Analysis method of veratraldehyde related substances

Abstract

The invention discloses an analysis method of veratraldehyde related substances. The method comprises the following steps: 1, solution preparation: respectively preparing a diluent, a test solution, a contrast solution, a mobile phase A and a mobile phase B; and 2, high performance liquid chromatography determination: respectively and precisely measuring the test solution and the contrast solution, injecting the test solution and the contrast solution into a high performance liquid chromatograph for detection, recording a chromatogram, and calculating the content of the veratraldehyde related substances. The analysis method for the veratraldehyde related substances is free of blank solvent interference, good in specificity, high in sensitivity and accuracy and capable of detecting one or more of 10 impurities in veratraldehyde at a time, so that the impurities of a sample can be measured more comprehensively, quality control over veratraldehyde is achieved, and the analysis method has good application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to an analysis method for veratraldehyde-related substances. Background Art

[0002] Veratraldehyde, also known as methyl vanillin, methyl vanillin, methyl vanillin, chemical name 3,4-dimethoxybenzaldehyde, molecular formula C 9 H 10 O 3 , whose structural formula is as follows:

[0003]

[0004] Veratraldehyde is a synthetic flavor with a vanilla-like aroma and a widely used pharmaceutical intermediate. As an important synthetic flavor, veratraldehyde is used in food flavors and can also be used in daily chemical flavors in small quantities; as an important pharmaceutical synthetic intermediate, veratraldehyde is mainly used to synthesize levodopa, carbidopa, methyldopa, methyldopa ethyl ester, diaminoveratridine and istradefylline, etc.

[0005] At present, a large number of literatures have disclosed the research on the preparation process of 3,4-dimethoxybenzaldehyde, but there are relatively few studies on the quality control of 3,4-dimethoxybenzaldehyde and its impurities. If the impurities introduced in the process of synthesizing veratraldehyde cannot be effectively controlled, they will cause side reactions and may remain in the finished product, directly affecting the safety and effectiveness of the drug. Patent application number 201910178634.9 discloses a high-performance liquid chromatography analysis and detection method for impurities in 3,4-dimethoxybenzaldehyde, but this method can only separate three impurities in veratraldehyde and cannot perform more comprehensive impurity detection and analysis.

[0006] Therefore, it is necessary to develop a faster and more accurate method for detecting veratraldehyde-related substances in order to achieve the goal of simply, quickly and accurately controlling product quality. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide an analysis method for veratraldehyde-related substances in view of the deficiencies of the above-mentioned prior art. The analysis method has no blank solvent interference, good specificity, high sensitivity and accuracy, and can detect one or more of the 10 impurities in veratraldehyde at one time, so that each impurity of the sample can be more comprehensively determined, and the quality control of veratraldehyde can be achieved, which solves the problem that the existing method cannot perform faster, more accurate and more comprehensive impurity detection and analysis.

[0008] In order to solve the above technical problems, the technical solution adopted by the present invention is: a method for analyzing veratraldehyde-related substances, characterized in that the method comprises the following steps:

[0009] Step 1: Solution preparation:

[0010] Diluent: Prepare 10mmol / L potassium dihydrogen phosphate solution, then mix it with acetonitrile in a volume ratio of 85:15, and adjust the pH value to 5.5 with 1mmol / L potassium hydroxide solution;

[0011] Preparation of test solution: weigh 20 mg of veratraldehyde into a 20 mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well;

[0012] Control solution: Accurately measure 1 mL of the test solution and place it in a 100 mL volumetric flask, add diluent to dilute to 100 mL, and shake well;

[0013] Mobile phase A: The preparation process is the same as that of the diluent;

[0014] Mobile phase B: Prepare 10mmol / L potassium dihydrogen phosphate solution, then mix it with acetonitrile in a volume ratio of 60:40, and adjust the pH value to 5.5 with 1mmol / L potassium hydroxide solution;

[0015] Step 2: High performance liquid chromatography determination: Accurately measure the test solution and control solution prepared in step 1 and inject them into a high performance liquid chromatograph for detection, and record the chromatogram. The specific chromatographic conditions are:

[0016] Chromatographic column: C18;

[0017] Detection wavelength: 230nm, 253nm;

[0018] Mobile phase: mobile phase A, mobile phase B;

[0019] Flow rate: 0.8mL / min~1.2mL / min;

[0020] Column temperature: 30°C;

[0021] The contents of veratraldehyde-related substances were calculated based on the chromatogram.

[0022] The above-mentioned method for analyzing veratraldehyde-related substances is characterized in that the chromatographic conditions in step 2 are:

[0023] Chromatographic column: XBridge C18, inner diameter 4.6 mm, length 250 mm, particle size 5 μm;

[0024] Flow rate: 1.0mL / min.

[0025] The above-mentioned method for analyzing veratraldehyde-related substances is characterized in that the detection in step 2 adopts the following procedure of gradient elution:

[0026] Time (min) Mobile phase A volume content (%) Volume content of mobile phase B (%) 0 100 0 20 100 0 35 0 100 50 0 100 51 100 0 60 100 0 .

[0027] Compared with the prior art, the present invention has the following advantages:

[0028] 1. The analytical method for veratraldehyde-related substances of the present invention has no blank solvent interference, good specificity, high sensitivity and accuracy, and provides an effective method for the detection of veratraldehyde-related substances.

[0029] 2. The analysis method of veratraldehyde-related substances of the present invention can detect one or more of the 10 impurities in veratraldehyde at one time, so that each impurity of the sample can be more comprehensively determined, the quality control of veratraldehyde can be achieved, the safety of veratraldehyde is ensured, and the detection is convenient and fast, and has a good application prospect.

[0030] 3. The reagents involved in the analysis method of veratraldehyde-related substances of the present invention are all conventional reagents, which are convenient and easy to obtain, thus reducing the cost of analysis and detection.

[0031] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is the HPLC chromatogram of the blank solution of the present invention.

[0033] Figure 2 It is the high performance liquid chromatogram of the system suitability solution in the specificity test of the present invention. DETAILED DESCRIPTION

[0034] The structures, names and reference substances of the drugs, reagents, instruments and impurities used in Example 1 and the methodological verification of the present invention are shown in Tables 1 to 3 below.

[0035] Table 1 Drugs and reagents

[0036] Reagents level source Veratraldehyde Industrial Grade Outsourcing Potassium dihydrogen phosphate Analytical level Aladdin Acetonitrile Analytical level Aladdin Purified water / self made

[0037] Table 2 Instruments

[0038] instrument model factory High performance liquid chromatography Agilent 1260infinityⅡ Agilent Technologies Electronic balance MS105 METTLER TOLEDO

[0039] Table 3 Impurity structure

[0040]

[0041]

[0042] Example 1

[0043] This embodiment includes the following steps:

[0044] Step 1: Solution preparation:

[0045] Diluent: Prepare 10mmol / L potassium dihydrogen phosphate solution, then mix it with acetonitrile in a volume ratio of 85:15, and adjust the pH value to 5.5 with 1mmol / L potassium hydroxide solution;

[0046] Preparation of test solution: weigh 20 mg of veratraldehyde into a 20 mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well;

[0047] Control solution: Accurately measure 1 mL of the test solution and place it in a 100 mL volumetric flask, add diluent to dilute to the mark, and shake well;

[0048] Mobile phase A: The preparation process is the same as that of the diluent;

[0049] Mobile phase B: prepare 10 mmol / L potassium dihydrogen phosphate solution, then mix it with acetonitrile at a volume ratio of 60:40, and adjust the pH value to 5.5 with 1 mmol / L potassium hydroxide solution to obtain mobile phase B;

[0050] Impurity stock solution: Take 25 mg of impurity 3,4-dimethoxybenzoic acid and N-methylformanilide reference substance, place them in 50 mL volumetric flasks respectively, add appropriate amount of acetonitrile and ultrasonically dissolve, dilute to scale with mobile phase A, and shake well;

[0051] Take 25 mg of the impurity 4,5-dimethoxy-1,2-dialdehyde benzene, place it in a 50 mL volumetric flask, add an appropriate amount of acetonitrile to dissolve and dilute to the scale, and shake well;

[0052] System suitability solution: Take 25 mg of Veratraldehyde reference substance, place it in a 25 mL volumetric flask, add mobile phase A to dissolve, accurately add 0.25 mL of each of the above two impurity stock solutions, dilute to the scale with mobile phase A, and shake well;

[0053] Step 2: High performance liquid chromatography determination: Accurately measure the test solution and control solution prepared in step 1 and inject them into a high performance liquid chromatograph for detection, and record the chromatogram. The specific chromatographic conditions are:

[0054] Chromatographic column: XBridge C18, inner diameter 4.6 mm, length 250 mm, particle size 5 μm;

[0055] Detection wavelength: 230nm, 253nm;

[0056] Mobile phase: mobile phase A, mobile phase B;

[0057] Flow rate: 1.0 mL / min;

[0058] Column temperature: 30°C;

[0059] The detection procedure using gradient elution is shown in Table 4 below:

[0060] Table 4 Gradient elution program

[0061] Time (min) Mobile phase A volume content (%) Volume content of mobile phase B (%) 0 100 0 20 100 0 35 0 100 50 0 100 51 100 0 60 100 0

[0062] The contents of veratraldehyde-related substances were calculated based on the chromatogram, and the results are shown in Table 5 below.

[0063] Table 5 Contents of related substances of veratraldehyde

[0064]

[0065] As can be seen from Table 5, the test results of the contents of related substances of the three batches of veratraldehyde in this example all meet the requirements.

[0066] A methodological investigation was conducted on the analytical method of the veratraldehyde-related substances of the present invention, and the specific process and results are as follows.

[0067] 1. Exclusive inspection

[0068] (1) Solution preparation

[0069] Blank solvent: i.e. diluent, weigh 1.36 g potassium dihydrogen phosphate, dissolve in water and filter to make 1000 mL of 10 mmol / L potassium dihydrogen phosphate solution, and measure 850 mL and mix with 150 mL acetonitrile, adjust the pH to 5.5 with 1 mmol / L potassium hydroxide solution, and filter;

[0070] Impurity reference substance stock solution: accurately weigh 25 mg of the impurity 4,5-dimethoxy-1,2-dialdehyde benzene reference substance and place it in a 50 mL volumetric flask, add acetonitrile to dissolve it by ultrasonication and dilute it to the scale, shake well, and use it as the impurity reference substance stock solution①;

[0071] Accurately weigh 25 mg of impurity reference substances of o-phthalic ether, 2,3-dimethoxybenzaldehyde, 4,5-dimethoxy-1,3-dialdehyde benzene, 3,4-dimethoxybenzyl alcohol, 3,4-dimethoxybenzoic acid, N-methylformanilide and N-methylaniline, respectively, place them in different 50 mL volumetric bottles, add acetonitrile and ultrasonically dissolve, dilute to the scale with diluent, shake well, and use as impurity reference substance stock solution②;

[0072] Accurately weigh 25 mg of impurities 3,4-dihydroxybenzaldehyde and 3-methoxy-4-hydroxybenzaldehyde, respectively, and place them in different 50 mL volumetric bottles. Add diluent, shake vigorously to dissolve and dilute to the scale, shake well, and use as impurity reference stock solution ③;

[0073] System suitability solution: accurately weigh 25 mg of 3,4-dimethoxybenzaldehyde reference substance and place it in a 25 mL volumetric flask, add diluent to dissolve, accurately add 0.25 mL of each of the above impurity reference substance stock solutions ①, ② and ③, dilute to the scale with diluent, and shake well;

[0074] Each impurity location solution: accurately pipette 0.1 mL of each impurity reference stock solution into a 10 mL volumetric flask, dilute to the mark with diluent, and shake well;

[0075] Test solution: accurately weigh 50 mg of veratraldehyde and place it in a 50 mL volumetric flask, add diluent to dissolve and dilute to the scale, and shake well.

[0076] (2) Experimental method: According to the detection method of veratraldehyde-related substances in Example 1, 10 μL of blank solution, each impurity location solution, test solution, and system suitability solution were accurately measured and injected respectively, and the corresponding chromatograms were recorded and analyzed. The chromatograms of the blank solution and the system suitability solution are shown in FIG. Figure 1 and Figure 2 The results are shown in Tables 6 to 8 below.

[0077] (3) Acceptable criteria: The blank solvent and other impurities do not interfere with the detection of the main peak; the retention time of the main peak of the impurity in each impurity location solution is consistent with that in the system suitability solution; in the system suitability solution, the separation between the main peak of each known impurity and the adjacent impurities meets the requirements.

[0078] (4) Experimental results:

[0079] Table 6 System suitability solution test results

[0080]

[0081] Table 7 Specificity results (230nm)

[0082]

[0083]

[0084] Table 8 Specificity results (253nm)

[0085]

[0086] (4) Conclusion: From Tables 6 to 8, it can be seen that the blank solvent has no interference, and the separation between the main peak and the specific impurity peak and the adjacent peak is ≥1.5, indicating that the analytical method of the present invention has good specificity.

[0087] 2. Limit of quantification (LOQ) and limit of detection (LOD) investigation

[0088] (1) Solution preparation

[0089] Impurity reference substance stock solution: accurately weigh 25 mg of the impurity 4,5-dimethoxy-1,2-dialdehyde benzene reference substance and place it in a 50 mL volumetric flask, add acetonitrile to dissolve it by ultrasonication and dilute it to the scale, shake well, and use it as the impurity reference substance stock solution①;

[0090] Accurately weigh 25 mg of impurity reference substances of o-phthalic ether, 2,3-dimethoxybenzaldehyde, 4,5-dimethoxy-1,3-dialdehyde benzene, 3,4-dimethoxybenzyl alcohol, 3,4-dimethoxybenzoic acid, N-methylformanilide and N-methylaniline respectively, place them in different 50 mL volumetric bottles, add acetonitrile and ultrasonically dissolve, dilute to the scale with diluent, shake well, and use as impurity reference substance stock solution②;

[0091] Accurately weigh 25 mg of impurities 3,4-dihydroxybenzaldehyde and 3-methoxy-4-hydroxybenzaldehyde, respectively, and place them in different 50 mL volumetric bottles. Add diluent, shake vigorously to dissolve and dilute to the scale, shake well, and use as impurity reference stock solution ③;

[0092] Accurately weigh 25 mg of 3,4-dimethoxybenzaldehyde reference substance, place it in a 50 ml volumetric flask, add diluent to dissolve and dilute to scale, shake well, and use it as the stock solution of impurity reference substance④;

[0093] Quantitative limit solution: Take the above impurity reference substance stock solutions ①, ②, ③ and ④, and dilute them with diluent to make a solution with S / N ≥ 10;

[0094] Detection limit solution: Take the above quantitation limit solution and dilute it with diluent to make a solution with S / N ≥ 3;

[0095] (2) Experimental method: According to the detection method of veratraldehyde-related substances in Example 1, 10 μL of the detection limit solution and the quantification limit solution were accurately measured and injected respectively, and the corresponding chromatograms were recorded and analyzed. The results are shown in Table 9 below.

[0096] (3) Acceptable criteria: The signal-to-noise ratio (S / N) of the quantification limit should be no less than 10; the signal-to-noise ratio (S / N) of the detection limit should be no less than 3;

[0097] (4) Experimental results:

[0098] Table 9 Results of limit of quantitation and limit of detection

[0099]

[0100]

[0101] (4) Conclusion: From Table 9, it can be seen that the S / N of each chromatographic peak at the detection limit concentration is ≥3, and the S / N of each chromatographic peak at the quantification limit concentration is ≥10, indicating that the method of the present invention has good sensitivity.

[0102] 3. Linearity and range

[0103] (1) Solution preparation

[0104] Blank solvent: i.e. diluent, weigh 1.36 g potassium dihydrogen phosphate, dissolve in water and filter to make 1000 mL of 10 mmol / L potassium dihydrogen phosphate solution, and measure 850 mL and mix with 150 mL acetonitrile, adjust the pH to 5.5 with 1 mmol / L potassium hydroxide solution, and filter;

[0105] Linear stock solution: accurately weigh 25 mg of the impurity 4,5-dimethoxy-1,2-dialdehyde benzene reference substance, place it in a 50 mL volumetric flask, add acetonitrile to dissolve by ultrasonication and dilute to the scale, shake well, and use it as the impurity reference substance stock solution①;

[0106] Accurately weigh 25 mg of impurity reference substances of o-phthalic ether, 2,3-dimethoxybenzaldehyde, 4,5-dimethoxy-1,3-dialdehyde benzene, 3,4-dimethoxybenzyl alcohol, 3,4-dimethoxybenzoic acid, N-methylformanilide and N-methylaniline, respectively, place them in 50 mL volumetric flasks, add acetonitrile and ultrasonically dissolve, dilute to the mark with diluent, shake well, and use as impurity reference substance stock solution②;

[0107] Accurately weigh 25 mg of impurities 3,4-dihydroxybenzaldehyde and 3-methoxy-4-hydroxybenzaldehyde respectively, place in different 50 mL volumetric flasks, add diluent, shake vigorously to dissolve and dilute to scale, shake well, and use as impurity reference stock solution ③;

[0108] Accurately weigh 25 mg of 3,4-dimethoxybenzaldehyde reference substance, place it in a 50 mL volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and use it as the impurity reference substance stock solution④;

[0109] 2.0% linear solution: Accurately pipette 1 mL each of impurity reference stock solution ①, impurity reference stock solution ②, impurity reference stock solution ③, and impurity reference stock solution ④ in the linear stock solution, place them in the same 25 mL volumetric flask, dilute to the scale with diluent, and shake well;

[0110] 1.0% linear solution: Accurately pipette 0.5 mL each of impurity reference stock solution ①, impurity reference stock solution ②, impurity reference stock solution ③, and impurity reference stock solution ④ in the linear stock solution, place them in the same 25 mL volumetric flask, dilute to the scale with diluent, and shake well;

[0111] 0.5% linear solution: Accurately pipette 0.5 mL of each of impurity reference stock solution ①, impurity reference stock solution ②, impurity reference stock solution ③, and impurity reference stock solution ④ in the linear stock solution, place them in the same 50 mL volumetric flask, dilute to the scale with diluent, and shake well;

[0112] 0.2% linear solution: accurately pipette 1 mL of 2.0% linear solution into the same 10 mL volumetric flask, dilute to the mark with diluent, and shake well;

[0113] 0.1% linear solution: Accurately pipette 1 mL of 1.0% linear solution into the same 10 mL volumetric flask, dilute to the mark with diluent, and shake well.

[0114] (2) Experimental method: According to the detection method of veratraldehyde-related substances in Example 1, blank solvent and each linear solution were injected respectively, and the corresponding chromatograms were recorded and analyzed. The results are shown in Table 10 below.

[0115] (3) Acceptable criteria: linear correlation coefficient r ≥ 0.990; |Y intercept| / 100% reference solution peak area ≤ 25%.

[0116] (4) Experimental results:

[0117] Table 10 Linearity results

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127] (4) Conclusion: From Table 10, it can be seen that the linear equation of 4,5-dimethoxy-1,2-dialdehyde benzene in the range of 0.0201 μg / mL to 20.0780 μg / mL is y=53.3731x-9.9744, r=0.9996, and the peak area of ​​|Y intercept| / 100% reference solution is 3.87%;

[0128] For o-phenylenedimethyl ether in the range of 0.0401 μg / mL to 20.0551 μg / mL, the linear equation is y=24.5884x+4.1171, and the peak area of ​​|Y intercept| / 100% reference solution is 3.23%;

[0129] 4,5-dimethoxy-1,3-dialdehyde benzene is in the range of 0.0191μg / mL to 19.0997μg / mL, the linear equation is y=50.8799x+6.3422, r=0.9996, |Y intercept| / 100% reference solution peak area is 2.54%;

[0130] 3,4-dimethoxybenzoic acid is in the range of 0.0100μg / mL to 19.9381μg / mL, the linear equation is y=32.4540x-0.2732, r=1.0000, |Y intercept| / 100% reference solution peak area is 0.17%;

[0131] N-methylaniline is in the range of 0.0324 μg / mL to 20.2556 μg / mL, the linear equation is y=35.8174x-1.4591, r=0.9997, |Y intercept| / 100% reference solution peak area is 0.81%;

[0132] 3,4-Dimethoxybenzaldehyde (253nm) is in the range of 0.1308μg / mL to 20.1177μg / mL, the linear equation is y=12.0925x-6.3340, r=0.9985, |Y intercept| / 100% reference solution peak area is 11.63%;

[0133] 3,4-Dimethoxybenzaldehyde (230nm) is in the range of 0.0322μg / mL to 20.1177μg / mL, the linear equation is y=53.6754x+3.0681, r=0.9999, |Y intercept| / 100% reference solution peak area is 1.12%;

[0134] 3,4-dihydroxybenzaldehyde is in the range of 0.0101μg / mL to 20.1398μg / mL, the linear equation is y=52.1131x+0.4100, r=1.0000, |Y intercept| / 100% reference solution peak area is 0.16%;

[0135] 2,3-Dimethoxybenzaldehyde is in the range of 0.0320μg / mL to 19.9760μg / mL, the linear equation is y=32.8265x+5.3137, r=0.9991, |Y intercept| / 100% reference solution peak area is 3.14%;

[0136] 3,4-dimethoxybenzyl alcohol is in the range of 0.0237μg / mL to 19.7541μg / mL, the linear equation is y=26.5431x+1.1390, r=1.0000, |Y intercept| / 100% reference solution peak area is 0.86%;

[0137] The linear equation of N-methylformanilide in the range of 0.0720 μg / mL to 20.5610 μg / mL is y=38.2223x+3.3152, r=0.9993, and the peak area of ​​|Y intercept| / 100% reference solution is 1.66%;

[0138] 3-methoxy-4-hydroxybenzaldehyde is in the range of 0.0200 μg / mL to 20.0189 μg / mL, the linear equation is y=52.5929x+3.9783, r=0.9999, |Y intercept| / 100% reference solution peak area is 1.49%;

[0139] In summary, the detection results of various substances meet the requirements, indicating that the method of the present invention has good linearity.

[0140] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for analyzing veratraldehyde-related substances, characterized in that: The method comprises the following steps: Step 1: Solution preparation: Diluent: Prepare 10mmol / L potassium dihydrogen phosphate solution, then mix it with acetonitrile in a volume ratio of 85:15, and adjust the pH value to 5.5 with 1mmol / L potassium hydroxide solution; Preparation of test solution: weigh 20 mg of veratraldehyde into a 20 mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well; Control solution: Accurately measure 1 mL of the test solution and place it in a 100 mL volumetric flask, add diluent to dilute to 100 mL, and shake well; Mobile phase A: The preparation process is the same as that of the diluent; Mobile phase B: Prepare 10mmol / L potassium dihydrogen phosphate solution, then mix it with acetonitrile in a volume ratio of 60:40, and adjust the pH value to 5.5 with 1mmol / L potassium hydroxide solution; Step 2: High performance liquid chromatography determination: Accurately measure the test solution and control solution prepared in step 1 and inject them into a high performance liquid chromatograph for detection, and record the chromatogram. The specific chromatographic conditions are: Chromatographic column: C18; Detection wavelength: 230nm, 253nm; Mobile phase: mobile phase A, mobile phase B; Flow rate: 0.8mL / min~1.2mL / min; Column temperature: 30°C; The contents of veratraldehyde-related substances were calculated based on the chromatogram.

2. The method for analyzing veratraldehyde-related substances according to claim 1, characterized in that: The chromatographic conditions in step 2 are: Chromatographic column: XBridge C18, inner diameter 4.6 mm, length 250 mm, particle size 5 μm; Flow rate: 1.0mL / min.

3. The method for analyzing veratraldehyde-related substances according to claim 1, characterized in that: The procedure for detection using gradient elution in step 2 is as follows: 。

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