Detection and analysis method of pinacol methyl borate used in crizotinib synthesis process

Abstract

The invention discloses a method for detecting and analyzing pinacol methyl borate used in a crizotinib synthesis process. The method is carried out in a gas chromatograph-headspace sampling manner, a capillary column taking cyanopropyl phenyl-dimethyl polysiloxane as a stationary liquid is adopted, nitrogen is taken as a carrier gas, and a hydrogen flame detector (FID) is taken as a detector. The pinacol methyl borate is easy to decompose and not easy to detect by directly adopting gas chromatography detection, and the method provided by the invention solves the problem that the pinacol methyl borate is difficult to detect by adopting gas chromatography, so that effective guarantee is provided for better controlling the quality of crizotinib, and clinical safety and effectiveness of crizotinib product medication are facilitated.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical chemicals, and more specifically to a detection and analysis method for methyl pinacol borate used in a crizotinib synthesis process. Background Art

[0002] Methyl pinacol borate (CAS RN: 1195-66-0), also known as methyl pinacol borate, has the chemical name of 2-methoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane and has the following structure:

[0003]

[0004] Methyl pinacol borate is an important organic reagent in the field of pharmaceutical synthesis. In this article, it participates in the synthesis reaction of crizotinib intermediate I, so the residual amount is controlled in intermediate I. The synthesis process of methyl pinacol borate participating in the reaction is as follows:

[0005]

[0006] Methyl pinacol borate has no obvious warning structure and no relevant toxicological data has been found, so according to ICHQ3A, the limit is set at 0.1%. Since the structure of methyl pinacol borate has no ultraviolet absorption and the boiling point is 120°C, it cannot be detected by HPLC; while using gas phase detection, methyl pinacol borate is prone to decomposition during gas phase detection, and there is no chromatographic peak when directly injected for detection, so a stable and reliable detection method for methyl pinacol borate is urgently needed in the prior art. Summary of the invention

[0007] The present invention aims to overcome the deficiencies of the prior art and provide a detection and analysis method for methyl pinacol borate used in a crizotinib synthesis process. The analysis method is performed on a gas chromatograph, using a 1 mol / L sodium hydroxide solution as a solvent to completely hydrolyze methyl pinacol borate into methanol and pinacol, using a headspace injection method, a DB624 chromatographic column, nitrogen as a carrier gas, and a headspace injection method FID as a detector for detection; by controlling the content of methanol, the control of methyl pinacol borate is achieved, and the conversion coefficient between methanol and methyl pinacol borate is 4.93.

[0008] In order to achieve the above technical objectives, the technical solution adopted by the present invention is:

[0009] The detection and analysis method of methyl pinacol borate used in the synthesis process of crizotinib adopts gas chromatography, and the chromatographic conditions are:

[0010] Preparation of the test solution: Take the crizotinib intermediate I test sample and use the diluent to prepare a 50-200 mg / ml solution, preferably 100-200 mg / ml, and most preferably 100 mg / ml; the methyl pinacol borate in the test sample is converted into pinacol and methanol in the above diluent, and the content of methanol is determined to achieve the purpose of determining the content of methyl pinacol borate.

[0011] Crizotinib intermediate I has the structure described below:

[0012]

[0013] Preparation of reference solution: Take methanol reference and dilute it with diluent to make a solution of 0.01mg-0.04mg / ml, preferably 0.02-0.04mg / ml, most preferably 0.02mg / ml;

[0014] The diluent is 0.8-1.2 mol / L sodium hydroxide solution;

[0015] Chromatographic column: capillary column, preferably a capillary column with 6% cyanopropylphenyl-dimethylpolysiloxane as the stationary liquid;

[0016] Carrier gas: nitrogen;

[0017] Inlet temperature: 195~205℃;

[0018] Detector (FID): 245~255℃;

[0019] Flow rate: 0.9ml~1.1ml per minute;

[0020] Initial column temperature: 35℃~45℃;

[0021] Injection method: headspace injection;

[0022] Headspace incubation temperature: 75-85°C;

[0023] Headspace equilibration time: 30 minutes;

[0024] The temperature was raised in the following manner:

[0025] 0~4min, column temperature 35~45℃;

[0026] 4-14 min, the column temperature is raised from 35-45°C to 200°C;

[0027] 14~19min, column temperature 200℃.

[0028] Further, the temperature is raised by the following method:

[0029] The initial temperature was 40°C, maintained for 4 minutes, then increased to 200°C at a heating rate of 20°C per minute and maintained for 5 minutes.

[0030] Furthermore, the diluent is 1 mol / L sodium hydroxide solution.

[0031] Furthermore, the specifications of the chromatographic column are 30m*0.32mm, 1.8μm.

[0032] Furthermore, the flow rate is 1 ml per minute.

[0033] Further, the headspace was incubated at 80°C for 30 minutes.

[0034] Furthermore, the injection port temperature is 200°C.

[0035] Furthermore, the detector temperature is 250°C.

[0036] Further, when the detection and analysis method is used for qualitative detection, if a methanol chromatographic peak appears, the test solution includes methyl pinacol borate; if no methanol chromatographic peak appears, the test solution does not contain methyl pinacol borate.

[0037] Further, when the detection and analysis method is used for quantitative detection, the content of methyl pinacol borate in the test solution is calculated as follows:

[0038]

[0039] Where:

[0040] W 对 :Weighing amount of reference substance

[0041] W 供 : Sample weight

[0042] A 对 : Peak area of ​​reference substance

[0043] A 供 : Methanol peak area in the test sample

[0044] C 对 : Purity of reference substance

[0045] V 供 : Dilution volume of test solution

[0046] V 对 : Dilution volume of reference solution

[0047] 4.93: Conversion factor between methanol and methyl pinacol borate.

[0048] In a specific embodiment, methyl pinacol borate is completely converted into methanol in 1 mol / L sodium hydroxide, and the conversion coefficient is 4.93.

[0049] The advantages of the present invention over the prior art are:

[0050] Methyl pinacol borate cannot be directly detected on liquid chromatography and gas chromatography. This method can decompose methyl pinacol borate into pinacol and methanol by using 1 mol / L sodium hydroxide solution, and control the residual amount of methanol to achieve the purpose of controlling methyl pinacol borate, which is simple and rapid. Gas chromatography is accurate and reliable in controlling methanol content, easy to implement, and is conducive to the control of the quality of crizotinib raw materials, thereby ensuring the safety of crizotinib clinical use.

[0051] The method provided by the present invention has been studied and verified through methodological studies on specificity, sensitivity, precision, etc. It is found that the method provided by the present invention is sensitive, accurate, and has good reproducibility, and can simply and quickly achieve qualitative and quantitative detection of methyl pinacol borate in crizotinib intermediate I.

[0052] This method has strong specificity and good durability. The instruments, equipment and reagents used are all conventional items, and the test parameters are also conventional parameters. There are no harsh conditions and it can be achieved in general laboratories. It is suitable for practical application and promotion and has high practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Blank solvent chromatogram under 2.1 in Example 3

[0054] Figure 2 The solvent chromatogram of the reference substance under 2.1 in Example 3

[0055] Figure 3 Chromatogram of methyl pinacol borate under item 2.1 in Example 3

[0056] Figure 4 Chromatogram of the test solution under 2.1 in Example 3

[0057] Figure 5 Chromatogram of the spiked test solution under 2.1 in Example 3

[0058] Figure 6 Chromatogram of 5-point reference solution in Comparative Example 1

[0059] Figure 7 Chromatogram of 5-point reference solution in Comparative Example 2 DETAILED DESCRIPTION

[0060] The present invention is further explained below with reference to the examples, but the examples do not limit the present invention in any form.

[0061] Embodiment 1:

[0062] (1) Solution preparation

[0063] Blank solution: 1 mol / L sodium hydroxide solution;

[0064] Methyl pinacol borate solution: Take an appropriate amount of methyl pinacol borate, weigh it accurately, add 1 mol / L sodium hydroxide solution to dissolve it and quantitatively dilute it to make a solution containing about 0.1 mg per 1 ml. Accurately measure 2 ml, place it in an empty bottle, and seal it.

[0065] Test solution: 0.2 g of crizotinib intermediate I, accurately weigh, accurately measure 2 ml of solvent, place an empty bottle on top, seal, and disperse by ultrasound.

[0066] Reference solution: Take an appropriate amount of methanol, weigh it accurately, add 1 mol / L sodium hydroxide solution to dissolve and quantitatively dilute to make a solution containing about 0.02 mg per 1 ml, accurately measure 2 ml, place it in an empty bottle, and seal it.

[0067] Spiked test solution: Take 0.2 g of crizotinib intermediate I, accurately weigh, accurately measure 2 ml of methyl pinacol borate solution, put an empty bottle on top, seal, and disperse by ultrasound.

[0068] (2) Detection method of methyl pinacol borate

[0069] Use a capillary column (DB624, 30m×0.32mm×1.8μm or a chromatographic column with similar polarity) with 6% cyanopropylphenyl-dimethylpolysiloxane as the stationary liquid; the starting temperature is 40°C, maintained for 2 minutes, then heated to 200°C at a rate of 20°C per minute, and maintained for 5 minutes; the injection port temperature is 200°C; the detector temperature is 250°C; the split ratio is 10:1; the headspace bottle equilibrium temperature is 80°C, and the equilibrium time is 30 minutes.

[0070] Example 2

[0071] Table 1 Test results of multiple batches of crizotinib intermediate I

[0072] batch Pinacol borate methyl ester content % 1 Not detected 2 Not detected 3 Not detected

[0073] Example 3

[0074] Method Validation:

[0075] 3.1 Exclusivity

[0076] Accurately measure the solvent, reference solution, methyl pinacol borate solution, and test solution, inject and analyze them respectively, and record the chromatograms. Figures 1 to 4 shown.

[0077] 3.2 Limit of detection and limit of quantification

[0078] Detection limit: The reference solution prepared in Example 1 was diluted step by step and injected into a gas chromatograph. The detection limit was obtained according to a signal-to-noise ratio of not less than 3:1. The results are shown in Table 2.

[0079] Limit of quantitation: The reference solution prepared in Example 1 was diluted step by step and injected into the gas chromatograph. The limit of quantitation was obtained according to a signal-to-noise ratio of not less than 10:1. The peak area RSD was repeated 6 times and was less than 10%. The results are shown in Table 3.

[0080] Table 2 Detection limit results

[0081]

[0082] Table 3 Quantitation limit results

[0083]

[0084]

[0085] The results showed that the detection limit concentration of methanol was 0.003 mg / ml, which was equivalent to 0.015 mg / ml of methyl pinacol borate, which was equivalent to a detectable concentration of 0.015% of the test sample.

[0086] The quantification limit of methanol is 0.006 mg / ml, which is equivalent to 0.03 mg / ml of methyl pinacol borate, which is equivalent to 0.03% of the test sample concentration. The quantification limit of 6 injections has good repeatability, which proves that this method has high sensitivity and meets the detection requirements of methyl pinacol borate in crizotinib intermediate I.

[0087] 3.3 Linearity and range

[0088] Accurately measure the quantitative limit concentration and linear solution, inject and analyze, record the chromatogram, and the linear results are shown in Table 4.

[0089] Table 4 Linearity test results

[0090]

[0091] The results show that methanol has a good linear relationship in the range of 0.006 mg / ml to 0.042 mg / ml (equivalent to methyl pinacol borate in the range of 0.03 mg / ml to 0.21 mg / ml), the linear equation is y=10.048x+0.021, r=0.996, which is greater than 0.995; the ratio of the intercept to the 100% concentration response value is 10%, which is no more than 10%, and the linearity is good.

[0092] 3.4 Precision

[0093] 3.4.1 Repeatability

[0094] Stock solution of methyl pinacol borate: take about 20 mg of methyl pinacol borate, accurately weigh it, place it in a 20 ml volumetric flask, dilute it to the mark with 1 mol / L sodium hydroxide solution, and shake well; accurately measure 5 ml, place it in a 50 ml volumetric flask, dilute it to the mark with 1 mol / L sodium hydroxide solution, and shake well.

[0095] Test sample solution: Take about 200 mg of the test sample and place it in a 20 ml headspace bottle. Add 2 ml of methyl pinacol borate stock solution to dissolve it. Seal it and prepare 6 portions in parallel.

[0096] 3.4.2 Intermediate precision

[0097] Take the same batch of this product and conduct experiments with the same method at different times, by different analysts, and on different instruments.

[0098] The above test solution was injected into a gas chromatograph, the chromatogram was recorded, and the content of methyl pinacol borate was calculated according to the above calculation method. The test results are shown in Table 5.

[0099] Table 5 Precision test results

[0100] name Content of methyl pinacol borate % Intermediate precision -1 0.092 Intermediate precision -2 0.081 Intermediate precision -3 0.089 Intermediate precision -4 0.093 Intermediate precision -5 0.087 Intermediate precision -6 0.094 RSD% (n=6) 5.4 Repeatability -1 0.089 Repeatability -2 0.088 Repeatability - 3 0.095 Repeatability - 4 0.086 Repeatability -5 0.082 Repeatability - 6 0.096 RSD% (n=6) 5.9 RSD% (n=12) 5.4

[0101] From the above table, we can know that the RSD of the content of methyl pinacol borate in 6 samples of each experimenter and a total of 12 samples is less than 10%, and the precision is good.

[0102] 3.5 Accuracy

[0103] Accuracy stock solution: Take about 50 mg of methyl pinacol borate, accurately weigh it, put it in a 50 ml volumetric flask, dilute it to the scale with 1 mol / L sodium hydroxide solution, shake well, and you have it.

[0104] 30% limit recovery solution: Accurately measure 3 ml of the accuracy stock solution, place it in a 100 ml volumetric flask, dilute to the mark with 1 mol / L sodium hydroxide, and shake well. Take 200 mg of the test sample, accurately weigh it, place it in a 20 ml headspace bottle, add 2 ml of the above solution to dissolve, seal it, and prepare 3 copies in parallel.

[0105] 50% limit recovery solution: Accurately measure 5 ml of the accuracy stock solution, place it in a 100 ml volumetric flask, dilute to the mark with 1 mol / L sodium hydroxide, and shake well. Take 200 mg of the test sample, accurately weigh it, place it in a 20 ml headspace bottle, add 2 ml of the above solution to dissolve, seal it, and prepare 3 copies in parallel.

[0106] 100% limit recovery solution: Accurately measure 5 ml of the accuracy stock solution, place it in a 50 ml volumetric bottle, dilute to the mark with 1 mol / L sodium hydroxide, and shake well. Take 200 mg of the test sample, accurately weigh it, place it in a 20 ml headspace bottle, add 2 ml of the above solution to dissolve, seal it, and prepare 3 copies in parallel.

[0107] 200% limit recovery solution: Accurately measure 5 ml of the accuracy stock solution, place it in a 25 ml volumetric bottle, dilute it to the mark with 1 mol / L sodium hydroxide, and shake it well. Take 200 mg of the test sample, accurately weigh it, place it in a 20 ml headspace bottle, add 2 ml of the above solution to dissolve, seal it, and prepare 3 copies in parallel.

[0108] The reference solution and each recovery rate solution were injected into the gas chromatograph and the chromatogram was recorded. The test results are shown in Table 6.

[0109] Recovery rate % = (measured amount - original amount) / added amount × 100%

[0110] Table 6 Recovery test results

[0111]

[0112] The results showed that the recoveries of methanol in the 12 accuracy solutions were all between 80% and 120%, and the RSD was no more than 10%, indicating that the accuracy of this method was good.

[0113] 3.6 Stability

[0114] Take the reference solution of Example 1, put it in an empty bottle, and seal it; prepare the test solution according to the repetitive solution preparation method as the solution stability solution. The test results are shown in Table 7.

[0115] Table 7 Stability test results

[0116] time Comparison time For testing 0h 0.278 0h 0.219 8h 0.253 3.5h 0.203 13h 0.235 8.5h 0.231 18h 0.25 13.5h 0.207 28h 0.225 23.5h 0.228 RSD% 8.1 RSD% 5.7

[0117] The results showed that the RSD of the peak area of ​​the reference solution was less than 10% when placed at room temperature for 28 hours, and the RSD of the peak area of ​​the test solution was less than 10% when placed at room temperature for 23.5 hours. The above solutions had good stability.

[0118] 3.7 Durability

[0119] Take the reference solution in Example 1, place it in an empty bottle, and seal it; prepare the test solution according to the repetitive solution preparation method, at a flow rate of ±0.1 ml / min and a starting temperature of ±5°C; take the above solution for sampling and analysis to examine its durability. The results are shown in Table 8.

[0120] Table 8 Durability test results

[0121] condition Content of methyl pinacol borate % Standard conditions 0.088

[0122] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for detecting and analyzing methyl pinacol borate used in the synthesis process of crizotinib, characterized in that: The method adopts gas chromatography, and the chromatographic conditions are: Preparation of test solution: Take the crizotinib intermediate I test sample and use the diluent to make a 50-200 mg / ml solution, preferably 100-200 mg / ml, most preferably 100 mg / ml; Preparation of reference solution: Take methanol reference and dilute it with diluent to make a solution of 0.01mg-0.04mg / ml, preferably 0.02-0.04mg / ml, most preferably 0.02mg / ml; The diluent is 0.8-1.2 mol / L sodium hydroxide solution; Chromatographic column: capillary column, preferably a capillary column with 6% cyanopropylphenyl-dimethylpolysiloxane as the stationary liquid; Carrier gas: nitrogen; Inlet temperature: 195~205℃; Detector (FID): 245~255℃; Flow rate: 0.9ml~1.1ml per minute; Initial column temperature: 35℃~45℃; Injection method: headspace injection; Headspace incubation temperature: 75-85°C; Headspace equilibration time: 30 minutes; The temperature was raised in the following manner: 0~4min, column temperature 35~45℃; 4-14 min, the column temperature is raised from 35-45°C to 200°C; 14~19min, column temperature 200℃.

2. The detection and analysis method of methyl pinacol borate used in the crizotinib synthesis process according to claim 1, characterized in that: The temperature was raised in the following manner: The initial temperature was 40°C, maintained for 4 minutes, then increased to 200°C at a heating rate of 20°C per minute and maintained for 5 minutes.

3. The detection and analysis method of methyl pinacol borate used in the crizotinib synthesis process according to claim 1, characterized in that: The diluent is 1 mol / L sodium hydroxide solution.

4. The detection and analysis method of methyl pinacol borate used in the crizotinib process according to claim 1, characterized in that: The specifications of the chromatographic column are 30m*0.32mm, 1.8μm.

5. The detection and analysis method of methyl pinacol borate used in the crizotinib synthesis process according to claim 1, characterized in that: The flow rate is 1 ml per minute.

6. The detection and analysis method of methyl pinacol borate used in the crizotinib synthesis process according to claim 1, characterized in that: Incubate at 80°C headspace for 30 minutes.

7. The detection and analysis method of methyl pinacol borate used in the crizotinib synthesis process according to claim 1, characterized in that: The injection port temperature was 200°C.

8. The detection and analysis method of methyl pinacol borate used in the crizotinib synthesis process according to claim 1, characterized in that: Detector temperature 250°C.

9. The detection and analysis method of methyl pinacol borate used in the crizotinib synthesis process according to claim 1, characterized in that: When the detection and analysis method is used for qualitative detection, if a methanol chromatographic peak appears, the test solution includes methyl pinacol borate; if no methanol chromatographic peak appears, the test solution does not contain methyl pinacol borate.

10. The detection and analysis method of methyl pinacol borate used in the crizotinib synthesis process according to claim 1, characterized in that: When the detection and analysis method is used for quantitative detection, the content of methyl pinacol borate in the test solution is calculated as follows: Where: W 对 :Weighing amount of reference substance W 供 : Sample weight A 对 : Peak area of ​​reference substance A 供 : Methanol peak area in the test sample C 对 : Purity of reference substance V 供 : Dilution volume of test solution V 对 : Dilution volume of reference solution 4.93: Conversion factor between methanol and methyl pinacol borate.

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