Method for detecting cyclohexyl isocyanate in gliquidone raw material medicine

Abstract

The invention discloses a method for detecting cyclohexyl isocyanate in a gliquidone raw material medicine. Diethylamine is adopted for derivation, cyclohexyl isocyanate and diethylamine are subjected to a derivation reaction, and the residual quantity of the impurity cyclohexyl isocyanate in the gliquidone raw material medicine is detected through a liquid chromatography-mass spectrometry method. The method has high system applicability, has incomparable advantages in specificity, quantitation limit, detection limit, linear range and repeatability, and has high precision.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of chemical medicine analysis and detection, and particularly relates to a method for detecting cyclohexyl isocyanate in gliclazide raw material. BACKGROUND

[0002] Gliclazide is a second-generation oral sulfonylurea hypoglycemic drug, which can bind to specific receptors on the membrane of pancreatic beta cells, induce the production of appropriate insulin, and thus reduce blood glucose concentration, and is mainly used for anti-diabetes. The gliclazide raw material generally contains residual starting material impurity cyclohexyl isocyanate. The detection method of cyclohexyl isocyanate in gliclazide was studied, and it was found that the existing technology directly uses liquid chromatography-mass spectrometry or gas chromatography-mass spectrometry for detection, and the parent ion or stable daughter ion peak cannot be obtained. Meanwhile, cyclohexyl isocyanate is easily hydrolyzed, and the hydrolysis product of the product is relatively complex, so that the specific hydrolysis degree cannot be determined, thereby causing deviation of the detection result.

[0003] The structural formula of gliclazide and cyclohexyl isocyanate is as follows:

[0004]

[0005] The present application detects the residual amount of impurity cyclohexyl isocyanate in gliclazide raw material by derivatizing cyclohexyl isocyanate with diethylamine and using liquid chromatography-mass spectrometry. The peak shape is good, the specificity is strong, it is not interfered by other impurities, the method is reliable, and the specific residual value of the impurity can be calculated. SUMMARY

[0006] The purpose of the present application is to provide a liquid chromatography-mass spectrometry detection method for cyclohexyl isocyanate in gliclazide raw material, so that the specificity, limit of quantification, detection limit, linear range, repeatability, accuracy and other aspects completely meet the standards and have high precision.

[0007] To achieve the above-mentioned purpose, the present application provides the following technical scheme: a method for detecting residual cyclohexyl isocyanate in gliclazide raw material, which uses diethylamine for derivatization, so that cyclohexyl isocyanate reacts with diethylamine to generate a derivative with a molecular weight [M+H]+199.2, and the residual amount of impurity cyclohexyl isocyanate in gliclazide raw material is detected by liquid chromatography-mass spectrometry;

[0008] The reaction equation of cyclohexyl isocyanate and diethylamine is as follows:

[0009]

[0010] Specifically, the method for detecting cyclohexyl isocyanate in gliclazide raw material provided by the present application comprises the following steps:

[0011] 1) Preparation of raw material sample solution

[0012] Take gliclazide tablet raw material sample dissolved in acetonitrile, then add derivative solution dilution and make the sample completely dissolved, continue to shake, ultrasonic, sample precipitation after a period of time, shake, take the filtrate, get test solution;

[0013] 2) Preparation of control stock solution

[0014] Take the appropriate amount of impurity cyclohexyl isocyanate control sample, add acetonitrile and dilute to prepare about 800 ng per 1 mL solution, shake, as control stock solution;

[0015] 3) Preparation of control solution

[0016] Take the control stock solution in step 2) in a flask, add acetonitrile, and then dilute to the mark with derivative solution, shake, as control solution;

[0017] 4) Precision test solution and control solution, inject liquid chromatography-mass spectrometry, record chromatogram, using external standard method with peak area calculation test solution cyclohexyl isocyanate residual amount.

[0018] Specifically, the derivative in step 1), step 3) is diethylamine, the molar ratio of diethylamine to gliclazide in step 1) is at least 1.5.

[0019] The present application by cyclohexyl isocyanate and diethylamine derivative stable derivative product, and gliclazide structure containing sulfonamide group, weak acid, consume part of the basic diethylamine, so the diethylamine involved in the reaction needs to be excessive.

[0020] Specifically, the solvent of the derivative solution in step 1), step 3) is acetonitrile, and the preparation method of the derivative solution is: diethylamine is dissolved in acetonitrile, and the derivative solution is prepared to a concentration of at least 2.6 mg / mL.

[0021] Preferably, the concentration of the derivative solution is 3 mg / mL.

[0022] Specifically, the acetonitrile: derivative solution = 1:4 in step 1), step 3).

[0023] Specifically, in step 1), take about 100 mg of gliclazide tablet raw material sample in a 10 mL flask, add 2 mL acetonitrile, dilute to the mark with derivative solution, ultrasonic, make the sample completely dissolved, continue to shake, ultrasonic, sample precipitation, after about 50 min, shake, take the filtrate sample;

[0024] In the step 3), 1 mL of the control sample stock solution was taken into a 10 mL volumetric flask, 1 mL of acetonitrile was precisely added, and then the solution was diluted to the mark with the derivatization reagent solution, shaken well, and allowed to stand for about 50 min, and then shaken well to obtain a control sample solution. Specifically, the calculation formula in the step 4) is as follows:

[0025]

[0026] wherein,

[0027] Astd: average peak area of the analyte in the control sample solution

[0028] Cstd: concentration of the control sample solution (ng / ml)

[0029] As: peak area of the analyte in the sample solution

[0030] Cs: concentration of the sample solution (mg / ml)

[0031] Preferably, the detection conditions of the liquid chromatography are as follows:

[0032] Chromatographic column: Agilent Poroshell 120EC-C18, with a size of 4.6*50 mm and a particle size of 2.7 μm; mobile phase A: 0.1% formic acid aqueous solution;

[0033] mobile phase B: acetonitrile;

[0034] elution mode: gradient elution;

[0035] flow rate: 0.6 ml / min;

[0036] injection volume: 2 μl.

[0037] The gradient elution program is as follows:

[0038]

[0039] The mass spectrometry parameters are as follows:

[0040] running time: 7.5 min, 2.2-3.5 min for collecting into the mass spectrometer, and the rest of the time period for waste liquid;

[0041] ion source: ESI source;

[0042] scanning mode: MRM positive ion mode;

[0043] capillary voltage: 3.0 kV;

[0044] cone voltage: 30 V;

[0045] desolvation temperature: 500℃;

[0046] Desolvation flow: 800 L / Hr;

[0047] Scan parameters:

[0048]

[0049] Preferably, the detection method can quantitatively detect 0.083 ppm of the impurity cyclohexyl isocyanate in the test sample.

[0050] Beneficial effects: (1) The present application uses acetonitrile solution of diethylamine as a derivatizing agent solution, so that the impurity cyclohexyl isocyanate reacts with diethylamine to form a stable derivative, and the derivative has a good peak shape. At the same time, using diethylamine as a derivative can prevent the hydrolysis of cyclohexyl isocyanate, so that the detection result is more accurate. (2) The present application shows incomparable advantages in specificity, quantitative limit, detection limit, linear range, accuracy and precision. BRIEF DESCRIPTION OF DRAWINGS

[0051] Figure 1 is the chromatogram of the blank solvent;

[0052] Figure 2 is the chromatogram of the cyclohexyl isocyanate reference solution;

[0053] Figure 3 is the chromatogram of the test sample solution;

[0054] Figure 4 is the chromatogram of the standard limit spiked test sample solution;

[0055] Figure 5 is the standard curve of the impurity cyclohexyl isocyanate. DETAILED DESCRIPTION

[0056] In order to deepen the understanding of the present application, the present application will be further described in combination with the embodiments and the accompanying drawings, which are only used to explain the present application and do not constitute a limitation on the protection scope of the present application.

[0057] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0058] According to the detection method described in the present application, the detection of the impurity cyclohexyl isocyanate is carried out by external standard method.

[0059] The instrument and reagent information used in the detection method described in the present application in the embodiments are shown in the following table:

[0060]

[0061] Example 1

[0062] Example 1-1

[0063] This example provides a method for detecting impurity cyclohexyl isocyanate in gliquidomide bulk drug.

[0064] 1. Detection conditions

[0065] (1) Chromatographic conditions:

[0066] Instrument: Waters TQD mass spectrometer

[0067] Chromatographic column: Agilent Poroshell 120EC-C18, 4.6*50mm, 2.7μm;

[0068] Mobile phase A: 0.1% formic acid aqueous solution;

[0069] Mobile phase B: acetonitrile;

[0070] Elution mode: gradient elution;

[0071] Flow rate: 0.6ml / min;

[0072] Injection volume: 2μl.

[0073] The gradient elution program is as follows:

[0074]

[0075] (2) Mass spectrometry parameters are as follows:

[0076] Run time: 7.5min, 2.2-3.5min collected into mass spectrometer, and the rest of the time period is cut off;

[0077] Ion source: ESI source;

[0078] Scan mode: MRM positive ion mode;

[0079] Capillary voltage: 3.0kV;

[0080] Cone voltage: 30V;

[0081] Desolvation temperature: 500℃;

[0082] Desolvation flow rate: 800L / Hr;

[0083] Scan parameters:

[0084]

[0085] 2. Detection method

[0086] (1) Preparation of derivatizing agent solution

[0087] Accurately weigh 2.6 g of diethylamine into 1 L of acetonitrile, ultrasonic, shake well, and prepare a 2.6 mg / mL derivative solution.

[0088] The molar ratio of diethylamine to gliclazide is theoretically 1:1. Since the sulfonamide group in the structure of gliclazide is weakly acidic, it consumes part of the basicity of diethylamine. In order to ensure that the sulfonamide group in the structure of gliclazide is not affected by the reaction of diethylamine and cyclohexyl isocyanate, the diethylamine needs to be excessive. In this embodiment 1-1, the molar ratio of diethylamine to gliclazide is 1.5.

[0089] (2) Preparation of raw drug test solution

[0090] Take about 100 mg of gliclazide tablet raw material sample into a 10 mL volumetric flask, add 2 mL of acetonitrile, and dilute to the mark with the derivative solution. Ultrasonic, make the sample completely dissolved, continue to shake, ultrasonic, sample precipitation, after about 50 min, shake well, take the filtrate sample.

[0091] (3) Preparation of control stock solution

[0092] Take an appropriate amount of impurity cyclohexyl isocyanate control sample, dissolve and dilute with acetonitrile to prepare a solution containing about 800 ng per 1 mL, shake well for use as a control stock solution.

[0093] (4) Preparation of control solution

[0094] Take 1 mL of control stock solution, place it in a 10 mL volumetric flask, accurately add 1 mL of acetonitrile, then dilute to the mark with the derivative solution, shake well, and stand for about 50 min, shake well.

[0095] (5) Accurately measure the test solution and control solution respectively, inject into the liquid chromatograph, record the chromatogram, and calculate the residual impurity cyclohexyl isocyanate in the test solution by peak area using the external standard method.

[0096] 3, Impurity cyclohexyl isocyanate residual amount calculation formula

[0097]

[0098] Among them,

[0099] Astd: Average peak area of the test substance in the control solution

[0100] Cstd: Concentration of the control solution (ng / ml)

[0101] As: Peak area of the test substance in the sample solution

[0102] Cs: Concentration of the sample solution (mg / ml)

[0103] Example 1-2

[0104] The detection method was the same as that in Example 1, except that in step (1), 3 g of diethylamine was precisely weighed into 1 L of acetonitrile, ultrasonicated, and shaken to prepare a 3 mg / mL derivative solution. The molar ratio of diethylamine to gliclazide in this Example 1-2 was 1.7.

[0105] Example 2: Specificity Test

[0106] (1) Derivative solution: The same as the derivative solution in Example 1-2, 3 g of diethylamine was precisely weighed into 1 L of acetonitrile, ultrasonicated, and shaken to prepare a 3 mg / mL derivative solution.

[0107] (2) Blank solvent: acetonitrile: derivative solution = 1:4.

[0108] (3) Control stock solution: an appropriate amount of impurity cyclohexyl isocyanate control was dissolved in acetonitrile and quantitatively diluted to prepare a solution containing about 800 ng per 1 mL, shaken and used as the control stock solution.

[0109] (4) Control solution: 1 mL of the control stock solution was taken and placed in a 10 mL volumetric flask, 1 mL of acetonitrile was precisely added, then diluted to the mark with the derivative solution, shaken and allowed to stand for about 50 min, and shaken again.

[0110] (5) Raw material test sample solution: about 100 mg of gliclazide tablet raw material sample was placed in a 10 mL volumetric flask, 2 mL of acetonitrile was added, diluted to the mark with the derivative solution, ultrasonicated to completely dissolve the sample, continued to shake, ultrasonicated, the sample was precipitated, allowed to stand for about 50 min, shaken, and the filtrate was taken for sampling.

[0111] (6) Standard limit spiked test sample solution: about 100 mg of gliclazide tablet raw material sample was placed in a 10 mL volumetric flask, 1 mL of acetonitrile was added, then 1 mL of the control stock solution was added, diluted to the mark with the derivative solution, ultrasonicated to completely dissolve the sample, continued to shake, ultrasonicated, the sample was precipitated, allowed to stand for about 50 min, shaken, and the filtrate was taken for sampling.

[0112] The blank solvent, control solution, raw material test sample solution, and standard limit spiked test sample solution were sampled according to the chromatographic conditions of Example 1-1, and the chromatograms were recorded, as shown in Figures 1-4 and Table 2.

[0113] Table 2: Specificity test results

[0114] Name Impurity Cyclohexyl isocyanate Retention time (min) Blank solvent / Reference solution 2.78 Drug substance test solution 2.79 Standard limit spiked test solution 2.78

[0115] The specificity test showed that the blank solvent and the test sample solution did not interfere with the detection of the impurity cyclohexyl isocyanate.

[0116] Example 3: Limit of quantitation and limit of detection test

[0117] The limit of detection (LOD) and limit of quantitation (LOQ) were determined according to the signal-to-noise method. The control solution was diluted step by step, the measured signal was compared with the baseline noise, and the lowest concentration that could be reliably detected was calculated, and the results are shown in Table 3.

[0118] Table 3: Limit of quantitation and limit of detection results

[0119]

[0120]

[0121] Note: Sensitivity = impurity cyclohexyl isocyanate limit of quantitation or limit of detection concentration / test sample concentration (10 mg / ml)

[0122] The test results show that the impurity cyclohexyl isocyanate in the bulk drug has a quality control limit of 8 ppm, and the sensitivity of the limit of quantitation and the limit of detection is lower than 1 / 10 of the impurity cyclohexyl isocyanate quality control limit, proving that the sensitivity of the present application is good.

[0123] Example 4: Linearity and range test

[0124] The control solution under the item of "specificity test" in Example 2 was taken and added to a certain volume of a volumetric flask. After adding an appropriate volume of acetonitrile, it was diluted to the mark with 3 mg / mL derivatization agent solution, shaken well, and allowed to stand for about 50 min, shaken well, and then 1.6 ppm, 4 ppm, 6.4 ppm, 8 ppm, 12 ppm and 16 ppm of the test sample solution were prepared as the test sample solutions of each linear gradient concentration. The linear relationship was plotted as the function of the measured peak area and the concentration of the analyte, and the linear regression was performed by the least squares method, and the value of the linear regression coefficient r should be not less than 0.999, and the results are shown in Table 4 and Figure 5 .

[0125] Table 4: Linear determination results

[0126]

[0127] Note: Intercept ratio = linear equation intercept / 8 ppm concentration peak area

[0128] From the above table results, the detection method described in the present application has a linear correlation coefficient r of 0.9991 for the impurity cyclohexyl isocyanate in the range of 1.6 ppm to 16 ppm relative to the test sample concentration, proving a good linear relationship.

[0129] Example 5: Precision test of control solution injection

[0130] The impurity cyclohexyl isocyanate reference solution under the item of Example 2 "Specificity test" was continuously determined for 6 times, and the relative standard deviation of peak area was investigated. The results are shown in Table 5.

[0131] Table 5: Results of injection precision test of reference solution

[0132]

[0133] From the above table results, the injection precision of the detection method of the application for the impurity cyclohexyl isocyanate reference solution was determined, and the RSD of peak area was less than 10%, and the precision was good.

[0134] Example 6: Stability detection of reference and standard limit spiked sample solution

[0135] The impurity cyclohexyl isocyanate reference solution and the standard limit spiked sample solution under the item of Example 2 "Specificity test" were respectively injected with 2 μl at different time points, the chromatogram was recorded, and the relative standard deviation of peak area of the impurity cyclohexyl isocyanate was calculated, and the test results are shown in Table 6.

[0136] Table 6: Solution stability results

[0137]

[0138] From the above table, the reference solution peak area RSD was less than 10% and the standard limit spiked sample solution peak area RSD was less than 10% after being placed at room temperature for 14 hours, and the solution stability was good.

[0139] Example 7: Reproducibility detection

[0140] The raw material was detected for 6 times according to the detection methods of Examples 1-2 of the application to verify the good precision of the method, and the results are shown in Table 7.

[0141] Table 5: Reproducibility test results

[0142]

[0143] From the above table, the impurity cyclohexyl isocyanate was less than 30% of the limit in 6 detections, and the residual amount RSD was less than 10%, which proved that the method had good precision.

[0144] Example 8: Accuracy detection

[0145] The recovery method was used to determine the ratio (recovery rate) between the actual determination amount and the theoretical amount of the impurity cyclohexyl isocyanate in the standard limit spiked sample solution under the item of Example 2 "Specificity test", expressed in percentage %, and the recovery rate was required to be between 80% and 115% to prove that the method had good accuracy, and the results are shown in Table 6.

[0146] Table 6: Accuracy test results

[0147]

[0148] From the above table, the recovery rate of impurity cyclohexyl isocyanate in the bulk drug is between 86.47% and 90.52%, which meets the verification requirement (80% to 115%), confirming that the method has good accuracy; the recovery rate RSD value is less than 10%, and the repeatability is good.

[0149] In summary, in the specificity test, the blank solvent and the test solution do not interfere with the detection of impurity cyclohexyl isocyanate.

[0150] In the quantitative limit test, the quantitative limit concentration of impurity cyclohexyl isocyanate is much lower than the quality control limit (8ppm); the detection limit concentration of impurity cyclohexyl isocyanate is less than 1 / 10 of the quality control limit (0.8ppm).

[0151] In the linear range test, impurity cyclohexyl isocyanate has a good linear relationship in the range of 1.6ppm to 16ppm relative to the test sample concentration, the correlation coefficient r is not less than 0.999, and the percentage of the y-axis intercept relative to the peak area of the limit concentration reference sample is much less than 25%.

[0152] In the accuracy test, the recovery rate of impurity cyclohexyl isocyanate is between 85% and 110%, and RSD≤10%.

[0153] In the repeatability test, the residual amount of impurity cyclohexyl isocyanate in the six samples is much lower than 30% of the quality control limit (8ppm).

[0154] Therefore, the diethylamine derivative system adopted in the present application can effectively separate impurity cyclohexyl isocyanate and gliquidomide bulk drug, and the peak shape has good symmetry, which is beneficial to the detection of impurity cyclohexyl isocyanate, and has high system applicability. At the same time, it has incomparable advantages in specificity, quantitative limit, detection limit, linear range and repeatability, and has high precision.

[0155] The above only describes the preferred embodiments of the present application, and it should be pointed out that for ordinary skilled persons in the technical field, some improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method for detecting cyclohexyl isocyanate in gliquidomel raw material drug, characterized in that, The cyclohexyl isocyanate is reacted with diethylamine to form a derivative with a molecular weight [M+H]+199.2, and the residual amount of cyclohexyl isocyanate in gliquidomide bulk drug is detected by liquid chromatography-mass spectrometry; The reaction equation of the cyclohexyl isocyanate and diethylamine is as follows:

2. The method of claim 1, wherein, Specifically comprising the following steps: 1) Preparation of bulk drug test sample solution The gliquidomide tablet bulk sample is dissolved in acetonitrile, then the derivative solution is added for dilution and complete dissolution of the sample, and the sample is shaken and ultrasonicated, then left to stand for a period of time after the sample is precipitated, shaken, and the filtrate is taken for the test sample solution; 2) Preparation of control sample stock solution An appropriate amount of impurity cyclohexyl isocyanate control sample is dissolved in acetonitrile and quantitatively diluted to prepare a solution containing about 800 ng per 1 mL, which is shaken and used as the control sample stock solution; 3) Preparation of control sample solution The control sample stock solution in step 2) is placed in a volumetric flask, acetonitrile is added, and then the derivative solution is added to the mark, which is shaken and used as the control sample solution; 4) The test sample solution and the control sample solution are precisely measured and injected into the liquid chromatography-mass spectrometer, and the chromatogram is recorded, and the residual amount of cyclohexyl isocyanate in the test sample solution is calculated by peak area using the external standard method.

3. The method of claim 2, wherein, The derivative in steps 1) and 3) is diethylamine, and the molar ratio of diethylamine to gliquidomide in step 1) is at least 1.

5.

4. The method of claim 3, wherein, The solvent of the derivative solution in steps 1) and 3) is acetonitrile, and the derivative solution is prepared by dissolving diethylamine in acetonitrile to prepare a derivative solution with a concentration of at least 2.6 mg / mL; the concentration of the derivative solution is preferably 3 mg / mL.

5. The method of claim 2, wherein, The ratio of acetonitrile to derivative solution in steps 1) and 3) is 1:

4.

6. The method of claim 5, wherein, In step 1), about 100 mg of gliquidomide tablet bulk sample is placed in a 10 mL volumetric flask, 2 mL of acetonitrile is added, and the derivative solution is added to the mark, which is ultrasonicated to completely dissolve the sample, and then shaken and ultrasonicated, and the sample is precipitated, left to stand for about 50 min, shaken, and the filtrate is taken for injection; In step 3), 1 mL of the control sample stock solution is placed in a 10 mL volumetric flask, 1 mL of acetonitrile is precisely added, and then the derivative solution is added to the mark, which is shaken, left to stand for about 50 min, shaken, and used as the control sample solution.

7. The method according to claim 1 or 2, characterized in that, The detection conditions of the liquid chromatography are as follows: Chromatographic column: Agilent Poroshell 120EC-C18, specifications: 4.6*50 mm, 2.7 μm; Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: acetonitrile; Elution mode: gradient elution; Flow rate: 0.6 ml / min; Injection volume: 2 μl.

8. The method of claim 7, wherein, During the gradient elution, the elution program is as follows:

9. The method of claim 7, wherein, Mass spectrometry parameters are as follows: Run time: 7.5 min, 2.2-3.5 min for mass spectrometry collection, and the rest of the time for waste liquid; Ion source: ESI source; Scanning mode: MRM positive ion mode; Capillary voltage: 3.0 kV; Cone hole voltage: 30 V; Desolvation temperature: 500℃; Desolvation flow rate: 800 L / Hr; Scan parameters:

10. The method of claim 1 or 2, wherein, The detection method can quantitatively detect 0.083 ppm of cyclohexyl isocyanate.

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