A method for detecting an erlotinib-related impurity and use thereof

Abstract

The present application relates to the field of pharmaceutical analysis, in particular to a method for detecting erlotinib related impurities and application thereof. The method adopts high performance liquid chromatography-mass spectrometry, the chromatographic column of the high performance liquid chromatography uses high-purity silica gel as the filler, and the mass spectrometry ion source is ESI source. The method provided by the present application realizes effective separation and detection of extremely low content of erlotinib related impurities. The method has the advantages of good specificity, high sensitivity and the like, realizes simultaneous detection of multiple erlotinib impurities, and has extremely low detection limit.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical analysis, in particular to a method for detecting related impurities of erlotinib and application thereof. BACKGROUND

[0002] Erlotinib is a kinase inhibitor, and is suitable for the treatment of patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) with sensitive mutations in the epidermal growth factor receptor (EGFR) gene. It is an important treatment drug for non-small cell lung cancer in clinical practice.

[0003]

[0004] During the production process, starting materials, intermediates, polymers, by-products, degradation products during storage, etc. may be brought into the final product as related substances and affect product quality. Erlotinib starting materials, intermediates, degradation products, process impurities, etc. may contain genetic warning structures, and their content should be strictly controlled. Therefore, developing a more sensitive and more scientific and reasonable method for detecting erlotinib impurities is of great significance for ensuring drug quality, safety and effectiveness, and thus realizing controllable drug quality.

[0005] CN107655983A discloses a method for detecting triethylamine and its potential genotoxic impurities by liquid chromatography. The detection sensitivity of 3-bromoaniline content is about 0.0022%, but the simultaneous detection of multiple erlotinib related impurities is not yet met, and the detection sensitivity needs to be further improved.

[0006] Therefore, the present application provides a method for detecting related impurities of erlotinib, which can improve the detection sensitivity and efficiency, facilitate the quality control of erlotinib, and thus improve the safety of clinical medication. SUMMARY

[0007] One of the purposes of the present application is to provide a method for detecting related impurities of erlotinib by high performance liquid chromatography-mass spectrometry.

[0008] In a preferred technical solution of the present application, the impurities are selected from any one or combination of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F or a pharmaceutically acceptable salt thereof.

[0009]

[0010] In a preferred embodiment of the present invention, the pharmaceutically acceptable salt is selected from any one or a combination of p-toluenesulfonate, methanesulfonate, sulfonate, benzoate, hydrochloride, hydrobromide, sulfate, phosphate, nitrate, tartrate, fumarate, maleate, citrate, formate, acetate, succinate, malonate, malate, and cinnamate.

[0011] In a preferred embodiment of the present invention, the chromatographic column of the high-performance liquid chromatography method uses high-purity silica gel as the packing material.

[0012] In a preferred embodiment of the present invention, the packing material of the chromatographic column for high-performance liquid chromatography is compatible with a high proportion of aqueous mobile phase.

[0013] In a preferred embodiment of the present invention, the column packing material for the high-performance liquid chromatography method is compatible with a 100% aqueous mobile phase.

[0014] In a preferred embodiment of the present invention, the chromatographic column has any one of the following specifications: 50*2.1mm, 100*2.1mm, 100*4.6mm, 150*3mm, 150*4.6mm, 250*3mm, and 250*4.6mm.

[0015] In a preferred embodiment of the present invention, the particle size of the chromatographic column packing is any one of 1.7μm, 1.8μm, 2μm, 2.1μm, 3μm, 3.5μm or 5μm.

[0016] In a preferred embodiment of the present invention, the mobile phase A of the high-performance liquid chromatography is a 0.01-0.2% formic acid solution, and the mobile phase B is acetonitrile, with gradient elution.

[0017] In a preferred embodiment of the present invention, the mobile phase A of the high-performance liquid chromatography is a 0.05-0.15% formic acid solution.

[0018] In a preferred embodiment of the present invention, the elution gradient of the high-performance liquid chromatography is:

[0019] 0min: The volume percentage of mobile phase A to mobile phase B is 70-80:20-30;

[0020] 12 min: The volume percentage of mobile phase A to mobile phase B is 50-70:30-50;

[0021] 14 min: The volume percentage of mobile phase A to mobile phase B is 15-25:75-85.

[0022] In a preferred embodiment of the present invention, the elution gradient, at 14 min, has a volume percentage of mobile phase A: mobile phase B of 15-25:75-85, and is maintained for 4-10 min.

[0023] In a preferred embodiment of the present invention, after the elution gradient detection is completed, elution is continued for 5-20 minutes with a mobile phase in which the volume percentage of mobile phase A to mobile phase B is 70-80:20-30.

[0024] In a preferred embodiment of the present invention, the column temperature of the high-performance liquid chromatography is 25-40℃.

[0025] In a preferred embodiment of the present invention, the flow rate of the high-performance liquid chromatography is 0.1-0.8 ml / min.

[0026] In a preferred embodiment of the present invention, the flow rate of the high-performance liquid chromatography is 0.2-0.5 ml / min.

[0027] In a preferred embodiment of the present invention, the injection volume of the high-performance liquid chromatography is 1-20 μl.

[0028] In a preferred embodiment of the present invention, the injection volume of the high performance liquid chromatography is 1-10 μl.

[0029] In a preferred embodiment of the present invention, the column temperature of the high-performance liquid chromatography is 25-50℃.

[0030] In a preferred embodiment of the present invention, the nebulization temperature of the mass spectrometer is 200-500℃.

[0031] In a preferred embodiment of the present invention, the temperature of the nebulizing gas of the mass spectrometer is 250-350°C.

[0032] In a preferred embodiment of the present invention, the nebulizing gas of the mass spectrometer is nitrogen.

[0033] In a preferred embodiment of the invention, the gas flow rate of the mass spectrometer is 2-10 L / min.

[0034] In the preferred embodiment of the invention, the gas flow rate of the mass spectrometer is 4-8 L / min.

[0035] In a preferred embodiment of the present invention, the mass spectrometry method is in positive ion mode (ESI+).

[0036] In a preferred embodiment of the present invention, the mass spectrometry detection ion pair is (m / z)

[0037] 298→58.9、313.1→197、118.1→73.9、402→304.1、398.1→276.

[0038] In a preferred embodiment of the invention, the gas flow rate of the mass spectrometer is 2-10 L / min.

[0039] In the preferred embodiment of the invention, the gas flow rate of the mass spectrometer is 4-8 L / min.

[0040] In a preferred embodiment of the present invention, the analyte, test sample, and reference standard are dissolved or diluted with a dissolving solvent, wherein the dissolving solvent is selected from any one or a combination of acetonitrile-water solution, acetonitrile-isopropanol, water, methanol, acetonitrile, and methanol-water solution.

[0041] In a preferred embodiment of the present invention, the dissolving solvent is a 40-60% acetonitrile-water solution, preferably a 50% acetonitrile-water solution.

[0042] Another object of the present invention is to provide the application of the high performance liquid chromatography method of the present invention for the detection of erlotinib-related impurities in the quality control of erlotinib raw material and / or its preparations.

[0043] In a preferred embodiment of the present invention, the erlotinib formulation is selected from erlotinib solid dosage form or liquid dosage form.

[0044] In a preferred embodiment of the present invention, the erlotinib solid dosage form is selected from any one of tablets, capsules, powders, granules, dry suspensions, and microcapsules.

[0045] In a preferred embodiment of the present invention, the erlotinib liquid formulation is selected from any one of oral liquid, syrup, injection, spray, and suspension.

[0046] Unless otherwise stated, when this invention relates to percentages between liquids, the percentage is volume / volume percentage; when this invention relates to percentages between liquids and solids, the percentage is volume / weight percentage; when this invention relates to percentages between solids and liquids, the percentage is weight / volume percentage; the remainder is weight / weight percentage.

[0047] Compared with the prior art, the present invention has the following beneficial technical effects:

[0048] 1. This invention achieves the effective separation and detection of genotoxic impurities in erlotinib at extremely low concentrations by scientifically screening and optimizing the detection conditions of high-performance liquid chromatography, including optimizing chromatographic conditions and gradient elution conditions. It can be used for monitoring genotoxic impurities in erlotinib raw materials and formulations.

[0049] 2. The high-performance liquid chromatography method of this invention has the advantages of high specificity and high sensitivity, enabling the simultaneous detection of multiple impurities in erlotinib with extremely low detection limits. This method is beneficial for the quality control of erlotinib raw materials and preparations, ensuring drug quality and safety. It is also simple and easy to implement, reducing production and testing costs, and is suitable for the requirements of industrialized and large-scale production. Attached Figure Description

[0050] Appendix Figure 1 Mass spectrum of impurity A;

[0051] Appendix Figure 2 Mass spectrum of impurity B;

[0052] Appendix Figure 3 Mass spectrum of impurity C;

[0053] Appendix Figure 4 Mass spectrum of impurity D;

[0054] Appendix Figure 5 Mass spectrum of impurity E;

[0055] Appendix Figure 6 Mass spectrum of impurity F;

[0056] Appendix Figure 7 Example 1: Blank solution diagram;

[0057] Appendix Figure 8 Figure 1 of the reference solution in Example 1;

[0058] Appendix Figure 9 Example 1: Sample solution diagram;

[0059] Appendix Figure 10 Example 4: Linear graph of impurity B;

[0060] Appendix Figure 11 Example 4: Linear graph of impurity C;

[0061] Appendix Figure 12 Example 4: Linear graph of impurity A;

[0062] Appendix Figure 13 Example 4: Linear graph of impurity D;

[0063] Appendix Figure 14 Example 5: Linear graph of impurity E;

[0064] Appendix Figure 15 Example 4: Linear graph of impurity F;

[0065] Appendix Figure 16 Chromatogram of Comparative Example 1;

[0066] Appendix Figure 17 The chromatogram of Comparative Example 2 obtained using column 1;

[0067] Appendix Figure 18 The chromatogram of Comparative Example 2 obtained using column 2. Detailed Implementation

[0068] The following specific embodiments illustrate the present invention. It should be noted that these specific embodiments are only for further explanation and do not represent a limitation on the scope of protection of the present invention. Non-essential modifications and adjustments made by others based on the present invention still fall within the scope of protection of the present invention.

[0069] In this specific implementation, the detection method follows high-performance liquid chromatography-mass spectrometry (General Chapters 0512 and 0431 of the 2020 edition of the Chinese Pharmacopoeia). The content calculation formula is as follows:

[0070]

[0071]

[0072] In the formula:

[0073] m 对 : Sample weight of the reference standard, mg; A 样 Peak area of ​​the test sample solution;

[0074] D 样 : Dilution factor of the test solution; A 对 Peak area of ​​the reference solution;

[0075] m 样 : Sample weight, mg; D 对 The dilution factor of the reference solution;

[0076] f: Response value; Average response value; P: content of reference standard

[0077] X: Response value (f) or content value; RD: Relative deviation, %;

[0078] Example 1 The detection method of the present invention

[0079] (1) Solution preparation (can be prepared at the same concentration or in the same proportion)

[0080] Mobile phase A: Measure 1.0 ml of formic acid, place it in 1000 ml of filtered water, mix well, and degas by sonication for 10 min to obtain the mobile phase A;

[0081] Mobile phase B: Take 1000 ml of acetonitrile, degas it by sonication for 10 min, and you will get the mobile phase B.

[0082] Diluent: Take 500ml of acetonitrile and 500ml of water, mix well, and sonicate to degas for 10 minutes to obtain the diluent;

[0083] Stock solution of impurity A: Weigh approximately 15 mg of impurity A reference standard, place it in a 25 ml volumetric flask, dissolve and dilute to the mark with diluent, shake well, take 1 ml and place it in a 50 ml volumetric flask, dilute to the mark with diluent, shake well, and the solution is ready. Prepare stock solutions of impurities B, C, and D hydrochloride in duplicate using the same method.

[0084] Impurity E stock solution: Weigh about 10 mg of impurity E hydrochloride reference standard, place it in a 25 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and prepare two parallel solutions.

[0085] Impurity F stock solution: Weigh about 20 mg of impurity F hydrochloride reference standard, place it in a 25 ml volumetric flask, add diluent to dissolve and dilute to the mark, and shake well; then measure 2 ml of the solution, place it in a 20 ml volumetric flask, add diluent to dilute to the mark, and prepare two parallel solutions.

[0086] Reference stock solution: Accurately measure 1 ml of each impurity stock solution, place it in a 100 ml volumetric flask, dilute to the mark with diluent, shake well, and prepare two parallel portions.

[0087] Reference solution: Accurately measure 5 ml of the reference stock solution, place it in a 100 ml volumetric flask, dilute to the mark with diluent, shake well, and prepare two parallel portions.

[0088] Test solution: Prepare fresh before use. Weigh approximately 40 mg of the test sample (erlotinib) accurately, place it in a 20 ml volumetric flask, add diluent to dissolve and dilute to the mark, and shake well.

[0089] (2) High performance liquid chromatography and mass spectrometry conditions

[0090] High performance liquid chromatography conditions:

[0091] Column: Waters ACQUITY UPLC HSS T3 (2.1*100mm, 1.8μm or equivalent column);

[0092] Mobile phase A: 0.1% formic acid solution; Mobile phase B: acetonitrile;

[0093] The flow rate was 0.2 ml / min; the column temperature was 30℃; the injection volume was 3 μl; gradient elution was performed according to Table 1:

[0094] Table 1

[0095] Time (min) 0 12 14 18 18.5 30 A(％) 75 60 20 20 75 75 B(％) 25 40 80 80 25 25

[0096] Mass spectrometry conditions: Triple quadrupole mass spectrometer detector, electrospray ionization positive ion mode (ESI+), multiple reaction monitoring (MRM), mass-to-charge ratios (m / z) of 298→58.9, 313.1→197, 118.1→73.9, 402→304.1, and 398.1→276 were selected as detection ion pairs, nebulizer gas temperature was 250℃, and other flow rates were 7 L / min; Timetable: 0 min, LC to MS; 6 min, LC to Washe; 9.5 min, LC to MS; The elution order and detection ion pairs are shown in Table 2.

[0097] Table 2

[0098]

[0099] (3) Determination: Inject the test solution and the reference solution into the liquid chromatography-mass spectrometry instrument and record the chromatograms;

[0100] (4) Precision test: Accurately measure 3 μl of the reference solution and inject it into the liquid chromatography-mass spectrometry instrument. Inject the sample 6 times consecutively, record the chromatogram, and examine the peak area RSD. The results are shown in the table below;

[0101] Sample injection precision test results

[0102] Impurity B Impurity A Impurity C Impurity D Impurity F Impurity E 1 4340 4851 9098 7815 43790 181433 2 4407.28 4966.81 8772.29 7925.34 45506.08 183618.33 3 4185.44 4967.96 9448.26 8095.5 45685.97 184186.11 4 4406.03 4913.73 9462.74 7953.43 45266.26 183387.11 5 4014.62 4800.75 8921.48 7861.79 44770.35 182119.66 6 3536.89 4545.82 8884.62 7599.62 41908.6 166608.48 RSD (%) 8.1 3.3 3.3 2.1 3.3 3.8

[0103] Conclusion: After six consecutive injections of the reference solution, the RSD values ​​of the peak areas of each impurity were all less than 10.0%, indicating that the injection precision of the system is good.

[0104] Example 2 Accuracy test of the detection method of the present invention

[0105] The diluent, reference solution, and test solution are the same as in Example 1;

[0106] 100% spiked test solution: Weigh approximately 40 mg of the test sample accurately, place it in a 20 ml volumetric flask, add an appropriate amount of solvent to dissolve it, accurately add 1.0 ml of the control stock solution, dilute to the mark with solvent, shake well, and you will get the solution. Prepare 6 parallel solutions.

[0107] Accurately measure 3 μl each of the diluent, reference solution, test solution, and 100% spiked test solution, and inject them into the liquid chromatography-mass spectrometry (LC-MS) instrument. Record the chromatograms. The results are shown in the table below.

[0108] Accuracy test results of impurity B

[0109]

[0110]

[0111] Accuracy test results of impurity C

[0112]

[0113] Accuracy test results of impurity A

[0114]

[0115] Accuracy test results of impurity D

[0116]

[0117] Accuracy test results of impurity F

[0118]

[0119] Accuracy test results of impurity E

[0120]

[0121] Conclusion: The recoveries of each impurity in the six spiked test solutions ranged from 60.0% to 140.0%, and the RSD values ​​of the recoveries were all less than 10.0%, indicating that the method has good accuracy.

[0122] Example 3 The detection limit (LOD) and quantitation limit (LOQ) of the detection method of this invention.

[0123] Limit of Quantitation Solution: Accurately measure 0.5 ml of the reference standard stock solution, place it in a 100 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution;

[0124] Detection limit solution: Accurately measure 1 ml of the reference solution, place it in a 20 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution;

[0125] Accurately measure 3 μl of each of the limit of quantitation solution and limit of detection solution, inject them into the liquid chromatography-mass spectrometry instrument, record the chromatograms, and the results are shown in the table below;

[0126] Limit of Quantification Test Results

[0127]

[0128] Detection limit test results

[0129]

[0130]

[0131] Conclusion: In the limit of quantitation solution, the S / N ratio of each impurity is greater than 10, and the concentration is less than 30% of the corresponding limit concentration, which can meet the quantitative detection requirements of each impurity; in the limit of detection solution, the S / N ratio of each impurity is greater than 3, and the concentration is less than 20% of the corresponding limit concentration, which can meet the detection requirements of each impurity.

[0132] Example 4 Linearity test of the detection method of the present invention

[0133] The preparation method for the reference standard stock solution is the same as in Example 1;

[0134] Linear stock solution: Take the reference stock solution and dilute it according to the table below;

[0135] Concentration equivalent to limit (%) Volume taken (ml) Dilution volume (ml) 1 1000 10 20 2 200 10 100 3 150 7.5 100 4 100 5 100 5 50 2.5 100 6 30 1.5 100 LOQ 10 0.5 100

[0136] Accurately measure 3 μl of each linear solution, inject it into the liquid chromatography-mass spectrometry instrument, record the chromatogram, and the results are as follows;

[0137] Impurity B linearity

[0138]

[0139]

[0140] impurity C linear

[0141]

[0142] Impurity A linear

[0143]

[0144] Impurity D linearity

[0145]

[0146]

[0147] Impurity E linearity

[0148]

[0149] Impurity F linearity

[0150]

[0151] Conclusion: For the above-mentioned impurities, within the corresponding limit concentration range of approximately 10% to 1000%, the linear relationship between concentration and peak area is good.

[0152] Comparative Example 1 HPLC-UV method for the detection of impurity AF in erlotinib

[0153] Chromatographic column: Agilent SB-C8 (4.6*250mm, 5μm); flow rate: 1.0ml / min; column temperature: 30℃; wavelength: 238nm; injection volume: 20μl;

[0154] Mobile phase A: 0.05 mol / L potassium dihydrogen phosphate (pH 6.7) - acetonitrile - methanol (70:30:5)

[0155] Mobile phase B: 0.05 mol / L potassium dihydrogen phosphate (pH 6.7) - acetonitrile - methanol (35:65:5)

[0156] Washing procedure:

[0157] Time (min) 0 22 23 35 40 60 61 70 Mobile phase A 100 100 70 70 30 30 100 100 Mobile phase B 0 0 30 30 70 70 0 0

[0158] The solution preparation method is as described in Example 1; the reference solution and test solution were prepared using this method, and the chromatograms of the reference solution and test solution are shown in the appendix. Figure 16 While meeting the detection requirements for each impurity, the concentration of the test solution is approximately 15 mg / ml, and the unknown impurity peaks interfere with the detection of known impurities, resulting in poor specificity.

[0159] Comparative Example 2 Detection of impurity AF in erlotinib using different chromatographic columns

[0160] The solution preparation method is the same as in Example 1;

[0161] The injection volume was 2 μl; the flow rate was 0.5 ml / min; the column temperature was 30 ℃; and the wavelength was 238 nm.

[0162] Mobile phase A: 0.1% formic acid in water; Mobile phase B: acetonitrile;

[0163] Column 1: Thermo Acclaim Polar Advatage II (3mm*150mm, 3μm);

[0164] Column 2: ES Epic C18 (2.1mm*100mm, 1.8μm);

[0165] The elution procedure is shown in Table 1;

[0166] Table 1

[0167] Time (min) 0 7 7.5 9.5 10 14 Mobile phase A 85 50 20 20 85 85 Mobile phase B 15 50 80 80 15 15

[0168] Mass spectrometry conditions:

[0169] Triple quadrupole mass spectrometer detector, electrospray ionization positive ion mode (ESI+), multiple reaction monitoring (MRM), nebulizer gas temperature 250℃, other flow rates 7 L / min. Detected ion pairs are shown in Table 2:

[0170] Table 2

[0171] Compound Parent ion Fragment ion Fragmentor (V) CE (V) Impurity B 298 58.9 140 12 Impurity A 313.1 197 140 40 Impurity C 118.1 73.9 120 60 Impurity D 402 304.1 180 40 Impurity E 398.1 276 180 40 Impurity F 398.1 276 180 40

[0172] The solution preparation method is the same as in Example 1; detection was performed using chromatographic column 1 and chromatographic column 2 under the conditions described above, and the results are shown in the appendix. Figures 17-18 Neither column 1 nor column 2 could effectively separate impurity F and impurity E.

Claims

1. A method for detecting impurities related to Erlotinib by high performance liquid chromatography-mass spectrometry, characterized in that, The impurities are impurity A, impurity C, impurity D, impurity E and impurity F. ， The high performance liquid chromatography mobile phase A is 0.01-0.2% formic acid solution, and the mobile phase B is acetonitrile, and gradient elution is performed. The chromatographic column is a Water ACQUITY UPLC HSS T3; 2.1*100mm, 1.8um. The gradient elution process is as follows: ； The mass spectrometry conditions are: triple quadrupole mass spectrometry detector, electrospray positive ion mode (ESI+), multiple reaction monitoring (MRM), atomization gas temperature 250°C, gas flow rate 7L / min; timetable: 0min, LC TO MS; 6min, LC TO WASTE; 9.5min, LC TO MS. 。 2. The method of claim 1, wherein, The chromatographic column filler of the high performance liquid chromatography is compatible with the high proportion of aqueous mobile phase.

3. The method of claim 2, wherein, The chromatographic column filler of the high performance liquid chromatography is compatible with the 100% aqueous solution mobile phase.

4. The method of claim 1, wherein, The high performance liquid chromatography mobile phase A is 0.05-0.15% formic acid solution.

5. The method of claim 1, wherein, The column temperature of the high performance liquid chromatography is 25-50°C.

6. The method of claim 1, wherein, The column temperature of the high performance liquid chromatography is 25-40°C.

7. The method of claim 1, wherein, The flow rate of the high performance liquid chromatography is 0.1-0.8ml / min.

8. The method of claim 1, wherein, The flow rate of the high performance liquid chromatography is 0.2-0.5ml / min.

9. The method according to claim 7 or 8, characterized in that, The injection amount of the high performance liquid chromatography is 1-20μl.

10. The method according to claim 7 or 8, characterized in that, The injection amount of the high performance liquid chromatography is 1-10μl.

11. The method of claim 1, wherein, The atomization gas of the mass spectrometry is nitrogen.

12. The method of claim 1, wherein, The test substance, the test sample and the control sample are dissolved or diluted with a dissolving solvent selected from any one or a combination of acetonitrile-water solution, acetonitrile-isopropanol, water, methanol, acetonitrile, methanol-water solution.

13. The method of claim 12, wherein, The dissolving solvent is 40-60% acetonitrile-water solution.

14. The method of any one of claims 1-13 for use in the quality control of erlotinib bulk drug and / or its preparation.

15. Use according to claim 14, characterized in that, The erlotinib preparation is selected from an erlotinib solid preparation or an erlotinib liquid preparation.

16. The use according to claim 15, characterized in that, The erlotinib solid preparation is selected from any one of tablets, capsules, powders, granules, dry suspensions, pellets.

17. The use according to claim 15, characterized in that, The erlotinib liquid preparation is selected from any one of oral liquids, syrups, injections, sprays, suspensions.

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