Method for measuring contents of auxiliary materials vanillin and ethyl vanillin in fudosteine oral solution

Abstract

The invention relates to a method for determining the content of vanillin and ethyl vanillin which are auxiliary materials in a fudosteine oral solution, which is simple to operate, has good sensitivity, specificity and accuracy, provides an effective prescription analysis method for drug development, and has practical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of analytical chemistry, and particularly relates to a method for determining the contents of excipients vanillin and ethyl vanillin in a fudosteine ​​oral solution. Background Art

[0002] Fudosteine ​​is S-(3-hydroxypropyl)-L-cysteine, a new type of expectorant, a compound with a steine ​​basic skeleton developed by SSP Pharmaceutical Co., Ltd. in 1988.

[0003] It was first launched in Japan on December 17, 2001. Experiments have shown that fudosteine's metabolites can reduce sputum viscosity, scavenge free radicals, prevent damage to lung elastin and neutrophils, reduce local inflammation, enhance and improve the penetration of antibiotics into the bronchial mucosa, and facilitate the treatment of various respiratory inflammatory conditions.

[0004] The prescription of the fudosteine ​​oral solution disclosed by the invention comprises: sorbitol, sodium benzoate, DL-malic acid, vanillin, ethyl vanillin and glycerol.

[0005] Currently, there is no description in the pharmacopoeias, patents and literatures of various countries on the detection methods of the excipients vanillin and ethyl vanillin in fudosteine ​​oral solution.

[0006] In order to effectively analyze drug quality and ensure drug safety, it is necessary to develop a method for determining the content of excipients that is highly sensitive, specific, durable, convenient, and effective. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a method for determining the content of vanillin and ethyl vanillin, excipients, in fudosteine ​​oral solution. The method is simple to operate and has good sensitivity, specificity, and accuracy. It provides an effective prescription analysis method for drug development and has practical significance.

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

[0009] A method for determining the content of vanillin and ethyl vanillin as excipients in a fudosteine ​​oral solution comprises liquid chromatography using a C18 column (CAPCELL PAK C18 4.6×250 mm, 5 μm); a mobile phase consisting of 0.5% formic acid solution: acetonitrile (80:20); a flow rate of 1.0 ml / min; a column temperature of 30° C.; a detection wavelength of 279 nm; and an injection volume of 20 μl.

[0010] Preferably, the chromatographic column is a C18 column. In the embodiment of the present invention, a chromatographic column with octadecylsilane bonded silica gel as a filler is used; the specification is 4.6×250 mm, 5 μm, and it is provided by OSAKA SODA.

[0011] Preferably, the mobile phase is 0.5% formic acid solution: acetonitrile (80:20).

[0012] Preferably, the flow rate is 1.0 ml / min.

[0013] Preferably, the column temperature is 30°C.

[0014] Preferably, the detection wavelength is 279 nm.

[0015] Preferably, the injection volume is 20 μl.

[0016] Preferably, the method comprises the following steps:

[0017] (1) Test solution: Accurately measure 2 ml of the product into a 20 ml volumetric flask, dilute to the mark with water, and shake well;

[0018] (2) Reference solution: Take 22 mg of vanillin, accurately weigh it, place it in a 20 ml volumetric flask, dissolve it with 0.1% formic acid in methanol solution and dilute it to the mark, shake well; take 20 mg of ethyl vanillin, accurately weigh it, place it in a 100 ml volumetric flask, dissolve it with 0.1% formic acid in methanol solution and dilute it to the mark, shake well; accurately measure 1 ml and 0.5 ml respectively, place them in the same 200 ml volumetric flask, dissolve it with 0.1% formic acid in methanol solution and dilute it to the mark, shake well, as the reference stock solution. Accurately measure 2 ml of the reference stock solution, place it in a 20 ml volumetric flask, dilute it with water to the mark, shake well;

[0019] (3) using liquid chromatography to measure the reference solution and the test solution respectively;

[0020] The detection conditions of the liquid chromatography method are as follows: chromatographic column C18 column (CAPCELL PAK C18 4.6×250 mm, 5 μm); mobile phase 0.5% formic acid solution: acetonitrile (80:20); flow rate 1.0 ml / min; column temperature 30° C.; detection wavelength 279 nm; injection volume 20 μl.

[0021] Calculation of results: Calculated according to the external standard method.

[0022] Due to the adoption of the above technical solution, the present invention has the following advantages compared with the prior art:

[0023] 1. The determination method of the present invention comprehensively analyzes the formulation of Fudosteine ​​oral solution and can effectively separate the excipients vanillin and ethyl vanillin;

[0024] 2. The determination method of the present invention has high sensitivity, strong specificity and good accuracy. It not only provides a simple and convenient detection method for the prescription analysis of fudosteine ​​oral solution, but also can strictly control the quality of fudosteine ​​oral solution, thereby ensuring its safety and reliability, and has practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a chromatogram of a blank solution in the present invention;

[0026] Figure 2 is a chromatogram of the blank matrix solution in the present invention;

[0027] Figure 3 Chromatogram of the solution positioning of each excipient in the present invention;

[0028] Figure 4 It is the chromatogram of the reference substance solution in the present invention;

[0029] Figure 5 It is the chromatogram of the test solution in the present invention; DETAILED DESCRIPTION

[0030] To make the technical solutions and beneficial effects of the present invention more clearly understood, the following is a detailed description with reference to the accompanying drawings and specific embodiments. It should be understood that these embodiments are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. Any product identical or similar to the present invention that is derived by anyone under the guidance of the present invention or by combining the features of the present invention with those of other prior arts shall fall within the scope of protection of the present invention.

[0031] The experimental methods in the following examples without specific conditions are generally based on conventional experimental conditions.

[0032] The fudosteine ​​oral solution, vanillin, ethyl vanillin and equipment used in the specific embodiments of the present invention are all known products, and the fudosteine ​​oral solution, vanillin and ethyl vanillin are obtained by purchasing commercially available products.

[0033] Table 1 Equipment information

[0034] Device Name model factory High-performance liquid chromatography Ultimate 3000 Thermo Fisher Scientific electronic balance 125SM-FR Prissex Weighing Equipment Co., Ltd. Pure water machine EUE-10UV Haisi Instrument Technology (Shanghai) Co., Ltd.

[0035] Table 2 Material information

[0036] name source batch number Formic acid AR 20231116 Methanol TEDIA 24046017 Acetonitrile TEDIA 24046019 Blank matrix Made by the pharmaceutical preparation R&D department JT002-240624-1 Fudosteine ​​oral solution Made by the pharmaceutical preparation R&D department JT002-240228-1

[0037] Table 3 Reference substance information

[0038] name factory batch number content% Vanillin Hunan Erkang Pharmaceutical Co., Ltd. 106020230601 99.4 Ethyl vanillin Guangzhou Daily Chemical Co., Ltd. BEV2208401 99.99

[0039] Example 1

[0040] Methodological research on the detection and analysis method of the present invention

[0041] Various tests in this embodiment all adopt the following conditions:

[0042] Chromatographic column: octadecylsilane bonded silica gel as filler (C18, 4.6×250 mm, 5 μm);

[0043] Mobile phase: 0.5% formic acid solution: acetonitrile (80:20);

[0044] Flow rate: 1.0 ml / min;

[0045] Detection wavelength: 279nm;

[0046] Column temperature: 30°C;

[0047] Injection volume: 20 μl.

[0048] Testing steps:

[0049] Blank solution: water.

[0050] Test solution: Accurately measure 2 ml of the product, place it in a 20 ml volumetric flask, dilute with water to the mark, and shake well.

[0051] Reference solution: Accurately weigh 22 mg of vanillin, place in a 20 ml volumetric flask, dissolve in 0.1% formic acid in methanol, dilute to the mark, and shake well. Accurately weigh 20 mg of ethyl vanillin, place in a 100 ml volumetric flask, dissolve in 0.1% formic acid in methanol, dilute to the mark, and shake well. Accurately measure 1 ml and 0.5 ml of each, place in the same 200 ml volumetric flask, dissolve in 0.1% formic acid in methanol, dilute to the mark, and shake well to prepare the reference stock solution. Accurately measure 2 ml of the reference stock solution, place in a 20 ml volumetric flask, dilute to the mark with water, and shake well.

[0052] 1. Specificity test

[0053] Solution preparation:

[0054] Blank solvent: water.

[0055] Blank matrix solution: Accurately measure 2 ml of blank matrix, place it in a 20 ml volumetric flask, dilute to the scale with water, and shake well.

[0056] Vanillin positioning solution: Take 22 mg of vanillin, accurately weigh it, place it in a 20 ml volumetric flask, dissolve it with 0.1% formic acid methanol solution and dilute it to the scale, and shake well.

[0057] Ethyl vanillin positioning solution: Take 20 mg of ethyl vanillin, accurately weigh it, place it in a 100 ml volumetric flask, dissolve it in 0.1% formic acid methanol solution and dilute it to the scale, and shake well.

[0058] Reference solution: Accurately weigh 22 mg of vanillin, place in a 20 ml volumetric flask, dissolve in 0.1% formic acid in methanol, dilute to the mark, and shake well. Accurately weigh 20 mg of ethyl vanillin, place in a 100 ml volumetric flask, dissolve in 0.1% formic acid in methanol, dilute to the mark, and shake well. Accurately measure 1 ml and 0.5 ml of each, place in the same 200 ml volumetric flask, dissolve in 0.1% formic acid in methanol, dilute to the mark, and shake well to prepare the reference stock solution. Accurately measure 2 ml of the reference stock solution, place in a 20 ml volumetric flask, dilute to the mark with water, and shake well.

[0059] Test solution: Accurately measure 2 ml of the product, place it in a 20 ml volumetric flask, dilute with water to the mark, and shake well.

[0060] Accurately measure 20 μl of blank solvent, blank matrix solution, each excipient positioning solution, reference solution and test solution and inject them into the high performance liquid chromatograph respectively, record the chromatogram, and the test results are shown in the attached Figure 1 ~Attachment Figure 5 And Table 4 below.

[0061] Table 4 Specificity test results

[0062]

[0063] From the above results, it can be seen that the unknown peaks in the blank solvent, blank matrix solution and test solution have no interference with the detection of vanillin and ethyl vanillin; the separation degree between vanillin and ethyl vanillin in the reference solution is 20.50, which meets the requirements, and the method has good specificity.

[0064] 2. Precision test

[0065] Solution preparation:

[0066] Reference solution: Accurately weigh 22 mg of vanillin, place in a 20 ml volumetric flask, dissolve in 0.1% formic acid in methanol, dilute to the mark, and shake well. Accurately weigh 20 mg of ethyl vanillin, place in a 100 ml volumetric flask, dissolve in 0.1% formic acid in methanol, dilute to the mark, and shake well. Accurately measure 1 ml and 0.5 ml of each, place in the same 200 ml volumetric flask, dissolve in 0.1% formic acid in methanol, dilute to the mark, and shake well to prepare the reference stock solution. Accurately measure 2 ml of the reference stock solution, place in a 20 ml volumetric flask, dilute to the mark with water, and shake well.

[0067] Take the reference solution and inject it 5 times continuously, record the peak area and retention time, and calculate the RSD values ​​of the peak area and retention time. The test results are shown in Tables 5 and 6 below.

[0068] Table 5 Vanillin injection precision test results

[0069]

[0070]

[0071] Table 6 Ethyl vanillin injection precision test results

[0072]

[0073] From the above results, we can see that: after 5 consecutive injections of the reference solution, the RSD values ​​of the peak areas of vanillin and ethyl vanillin were 0.31 and 4.07%, respectively, both of which were no more than 5.0%; the RSD values ​​of the retention times of vanillin and ethyl vanillin were 0.02% and 0.09%, respectively, both of which were no more than 5.0%. This method has good injection precision.

[0074] 3. Linearity and range test

[0075] Solution preparation:

[0076] Reference substance stock solution: Take 22 mg of vanillin, accurately weigh it, place it in a 20 ml volumetric flask, dissolve it with 0.1% formic acid methanol solution and dilute it to the scale, shake well; take 20 mg of ethyl vanillin, accurately weigh it, place it in a 100 ml volumetric flask, dissolve it with 0.1% formic acid methanol solution and dilute it to the scale, shake well; accurately measure 1 ml and 0.5 ml respectively, place them in the same 200 ml volumetric flask, dissolve it with 0.1% formic acid methanol solution and dilute it to the scale, shake well, as the reference substance stock solution.

[0077] Take an appropriate amount of reference substance stock solution to prepare linear solutions of various concentrations according to the table below.

[0078] Linear concentration Volume of reference stock solution (ml) Dilute to (ml) 25％ 0.25 10 50％ 0.5 10 75％ 0.75 10 100％ 1 10 150％ 1.5 10 200％ 2 10

[0079] Accurately measure 20 μl of each linear solution, inject it into the high performance liquid chromatograph, record the chromatogram, and perform linear regression analysis on the concentration using the peak area. The test results are shown in Tables 7 and 8 below.

[0080] Table 7 Vanillin linearity test results

[0081]

[0082] Table 8 Ethyl vanillin linearity test results

[0083]

[0084] From the above results, we can see that the correlation coefficient R 2 is 0.9997, not less than 0.998; ethyl vanillin is in the concentration range of 0.01268μg / ml to 0.1014μg / ml, the correlation coefficient R 2 It is 0.9988, not less than 0.998, and the method has a good linear relationship.

[0085] 4. Detection limit test

[0086] Solution preparation:

[0087] Solvent: water.

[0088] Take each linear point solution prepared under 3. Linear range test and dilute it step by step. The solution with a signal-to-noise ratio of not less than 10:1 is used as the quantification limit solution, and the solution with a signal-to-noise ratio of not less than 3:1 is used as the detection limit solution.

[0089] The above diluted solutions were respectively injected into a high performance liquid chromatograph, and the chromatograms were recorded. The test results are shown in Tables 9 and 10 below.

[0090] Table 9 Results of detection limits of vanillin

[0091] Vanillin <![CDATA[Concentration( μg / ml )]]> S / N <![CDATA[Reference substance concentration( μg / ml )]]> Ratio of concentration to reference substance (%) Detection limit 0.01114 14.5 0.5571 2.00 Limit of quantification 0.02229 22.5 0.5571 4.00

[0092] Table 10 Detection limit results of ethyl vanillin

[0093]

[0094] From the above results, it can be seen that the ratios of the detection limit concentrations of vanillin and ethyl vanillin to the reference concentrations are 2.00% and 20.00%, respectively, both of which are not greater than 20% of the reference concentrations; the ratios of the quantitative limit concentrations of vanillin and ethyl vanillin to the reference concentrations are 4.00% and 50.00%, respectively, both of which are not greater than 50% of the reference concentrations. This method has high detection sensitivity.

[0095] 5. Repeatability test

[0096] Solution preparation:

[0097] Solvent: water.

[0098] Blank matrix solution: Prepare the same as in 1. Specificity test.

[0099] Test solution: Prepare the same as in 1. Specificity test.

[0100] Reference solution: Prepare the same as in 1. Specificity test.

[0101] 20 μl of each of the above solutions was injected into a high performance liquid chromatograph, the chromatogram was recorded, and the amount of vanillin and ethyl vanillin in the test sample was calculated using the external standard method. The test results are shown in Tables 11 and 12 below.

[0102] Table 11 Vanillin repeatability test results

[0103]

[0104] Table 12 Ethyl vanillin repeatability test results

[0105]

[0106] From the above results, we can see that the RSD values ​​of the measured amounts of vanillin and ethyl vanillin in the 6 test solutions are 0.92% and 2.34%, respectively, both of which are no more than 5.0%. The method has good repeatability.

[0107] 6. Accuracy test

[0108] Solution preparation:

[0109] Solvent: water.

[0110] Reference substance stock solution: Take 22 mg of vanillin, accurately weigh it, place it in a 20 ml volumetric flask, dissolve it with 0.1% formic acid methanol solution and dilute it to the scale, shake well; take 20 mg of ethyl vanillin, accurately weigh it, place it in a 100 ml volumetric flask, dissolve it with 0.1% formic acid methanol solution and dilute it to the scale, shake well; accurately measure 1 ml and 0.5 ml respectively, place them in the same 200 ml volumetric flask, dissolve it with 0.1% formic acid methanol solution and dilute it to the scale, shake well, as the reference substance stock solution.

[0111] Reference substance solution: Accurately measure 2 ml of reference substance stock solution, place it in a 20 ml volumetric flask, dilute to the scale with water, and shake well.

[0112] Blank matrix solution: Accurately measure 2 ml of blank matrix, place it in a 20 ml volumetric flask, dilute to the scale with water, and shake well.

[0113] 50% recovery solution: Accurately measure 2 ml of blank matrix and 1 ml of reference stock solution, place them in the same 20 ml volumetric flask, dilute to the mark with water, shake well, and prepare 3 parallel aliquots.

[0114] 100% recovery solution: Accurately measure 2 ml of blank matrix and 2 ml of reference stock solution, place them in the same 20 ml volumetric flask, dilute to the mark with water, shake well, and prepare 3 parallel aliquots.

[0115] 150% recovery solution: Accurately measure 2 ml of blank matrix and 3 ml of reference stock solution, place them in the same 20 ml volumetric flask, dilute to the mark with water, shake well, and prepare 3 parallel aliquots.

[0116] Accurately measure 20 μl of each of the above solutions for injection, record the chromatogram, calculate using the external standard method, compare the measured amount with the added amount, and calculate the recovery rate. The test results are shown in Tables 13 and 14 below.

[0117] Table 13 Vanillin accuracy test results

[0118]

[0119] Table 14 Ethyl vanillin accuracy test results

[0120]

[0121] From the above results, it can be seen that the average value of the 9 recovery rate data at various vanillin concentrations is 98.6%, all ranging from 95.0% to 105.0%, and the RSD value of the 9 recovery rate data is 1.23%, which is no more than 5.0%; the average value of the 9 recovery rate data at various ethyl vanillin concentrations is 98.1%, all ranging from 95.0% to 105.0%, and the RSD value of the 9 recovery rate data is 2.20%, which is no more than 5.0%; this method has good accuracy.

[0122] 7. Solution stability test

[0123] Solution preparation:

[0124] Solvent: water.

[0125] Take the reference solution (prepared in the same way as in 1. Specificity test) and place them at room temperature.

[0126] 20 μl of the above-mentioned reference solution was respectively injected into the liquid chromatograph, and the chromatogram was recorded. The test results are shown in Table 15 below.

[0127] Table 15 Reference solution storage stability test results

[0128] Placement time 3 days Content of vanillin reference solution (%) 99.7 Ethyl vanillin reference solution content (%) 98.8

[0129] From the above results, it can be seen that the contents of vanillin and ethyl vanillin reference solutions after being placed at room temperature for 3 days are 99.7% and 98.8% respectively, both between 98.0% and 102.0%. The reference solutions are stable after being placed at room temperature for at least 3 days.

[0130] In summary, the present invention provides a method suitable for determining the content of vanillin and ethyl vanillin, excipients, in fudosteine ​​oral solution. The method has good specificity, and baseline separation can be achieved between the vanillin and ethyl vanillin peaks; the system applicability is good; the method is extremely sensitive, and the detection limit can reach as low as 0.01014 μg / ml; the method has good repeatability and accuracy, and provides a convenient detection method for the prescription analysis of fudosteine ​​oral solution, so as to prepare products with quality and efficacy consistent with the original drug.

[0131] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations of the claims. Various modifications and variations may be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form additional embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments merely illustrate several implementations of the present invention and do not limit the scope of protection of the patent of this invention.

Claims

1. A method for determining the content of vanillin and ethyl vanillin in fudosteine ​​oral solution, characterized in that: High performance liquid chromatography was used for detection, and the chromatographic conditions were as follows: the chromatographic column was filled with octadecylsilane bonded silica gel (C18, 4.6×250 mm, 5 μm); the mobile phase was 0.5% formic acid solution: acetonitrile (80:20) for isocratic elution; the flow rate was 1.0 ml / min; the detection wavelength was 279 nm; the column temperature was 30°C; and the injection volume was 20 μl.

2. The method for determining the content of excipients vanillin and ethyl vanillin in the fudosteine ​​oral solution according to claim 1, characterized in that: The chromatographic column is a chromatographic column using octadecylsilane bonded silica gel as a filler; the specification is 4.6 mm×250 mm, the film thickness is 5 μm, and it is provided by OSAKA SODA Company.

3. The measuring method according to claim 1, wherein The C18 column is CAPCELL PAK C18, and the specifications of the C18 chromatographic column are 4.6×250 mm×5 μm.

4. The measuring method according to claim 1, wherein The mobile phase is 0.5% formic acid solution: acetonitrile (55-80:45-20). Preferably, the mobile phase is 0.5% formic acid solution: acetonitrile (80:20).

5. The measuring method according to claim 1, wherein The flow rate is 0.5 ml / min to 1.5 ml / min, preferably, the flow rate is 1.0 ml / min.

6. The measuring method according to claim 1, wherein The detection wavelength is 270nm to 290nm, preferably, the detection wavelength is 279nm.

7. The measuring method according to claim 1, wherein The column temperature is 25°C to 35°C, preferably, the column temperature is 30°C.

8. The method for determining the content of vanillin and ethyl vanillin as excipients in the fudosteine ​​oral solution according to any one of claims 1 to 7, characterized in that: The following steps are involved: (1) Test solution: Accurately measure 2 ml of the product into a 20 ml volumetric flask, dilute to the mark with water, and shake well; (2) Reference solution: Take 22 mg of vanillin, accurately weigh it, place it in a 20 ml volumetric flask, dissolve it with 0.1% formic acid in methanol solution and dilute it to the mark, shake well; take 20 mg of ethyl vanillin, accurately weigh it, place it in a 100 ml volumetric flask, dissolve it with 0.1% formic acid in methanol solution and dilute it to the mark, shake well; accurately measure 1 ml and 0.5 ml respectively, place them in the same 200 ml volumetric flask, dissolve it with 0.1% formic acid in methanol solution and dilute it to the mark, shake well, as the reference stock solution. Accurately measure 2 ml of the reference stock solution, place it in a 20 ml volumetric flask, dilute it with water to the mark, shake well; (3) Accurately pipette 20 μl of the reference solution and the test solution respectively and inject them into the high performance liquid chromatograph for determination.

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