CN108226326B - Application and method of tetrahydroquinoxaline derivative of o-phenylenediamine in detecting content of o-dicarbonyl compound - Google Patents

Abstract

The invention provides application of a tetrahydroquinoxaline derivative of o-phenylenediamine in detecting the content of an o-dicarbonyl compound and a method thereof, and particularly relates to a method for detecting the content of the o-dicarbonyl compound (alpha-DCs) in different food samples by using the tetrahydroquinoxaline derivative of the o-phenylenediamine, such as 4- (1,2,3, 4-tetrahydro-6-quinoxalinyl) -1, 2-o-phenylenediamine (I), 4- (1,2,3, 4-tetrahydro-2, 3-dimethyl-6-quinoxalinyl) -1, 2-o-phenylenediamine (II), analogs III, IV and the like as derivatization reagents and adopting a pre-column derivatization-HPLC method. The establishment of a pre-column derivatization-HPLC method for the determination of various α -DCs contents in different food samples using compounds I-IV as derivatization reagents for the first time. Compared with the existing method for determining alpha-DCs in food by pre-column derivatization-HPLC, the method has the advantages of mild and stable derivatization reaction conditions, and simple and quick operation.

Description

Application and method of tetrahydroquinoxaline derivative of o-phenylenediamine in detecting content of o-dicarbonyl compound

Technical Field

The invention relates to a reagent for detecting the content of ortho-dicarbonyl compounds (alpha-DCs) and a detection method thereof.

Background

Ortho-dicarbonyl compounds (α -DCs), such as glyoxal (Gly), methylglyoxal (MGly), phenylglyoxal (PGly), butanedione (DA), 2, 3-Pentanedione (PD), D-glucurone (2-KG), 1-deoxy-D-glucurone (1-DG), 3-deoxy-D-glucurone (3-DG), are common degradation products formed during processing of food products, and the chemical formulae are specifically as follows:

generally, sugars in food, such as glucose, and free amino groups in proteins during food processing first undergo maillard reactions to form early glycosylation products, which in turn translate into highly reactive α -DCs; meanwhile, in some food fermentation processes and some biological processes, α -DCs can also be produced through a lipid oxidation pathway. Previous studies have shown that these compounds are present in many types of food products, such as fruit juices, tea, coffee, beer, soy sauce, milk, honey, some baby food and wine, etc. Generally, foods containing larger amounts of carbohydrates, lipids or proteins are more likely to produce α -DCs. On the one hand, these α -DCs play an important role in the organoleptic characteristics of foods, for example, their content in various foods affects the flavor, taste, color, etc. of the foods, thereby determining the quality of the foods; and some alpha-DCs have the function of improving the flavor and color of the food, so that the alpha-DCs can be widely used as food additives in the food processing process. On the other hand, the presence of these compounds may mediate cross-linking reactions of food proteins, leading to an impaired nutritional value of the food. Therefore, the content of α -DCs, particularly Gly and MGly, in food products has been used as a toxicological characteristic parameter of glucostoxins in western countries as an index for food safety quality evaluation. The existing research also proves that some effective mechanisms exist in the human body to metabolize the carbonyl compounds, but when excessive compounds are ingested from exogenous diet, the human body cannot completely eliminate the compounds, and residual alpha-DCs are accumulated in various tissues of the human body, so that the proteins lose activity through interaction with various proteins in the tissues, thereby generating pathological effect on the body and causing various diseases. In fact, pharmacological studies have shown that α -DCs are associated with many chronic and age-related diseases, such as chronic complications of diabetes, cardiovascular diseases, alzheimer's disease, parkinson's disease, and aging, among others. Therefore, it is important to establish an effective and practical detection means for quantifying α -DCs in various foods based on quality control of foods and protection of human health.

Most of the existing methods for measuring alpha-DCs are pre-column derivatization-High Performance Liquid Chromatography (HPLC), which is mainly because alpha-DCs are compounds which are highly water-soluble and lack of chromophores, direct detection not only has complex pretreatment process but also has unsatisfactory sensitivity of the method, but also can improve the detectability of the alpha-DCs and increase the sensitivity of the detection by derivatizing the alpha-DCs to introduce some groups with ultraviolet absorption or fluorescence. The derivatization reagents commonly used in the prior pre-column derivatization-HPLC methods are o-phenylenediamine, its derivatives, and analogs thereof, which have the major disadvantage that the derivatization reaction needs to be performed under relatively severe conditions, e.g., almost all of the prior derivatization processes require heating to promote the reaction process, which may enhance autooxidation of the α -DCs precursor species in the sample, resulting in increased α -DCs concentration, thereby affecting the accuracy of the assay. In addition, studies have shown that heating the sample for extended periods of time in the presence of a derivatizing agent, such as o-phenylenediamine, alters the autoxidation pathway of the glucose, which in turn affects the actual content of α -DCs in the sample, making the measurement completely unreliable. Although studies have shown that the reaction can also be carried out at room temperature when o-phenylenediamine is used as a derivatizing agent, the disadvantage is that the reaction takes several hours to complete, which not only makes the determination process tedious, but also results in inaccurate determination of some alpha-DCs, especially DA, because acetolactate, a precursor compound of DA, is converted to DA by a non-enzymatic oxidation process during a long period of time. Therefore, it is required to develop a novel derivatization reagent for α -DCs to allow accurate determination of α -DCs in various food samples under mild conditions (derivatization reaction is completed at room temperature in a short time).

In previous studies, the applicant established an HPLC method for measuring DA in white spirit and beer by using 3,3' -Diaminobenzidine (DAB) and 4- (2, 3-dimethyl-6-quinoxalinyl) -1, 2-o-phenylenediamine (DQB) as pre-column derivatization reagents (article: WangXinjie et al, chromatography, 2017,35,837; Ji-Yu Wang et al, J.Agric.food chem., 2017,65,2635; patent application: Gao Wen Yun et al, invention patent application No. 201610497512.2). The main advantages of these methods are that the conditions of the pre-column derivatization process are mild and can be completed within 10 minutes at room temperature. However, these two compounds have their own disadvantages as derivatizing agents: the compound DAB has two groups of o-diaminobenzene structural units in the structure, so when the compound DAB reacts with a mixture of a plurality of alpha-DCs, the product is complex, and only a single alpha-DC compound can be separately measured, but a plurality of alpha-DCs components cannot be simultaneously measured. Although the compound DQB and the analogue overcome the defects of the compound DAB, the compound DQB and the analogue are unstable under stronger acid or alkaline conditions (pH <2 or >10) due to the quinoxaline structural unit; meanwhile, DQB and the analogue thereof have lower sensitivity when detecting certain alpha-DCs, such as 2-KG, 1-DG, 3-DG and the like, and the defects limit the application of the DQB and the analogue as a derivatization reagent in the aspect of simultaneously measuring various alpha-DCs.

Disclosure of Invention

The invention researches a suitable pre-column derivatization reagent for simultaneously detecting the content of a plurality of ortho-dicarbonyl compounds (alpha-DCs), and provides a sensitive, rapid, stable and reliable method for measuring the content of the alpha-DCs in various foods by an HPLC method.

The scheme determined by the invention is as follows:

the method comprises the steps of taking tetrahydroquinoxaline derivatives of o-phenylenediamine as derivatization reagents, and detecting the content of a plurality of alpha-DCs in different foods by adopting a pre-column derivatization-HPLC method. The reaction principle is as follows (taking the compounds I/II as an example):

the tetrahydroquinoxaline derivative of the above-mentioned o-phenylenediamine is preferably: 4- (1,2,3, 4-tetrahydro-6-quinoxalinyl) -1, 2-o-phenylenediamine (I), 4- (1,2,3, 4-tetrahydro-2, 3-dimethyl-6-quinoxalinyl) -1, 2-o-phenylenediamine (II) and their analogs III, IV, the structures of which are as follows:

the synthesis methods of the above 4- (1,2,3, 4-tetrahydro-6-quinoxalinyl) -1, 2-o-phenylenediamine (I) and 4- (1,2,3, 4-tetrahydro-2, 3-dimethyl-6-quinoxalinyl) -1, 2-o-phenylenediamine (II): 1.0mmol of 3,3' -Diaminobenzidine (DAB) and 0.3mmol of an o-diketone compound of glyoxal or butanedione are reacted in water at room temperature to obtain an intermediate product, and the intermediate product is reduced by lithium aluminum hydride (LiAlH4) to obtain a target product; the reaction formula is as follows:

the synthesis method of the analogues III and IV comprises the following steps: 1.0mmol of 3,3' -Diaminobenzidine (DAB) and 0.3mmol of the cyclo-o-diketone compound 1, 2-cyclopentadione or 1, 2-cycloReaction of hexanedione in methanol at room temperature gives an intermediate product which is lithium aluminum hydride (LiAlH)₄) Reducing to obtain a target product, wherein the reaction formula is as follows:

the method for detecting the content of alpha-DCs in food by taking the tetrahydroquinoxaline derivative of o-phenylenediamine as a derivatization reagent comprises the following steps:

(1) 0.1-1 mM 20-50% methanol solution of o-phenylenediamine tetrahydroquinoxaline derivative is added into 1.5mL of sample to be tested, pH is adjusted to 7.0-10.0 after uniform mixing, and reaction is carried out for 30-60 minutes at room temperature;

(2) the reaction-completed sample was filtered through a 0.22 μm filter and the filtrate was examined by HPLC.

Wherein, the HPLC optimum configuration carried out in the step (2) is as follows:

a chromatographic column: a Shim-Pack VP-ODS C18 column; mobile phase: gradient eluting with methanol-water for 0min, 60% (volume fraction) methanol; 10 min: 100% methanol; 15 min: 60% (volume fraction) methanol; and (17 min): 60% (volume fraction) methanol; column temperature: room temperature; sample introduction amount: 20-100 mu L; the flow rate is 0.7-1.5 mL/min; detection wavelength: 254 nm.

The invention has the following technical effects:

the new derivatization reagents, namely 4- (1,2,3, 4-tetrahydro-6-quinoxalinyl) -1, 2-o-phenylenediamine (I), 4- (1,2,3, 4-tetrahydro-2, 3-dimethyl-6-quinoxalinyl) -1, 2-o-phenylenediamine (II) and analogues III-IV thereof are used as the derivatization reagents for simultaneously measuring a plurality of alpha-DCs in various foods, the derivatization reaction is mild (the reaction can be completely carried out at the pH value of 7.0-9.0 and the room temperature for 30 min), the operation is convenient, and the HPLC measurement result is accurate and reliable.

Drawings

FIG. 1 is an HPLC chromatogram in the measurement of a compound 4- (1,2,3, 4-tetrahydro-6-quinoxalinyl) -1, 2-o-phenylenediamine (I) as a derivatization agent.

Wherein: A. HPLC chromatogram of compound I; B. HPLC chromatogram of underivatized black tea extract; C. determining HPLC chromatograms of α -DCs (α -DCs include 2-KG (a), 3-DG (b), 1-Gly (c), 2-MGly (d), 3-DA (e) and 4-PD (f)) for compound I; D. compound I determination of alpha-DCs content in black tea (not labeled); E. compound I the alpha-DCs content of black tea was determined (after labelling).

FIG. 2 is a graph showing the working curves of compounds I and II for determining alpha-DCs.

Wherein: dotted line: i is a working curve of each compound when a derivatization reagent is determined; solid line: II is the working curve of each compound when the derivatization reagent is determined.

FIG. 3 is an HPLC chromatogram when compound 4- (1,2,3, 4-tetrahydro-2, 3-dimethyl-6-quinoxalinyl) -1, 2-o-phenylenediamine (II) is used as a derivatization reagent for determination.

Wherein: A. HPLC chromatogram of compound II; B. determining HPLC chromatograms of α -DCs for Compound II (α -DCs include 2-KG (a), 3-DG (b), 1-Gly (c), 2-MGly (d), 3-DA (e), and 4-PD (f)); C. HPLC chromatogram of underivatized dark tea extract; D. the content of alpha-DCs in the dark tea is determined by the compound II (the chromatogram 1 is a dark tea sample without a standard, and the chromatogram 2 is a dark tea sample after a standard is added).

Detailed Description

Firstly, preparing a derivatization reagent:

1. synthesis of compounds I and II:

reaction of 3,3' -Diaminobenzidine (DAB) (1.0mmol) with an ortho-diketone compound glyoxal or butanedione (0.3mmol) in water at room temperature gives an intermediate product which is reacted with lithium aluminium hydride (LiAlH)₄) Or reducing to obtain target molecules I and II. The reaction formula is as follows:

the specific process is as follows:

1) preparation of I-a/II-a: 3,3' -diaminobenzidine tetrahydrochloride (DAB-4HCl) (108mg, 0.3mmol) was weighed into a 25mL round-bottomed flask, 10mL of distilled water was added, and DAB was dissolved well with gentle stirring at room temperature. After about 10-20 minutes, addStirring at room temperature for half an hour with glyoxal (24mg, 0.4mmol) or butanedione (35mg, 0.4mmol), filtering the reaction solution, washing the insoluble substance with distilled water three times (5 mL. times.3) to obtain a washing solution, mixing the washing solutions with the previous filtrate, extracting with chloroform (20 mL. times.5), mixing the 3 rd to 5 th extracts, and adding anhydrous MgSO 2₄After drying for 2 hours, the drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure to give a dark red powder as the objective compound (I-a: 63.5mg, 86.1%; II-a: 65.2mg, 82.3%).

I-a：¹H-NMR(CD₃OD, 400 MHz): 8.41(2H, d, J ═ 6), 8.11(1H, s), 7.99(2H, m), 7.26(1H, s), 7.18(1H, d, J ═ 6), 6.95(1H, d, J ═ 6). HRESI-MS (positive ion mode) m/z: calculated value 237.1140, measured value: 237.1151[ M + H]⁺。

II-a：¹H-NMR(CD₃OD, 400 MHz): 8.04(1H, s), 7.94(2H, m), 7.19(1H, brs), 7.08(1H, d, J ═ 6), 6.81(1H, d, J ═ 6), 2.78(3H, s, Me), 2.73(3H, s, Me). ESI-MS (positive ion mode) m/z: calculated value 265.1453, measured value: 265.1439[ M + H]⁺。

2) I/II preparation: a dry, nitrogen-filled 10mL round bottom flask was charged with LiAlH₄(38mg, 1.0mmol), 5mL of dry acetonitrile and a compound I-a (24mg, 0.1mmol) or a compound II-a (27mg, 0.1mmol), reacting at room temperature for 1 hour under the protection of nitrogen, slowly adding methanol dropwise into the system until no bubbles are generated, transferring the reaction mixture into a centrifuge tube, centrifuging at 4 ℃, 6000rpm for 10 minutes, transferring the supernatant into a round-bottom flask, washing the solid with 5mL of multiplied by 2 methanol, centrifuging, combining the supernatants, and distilling under reduced pressure to remove the solvent to obtain dark red powder, namely the target compound (I: 21.3mg, 88.1%; II: 22.5mg, 83.9%).

I：¹H-NMR(CD₃OD, 400 MHz): 7.13(2H, d, J ═ 6), 6.94(2H, brs), 6.69(2H, d, J ═ 6), 3.31(4H, t, J ═ 7). HRESI-MS (positive ion mode) m/z: calculated value 241.1453, measured value: 241.1451[ M + H]⁺。

II：¹H-NMR(CD₃OD，400MHz)：6.93(2H，d，J＝6)，6.84(2H，brs)，6.59(2H， d, J ═ 6), 3.08(2H, m), 1.43(6H, d, J ═ 8). ESI-MS (positive ion mode) m/z: calculated value 269.1766, measured value: 269.1759[ M + H]⁺。

2. Synthesis of compounds III and IV: 3,3' -Diaminobenzidine (DAB) (1.0mmol) and 1, 2-cyclopentanedione or 1, 2-cyclohexanedione (0.3mmol) as the cyclic o-diketone compound are reacted in methanol at room temperature to give an intermediate product which is reacted with lithium aluminum hydride (LiAlH)₄) And reducing to obtain target molecules III and IV. The reaction formula is as follows:

the specific process is as follows:

1) preparation of III-a/IV-a: 3,3' -diaminobenzidine tetrahydrochloride (DAB-4HCl) (108mg, 0.3mmol) was weighed into a 25mL round-bottomed flask, 8mL distilled water and 2mL methanol were added, and DAB was dissolved well with gentle stirring at room temperature. After about 10-20 minutes, 1, 2-cyclopentanedione (40mg, 0.4mmol) or 1, 2-cyclohexanedione (45mg, 0.4mmol) was added, stirring was continued at room temperature for half an hour, the reaction solution was filtered, the insoluble matter was washed with distilled water three times (5 mL. times.3) to obtain a washing solution, which was combined with the previous filtrate, extracted with chloroform (20 mL. times.5), and the 3 rd to 5 th extracts were combined, over anhydrous MgSO₄After drying for 2 hours, the drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure to give a dark red powder as the target compound (III-a: 53.9mg, 65.1%; IV-a: 65.4mg, 75.2%).

III-a：¹H-NMR(CD₃OD, 400 MHz): 8.01(1H, s), 7.80(2H, m), 7.30(1H, s), 7.14(1H, d, J ═ 6), 6.91(1H, d, J ═ 6), 2.15(4H, t, J ═ 8), 1.49(2H, m). HRESI-MS (positive ion mode) m/z: calculated value 277.1453, measured value: 277.1441[ M + H]⁺。

IV-a：¹H-NMR(CD₃OD, 400 MHz): 8.02(1H, s), 7.91(2H, m), 7.11(1H, brs), 7.03(1H, d, J ═ 6), 6.73(1H, d, J ═ 6), 2.31(4H, t, J ═ 8), 1.61(4H, m). ESI-MS (positive ion mode) m/z: calculated value 291.1610, measured value: 291.1629[M+H]⁺。

2) III/IV preparation: a dry, nitrogen-filled 10mL round bottom flask was charged with LiAlH₄(38mg, 1.0mmol), 5mL of dry acetonitrile and a compound III-a (28mg, 0.1mmol) or a compound IV-a (29mg, 0.1mmol), reacting at room temperature for 1 hour under the protection of nitrogen, slowly adding methanol dropwise into the system until no bubbles are generated, transferring the reaction mixture into a centrifuge tube, centrifuging at 4 ℃ and 6000rpm for 10 minutes, transferring the supernatant into a round-bottom flask, washing the solid with 5mL of multiplied by 2 methanol, merging the supernatants after centrifuging, and distilling under reduced pressure to remove the solvent to obtain dark red powder, namely the target compound (III: 23.3mg, 84.1%; IV: 24.1mg, 83.1%).

III：¹H-NMR(CD₃OD, 400 MHz): 8.07(1H, s), 7.89(2H, m), 7.32(1H, s), 7.21 (1H, d, J ═ 6), 6.99(1H, d, J ═ 6), 2.65(2H, m)1.65(4H, m), 1.49(2H, m). HRESI-MS (positive ion mode) m/z: calculated value 281.1766, measured value: 281.1751[ M + H]⁺。

IV：¹H-NMR(CD₃OD, 400 MHz): 8.02(1H, s), 7.91(2H, m), 7.11(1H, brs), 7.03(1H, d, J ═ 6), 6.73(1H, d, J ═ 6), 2.71(2H, m), 1.61(4H, m), 1.41(4H, m). ESI-MS (positive ion mode) m/z: calculated value 295.1923, measured value: 295.1919[ M + H]⁺。

Secondly, the compounds I-IV are used as derivatization reagents to measure alpha-DCs in different foods by a pre-column derivatization-HPLC method (standard addition method)

Since the compounds III and IV are analogues of the compounds I and II, and the properties and application effects thereof can be expected to be similar to those of the compounds I and II, the following mainly take the compounds I and II as derivatization reagents as examples:

1. pretreatment of different food samples: ultrasonic degassing carbonated beverage and beer samples (10.0 mL each) at room temperature for 10min, and storing at 4 deg.C before analysis; coffee (1.0g) was dissolved well by adding 8.0mL of boiling water and left at 85 ℃ for 20min, cooled and diluted to 10.0mL with Millipore water and stored at 4 ℃ before analysis; adding 8.0mL of boiling water into tea (1.0g), standing at 80 deg.C for 25min to dissolve out components, cooling, diluting with Millipore water to 10.0mL, and storing the supernatant at 4 deg.C before analysis; after shaking the yogurt sample well, 10.0mL of the mixture was mixed well with 2mL of methanol, the mixture was centrifuged at 8000rpm for 30min at 4 ℃ and the supernatant (about 1.5mL) was diluted to 2.0mL with Millipore water and stored at 4 ℃ prior to analysis. The remaining food samples, such as whisky, fruit drinks and wine, did not require special handling.

2. The compound I is used as a derivatization reagent for detecting the content of alpha-DCs in a food sample: the pretreated food sample (0.67 mL) was added to 0.05mL of the methanol solution of II (5mM), 0.08mL of Millipore water and 0.20mL of methanol were added thereto, and after thoroughly mixing, the mixture was adjusted to pH 8.0 and reacted at room temperature for 30 minutes. In the labeling experiment, four standard solutions were added to each food sample: gly (0.5, 1.0 and 5.0. mu.M), MGly (0.5, 1.0 and 5.0. mu.M), DA (0.5, 1.0 and 5.0. mu.M) and PD (0.5, 1.0 and 5.0. mu.M) were thoroughly mixed by a vortex mixer, the pH of the mixture was adjusted to 8.0, and the reaction was carried out at room temperature for 30 minutes. The reaction mixture (with no standard added and with standard added) was filtered through 0.22 μm filter, and the filtrate was subjected to HPLC analysis to determine the peak area of the derivative. The chromatographic conditions are as follows: column chromatography, Shim-Pack VP-ODS C18 column (250X 4.6mm,5 μm); mobile phase, methanol-water gradient elution, 0min, 60% methanol; 10 min: 100% methanol; 15 min: 60% methanol; and (17 min): 60% methanol; column temperature, room temperature; sample size, 20 μ L; the flow rate is 0.7 mL/min; detection wavelength, 254 nm. The HPLC chromatogram and the working curve of each compound are respectively shown in FIG. 1 and FIG. 2; the results of the LC-MS measurements are shown in Table I.

3. The compound II is used as a derivatization reagent to detect the content of alpha-DCs in a food sample: the determination process is consistent with the process of detecting the content of the alpha-DCs in the food sample by taking the compound I as a derivatization reagent. The HPLC chromatogram and the working curve of each compound are respectively shown in FIG. 2 and FIG. 3; the results of the LC-MS measurements are shown in Table II.

The detection results of the content of various alpha-DCs in different foods are shown in the third table.

TABLE I LC-MS identification of derivatives obtained when assays are performed with Compound I as derivatizing agent

^aESI-MS data for each derivative was collected from positive ion mode;^bthe structures of the respective derivatives are described in the aforementioned "background art".

TABLE II LC-MS identification of derivatives obtained in assays with Compound II as derivatizing agent^a

^aESI-MS data for each derivative was collected from positive ion mode;^bthe structures of the respective derivatives are described in the aforementioned "background art". Table III, results of content detection of various alpha-DCs in different foods

Measuring by using compound I as a derivatization reagent; measuring with compound II as derivative reagent;

^athe concentration unit of the object to be measured is mu M when the liquid sample is used, and mu g/g when the solid sample is used;^brelative standard deviation at six replicates;^cnot detected;^dthe concentration of the substance to be detected is higher than the detection limit but lower than the quantification limit.

Claims (3)

1. The application of tetrahydroquinoxaline derivative of o-phenylenediamine as a derivatization reagent for simultaneously detecting the contents of various o-dicarbonyl compounds in food by a pre-column derivatization-HPLC method;

the tetrahydroquinoxaline derivative of the o-phenylenediamine is selected from 4- (1,2,3, 4-tetrahydro-6-quinoxalinyl) -1, 2-o-phenylenediamine (I), 4- (1,2,3, 4-tetrahydro-2, 3-dimethyl-6-quinoxalinyl) -1, 2-o-phenylenediamine (II) and analogues III and IV thereof, and has the following structures:

2. a method for simultaneously detecting the contents of a plurality of ortho-dicarbonyl compounds in food using the tetrahydroquinoxaline derivative of o-phenylenediamine described in claim 1 as a derivatization reagent, comprising the steps of:

(1) 0.1-1 mM 20-50% methanol solution of o-phenylenediamine tetrahydroquinoxaline derivative is added into 1.5mL of sample to be tested, pH is adjusted to 7.0-10.0 after uniform mixing, and reaction is carried out for 30-60 minutes at room temperature;

(2) the reaction-completed sample was filtered through a 0.22 μm filter and the filtrate was examined by HPLC.

3. The method of claim 2, wherein: HPLC performed in step (2) is as follows:

a chromatographic column: a Shim-Pack VP-ODS C18 column; mobile phase: gradient eluting with methanol-water for 0min, 60% (volume fraction) methanol; 10 min: 100% methanol; 15 min: 60% (volume fraction) methanol; and (17 min): 60% (volume fraction) methanol; column temperature: room temperature; sample introduction amount: 20-100 mu L; the flow rate is 0.7-1.5 mL/min; detection wavelength: 254 nm.

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