A method for detecting impurity content in vanillin acetone samples

Abstract

The present invention provides a method for detecting the impurity content in a vanillin ethyl ketone sample. The method for detecting the impurity content in the vanillin ethyl ketone sample of the present invention can detect the contents of three main impurities, namely, guaiacol, isovanillin ethyl ketone and 2-methoxyphenyl acetate, in the vanillin ethyl ketone sample by high performance liquid chromatography; specifically, water is used as mobile phase A and methanol is used as mobile phase B for gradient elution, and area normalization method is used for quantitative calculation to detect the content of each impurity in the vanillin ethyl ketone.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to a method for detecting impurity content in a vanillyl acetone sample. Background Art

[0002] Vanillyl ethyl ketone (HMP) is also known as acetovanillin, CAS number is 489-02-2, molecular formula is C9H 10 O3, molecular weight: 166.1739. English name: Acetovanillone. The structural formula of vanillone ethyl ketone (HMP) is

[0003]

[0004] HMP is a substance with a light vanilla flavor. In organic synthesis, HMP is a commonly used antioxidant that can be used to synthesize a wide variety of new antimalarial drugs and the psychotropic drug iloperidone. HMP is also an oxidase inhibitor of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and a xanthine oxidase inhibitor. HMP can be used as a drug for common cardiovascular diseases to reduce the number of free radicals produced and myocardial cell apoptosis, ultimately achieving the effect of alleviating reperfusion injury.

[0005] A new synthetic route for vanillyl acetone is to use guaiacol as raw material, first react with acetyl chloride to form an ester, and then react with Lewis acid such as AlCl3 to form vanillyl acetone. The synthetic route is as follows:

[0006]

[0007] Guaiacol (CAS No. 90-05-1, molecular formula C7H8O2, molecular weight: 124.14, colorless or slightly yellow crystals (or oily liquids) at room temperature, with an aromatic odor. Guaiacol is the main raw material in the synthesis of ethyl ketone and one of the main impurities in the final product; at the same time, the synthesized ethyl ketone also contains impurities 1-(2-hydroxy-3-methoxyphenyl) ethyl ketone and 2-methoxyphenyl acetate. Therefore, it is necessary to detect the impurity content in the ethyl ketone product, formulate relevant detection methods and standards, and then provide a theoretical basis for the quality control of the industrial production of ethyl ketone. However, there is currently no detection method for impurities related to ethyl ketone. Summary of the invention

[0008] In view of this, the present invention proposes a method for detecting the impurity content in a vanillin sample to solve or partially solve the defects in the prior art.

[0009] The present invention provides a method for detecting the impurity content in a vanillin ethyl ketone sample, and adopts high performance liquid chromatography to detect the impurity content in the vanillin ethyl ketone;

[0010] The HPLC conditions include:

[0011] The chromatographic column is a reverse phase chromatographic column bonded with a non-polar octadecyl functional group;

[0012] The mobile phase includes mobile phase A and mobile phase B, mobile phase A is water, and mobile phase B is methanol; mobile phase A and mobile phase B are used as eluents for gradient elution, and the elution gradient program includes:

[0013] 0min: Mobile phase B accounts for 40% by volume and is maintained for 3min, while mobile phase A accounts for 60% by volume and is maintained for 3min;

[0014] 3-13min: the volume proportion of mobile phase B changes from 40% to 100%, and the volume proportion of mobile phase A changes from 60% to 0%;

[0015] 13-16min: the volume proportion of mobile phase B is maintained at 100%, and the volume proportion of mobile phase A is maintained at 0%;

[0016] 16-16.1min: the volume proportion of mobile phase B changes from 100% to 40%, and the volume proportion of mobile phase A changes from 0% to 60%;

[0017] 16.1~19min: the volume proportion of mobile phase B is maintained at 40%, and the volume proportion of mobile phase A is maintained at 60%.

[0018] Preferably, in the method for detecting the impurity content in the vanillyl acetone sample, the column temperature is 20 to 30°C.

[0019] Preferably, in the method for detecting the impurity content in the vanillyl acetone sample, the injection volume is 10 to 20 μL.

[0020] Preferably, in the method for detecting the impurity content in the vanillyl acetone sample, the flow rate of the mobile phase is 0.5 to 2 mL / min.

[0021] Preferably, the method for detecting the impurity content in the vanillyl acetone sample has a detection wavelength of 225 to 230 nm.

[0022] Preferably, the method for detecting the impurity content in the vanillone sample further comprises structural identification of the vanillone sample by nuclear magnetic resonance detection, infrared spectroscopy detection, and mass spectrometry detection before detecting the impurity content in the vanillone sample.

[0023] Preferably, the method for detecting the impurity content in the vanillin ethyl ketone sample further comprises measuring the melting point of the vanillin ethyl ketone sample before detecting the impurity content in the vanillin ethyl ketone.

[0024] Preferably, the method for detecting the impurity content in the vanillin ethyl ketone sample further comprises detecting the water content in the vanillin ethyl ketone sample before detecting the impurity content in the vanillin ethyl ketone sample.

[0025] Preferably, the method for detecting the impurity content in the vanillone sample adopts the Karl Fischer method to detect the water content.

[0026] The method for detecting impurity content in a vanillyl acetone sample of the present invention has the following beneficial effects:

[0027] 1. The method for detecting the impurity content in the vanillin ethyl ketone sample of the present invention can detect the contents of three main impurities, namely guaiacol, isovillarin ethyl ketone and 2-methoxyphenyl acetate, in the vanillin ethyl ketone sample by high performance liquid chromatography; specifically, gradient elution is performed with water as mobile phase A and methanol as mobile phase B, and quantitative calculation is performed using the area normalization method to detect the content of each impurity in the vanillin ethyl ketone.

[0028] 2. The method for detecting the impurity content in the vanillin ethyl ketone sample of the present invention, before the detection, adopts nuclear magnetic resonance detection, infrared spectrum detection, and mass spectrometry detection to carry out structural identification of the vanillin ethyl ketone sample. At the same time, the water content in the vanillin ethyl ketone sample is detected, and the melting point of the vanillin ethyl ketone is measured to determine whether the vanillin ethyl ketone sample is a qualified sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the prior art descriptions. Obviously, the drawings described below are only some embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0030] Figure 1 The chromatogram is a chromatogram of impurities in the vanillyl acetone sample detected by high performance liquid chromatography in Example 1;

[0031] Figure 2 is the retention time of different impurities in the vanillyl acetone sample;

[0032] Figure 3 is the vanillone sample in Example 2 1 HNMR spectrum;

[0033] Figure 4 is the vanillone sample in Example 2 13 CNMR spectrum;

[0034] Figure 5 This is the infrared spectrum of the vanillyl acetone sample in Example 2;

[0035] Figure 6 This is the mass spectrum of the vanillone sample in Example 2. DETAILED DESCRIPTION

[0036] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] The embodiment of the present application provides a method for detecting the impurity content in a vanillin ethyl ketone sample, using high performance liquid chromatography to detect the impurity content in the vanillin ethyl ketone;

[0038] Among them, the high performance liquid chromatography conditions include:

[0039] The chromatographic column is a reverse phase chromatographic column bonded with a non-polar octadecyl functional group;

[0040] The mobile phase includes mobile phase A and mobile phase B, mobile phase A is water, and mobile phase B is methanol; mobile phase A and mobile phase B are used as eluents for gradient elution, and the elution gradient program includes:

[0041] 0min: Mobile phase B accounts for 40% by volume and is maintained for 3min, while mobile phase A accounts for 60% by volume and is maintained for 3min;

[0042] 3-13min: the volume proportion of mobile phase B changes from 40% to 100%, and the volume proportion of mobile phase A changes from 60% to 0%;

[0043] 13-16min: the volume proportion of mobile phase B is maintained at 100%, and the volume proportion of mobile phase A is maintained at 0%;

[0044] 16-16.1min: the volume proportion of mobile phase B changes from 100% to 40%, and the volume proportion of mobile phase A changes from 0% to 60%;

[0045] 16.1~19min: the volume proportion of mobile phase B is maintained at 40%, and the volume proportion of mobile phase A is maintained at 60%.

[0046] It should be noted that the method for detecting the impurity content in the vanillin ethyl ketone sample of the present application can detect the contents of the three main impurities guaiacol, isovillarin ethyl ketone, and 2-methoxyphenyl acetate in the vanillin ethyl ketone sample by high performance liquid chromatography; specifically, gradient elution is performed with water as mobile phase A and methanol as mobile phase B, and quantitative calculation is performed using the area normalization method, so as to detect the content of each impurity in the vanillin ethyl ketone.

[0047] Specifically, if the mass content of vanillin in the vanillin sample is greater than 98%, it is a qualified product.

[0048] At the same time, it should be pointed out that the mobile phase B of the present application is methanol, and ethanol is not used as the mobile phase B. This is because the viscosity of ethanol is too large, and the column pressure is too large when used as a mobile phase. Taking the viscosity of water as 1, the viscosity of ethanol is 1.2, and the viscosity of methanol is 0.6; in addition, the cost of using ethanol is higher than that of methanol, so ethanol is not used as the mobile phase B in the present application.

[0049] In some embodiments, the column temperature is 20-30°C.

[0050] In some embodiments, the injection volume is 10-20 μL.

[0051] In some embodiments, the mobile phase flow rate is 0.5-2 mL / min.

[0052] In some embodiments, the detection wavelength is 225-230 nm.

[0053] Specifically, the instrument is a Thermo Fisher high performance liquid chromatograph, and the high performance liquid chromatography conditions include: the chromatographic column is a reverse phase chromatographic column with a highly water-resistant bonded non-polar octadecyl functional group (ODS) (length 150 mm, inner diameter 4.6 mm, filler particle size 5 μm; Thermo, USA); the mobile phase includes mobile phase A and mobile phase B, mobile phase A is water, and mobile phase B is methanol; gradient elution is performed using mobile phase A and mobile phase B as eluents; detection wavelength: 230 nm; detection temperature: 25°C; flow rate: 1.0 mL / min; injection volume: 10 μL; time: 0 to 19 min; elution method: gradient elution.

[0054] In some embodiments, before detecting the impurity content in the vanillone sample, the structure of the vanillone is identified by nuclear magnetic resonance detection, infrared spectroscopy detection, and mass spectrometry detection.

[0055] In the above embodiment, the structure of the vanillone acetone sample was identified by nuclear magnetic resonance detection, infrared spectroscopy detection, and mass spectrometry detection. Specifically, the structure of the vanillone acetone sample was identified by nuclear magnetic resonance detection, infrared spectroscopy detection, and mass spectrometry detection. 1 HNMR spectroscopy and 13CNMR spectral measurement showed that the chemical shift and peak area of ​​each peak of the hydrogen spectrum and carbon spectrum were consistent with the theoretical peak of vanillin, indicating that the main component of the vanillin sample was vanillin; infrared spectral detection showed that the absorption peaks of each functional group were consistent with the theoretical peak of vanillin, indicating that the main component of the vanillin sample was vanillin; the test mass spectrum showed that the result was consistent with the theoretical value [M+H]+167.1.

[0056] In some embodiments, the detection of impurity content in the ethyl phenoxide sample also includes measuring the melting point of the ethyl phenoxide, and the result is between 114.4-115.3°C.

[0057] In some embodiments, the method further includes detecting the water content in the vanillone before detecting the impurity content in the vanillone.

[0058] Specifically, the moisture content is detected by Karl Fischer method, and the moisture content is measured, and the result obtained is less than 2%.

[0059] The above different methods are used to mutually verify the structures of the vanillin ethyl ketone samples to determine whether the vanillin ethyl ketone samples are qualified samples.

[0060] The following further illustrates the method for detecting impurity content in the vanillin acetone sample of the present application with specific examples. This section further illustrates the content of the present invention in conjunction with specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means adopted in the examples are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0061] Example 1

[0062] The present application also provides a method for detecting the impurity content in a vanillin ethyl ketone sample, using high performance liquid chromatography to detect the impurity content in the vanillin ethyl ketone;

[0063] The instrument is a Thermo Fisher high performance liquid chromatograph, and the high performance liquid chromatography conditions include:

[0064] The chromatographic column was a highly water-resistant reversed-phase column bonded with nonpolar octadecyl functional groups (ODS) (length 150 mm, inner diameter 4.6 mm, filler particle size 5 μm; Thermo, USA);

[0065] The mobile phase includes mobile phase A and mobile phase B, mobile phase A is water, and mobile phase B is methanol; gradient elution is performed using mobile phase A and mobile phase B as eluents;

[0066] Detection wavelength: 230nm;

[0067] Detection temperature: 25℃;

[0068] Flow rate: 1.0 mL / min;

[0069] Injection volume: 10 μL; Specifically, when injecting, take an appropriate amount of vanillyl ethyl ketone sample, dissolve and dilute it with methanol to make a solution containing 1 mg of vanillyl ethyl ketone per 1 mL, filter, take the filtrate as the test solution, and accurately measure 10 μL of the test solution for injection;

[0070] Time: 0-19 minutes;

[0071] Elution mode: gradient elution;

[0072] The elution gradient program included:

[0073] 0min: Mobile phase B accounts for 40% by volume and is maintained for 3min, while mobile phase A accounts for 60% by volume and is maintained for 3min;

[0074] 3-13min: the volume proportion of mobile phase B changes from 40% to 100%, and the volume proportion of mobile phase A changes from 60% to 0%;

[0075] 13-16min: the volume proportion of mobile phase B is maintained at 100%, and the volume proportion of mobile phase A is maintained at 0%;

[0076] 16-16.1min: the volume proportion of mobile phase B changes from 100% to 40%, and the volume proportion of mobile phase A changes from 0% to 60%;

[0077] 16.1~19min: the volume proportion of mobile phase B is maintained at 40%, and the volume proportion of mobile phase A is maintained at 60%.

[0078] The chromatogram obtained by detecting impurities in vanillyl acetone by high performance liquid chromatography in Example 1 is as follows: Figure 1 shown.

[0079] Specifically, Figure 1 In the figure, the peaks are attributed to:

[0080] Retention time 7.390min-Main product-vanillyl acetone

[0081] Retention time 8.083min-1-(2-hydroxy-3-methoxyphenyl)ethanone

[0082] Retention time 6.063min-guaiacol

[0083] Retention time 11.070min 2-Methoxyphenyl acetate

[0084] Figure 1The retention times of different substances in Figure 2 shown.

[0085] from Figure 2 It can be seen that the retention time of vanillone is 7.390min, the retention time of 1-(2-hydroxy-3-methoxyphenyl)ethanone is 8.083min, the retention time of guaiacol is 6.063min, and the retention time of 2-methoxyphenyl acetate is 11.070min; Figure 2 The substances corresponding to the retention times of 4.647min and 13.703min are unknown substances produced in the synthesis of vanillyl acetone, which will decrease as the amount of known impurities decreases.

[0086] Figure 2 In it, Area means peak area, Height means peak height, and Relative Area means relative peak area.

[0087] Relative Height indicates relative peak height. Based on the peak area and peak height, the specific parameters set within the software can directly calculate the amount of substance represented by each chromatographic peak, which is represented by the Amount value. It is generally not used, so it is displayed. Figure 1 The area normalization method is used for quantitative calculation, and the content of each impurity in the vanillone acetonide sample can be detected. Specifically, the mass content of guaiacol in the vanillone acetonide sample is 5.05%, the mass content of 1-(2-hydroxy-3-methoxyphenyl)ethanone is 0.73%, and the mass content of 2-methoxyphenyl acetate is 5.30%.

[0088] Example 2

[0089] The present application example provides a structural identification of a vanillyl acetone sample, specifically comprising:

[0090] A. Dissolve 15 mg of vanillyl acetone sample (batch 1) and 0.6 ml of deuterated DMSO reagent in a nuclear magnetic resonance tube and place it in a nuclear magnetic resonance spectrometer for 1HNMR spectrum (400 MHz) and 13CNMR spectrum (101 MHz) determination. The obtained spectra are as follows: Figure 3 , Figure 4 shown.

[0091] B. Accurately weigh 2 mg of vanillin acetone sample (batch number 1), weigh potassium bromide drying reagent, grind and stir in a mortar until the particle size is less than 2.5 μm, then put the sample in a 150°C oven to dry for 30 minutes, take out and press the slice, and finally put the pressed transparent slice into a Fourier transform infrared spectrometer for measurement. The obtained infrared spectrum is as follows: Figure 5 shown.

[0092] C. Accurately weigh 15 mg of vanillyl acetone sample (batch number 1), dissolve it in methanol, flow inject it into the mass spectrometer, and detect it in ESI positive ion mode. The obtained mass spectrum is as follows Figure 6 shown.

[0093] D. Add vanillin acetone sample (batch number 1) to a mortar and grind it. Place the capillary vertically on a watch glass. Fill the sample with a sealed melting point capillary and allow the capillary to fall freely from the top opening several times. This allows the powder to be tightly concentrated at a height of 1-2 mm at the end of the capillary seal. When the temperature of the melting point meter rises to 10°C (the minimum melting point specified for the sample), place the sample in a melting point tester and measure it. Record the melting point and obtain the melting range. The average value of 5 measurements is 114.9-115.7°C.

[0094] E. Determination of moisture content: The moisture content of the vanillyl ethyl ketone sample (batch 1) was determined by Karl Fischer moisture determination method. The average value of the three measurements was 1.08% (mass content).

[0095] Since the test results in Example 2 are consistent with the theoretical values, the quality of the vanillyl ethyl ketone sample (batch number 1) in Example 2 is qualified.

[0096] Example 3

[0097] The present application example provides a structural identification of a vanillyl acetone sample, specifically comprising:

[0098] A. Dissolve 15 mg of vanillyl ethyl ketone sample (batch 2) and 0.6 ml of deuterated DMSO reagent in a nuclear magnetic resonance tube and place it in a nuclear magnetic resonance spectrometer for 1HNMR spectrum (400 MHz) and 13CNMR spectrum (101 MHz) determination. The obtained hydrogen spectrum and carbon spectrum are the same as the results of the test sample (batch 1) in Example 1.

[0099] B. Accurately weigh 2 mg of vanillyl acetone sample (batch number 2), weigh potassium bromide drying reagent, grind and stir in a mortar until the particle size is less than 2.5 μm, then put the sample into a 150°C oven for drying for 30 minutes, take out and press the sheet, and finally place the pressed transparent sheet into a Fourier transform infrared spectrometer for determination. The obtained infrared spectrum is the same as the result of the test sample (batch number 1) in Example 1.

[0100] C. Accurately weigh 15 mg of vanillyl ethyl ketone sample (batch number 2), dissolve it in methanol, flow inject it into the mass spectrometer, and detect it in ESI positive ion mode. The obtained mass spectrum is the same as the result of the test sample (batch number 1) in Example 1.

[0101] D. Add vanillin acetone sample (batch 2) to the mortar and grind it. Place the capillary vertically on the watch glass. Fill the sample with the sealed melting point capillary and let the capillary fall freely from the top opening several times. This allows the powder to be tightly concentrated at a height of 1-2 mm at the end of the capillary seal. When the temperature of the melting point meter rises to 10°C (the minimum melting point specified by the sample), place the sample in the melting point tester for measurement, record the melting point, and obtain the melting range. The average value of 5 measurements is 113.9-115.1°C.

[0102] E. Determination of moisture content: The moisture content of the vanillyl ethyl ketone sample (batch 2) was determined by Karl Fischer moisture determination method. The average value of the three measurements was 2.58% (mass content).

[0103] As the water content exceeded the quality control limit, the test sample (Batch No. 2) failed the quality test.

[0104] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting impurity content in a vanillyl acetone sample, characterized in that: The impurity content in vanillyl acetone was determined by high performance liquid chromatography; Among them, the high performance liquid chromatography conditions include: The chromatographic column is a reverse phase chromatographic column bonded with a non-polar octadecyl functional group; The mobile phase includes mobile phase A and mobile phase B, mobile phase A is water, and mobile phase B is methanol; mobile phase A and mobile phase B are used as eluents for gradient elution, and the elution gradient program includes: 0min: Mobile phase B accounts for 40% by volume and is maintained for 3min, while mobile phase A accounts for 60% by volume and is maintained for 3min; 3-13min: the volume proportion of mobile phase B changes from 40% to 100%, and the volume proportion of mobile phase A changes from 60% to 0%; 13-16min: the volume proportion of mobile phase B is maintained at 100%, and the volume proportion of mobile phase A is maintained at 0%; 16-16.1min: the volume proportion of mobile phase B changes from 100% to 40%, and the volume proportion of mobile phase A changes from 0% to 60%; 16.1-19min: the volume proportion of mobile phase B is maintained at 40%, and the volume proportion of mobile phase A is maintained at 60%; The impurities are 1-(2-hydroxy-3-methoxyphenyl)ethanone, guaiacol and 2-methoxyphenyl acetate.

2. The method for detecting impurity content in a vanillyl acetone sample according to claim 1, characterized in that: The column temperature is 20-30℃.

3. The method for detecting impurity content in a vanillyl acetone sample according to claim 1, characterized in that: The injection volume is 10-20 μL.

4. The method for detecting impurity content in a vanillyl acetone sample according to claim 1, characterized in that: The flow rate of the mobile phase is 0.5~2mL / min.

5. The method for detecting impurity content in a vanillyl acetone sample according to claim 1, characterized in that: The detection wavelength is 225~230nm.

6. The method for detecting impurity content in a vanillyl acetone sample according to claim 1, characterized in that: Before the impurity content in vanillic ethyl ketone is detected, the structure of the vanillic ethyl ketone sample is identified by nuclear magnetic resonance detection, infrared spectroscopy detection, and mass spectrometry detection.

7. The method for detecting impurity content in a vanillyl acetone sample according to claim 1, characterized in that: The impurity content detection in ethyl phenoxide also includes measuring the melting point of the ethyl phenoxide sample.

8. The method for detecting impurity content in a vanillyl acetone sample according to claim 1, characterized in that: Before testing the impurity content in the vanillin ethyl ketone sample, the water content in the vanillin ethyl ketone sample is also tested.

9. The method for detecting impurity content in a vanillyl acetone sample according to claim 8, characterized in that: The moisture content was tested using the Karl Fischer method.

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