HPLC (High Performance Liquid Chromatography) detection method for measuring content of malonic acid in ferulic acid

Abstract

The invention provides an HPLC (High Performance Liquid Chromatography) detection method for determining the content of malonic acid in ferulic acid, which comprises the following steps: preparing a to-be-detected sample solution and a reference substance solution, carrying out gradient elution on the solutions by adopting a high performance liquid chromatography method, controlling the mobile phase A to be composed of acetonitrile, methanol and a buffer solution, controlling the mobile phase B to be any one of acetonitrile, methanol, ethanol and tetrahydrofuran, and controlling the mobile phase C to be the same as the mobile phase D; and setting parameters of a high-performance chromatograph for gradient elution. The HPLC detection method for measuring the content of malonic acid in ferulic acid provided by the invention is relatively stable, the detection accuracy of malonic acid is improved, the operation is simple, and the detection result is reliable.

Description

Technical Field

[0001] The invention relates to the field of drug detection, and particularly relates to an HPLC detection method for determining the content of malonic acid in ferulic acid. Background Art

[0002] The chemical name of ferulic acid is 3-methoxy-4-hydroxycinnamic acid, which is one of the effective ingredients of the traditional Chinese medicines Ferula, Angelica, Chuanxiong, Cimicifuga, and Ziziphus jujuba seeds. Ferulic acid has two isomers, cis and trans, both of which are light yellow solids. As a phenolic plant component, ferulic acid has strong antioxidant activity and radiation protection effects, and has a great promoting effect on human health. Ferulic acid can significantly increase the content of glutathione and nicotinamide adenine dinucleotide phosphate in irradiated cells, and has a protective effect on irradiated endothelial cells. Heme oxygenase is an enzyme with antioxidant effects. It can convert heme into biliverdin and finally into the antioxidant bilirubin. Ferulic acid can regulate the expression of this enzyme well, thereby playing a protective role in radiation protection. The main preparation method of ferulic acid is chemical synthesis, including the synthesis of ferulic acid by Wittig-Horner reaction and the synthesis of ferulic acid by Kneoevenagel reaction. The industrial synthesis method improves the production efficiency of ferulic acid, resulting in impurities such as malonic acid in ferulic acid, which need to be quantitatively removed. Therefore, it is particularly important to accurately determine the content of malonic acid in ferulic acid. At present, the main determination method has a high performance liquid chromatography detection method, but the detection conditions such as mobile phase, flow velocity, column temperature, etc. in the detection affect the determination accuracy of malonic acid, including the selection of mobile phase solvent, the control of flow velocity and the adjustment of column temperature. Therefore, it is of application value to provide a HPLC detection method for determining the content of malonic acid in ferulic acid. Summary of the invention

[0003] In view of this, the present invention proposes an HPLC detection method for determining the content of malonic acid in ferulic acid to solve the above problems.

[0004] The technical solution of the present invention is achieved in this way:

[0005] A HPLC detection method for determining the content of malonic acid in ferulic acid comprises the following steps:

[0006] S1. Prepare the sample solution: weigh the ferulic acid sample, use methanol as solvent, ultrasonically dissolve it, cool it, and make up the volume to prepare the test solution;

[0007] S2. Prepare a control solution: weigh a malonic acid reference substance, use methanol as a solvent, ultrasonically dissolve, cool, and dilute to obtain a control solution;

[0008] S3. Use high performance liquid chromatography to perform gradient elution on the sample solution and the reference solution. The mobile phase A is composed of acetonitrile, methanol and buffer solution, and the mobile phase B is any one of acetonitrile, methanol, ethanol and tetrahydrofuran. After elution, the malonic acid content is determined.

[0009] Furthermore, in step S1 and step S2, the mass ratio of ferulic acid to methanol is 2.5-3.5:1.

[0010] Furthermore, in step S1 and step S2, the frequency of the ultrasonic dissolution is 20-30 kHz, the power is 100-120 W, and the time is 8-12 min.

[0011] Furthermore, in step S3, the buffer solution is a phosphoric acid solution with a mass fraction of 0.1%-0.2%.

[0012] Furthermore, in step S3, the volume ratio of acetonitrile, methanol and buffer solution is 1:1:0.5-0.8.

[0013] Further, in step S3, the gradient elution, the elution procedure is:

[0014] Time min Mobile phase A (v / v) Mobile phase B (v / v) 0-5 90％ 10％ 5.1-15 70％ 30％ 15.1-25.0 50％ 50％ 25.1-45.0 70％ 30％ 45.1-55 90％ 10％ .

[0015] Furthermore, in step S3, the high-efficiency chromatograph uses a C18 chromatographic column with a length of 240-250 mm, an inner diameter of 2-4.6 mm, and a filler particle size of 3-5 μm.

[0016] Further, in step S3, the detection wavelength is 210-250 nm; the elution flow rate is 1.0-1.4 mL / min, and the time is 50-60 min.

[0017] Furthermore, the injection volume was 14-16 μL.

[0018] Furthermore, the detection limit of malonic acid is 100.0-102.0 ppm.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The high performance liquid chromatography analysis method of the present invention is adopted, the mobile phase is optimized, the test peak shape is good, and the linear relationship, recovery rate, repeatability and stability are all good. The inventive method can accurately detect the content of malonic acid in ferulic acid. The method can stably detect samples and maintain the stability of column efficiency. Moreover, the separation degree of malonic acid in the test sample, system suitability, specificity, detection limit and accuracy all meet the requirements, and the content of malonic acid in ferulic acid can be accurately detected, the operation is relatively simple, and the practicability is relatively strong. DETAILED DESCRIPTION

[0021] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0022] Unless otherwise specified, the experimental methods used in the examples of the present invention are all conventional methods. The materials, reagents, etc. used in the examples of the present invention, unless otherwise specified, can all be obtained from commercial channels.

[0023] Example 1

[0024] A HPLC detection method for determining the content of malonic acid in ferulic acid comprises the following steps:

[0025] Step (a), preparing a sample solution to be tested: weighing a ferulic acid sample, using methanol as a solvent, with a mass ratio of ferulic acid to methanol of 2.5:1, ultrasonically dissolving at a frequency of 20 kHz, a power of 100 W, and a time of 8 min, cooling, and fixing the volume to obtain a sample solution;

[0026] Step (ii), preparing a control solution: weighing a malonic acid reference substance, using methanol as a solvent, with a mass ratio of the reference substance to methanol of 2.5:1, ultrasonically dissolving at a frequency of 20 kHz, a power of 100 W, and a time of 8 min, cooling, and fixing the volume to obtain a control solution;

[0027] Step (iii), using high performance liquid chromatography to perform gradient elution on the sample solution and the reference solution, the mobile phase A is composed of acetonitrile, methanol and a buffer solution, the buffer solution is a phosphoric acid solution with a mass fraction of 0.1%, and the volume ratio of acetonitrile, methanol and the buffer solution is 1:1:0.5; the mobile phase B is acetonitrile, and the malonic acid content is determined after elution.

[0028] The gradient elution program was:

[0029] Time min Mobile phase A (v / v) Mobile phase B (v / v) 0-5 90％ 10％ 5.1-15 70％ 30％ 15.1-25.0 50％ 50％ 25.1-45.0 70％ 30％ 45.1-55 90％ 10％

[0030] High-efficiency chromatograph parameters: C18 chromatographic column, 240 mm long, 4.6 mm inner diameter, 5 μm filler particle size. Detection wavelength is 250 nm; elution flow rate is 1.4 mL / min, elution time is 55 min. Injection volume is 14 μL. The detection limit of malonic acid is 101 ± 1 ppm.

[0031] Example 2

[0032] A HPLC detection method for determining the content of malonic acid in ferulic acid comprises the following steps:

[0033] Step (a), preparing a sample solution to be tested: weighing a ferulic acid sample, using methanol as a solvent, with a mass ratio of ferulic acid to methanol of 3.5:1, dissolving by ultrasonication at a frequency of 30 kHz, a power of 120 W, and a time of 12 min, cooling, and fixing the volume to obtain a sample solution;

[0034] Step (ii), preparing a control solution: weighing a malonic acid reference substance, using methanol as a solvent, with a mass ratio of the reference substance to methanol of 3.5:1, dissolving by ultrasonication at a frequency of 30 kHz, a power of 120 W, and a time of 12 min, cooling, and fixing the volume to obtain a control solution;

[0035] Step (iii), using high performance liquid chromatography to perform gradient elution on the sample solution and the reference solution, the mobile phase A is composed of acetonitrile, methanol and a buffer solution, the buffer solution is a phosphoric acid solution with a mass fraction of 0.2%, and the volume ratio of acetonitrile, methanol and the buffer solution is 1:1:0.8; the mobile phase B is methanol, and the malonic acid content is determined after elution.

[0036] The gradient elution program was:

[0037] Time min Mobile phase A (v / v) Mobile phase B (v / v) 0-5 90％ 10％ 5.1-15 70％ 30％ 15.1-25.0 50％ 50％ 25.1-45.0 70％ 30％ 45.1-55 90％ 10％

[0038] High-efficiency chromatograph parameters: C18 chromatographic column, 250 mm long, 2-4.6 mm inner diameter, 5 μm filler particle size. Detection wavelength is 240 nm; elution flow rate is 1.4 mL / min, elution time is 55 min. Injection volume is 14-16 μL. The detection limit of malonic acid is 101 ± 1 ppm.

[0039] Example 3

[0040] A HPLC detection method for determining the content of malonic acid in ferulic acid comprises the following steps:

[0041] Step (a), preparing a sample solution to be tested: weighing a ferulic acid sample, using methanol as a solvent, with a mass ratio of ferulic acid to methanol of 3:1, ultrasonically dissolving at a frequency of 25 kHz, a power of 110 W, and a time of 10 min, cooling, and constant volume to prepare a test solution;

[0042] Step (ii), preparing a control solution: weighing a malonic acid reference substance, using methanol as a solvent, with a mass ratio of the reference substance to methanol of 3:1, dissolving by ultrasonication at a frequency of 25 kHz, a power of 110 W, and a time of 10 min, cooling, and fixing the volume to obtain a control solution;

[0043] Step (iii), using high performance liquid chromatography to perform gradient elution on the sample solution and the reference solution, the mobile phase A is composed of acetonitrile, methanol and a buffer solution, the buffer solution is a phosphoric acid solution with a mass fraction of 0.15%, and the volume ratio of acetonitrile, methanol and the buffer solution is 1:1:0.7; the mobile phase B is tetrahydrofuran, and the malonic acid content is determined after elution.

[0044] The gradient elution program was:

[0045] Time min Mobile phase A (v / v) Mobile phase B (v / v) 0-5 90％ 10％ 5.1-15 70％ 30％ 15.1-25.0 50％ 50％ 25.1-45.0 70％ 30％ 45.1-55 90％ 10％

[0046] High-efficiency chromatograph parameters: C18 chromatographic column, 250 mm long, 4.6 mm inner diameter, 5 μm filler particle size. Detection wavelength is 230 nm; elution flow rate is 1.2 mL / min, elution time is 55 min. Injection volume is 15 μL. The detection limit of malonic acid is 101 ± 1 ppm.

[0047] Comparative Example 1

[0048] Based on Example 3, in step (iii), mobile phase A was replaced with methanol, and the other steps were the same as in Example 3.

[0049] Comparative Example 2

[0050] Based on Example 3, in step (iii), mobile phase B was replaced with pure water, and the other steps were the same as in Example 3.

[0051] Comparative Example 3

[0052] Based on Example 3, in step (1) and step (2), methanol is replaced by ethanol; in step (3), the buffer solution is removed from mobile phase A.

[0053] Comparative Example 4

[0054] Based on Example 1, the elution time and volume ratio of the gradient elution were adjusted to 30 min, and the other steps were the same as in Example 1.

[0055] Time min Mobile phase A (v / v) Mobile phase B (v / v) 0-10.0 100％ 0％ 10.1-20.0 50％ 50％ 20.1-30.0 0％ 100％

[0056] test:

[0057] Chromatographic instrument model: Waters e2695 high performance liquid chromatograph, methanol (chromatographic grade), acetone (chromatographic grade), phosphoric acid (chromatographic grade), acetonitrile (chromatographic grade), ethanol (chromatographic grade), tetrahydrofuran (chromatographic grade), ultrapure water (resistivity ≥ 18.0 MΩ). According to the chromatographic conditions of Examples 1-3, the solution was drawn and injected, the chromatogram was recorded, and the RSD of the peak area was calculated, which was required to be ≤ 10.0%.

[0058] Before the experiment, the analytical system was verified. Referring to the requirements for system suitability test in Part IV 0512 of the 2020 edition of the Chinese Pharmacopoeia, the test solution and reference solution prepared in Examples 1-3 were subjected to system suitability test, and the verification parameters were: number of theoretical plates, resolution, tailing factor, and repeatability. The resolution between the main peak and the phase impurity peak should be no less than 1.5, and the number of theoretical plates, resolution, tailing factor, and repeatability all meet the requirements for system suitability test in Part IV 0512 of the 2020 edition of the Chinese Pharmacopoeia.

[0059] Preparation of standard solution: accurately weigh the malonic acid standard, dissolve it in methanol and dilute it to 100 μg / mL; accurately weigh the ferulic acid sample, dissolve it in methanol and dilute it. For HPLC analysis, balance the system with the mobile phase until the baseline is stable; inject different concentrations of malonic acid standard solutions, record the peak area, and draw a standard curve; inject the treated ferulic acid sample solution and record the peak area of ​​malonic acid. Calculate the content of malonic acid in the sample according to the standard curve. Each sample was measured three times and the average value was taken. Precision: Calculate the relative standard deviation (RSD) by measuring the same sample multiple times.

[0060] Stability test: the test solution was prepared according to the method of Example 1, Example 2 and Example 3, placed at 25°C for 12 hours, and injected after 0, 2, 4, 6, 8, 10 and 12 hours, and the peak area was measured to observe the stability. The results showed that RSD < 1%, that is, the test solution was stable for 12 hours.

[0061] Calculation method of malonic acid content in sample: Peak area substitution: Substitute the peak area of ​​malonic acid in the sample into the regression equation to calculate the concentration; Content calculation: Calculate the content of malonic acid in ferulic acid based on sample weight and dilution multiple.

[0062] The formula is: Malonic acid content (%) = (C × V × D) × 100 / (W × 10 6 )

[0063] C: Measured malonate concentration (μg / mL)

[0064] V: Sample solution volume (mL)

[0065] D: dilution factor

[0066] W: sample weight (g)

[0067] Recovery rate % = sample measurement value / spiked sample measurement value × 100%

[0068] Recovery test

[0069] Accurately weigh the ferulic acid sample, place it in a 50mL volumetric flask, add a blank auxiliary material solution to dissolve and dilute to the scale, shake well, prepare a solution of 90%, 100%, and 110% of the ferulic acid sample, test it according to the determination method of Examples 1-3 and Comparative Examples 1-4, record the chromatogram, and calculate the average recovery rate. The results show that the recovery rate of malonic acid in Examples 1-3 ranges from 99.83% to 99.90%, which meets the requirements of a recovery rate of 95% to 105% and RSD < 5%. The recovery rate of the comparative example is reduced compared with that of Examples 1-3, indicating that the changed determination method and conditions reduce the recovery rate of malonic acid. In Comparative Example 1, on the basis of Example 3, mobile phase A is replaced with methanol, and mobile phase B in Comparative Example 2 is replaced with pure water, and the recovery rate is reduced, indicating that the selection of mobile phase A and mobile phase B substances is crucial; after changing the dissolved substance of the sample in Comparative Example 3 and the elution time and volume ratio in Comparative Example 4, the recovery rate is reduced. Therefore, the method of the present invention has a significant effect on improving the recovery rate of malonic acid.

[0070] Table 1 Accuracy comparison table

[0071]

[0072] Table 2 Recovery rate table

[0073]

[0074] Summary: The present invention uses methanol as a solvent to prepare a test solution and a control solution, and uses high performance liquid chromatography for liquid chromatography analysis. Mobile phase A is composed of acetonitrile, methanol and a buffer solution, and mobile phase B is any one of acetonitrile, methanol, ethanol, and tetrahydrofuran. The parameters are set for gradient elution, and the malonic acid content is determined after elution. System suitability tests and stability tests were carried out in accordance with Part IV 0512 of the 2015 edition of the Chinese Pharmacopoeia, indicating that the method has good durability and stability. The recovery test results show that the recovery rate of malonic acid determined by the method of the embodiment of the present invention is high, and changing the dissolving solution, the mobile phase substance and the determination conditions significantly reduces the recovery rate. Therefore, the method of the present invention improves the detection accuracy, the detection results of this method are reliable, the stability is strong, and it has a significant effect on improving the recovery rate of malonic acid.

[0075] 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. An HPLC detection method for determining the content of malonic acid in ferulic acid, characterized in that: The following steps are involved: S1. Prepare the sample solution: weigh the ferulic acid sample, use methanol as solvent, ultrasonically dissolve it, cool it, and make up the volume to prepare the test solution; S2. Prepare a control solution: weigh a malonic acid reference substance, use methanol as a solvent, ultrasonically dissolve, cool, and dilute to obtain a control solution; S3. Use high performance liquid chromatography to perform gradient elution on the sample solution and the control solution, where the mobile phase A is composed of acetonitrile, methanol and buffer solution, and the mobile phase B is any one of acetonitrile, methanol, ethanol and tetrahydrofuran, and the malonic acid content is detected after elution.

2. The HPLC detection method for determining the content of malonic acid in ferulic acid according to claim 1, characterized in that: In step S1 and step S2, the mass ratio of ferulic acid to methanol is 2.5-3.5:

1.

3. The HPLC detection method for determining the content of malonic acid in ferulic acid according to claim 1, characterized in that: In step S1 and step S2, the frequency of the ultrasonic dissolution is 20-30 kHz, the power is 100-120 W, and the time is 8-12 min.

4. The HPLC detection method for determining the content of malonic acid in ferulic acid according to claim 1, characterized in that: Step S3, the buffer solution is a phosphoric acid solution with a mass fraction of 0.1%-0.2%.

5. The HPLC detection method for determining the content of malonic acid in ferulic acid according to claim 1, characterized in that: In step S3, the volume ratio of the mobile phase B, acetonitrile, methanol and buffer solution is 1:1:0.5-0.

8.

6. The HPLC detection method for determining the content of malonic acid in ferulic acid according to claim 1, characterized in that: Step S3, the gradient elution, the elution procedure is: 。 7. The HPLC detection method for determining the content of malonic acid in ferulic acid according to claim 1, characterized in that: Step S3, the high-efficiency chromatograph uses a C18 chromatographic column with a length of 240-250 mm, an inner diameter of 2-4.6 mm, and a filler particle size of 3-5 μm.

8. The HPLC detection method for determining the content of malonic acid in ferulic acid according to claim 1, characterized in that: In step S3, the detection wavelength is 210-250 nm; the elution flow rate is 1.0-1.4 mL / min, and the time is 50-60 min.

9. The HPLC detection method for determining the content of malonic acid in ferulic acid according to claim 1, characterized in that: The injection volume was 14-16 μL.

10. The HPLC detection method for determining the content of malonic acid in ferulic acid according to claim 1, characterized in that: The detection limit of malonic acid is 100.0-102.0 ppm.

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