Method for detecting dissolution rate or release rate of gallic acid in alkaline buffer solution

Abstract

The invention belongs to the field of detection, and discloses a method for detecting the dissolution rate or the release rate of gallic acid in an alkaline buffer solution, gallic acid is dissolved in a phosphate buffer solution containing vitamin C and EDTA-2Na, and the dissolution rate or the release rate of gallic acid at different time points is detected through high performance liquid chromatography. The method solves the problem that gallic acid cannot be effectively detected under a slightly neutral or alkaline condition (simulating the gastrointestinal tract of an animal).

Description

Technical Field

[0001] This invention relates to the field of detection, and more particularly to a method for detecting the dissolution or release rate of gallic acid-base buffer solutions. Background Technology

[0002] Dissolution rate refers to the rate and extent to which an active drug dissolves from common formulations such as tablets, capsules, or granules under specified conditions. In formulations such as sustained-release, controlled-release, enteric-coated, and transdermal patches, it is also called release rate. The specific test method involves transferring a certain amount of drug through a rotating basket or directly into a dissolution apparatus containing the dissolution medium. The mixture is stirred at a specific temperature and speed, and after a certain time, a specific volume of the dissolution solution is collected. The drug concentration in the dissolution solution is then detected using a UV spectrophotometer or high-performance liquid chromatography (HPLC), and the dissolution rate or release rate of the drug in the formulation is calculated. The detection of drug dissolution rate or release rate can simulate the dissolution or release process of a formulation in the gastrointestinal tract, avoiding the risks of clinical trials and saving drug development costs. However, this requires accurate determination of drug concentration in in vitro dissolution or release tests.

[0003] Gallic acid is a class of phenolic compounds, also known as 3,4,5-trihydroxybenzoic acid. It is a naturally occurring secondary metabolite and the earliest organic acid obtained in the world. It is a major component of the traditional Chinese medicine "Baiyao Jian" and has the effects of clearing heat and resolving phlegm, promoting body fluid and quenching thirst. It is used for lung heat cough, toothache due to wind-heat, oral ulcers, and chronic dysentery with rectal prolapse. Modern research shows that gallic acid has anti-inflammatory, antioxidant, antibacterial, and antitumor effects.

[0004] Gallic acid contains carboxyl and phenolic hydroxyl groups, exhibiting properties similar to carboxylic acids (weak acidity, decarboxylation reaction) and phenols (easily oxidized, undergoes a color reaction with ferric chloride, etc.). It possesses strong reducing properties, and the hydroxyl group (-OH) ionizes into an oxonium (-O) ion. - Gallic acid has an increased electron density and is more easily oxidized. It is unstable under neutral, alkaline and strong oxidizing conditions. Alkali can catalyze and accelerate oxidation reactions (such as the formation of quinones). At pH 7.0~10.0, it can be completely degraded in 3 hours. The oxidation equation of gallic acid can be seen in Equation 1 below.

[0005] Formula 1 During the experiment, we found that even if we controlled the oxidation of gallic acid, we could not achieve a good peak elution of gallic acid, thus making it impossible to achieve accurate detection. Summary of the Invention

[0006] The purpose of this invention is to provide a method for detecting the dissolution or release of gallic acid in an acid-base buffer solution. This method solves the problem that gallic acid cannot be effectively detected under slightly neutral or alkaline conditions (simulating the gastrointestinal tract of animals).

[0007] To achieve the above objectives, this application discloses: A method for detecting the dissolution or release rate of gallic acid in an acid-base buffer solution involves dissolving gallic acid in a phosphate buffer solution containing vitamin C and EDTA-2Na, and detecting the dissolution or release rate of gallic acid at different time points using high-performance liquid chromatography (HPLC). The pH of the phosphate buffer solution is 6.0–6.8. Preferably, before sample injection, 5 μL of a 6 mol / L hydrochloric acid solution is added per milliliter of phosphate buffer solution. This invention has confirmed through preliminary experiments that gallic acid can exist relatively stably in an acidic environment. When the pH reaches 6 or above, the chromatographic peak is doubled when detected by high performance liquid chromatography. Furthermore, with the extension of time, oxidative degradation occurs, making it impossible to accurately detect gallic acid.

[0008] However, pH 6 or above is the most commonly used pH environment for simulating the animal gut. Based on this dilemma, this case is proposed.

[0009] After careful analysis, we believe the main reason is: 1. Gallic acid is easily oxidized. It contains carboxyl and phenolic hydroxyl groups, exhibiting properties similar to carboxylic acids (weak acidity, decarboxylation reaction) and phenols (easily oxidized, undergoes a color reaction with ferric chloride, etc.). It possesses strong reducing properties; the hydroxyl group (-OH) ionizes into an oxonium (-O) ion. - With increased electron density, it is more easily oxidized and is unstable under neutral, alkaline and strong oxidizing conditions. Alkali can catalyze and accelerate oxidation reactions (such as the formation of quinones). It can be completely degraded in 3 hours at pH 7.0~10.0. 2. The water used to prepare the buffer solution contains trace amounts of the metal Fe. 2+ Cu 2+ Plasma induces catalysis, such as chelating with the catechol group of gallic acid to form a highly active intermediate, accelerating electron transfer, and catalyzing the oxidation of gallic acid by dissolved oxygen to generate quinone and superoxide radicals (•O2). - Furthermore, dissolved oxygen in the medium can also lead to direct oxidation, oxidizing gallic acid to produce quinones and peroxides; Based on the above two main reasons, this invention uses vitamin C, which has a stronger reducing power than gallic acid, for protection, and at the same time uses a chelating agent to remove metal ions to protect gallic acid from oxidative degradation, so that the gallic acid released in the preparation can exist stably in buffer solutions of different pH values, providing a prerequisite for accurate drug detection.

[0010] Furthermore, there are some different mechanisms when simulating pH values ​​of 6.0 and 6.8, resulting in some different results.

[0011] In some embodiments of the present invention, we found that when the pH is 6.8, only a certain amount of vitamin C needs to be added to reduce the detected degradation from 26% to 0~2wt%; however, when the pH is 6.0, if only a certain amount of vitamin C is added, the detected degradation increases instead, and the chelating agent EDTA-2Na must be added to significantly eliminate the degradation. Before explaining the specific mechanisms underlying the different results at different pH values, it is necessary to elucidate a portion of the oxidation pathway of gallic acid: the phenolic hydroxyl group of gallic acid is easily attacked by hydroxyl radicals, initiating the oxidation of gallic acid, as shown in the following equation: RH + •OH → R • + H2O; R• + O2 → ROO• → further oxidation products; Therefore, hydroxyl radicals are key to promoting the entire oxidation process; The generation of hydroxyl radicals originates from the Fenton reaction: Fe 2+ + H2O2→ Fe 3+ + •OH+OH - ; The role of vitamin C in this process is: Fe 3+ +Ascorbic acid ions (AH) - →Fe 2+ +Dehydroascorbic acid+H + ; 2AH - +O2→2ascorbic acid+H2O2; In other words, vitamin C acts as an oxidation accelerator under specific conditions. These "specific conditions" refer to pH; see the analysis below for details. We believe the reason for its inner layer is: Fe 3+ Cu 2+ Ions, especially Fe, have extremely poor solubility at pH 6.8. 3+ It exists in the form of colloidal or amorphous Fe(OH)3 and does not affect gallic acid; therefore, when the pH is 6.8, the above oxidation cycle produces Fe. 3+ When the vitamin C is cut off, it only plays an antioxidant role.

[0012] Fe 3+ Cu 2+ At pH 6.0, the ions are more soluble and exist as free ions. At this pH, the vitamin C-induced oxidation cycle continues to operate. Therefore, in some cases of this invention, the addition of vitamin C actually increases gallic acid degradation. To disrupt this cycle, we used EDTA-2Na, which causes the Fe produced in the Fenton reaction to...3+ By chelating and inactivating it as much as possible, we eventually found that its gallic acid degradation decreased rapidly.

[0013] In addition, since gallic acid has a pKa of approximately 4.0, it exists in both anionic and molecular form under alkaline conditions, resulting in double peaks during high-performance liquid chromatography (HPLC) detection. To avoid the occurrence of double peaks, a small amount of hydrochloric acid is added to the test solution before injection to adjust the pH of the test solution, ensuring that gallic acid exists entirely in molecular form, thereby improving the accuracy of detection.

[0014] In the above detection method, not less than 54 mg of vitamin C is added to every 900 ml of the phosphate buffer solution; the concentration of EDTA-2Na in the phosphate buffer solution is 0.01~0.02 wt%.

[0015] In this invention, the objective can be achieved with a very small amount of vitamin C; particularly preferably, 54-90 mg of vitamin C is added to every 900 ml of the phosphate buffer solution. Excessive vitamin C will change the pH of the solution, reducing the reliability and accuracy of the simulated release. 54-90 mg of vitamin C, during the verification process of this invention, hardly changes the pH of the solution, meeting the accuracy requirements of the model.

[0016] In the above detection method, 1-5 ml of gallic acid aqueous solution is added to every 900 ml of the phosphate buffer solution; the concentration of the gallic acid aqueous solution is 0.5-1 wt%.

[0017] In the above detection method, the detection conditions for the high-performance liquid chromatography (HPLC) method are as follows: Column: WondaSil, C18 Superb 5μm, 4.6×150mm; Mobile phase: 0.05% aqueous phosphoric acid solution and methanol, with a volume ratio of 93:7; Detection wavelength: 271 nm; Flow rate: 1.0 mL / min; Column temperature: 35℃; Injection volume: 20 μL.

[0018] This application has at least the following beneficial effects: The detection method of this invention can test the release of gallic acid in a simulated environment close to the pH value of animal intestines, with high detection accuracy and low degradation. Attached Figure Description

[0019] Figure 1 The graph shows the detection results of gallic acid in the buffer solution of Example 1 with a pH of 4.5 to 6.2; Figure 2 The graph shows the detection results of gallic acid in the buffer solution of Example 1 with a pH of 6.2 to 7.2; Figure 3 The graph shows the detection results of gallic acid in the buffer solution of Example 1 with a pH of 7.2 to 8.0; Figure 4 The graph shows the detection results of gallic acid in a buffer solution with 1 to 20 μL of hydrochloric acid added at pH 6.8. Figure 5 The graph shows the detection results of gallic acid in a buffer solution with 10 to 20 μL of hydrochloric acid added at pH 8.0. Detailed Implementation

[0020] The present invention will now be clearly and completely described in conjunction with embodiments thereof. It should be noted that, unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products. Unless otherwise specified, all parts used in the embodiments of the present invention are parts by weight.

[0021] Reagent Introduction Preparation of the main solution: pH 1.2 hydrochloric acid solution: Accurately transfer 7.65 mL of hydrochloric acid into a 1000 mL volumetric flask, dilute with water to the mark, and shake well. pH 4.6 acetate buffer solution: Accurately weigh 2.99 g of sodium acetate trihydrate, place it in a 1000 mL volumetric flask, add 14.5 mL of glacial acetic acid, dissolve and dilute with water to the mark, and shake well to obtain the solution; pH 6.0 phosphate buffer solution (containing 0.01% EDTA-2Na): Accurately weigh 6.80 g of potassium dihydrogen phosphate and place it in a 1000 mL volumetric flask. Add 28.0 mL of 0.2 mol / L sodium hydroxide solution, dissolve and dilute to the mark with water, and shake well to obtain a pH 6.0 phosphate buffer solution. Then add 0.1 g of EDTA-2Na to prepare a pH 6.0 phosphate buffer solution containing 0.01% EDTA-2Na. Add 54 mg of vitamin C per 900 mL before use. pH 6.8 phosphate buffer solution (containing 0.01% EDTA-2Na): Accurately weigh 6.80 g of potassium dihydrogen phosphate and place it in a 1000 mL volumetric flask. Add 112.0 mL of 0.2 mol / L sodium hydroxide solution, dissolve and dilute to the mark with water, and shake well to obtain a pH 6.8 phosphate buffer solution. Then add 0.1 g of EDTA-2Na to prepare a pH 6.8 phosphate buffer solution containing 0.01% EDTA-2Na. Before use, add 54 mg of vitamin C per 900 mL. High-performance liquid chromatography (HPLC) detection conditions: Column: WondaSil, C18 Superb 5μm, 4.6×150mm; Mobile phase: 0.05% aqueous phosphoric acid solution - methanol (V:V) = 93:7; Detection wavelength: 271 nm; Flow rate: 1.0 mL / min; Column temperature: 35℃; Injection volume: 20 μL.

[0022] Example 1 High-performance liquid chromatography detection of gallic acid in solutions with different pH values Accurately weigh 0.1 g of gallic acid into a 100 mL volumetric flask, dilute to the mark with buffer solutions of different pH values, accurately pipette 1 mL into a 50 mL volumetric flask, dilute and bring to volume with the corresponding pH buffer solution to obtain a solution with a concentration of 20 μg / mL, and detect it on the instrument according to the high performance liquid chromatography conditions.

[0023] The pH values ​​of the buffer solutions were 4.5, 5.5, 5.8, 6.0, 6.2, 6.4, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, and 8.0, respectively.

[0024] Note: The buffer solution used in this embodiment is a phosphate buffer solution, which is achieved by adjusting the molar ratio of NaOH and potassium dihydrogen phosphate in the phosphate buffer solution. For specific methods, please refer to the "Guidelines for Determination and Comparison of Dissolution Curves of Common Oral Solid Dosage Forms".

[0025] Test results reference Figures 1 to 3 , Figure 1 The results are for pH values ​​ranging from 4.5 to 6.2. Figure 2 The results are for pH values ​​between 6.2 and 7.2. Figure 3 The results are for pH values ​​between 7.2 and 8.0; through... Figures 1 to 3 It can be seen that a double peak appears when the pH rises to 6.2; when the pH rises further to 7.2, the double peaks separate; above pH 7.2, the double peaks stabilize and show almost no change. Gallic acid exhibited biphasic peaks in liquid chromatography starting at pH 6.2, making accurate quantification impossible. The phenomenon was further observed after adding 2 mol / L hydrochloric acid to pH 6.8 and pH 8.0 buffer solutions, with the following results: Figure 4 and Figure 5 ; Figure 4 In the buffer solution with pH 6.8, the addition of 1 to 20 μL of hydrochloric acid resulted in a single peak. Figure 5 In the sample, when 10 to 20 μL of hydrochloric acid is added to a buffer solution with a pH of 8.0, the elution peak appears as a single peak.

[0026] The results above show that it is difficult to simulate the release of gallic acid at pH values ​​greater than 6.0. Although adding hydrochloric acid to the buffer solution in this embodiment can eliminate the bimodal phenomenon, subsequent tests show that excessive degradation still exists.

[0027] Example 2 Stability of gallic acid in hydrochloric acid solution at pH 1.2, acetic acid solution at pH 4.6, phosphoric acid solution at pH 6.0, and phosphoric acid solution at pH 6.8 without the addition of EDTA-2Na and vitamin C. Test methods Accurately weigh approximately 0.9 g of gallic acid raw material and place it in a 100 mL volumetric flask. Dissolve the raw material in water and dilute to the mark, then shake well. Accurately transfer 2 mL of the above solution to three replicates in each of the following dissolution media: pH 1.2 hydrochloric acid solution, pH 4.6 acetate buffer solution, pH 6.0 phosphate solution, and pH 6.8 phosphate solution. Under the conditions of a medium temperature of 37 ℃ ± 0.5 ℃ and a stirring speed of 100 r / min, take 2 mL samples at 0 h, 2 h, 4 h, 6 h, 8 h, and 10 h. For the samples taken from pH 6.0 and pH 6.8 phosphate buffer solutions, add 10 μL of 6 mol / L hydrochloric acid, vortex to mix, filter through a syringe filter, and analyze under high performance liquid chromatography (HPLC) conditions.

[0028] The test results are shown in Table 1; Table 1. Stability of gallic acid in different pH release media (without EDTA-2Na and VC)

[0029] As can be seen from the experimental results in Table 1, degradation is minimal in buffer solutions with pH below 6.0, but significantly increases at pH 6.0 and above, and the degradation tends to amplify with increasing pH.

[0030] Example 3: Stability of gallic acid in pH 6.0 and pH 6.8 phosphate solutions containing vitamin C. Accurately weigh approximately 0.9 g of gallic acid raw material and place it in a 100 mL volumetric flask. Dissolve the raw material in water and dilute to the mark, then shake well. Accurately transfer 2 mL of the above solution into 900 mL of pH 6.0 phosphate solution and pH 6.8 phosphate solution (with 54 mg VC) in triplicate for each dissolution medium. Under the conditions of medium temperature of 37 ℃ ± 0.5 ℃ and stirring speed of 100 r / min, take 2 mL samples at 0 h, 2 h, 4 h, 6 h, 8 h, and 10 h. Add 10 μL of 6 mol / L hydrochloric acid to the samples taken from pH 6.0 and pH 6.8 phosphate buffer, vortex to mix, filter through a syringe filter, and analyze under high performance liquid chromatography (HPLC) conditions. The results are shown in Table 2.

[0031] Table 2 Test Results

[0032] As can be seen from the data in Tables 1 and 2, when vitamin C is added, the degradation of the buffer solution at pH 6.8 decreases rapidly, while the degradation of the buffer solution at pH 6.0 tends to increase.

[0033] The fundamental reason can be found in the description above regarding the mechanism of the amplified oxidation reaction of vitamin C.

[0034] Example 4: Stability of gallic acid in pH 6.0 and pH 6.8 phosphate solutions containing 0.01% EDTA-2Na with different amounts of vitamin C. Accurately weigh approximately 0.9 g of gallic acid raw material and place it in a 100 mL volumetric flask. Dissolve the raw material in water and dilute to the mark. Shake well. Accurately transfer 2 mL of the above solution and place it in 900 mL of pH 6.0 phosphate solution and pH 6.8 phosphate solution (with 54 mg VC) containing different amounts of 0.01% EDTA-2Na for each dissolution medium (3 replicates). Under the conditions of medium temperature of 37 ℃ ± 0.5℃ and stirring speed of 100 r / min, take 2 mL samples at 0 h, 2 h, 4 h, 6 h, 8 h, and 10 h. Add 10 μL of 6 mol / L hydrochloric acid to the samples taken from pH 6.0 and pH 6.8 phosphate buffer, vortex to mix, filter through a syringe filter, and analyze under high performance liquid chromatography (HPLC) conditions. The results are shown in Tables 3 and 4.

[0035] Table 3 Test Results

[0036] Table 4 Test Results

[0037] As shown in Tables 3 and 4, the degradation of EDTA-2Na is reduced in buffer solutions with added EDTA-2Na, regardless of whether the buffer solution is at pH 6.0 or pH 6.8. This indicates that EDTA-2Na, based on its chelating effect, can reduce the influence of metal ions on detection errors.

[0038] In particular, it should be noted that: In a pH 6.8 buffer solution, if either EDTA-2Na or vitamin C is added alone, degradation occurs within 10 hours. If only EDTA-2Na is added, there is no substantial difference compared to the pH 6.8 buffer solution without any treatment. However, if EDTA-2Na and vitamin C are present simultaneously, degradation almost disappears. In a buffer solution at pH 6.0, if EDTA-2Na or vitamin C is added alone, degradation occurs after 10 hours. Although there are differences between the two, they are not as significant as the differences at pH 6.8. Neither can significantly eliminate the degradation phenomenon. When the two are used in combination, the degradation almost disappears.

[0039] Based on the data in Tables 1, 2, and 3, we can conclude that in a buffer system with pH 6.0, using vitamin C alone is not conducive to degradation and elimination; using EDTA-2Na alone is conducive to degradation and elimination; and using both simultaneously significantly controls degradation. In a buffer system with pH 6.8, vitamin C alone is beneficial for degradation and elimination; EDTA-2Na alone does not significantly help with degradation and elimination; when both are used together, degradation is significantly controlled.

[0040] The above phenomena further illustrate that the mechanisms by which vitamin C and EDTA-2Na reduce degradation differ in different pH buffer systems.

[0041] Example 5: Stability of gallic acid in pH 6.0 and pH 6.8 phosphoric acid solutions containing EDTA-2Na and vitamin C. Accurately weigh approximately 0.9 g of gallic acid raw material and place it in a 100 mL volumetric flask. Dissolve the raw material in water and dilute to the mark, then shake well. Accurately transfer 2 mL of the above solution into 900 mL of pH 6.0 phosphate solution and pH 6.8 phosphate solution (with 54 mg VC and 0.01% EDTA-2Na) in triplicate for each dissolution medium. Under the conditions of medium temperature of 37 ℃ ± 0.5 ℃ and stirring speed of 100 r / min, take 2 mL samples at 0 h, 2 h, 4 h, 6 h, 8 h, and 10 h. Add 10 μL of 6 mol / L hydrochloric acid to the samples taken from pH 6.0 and pH 6.8 phosphate buffer, vortex to mix, filter through a syringe filter, and analyze under high performance liquid chromatography (HPLC) conditions. The results are shown in Table 5.

[0042] Table 5 Test Results

[0043] Example 6 The injection stability of the sample from Example 5 after being placed in the injection tray for different times is shown in Table 6. Table 6 Test Results

[0044] Summarize: Through the different cases mentioned above, we can draw the following conclusions: 1. For pH values ​​above 6.0, adding hydrochloric acid can eliminate the bimodal phenomenon; 2. Adding vitamin C can inhibit the degradation of gallic acid in high pH buffer solutions, but has a negative effect on the degradation of gallic acid in slightly lower pH buffer solutions. 3. By adding EDTA-2Na, the detection and degradation of gallic acid in buffer solutions of different pH values ​​can be inhibited. The inhibition is most significant in buffer solutions with low pH values. The reason is that iron ions will form a precipitate at pH 6.8. The reason for the degradation is not whether chelation occurs, but the easy oxidation and decomposition characteristics of gallic acid itself at this pH. Therefore, it can be found that vitamin C is more suitable for inhibiting the degradation of gallic acid in high pH buffer solutions.

[0045] 4. By comparing different data, we can find that regardless of the pH value, adding EDTA-2Na and vitamin C can inhibit the degradation of gallic acid; However, based on the above experiments, we can prove that the inhibition mechanisms are completely different; For a buffer solution with a pH of 6.8, the main cause of degradation is the easy oxidation of gallic acid under alkaline conditions. Therefore, by using vitamin C as the main degradation eliminator and combining it with the chelating effect of EDTA-2Na, the degradation can be controlled to a satisfactory level. For a buffer solution with a pH of 6.0, the main cause of degradation is the decomposition of gallic acid by hydroxyl radicals generated by the Fenton reaction. For this type of buffer solution, EDTA-2Na and vitamin C are the main degradation eliminators. Although vitamin C is an accelerator of the Fenton reaction, its inhibitory effect on the oxidative decomposition of gallic acid cannot be denied. By inhibiting the iron ions in the Fenton reaction involving vitamin C with EDTA-2Na, the Fenton reaction is interrupted, thereby reducing degradation.

[0046] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description; thus, all changes falling within the meaning and scope of the claims are intended to be embraced within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

Claims

1. A method for detecting the dissolution or release rate of gallium in an acid-base buffer solution, characterized in that, Gallic acid was dissolved in a phosphate buffer solution containing vitamin C and EDTA-2Na, and the dissolution or release rate of gallic acid at different time points was detected by high performance liquid chromatography. The pH of the phosphate buffer solution was 6.0-6.

8.

2. The detection method according to claim 1, characterized in that, Add at least 54 mg of vitamin C to every 900 mL of the phosphate buffer solution; the concentration of EDTA-2Na in the phosphate buffer solution is 0.01~0.02 wt%.

3. The detection method according to claim 1, characterized in that, Add 54-90 mg of vitamin C to every 900 mL of the phosphate buffer solution.

4. The detection method according to claim 1, characterized in that, Add 1-5 mL of gallic acid aqueous solution to every 900 mL of the phosphate buffer solution; the concentration of the gallic acid aqueous solution is 0.5-1 wt%.

5. The detection method according to claim 1, characterized in that, Before sample injection and testing, add 5 μL of 6 mol / L hydrochloric acid solution to each milliliter of phosphate buffer solution.

6. The detection method according to claim 1, characterized in that, The detection conditions for the high-performance liquid chromatography method are as follows: Column: WondaSil, C18 Superb 5μm, 4.6×150mm; Mobile phase: 0.05% aqueous phosphoric acid solution and methanol, with a volume ratio of 93:7; Detection wavelength: 271 nm; Flow rate: 1.0 mL / min; Column temperature: 35℃; Injection volume: 20 μL.

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