results

[0060]

[0061] Table 2 Specificity test results

[0062] solution Retention time min Peak area Separation Theoretical plates Reference solution 18.071 10222 / /

[0063] (3) Test results:

[0064] The system suitability requirements were met, and other components in the blank solution and the test solution had no interference at the peak position of impurity I; the reference solution was injected continuously for 7 times, and the peak area RSD was 17.51%, less than 20.0%, and the relative retention time RSD was 0.01%, less than 1.0%.

[0065] Example 2 Linearity and Range

[0066] (1) Solution preparation

[0067] Linear stock solution: Accurately measure 2.5 ml of the reference stock solution, place it in a 200 ml volumetric flask, add acetonitrile to dilute to the scale, shake well, and obtain.

[0068] Linearity solution: Accurately measure an appropriate amount of linearity stock solution and quantitatively dilute it to the scale with acetonitrile according to Table 3 to prepare 20%, 40%, 80%, 100%, 120%, and 200% linearity solutions.

[0069] Table 3 Linear solution configuration

[0070] Concentration level Dilution multiple Concentration (μg / ml) 20％(L1) 1ml linear stock solution to 50ml 0.0117 40％(L2) 2ml linear stock solution to 50ml 0.0234 80％(L3) 2ml linear stock solution to 25ml 0.0467 100％(L4) 1ml linear stock solution to 10ml 0.0584 120％(L5) 3ml linear stock solution to 25ml 0.0701 200％(L6) 2ml linear stock solution to 10ml 0.1168

[0071] (2) Sample injection: The chromatographic condition parameters remain unchanged and are the same as in Example 1. The test results are shown in Table 4.

[0072] Table 4 Summary of linear results

[0073]

[0074] (3) Results:

[0075] The peak area of ​​impurity I has a good linear relationship with the concentration in the concentration range of 0.0117-0.1168 μg / ml, and the linear equation is y=122286x+361.95, R 2 The value of the intercept is 0.9993 and the intercept deviation is 0.049, which meets the requirements.

[0076] The lowest concentration of the linear solution in the linear test, which is the quantitative line of the impurity, is 0.0117 ppm. Figure 1 .

[0077] Example 3 Accuracy

[0078] (1) Solution configuration:

[0079] Reference substance solution: Accurately measure 1.0 ml of the reference substance mother solution, place it in a 10 ml volumetric flask, dilute to the scale with acetonitrile, and shake well.

[0080] Test solution: Accurately weigh 512.11 mg of the test sample (crisiborol API) into a 10 ml volumetric flask, dissolve it in acetonitrile and dilute to the mark, shake well, and obtain the solution.

[0081] Spiked test solution 1: Accurately weigh 2.49238 g of the test sample, place it in a 50 ml volumetric flask, accurately add 1.0 ml of the reference sample stock solution, dissolve it in acetonitrile and dilute to the scale, shake well, and obtain.

[0082] Spiked test solution 2: Accurately weigh 509.80 mg of the test sample and place it in a 10 ml volumetric flask. Accurately add 1.0 ml of the reference sample stock solution, dissolve it in acetonitrile and dilute to the scale, shake well, and obtain the product.

[0083] (2) Sampling: Accurately measure each test solution and inject it into the gas chromatography-mass spectrometry instrument respectively. The results are shown in Tables 5 and 6.

[0084] Table 5 Calculation table of recovery rate of criborol impurity I-20%

[0085]

[0086] Table 6 Calculation table of 100% recovery rate of criborol impurity I

[0087]

[0088]

[0089] (3) Results:

[0090] The test results show that the accuracy of the solution recovery of impurity Ⅰ is 116.4% and 117.2%, both within 80% to 120%, proving that the method has good accuracy.

[0091] Example 4 Precision

[0092] (1) Solution preparation:

[0093] Blank solution: acetonitrile

[0094] Reference substance stock solution: Accurately weigh 4.856 mg of impurity I reference substance, place it in a 100 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and obtain.

[0095] Reference substance mother solution: Accurately measure 2.5 ml of reference substance stock solution, place it in a 200 ml volumetric flask, add acetonitrile to dilute to the scale, shake well, and obtain.

[0096] Reference substance solution: Accurately measure 1.0 ml of the reference substance mother solution, place it in a 10 ml volumetric flask, dilute to the scale with acetonitrile, and shake well.

[0097] Test solution: Accurately weigh 497.43 mg of the test sample (crisiborol API) into a 10 ml volumetric flask, dissolve it in acetonitrile and dilute to the mark, shake well, and obtain the solution.

[0098] Spiked test solution: Accurately weigh 509.80 mg of the test sample and place it in a 10 ml volumetric flask. Accurately add 1.0 ml of the reference sample stock solution, dissolve it in acetonitrile and dilute to the scale, shake well, and obtain the solution.

[0099] (2) Injection: Analyze the blank solution, test solution, and reference solution 7 times and record the chromatograms. The specific test results are shown in Table 7.

[0100] Table 7. Injection precision of crisoborole impurities

[0101]

[0102] (3) Results:

[0103] The RSD of the peak area of ​​impurity I was 17.51%, which was less than 20.0%, and the precision was good.

[0104] Comparative Example 1

[0105] Reagents and materials

[0106] Acetonitrile: chromatography grade

[0107] Trifluoroacetic acid: chromatography grade

[0108] Instruments and Equipment

[0109] High-performance liquid chromatography

[0110] Mettler XS205DU Analytical Balance

[0111] Mettler XP6 Analytical Balance

[0112] Testing conditions:

[0113] Chromatographic column: Pentafluorophenyl bonded silica gel as the filler (recommended column: Ultimate PFP, 250 × 4.6 mm, 5 μm or equivalent performance column)

[0114] Mobile phase A: 0.1% acetic acid in water; Mobile phase B: 0.1% acetic acid in acetonitrile

[0115] Flow rate: 1.0 ml / min;

[0116] Detection wavelength: 250nm;

[0117] Column temperature: 35°C;

[0118] Injection volume: 10 μl; diluent: acetonitrile

[0119] Perform gradient elution according to the following table:

[0120] Table 8 Gradient elution table

[0121] Time (min) Mobile phase A (%) Mobile phase B (%) 0 50 50 5 50 50 35 10 90 40 10 90 41 50 50 50 50 50

[0122] Limit of quantification and limit of detection

[0123] Blank solution: water: acetonitrile = 10:90 (same as diluent).

[0124] Limit of Quantitation Solution: Using the signal-to-noise ratio method, the concentration corresponding to a signal-to-noise ratio of 10:1 is used as the limit of quantitation solution for the analyte. Solution Preparation: Accurately weigh approximately 5 mg of impurity I into a 10 ml volumetric flask. Dissolve with an appropriate amount of acetonitrile, dilute to volume, and shake well. Accurately pipette 1 ml into a 50 ml volumetric flask, dissolve with an appropriate amount of acetonitrile, dilute to volume, and shake well. Accurately pipette 4 ml into a 250 ml volumetric flask, dissolve with an appropriate amount of acetonitrile, dilute to volume, and shake well to prepare a solution containing approximately 0.16 μg / ml of impurity I.

[0125] Detection Limit Solution: Using the signal-to-noise ratio method, the concentration corresponding to a signal-to-noise ratio of 3:1 is used as the detection limit solution for the analyte. Solution Preparation: Accurately measure 3 ml of the quantitation limit solution into a 10 ml volumetric flask. Dissolve it with an appropriate amount of acetonitrile, dilute to volume, and shake well to prepare a solution containing approximately 0.05 μg / ml of impurity I.

[0126] The results of the injection test are shown in Table 9, Table 10 and Figure 2 .

[0127] Table 9 Summary of quantification limits and detection limits

[0128]

[0129] Table 10 Quantitation limit precision results

[0130]

[0131] The results showed that the limit of quantification of impurity I in liquid chromatography-mass spectrometry was 0.1590 ppm, which failed to meet the required limit of 0.0625 ppm. The present invention used gas chromatography-mass spectrometry to reduce the limit of quantification of impurity I from 0.1590 ppm to 0.0117 ppm. This nearly 13-fold increase in the lower limit of detection significantly meets the detection requirements for genotoxic impurities, while also providing enhanced mass spectrometry specificity.

[0132] The specific embodiments of the present invention described above do not limit the scope of protection of the present invention. Any other corresponding changes and modifications made based on the technical concept of the present invention should be included in the scope of protection of the claims of the present invention.

Claims

1. A method for detecting genotoxic impurity I in crisaborole raw materials by gas chromatography-mass spectrometry, characterized in that: The impurity I is 2-bromo-5-(4-cyanophenoxy)benzyl acetate, and its specific structural formula is shown in Formula I:

2. The method according to claim 1, characterized in that A chromatographic column with (5%-phenyl)-methylpolysiloxane as filler was used, with an initial column temperature of 40°C to 45°C, maintained for 0 to 2 minutes, then heated to 220 to 260°C at a rate of 5 to 15°C per minute, and maintained for 1 to 4 minutes.

3. The method according to claim 1, characterized in that The temperature of the chromatographic column injection port is 270-290°C.

4. The method according to claim 1, wherein The column flow rate temperature of the chromatographic column is 1.40 ml / min to 1.60 ml / min.

5. The method according to claim 1, wherein The split ratio is 9:1 to 11:

1.

6. The method according to claim 1, characterized in that The detector ion source temperature is 220℃~240℃.

7. The method according to claim 1, characterized in that When quantitatively determining genotoxic impurities, calculate using the peak area external standard method.

8. The method according to claim 1, characterized in that When quantitatively determining genotoxic impurities, the control limit is the quantification limit solution, and the signal-to-noise ratio must not be less than 10:1.

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