Method for determining impurities in gastrodin through gradient method

Abstract

The invention discloses a method for determining impurities in gastrodin by a gradient method. The method comprises the following steps: (1) preparing a test solution; (2) preparing a contrast solution; (3) high performance liquid chromatography elution test: gradient elution and spectrogram comparison are carried out, and the impurity content in the test solution is calculated; a mobile phase A is an acetonitrile-0. 1% phosphoric acid solution with the volume ratio of 3: 97; an acetonitrile-0. 1% phosphoric acid solution with the volume ratio of 45: 55 is used as a mobile phase B; according to the method for determining the impurities in the gastrodin through the gradient method, through gradient testing, fat-soluble impurities in the gastrodin can be in time and all peaks appear, all impurity peaks are effectively separated, the accuracy of a testing result is improved, and the method has beneficial effects on improving the purity and the quality of the gastrodin.

Description

Technical Field

[0001] The invention relates to the technical field of organic compound purification, in particular to a method for determining impurities in gastrodin using a gradient method. Background Art

[0002] Gastrodin is an organic compound extracted from the dried root of Gastrodia elata, an orchid plant. The raw material is produced in Yunnan. Gastrodin has a good sedative and hypnotic effect, and can relieve symptoms of neurasthenia, insomnia, and headache. The traditional Chinese medicine Gastrodia elata can treat headache, dizziness, limb numbness, and convulsions. In clinical applications, it can be used to treat vertebral-basilar artery insufficiency, vestibular neuronitis, and vertigo.

[0003] In the prior art, the following problems exist in the process of gastrodin purity inspection: fat-soluble impurities are difficult to elute and the peaks are incomplete, resulting in a low impurity content in the collected spectrum; the separation degree between fat-soluble impurities and water-soluble impurities is poor, affecting the accuracy of the analysis results. Summary of the invention

[0004] The present invention solves the above problems existing in the prior art by providing a method for determining impurities in gastrodin by a gradient method.

[0005] In order to solve the above technical problems, the present invention provides a method for determining impurities in gastrodin by a gradient method, comprising the following steps:

[0006] (1) Prepare the test solution: weigh a certain amount of gastrodin sample and place it in a volumetric flask, add water to dissolve it, and make up to volume to obtain the test solution;

[0007] (2) Prepare the control solution: measure a certain amount of the above test solution into a volumetric flask, dilute it with water and make up to volume to obtain the control solution;

[0008] (3) HPLC elution test: The test solution and control solution are injected into a liquid chromatograph, gradient elution is performed, spectra are compared, and the impurity content in the test solution is calculated;

[0009] The conditions used in the HPLC test include:

[0010] Filler: octadecylsilane bonded silica gel;

[0011] Mobile phase: acetonitrile-0.1% phosphoric acid solution with a volume ratio of 3:97 as mobile phase A; acetonitrile-0.1% phosphoric acid solution with a volume ratio of 45:55 as mobile phase B;

[0012] The detection wavelength is 220 nm.

[0013] In a preferred embodiment of the present invention, the concentration of the test solution is 0.5 mg / L.

[0014] In a preferred embodiment of the present invention, the concentration of the control solution is 5 μg / ml.

[0015] In a preferred embodiment of the present invention, the specifications of the filler are 4.6 μm and 150 mm.

[0016] In a preferred embodiment of the present invention, the gradient elution conditions are as follows:

[0017] 0-6min, the proportion of mobile phase A is 100%, and the proportion of mobile phase B is 0%;

[0018] From 6 to 10 min, the proportion of mobile phase A changed from 100% to 0%, and the proportion of mobile phase B changed from 0% to 100%;

[0019] From 10 to 16 min, the proportion of mobile phase A remained unchanged at 0%, and the proportion of mobile phase B remained unchanged at 100%;

[0020] 16-18 min, the proportion of mobile phase A changed from 0% to 100%, and the proportion of mobile phase B changed from 100% to 0%;

[0021] From 18 to 25 min, the proportion of mobile phase A remained unchanged at 100%, and the proportion of mobile phase B remained unchanged at 0%.

[0022] In a preferred embodiment of the present invention, in gradient elution, the flow rates of the mobile phase A and the mobile phase B are both 1 ml / min.

[0023] The beneficial effects of the present invention are as follows: the present invention provides a method for determining impurities in gastrodin by a gradient method. Through gradient testing, the fat-soluble impurities in gastrodin can be timely and completely peaked, and the impurity peaks can be effectively separated, thereby improving the accuracy of the test results and having a beneficial effect on improving the purity and quality of gastrodin. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a liquid chromatogram of the impurities in gastrodin determined by the gradient method of the present invention;

[0025] Figure 2 It is a liquid chromatogram for determining impurities in gastrodin in the prior art. DETAILED DESCRIPTION

[0026] The preferred embodiments of the present invention are described in detail below in conjunction with the accompanying drawings so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention.

[0027] In the existing gastrodin detection process, it was found that some batches of gastrodin had low impurity content. In this case, after excluding factors such as solvent and water, some impurities, such as impurities with strong fat solubility, had peaks that were not eluted under the detection conditions. This was because they were not eluted or the elution time was very late under the low proportion of acetonitrile liquid phase conditions, resulting in a low impurity content in the collected impurity spectrum. If the acetonitrile ratio is adjusted too high, although the fat-soluble impurities can be peaked earlier, the water-soluble impurities will also be peaked too early, resulting in poor separation between fat-soluble impurities and water-soluble impurities.

[0028] Therefore, in view of the above findings, the present invention discloses a method for determining impurities in gastrodin by a gradient method. The process of the method is specifically described below through examples.

[0029] Example 1

[0030] A method for determining impurities in gastrodin by a gradient method comprises the following steps:

[0031] (1) Prepare the test solution: accurately weigh 50 mg of gastrodin sample and place it in a 100 ml volumetric flask, dissolve it with purified water, and dilute to obtain a test solution with a concentration of 0.5 mg / ml;

[0032] (2) Prepare the control solution: Accurately measure 1 ml of the above test solution into a 100 ml volumetric flask, dilute with water and make up to volume to obtain a control solution with a concentration of 5 μg / ml;

[0033] (3) HPLC elution test: The test solution and control solution are injected into a liquid chromatograph, gradient elution is performed, spectra are compared, and the impurity content in the test solution is calculated;

[0034] Specifically, the conditions used in the high performance liquid chromatography test include:

[0035] Filler: octadecylsilane bonded silica gel, 4.6μm, 150mm;

[0036] Mobile phase: acetonitrile-0.1% phosphoric acid solution with a volume ratio of 3:97 as mobile phase A; acetonitrile-0.1% phosphoric acid solution with a volume ratio of 45:55 as mobile phase B;

[0037] Mobile phase flow rate: 1 ml / min;

[0038] The detection wavelength is 220 nm.

[0039] The gradient elution conditions are:

[0040] 0-6min, the proportion of mobile phase A is 100%, and the proportion of mobile phase B is 0%;

[0041] From 6 to 10 min, the proportion of mobile phase A changed from 100% to 0%, and the proportion of mobile phase B changed from 0% to 100%;

[0042] From 10 to 16 min, the proportion of mobile phase A remained unchanged at 0%, and the proportion of mobile phase B remained unchanged at 100%;

[0043] 16-18 min, the proportion of mobile phase A changed from 0% to 100%, and the proportion of mobile phase B changed from 100% to 0%;

[0044] From 18 to 25 minutes, the proportion of mobile phase A remained unchanged at 100%, and the proportion of mobile phase B remained unchanged at 0%; the details are shown in Table 1 below.

[0045] Table 1

[0046] Time / min Flow rate (ml / min) ％A ％B 0 1 100 0 6 1 100 0 10 1 0 100 16 1 0 100 18 1 100 0 25 1 100 0

[0047] The chromatogram obtained according to the above test conditions is as follows Figure 1 The peak results are shown in Table 2 below.

[0048] Table 2

[0049] Retention time (minutes) Area(μV*sec) Height (microvolt) area% 1 2.279 740 184 0.01 2 3.535 11969617 1740808 98.96 3 12.179 14622 2457 0.12 4 15.138 101019 10869 0.84 5 17.158 9325 901 0.08

[0050] Comparative Example 1

[0051] (1) Prepare the test solution: accurately weigh 50 mg of gastrodin sample and place it in a 100 ml volumetric flask, dissolve it with purified water, and dilute to obtain a test solution with a concentration of 0.5 mg / ml;

[0052] (2) Prepare the control solution: Accurately measure 1 ml of the above test solution into a 100 ml volumetric flask, dilute with water and make up to volume to obtain a control solution with a concentration of 5 μg / ml;

[0053] (3) HPLC elution test: The test solution and control solution are injected into a liquid chromatograph for elution and spectrum comparison, and the impurity content in the test solution is calculated;

[0054] Specifically, the conditions used in the high performance liquid chromatography test include:

[0055] Filler: octadecylsilane bonded silica gel, 4.6μm, 150mm;

[0056] Mobile phase: acetonitrile-0.1% phosphoric acid solution with a volume ratio of 3:97 was used as the mobile phase;

[0057] Mobile phase flow rate: 1 ml / min;

[0058] The detection wavelength is 220 nm.

[0059] The chromatogram obtained according to the above test conditions is as follows Figure 2 The peak results are shown in Table 3 below.

[0060] Table 3

[0061] Retention time (minutes) Area(μV*sec) Height (microvolt) area% 1 2.295 627 184 0.01 2 3.666 12095973 1740808 98.96 3 10.351 2305 2457 0.12

[0062] Depend on Figure 1 and Figure 2 By comparison, it can be seen that the peak at about 3.5 minutes is the gastrodin peak, and the peak after 12 minutes is the impurity peak that is not eluted and separated by the method in Comparative Document 1. It can be seen that the method of the present invention can make all impurities emerge in time, and realize the effective separation of impurity peaks, with excellent results.

[0063] According to the requirements of the Chinese Pharmacopoeia, the reliability of the present invention was verified by methodology, and the structure is shown in Table 3 below.

[0064] Table 3

[0065]

[0066] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for determining impurities in gastrodin by gradient method, characterized in that: The steps include: (1) Prepare the test solution: weigh a certain amount of gastrodin sample and place it in a volumetric flask, add water to dissolve it, and make up to volume to obtain the test solution; (2) Prepare the control solution: measure a certain amount of the above test solution into a volumetric flask, dilute it with water and make up to volume to obtain the control solution; (3) HPLC elution test: The test solution and control solution are injected into a liquid chromatograph, gradient elution is performed, spectra are compared, and the impurity content in the test solution is calculated; The conditions used in the HPLC test include: Filler: octadecylsilane bonded silica gel; Mobile phase: acetonitrile-0.1% phosphoric acid solution with a volume ratio of 3:97 as mobile phase A; acetonitrile-0.1% phosphoric acid solution with a volume ratio of 45:55 as mobile phase B; The detection wavelength is 220 nm.

2. The method for determining impurities in gastrodin by a gradient method according to claim 1, characterized in that: The concentration of the test solution is 0.5 mg / L.

3. The method for determining impurities in gastrodin by a gradient method according to claim 1, characterized in that: The concentration of the control solution was 5 μg / ml.

4. The method for determining impurities in gastrodin by a gradient method according to claim 1, characterized in that: The specifications of the filler are 4.6 μm and 150 mm.

5. The method for determining impurities in gastrodin by a gradient method according to claim 1, characterized in that: The gradient elution conditions are as follows: 0-6min, the proportion of mobile phase A is 100%, and the proportion of mobile phase B is 0%; From 6 to 10 min, the proportion of mobile phase A changed from 100% to 0%, and the proportion of mobile phase B changed from 0% to 100%; From 10 to 16 min, the proportion of mobile phase A remained unchanged at 0%, and the proportion of mobile phase B remained unchanged at 100%; 16-18 min, the proportion of mobile phase A changed from 0% to 100%, and the proportion of mobile phase B changed from 100% to 0%; From 18 to 25 min, the proportion of mobile phase A remained unchanged at 100%, and the proportion of mobile phase B remained unchanged at 0%.

6. The method for determining impurities in gastrodin by a gradient method according to claim 5, characterized in that: In the gradient elution, the flow rates of the mobile phase A and the mobile phase B were both 1 ml / min.

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