A HPLC detection method for norepinephrine bitartrate intermediate NEL-SIP-02 and its related impurities

Abstract

The present invention belongs to the technical field of drug analysis, and in particular to a HPLC detection method for a norepinephrine bitartrate intermediate NEL‑SIP‑02 and its related impurities. The detection method is: using octadecylsilane chemically bonded porous silica gel embedded in a carbamate group in a ligand as a reverse phase chromatographic column of a filler, with a weak elution mobile phase: strong elution mobile phase = (95-35): (5-65), v / v, gradient elution, the weak elution mobile phase is 10mM potassium dihydrogen phosphate and 100mM sodium perchlorate, pH 2.1; the strong elution mobile phase is methanol: acetonitrile volume ratio is 20: 80; its chromatographic conditions: mobile phase flow rate is 0.8-1.2ml / min; the chromatographic column temperature is 25-35°C; the detection wavelength is 200-230nm. The detection method is simple to operate and low in cost. It can effectively separate the intermediate NEL-SIP-02 and its related impurities. The peak shape of the main peak is symmetrical, which can better achieve the quality control of the intermediate NEL-SIP-02 and the raw material norepinephrine bitartrate, improve the quality of the final product, and ensure its safety and effectiveness in clinical use.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to an HPLC detection method for a norepinephrine bitartrate intermediate NEL-SIP-02 and related impurities thereof. Background Art

[0002] Norepinephrine bitartrate is an adrenergic receptor agonist. The injection was developed by HOSPIRA and was first approved for marketing in the United States in July 1950 under the trade name LEVOPHED. It is a strong α receptor agonist and also stimulates β receptors. By stimulating α receptors, it can cause extreme vasoconstriction, increase blood pressure, and increase coronary blood flow; by stimulating β receptors, it can strengthen myocardial contraction and increase cardiac output. As early as the 1950s, norepinephrine was the main pressor for shock treatment. The results of a domestic meta-analysis showed that in patients with septic shock, compared with dopamine, norepinephrine can reduce the risk of death during hospitalization by 12%, and the difference is statistically significant; the risk of arrhythmia events in patients with septic shock in the dopamine group is 2.63 times that in the norepinephrine group; the cardiac index in the dopamine group is higher than that in the norepinephrine group; but in terms of heart rate and mean arterial pressure, there is no statistically significant difference between the two groups. According to the results of a systematic review, norepinephrine is superior to dopamine in improving hemodynamics, reducing mortality, and safety in patients with septic shock. Some recent studies have also shown that for patients with septic shock who do not respond well to volume resuscitation, the combination of norepinephrine and dobutamine can improve tissue perfusion and oxygen delivery, increase coronary and renal blood flow and creatinine clearance, and reduce lactate levels without aggravating organ ischemia.

[0003] The intermediate (code name NEL-SIP-02) is a key intermediate in the synthesis of norepinephrine bitartrate. Its Chinese chemical name is 2-dibenzylamino-1-(3,4-dihydroxy-phenyl)-ethanone hydrochloride, its English name is 2-dibenzylamino-1-(3,4-dihydroxy-phenyl)-ethanone hydrochloride, and its molecular weight is 347.41. The structural formula of the intermediate (code name NEL-SIP-02) and its related impurities are shown in Table 1. The intermediate (code name NEL-SIP-02) is in the second step of the synthesis of the raw material drug, which is a key step in quality control. The purity of the key intermediate needs to be controlled to reduce the occurrence of side reactions and the generation of impurities, thereby improving the yield and purity of the final product. For this intermediate, the analytical detection methods of related substances are not included in the pharmacopoeias of various countries. In order to better control the quality of norepinephrine bitartrate raw materials, it is necessary to control the quality of the intermediate during the synthesis process, and it is necessary to develop a specific analytical method with stability indication capability to detect the related substances of the intermediate (codenamed NEL-SIP-02).

[0004] The applicant has consulted a large number of Chinese and foreign patents and non-patent literatures and found that there is currently no HPLC analysis method for the impurities related to the intermediate NEL-SIP-02, which is not conducive to the company's control of product quality. The intermediate is an alkaline compound, and there may be a peak tailing phenomenon in the reverse phase analysis method; secondly, the related impurities of the intermediate are both acidic and neutral, and alkaline, and there are also positional isomers on the benzene ring, so there are certain difficulties in the HPLC analysis method for analyzing the intermediate and its related impurities. However, controlling the chemical purity of the norepinephrine bitartrate intermediate has important practical significance in the synthesis of the final product norepinephrine bitartrate and its quality control. Therefore, it is necessary to systematically develop an HPLC analysis method for the intermediate NEL-SIP-02 and its related impurities to provide a certain basis for the quality of the intermediate and the final product.

[0005] Table 1 Structural formula of intermediate NEL-SIP-02 and its related impurities

[0006]

[0007] Summary of the invention

[0008] In order to solve the problems existing in the prior art, the inventors of the present application have provided a HPLC detection method for separating and analyzing the norepinephrine bitartrate intermediate NEL-SIP-02 and its related impurities through a large amount of theoretical research and experimental exploration, so as to better control the quality of norepinephrine bitartrate products.

[0009] In order to achieve the purpose of the present invention, the technical solution provided by the present invention is:

[0010] A high performance liquid chromatography detection method for a norepinephrine bitartrate intermediate NEL-SIP-02 and related impurities thereof: a reverse phase chromatography column using octadecylsilane chemically bonded porous silica gel with a carbamate group embedded in a ligand as a filler, a mixture of potassium dihydrogen phosphate and sodium perchlorate as a weak elution mobile phase, methanol and acetonitrile mixed in a certain proportion as a strong elution mobile phase, and a gradient elution with weak elution mobile phase: strong elution mobile phase = (95-35): (5-65) (v / v), and the high performance liquid chromatography analysis method adopts the following chromatographic conditions:

[0011] The flow rate of the mobile phase is 0.8-1.2 mL / min;

[0012] The column temperature is 25-35°C;

[0013] The UV detector used in the detector has a detection wavelength of 200 to 230 nm;

[0014] Preferably, the chromatographic column is Waters Shield RP18 (150mm*4.6mm, 3.5um);

[0015] Preferably, the weak elution mobile phase is a mixture of 10 mM potassium dihydrogen phosphate and 100 mM sodium perchlorate, pH = 2.1 (adjusted with phosphoric acid);

[0016] Preferably, the strong elution mobile phase is a mixture of methanol and acetonitrile in a volume ratio of 20:80;

[0017] Preferably, the chromatographic column temperature is 30°C;

[0018] Preferably, the detection wavelength is 210 nm;

[0019] Preferably, the mobile phase flow rate is 1.0 mL / min;

[0020] Preferably, the injection volume is 10 μL.

[0021] Furthermore, the detection method comprises the following steps:

[0022] 1.1 Prepare the sample solution with a diluent, wherein the diluent is a mixture of acetonitrile and 0.1 v / v% phosphoric acid in a volume ratio of 20:80. The preparation method of 0.1 v / v% phosphoric acid is as follows: dilute 85 wt% concentrated phosphoric acid 1000 times with water;

[0023] 1.2 Inject the sample solution obtained in step 1.1 into the chromatographic column of a high performance liquid chromatograph for detection.

[0024] Compared with the prior art, the method of the present invention has the following advantages and beneficial effects:

[0025] 1. Effective separation and detection of the norepinephrine bitartrate intermediate NEL-SIP-02 and its related impurities are achieved. Therefore, the detection method of the present invention can better achieve the quality control of the intermediate NEL-SIP-02 and the raw material drug norepinephrine bitartrate, improve the quality of the final product, and ensure the safety and effectiveness of its clinical use.

[0026] 2. The detection method is simple to operate and low in cost. It can effectively separate the intermediate NEL-SIP-02 and its related impurities. The separation degree between the main peak NEL-SIP-02 and adjacent impurities is greater than 1.5, the separation degree between known impurities is greater than 1.5, and the separation degree between known impurities and unknown impurities is greater than 1.2. The peak shape of the main peak is symmetrical, which provides a simple, stable and reliable analytical detection method for product research and development and production.

[0027] It should be noted that the unit "mM" in the present invention refers to mmol / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Separation results of NEL-SIP-02 and its related impurities using acetonitrile:methanol = 50:50, v / v for phase B;

[0029] Figure 2 Separation results of NEL-SIP-02 and its related impurities using acetonitrile:methanol = 75:25, v / v for phase B;

[0030] Figure 3 Separation results of NEL-SIP-02 and its related impurities using acetonitrile:methanol=80:20, v / v for phase B;

[0031] Figure 4 The separation results of NEL-SIP-02 and its related impurities when acetonitrile:methanol=100:0, v / v, was used for phase B;

[0032] Figure 5 The results of separation of NEL-SIP-02 and its related impurities at a column temperature of 20°C;

[0033] Figure 6 The results of separation of NEL-SIP-02 and its related impurities at a column temperature of 25°C;

[0034] Figure 7 The results of separation of NEL-SIP-02 and its related impurities at a column temperature of 30°C;

[0035] Figure 8 The results of separation of NEL-SIP-02 and its related impurities at a column temperature of 35°C;

[0036] Fig. 9 The results of separation of NEL-SIP-02 and its related impurities at a column temperature of 40°C;

[0037] Fig.10 The figure shows the separation results of NEL-SIP-02 and its related impurities at a column temperature of 45°C. DETAILED DESCRIPTION

[0038] To facilitate those skilled in the art to understand the contents of the present invention, the technical solution of the present invention is further described below in conjunction with specific embodiments, but the following content should not limit the scope of protection requested by the claims of the present invention in any form.

[0039] The adjustment test of the parameter items involved in the embodiment of the present invention, including the screening of the chromatographic column and the mobile phase A phase, the screening of the mobile phase B phase, and the optimization of the column temperature, are described one by one as follows:

[0040] In the following examples, the instruments and reagents used are as follows:

[0041] Instrument: Thermo Fisher U3000 HPLC.

[0042] Reagents: Methanol, acetonitrile, phosphoric acid, and potassium dihydrogen phosphate were all of chromatography grade;

[0043] Sodium perchlorate was of analytical grade;

[0044] The reference substances NEL-SI-01, NEL-SIP-02-IMP-01, NEL-SIP-02-IMP-02, NEL-SIP-02-IMP-13 and NEL-SIP-02 were prepared by the applicant according to the prior art.

[0045] The NEL-SIP-02-IMP-03 control substance was purchased from Anergy, the NEL-SIP-02-IMP-05 control substance was purchased from Aladdin, and the ADR-3-IMP-08 control substance was purchased from McLean.

[0046] The chromatographic columns Waters Atlantis T3 (150mm*4.6mm, 3um) and Waters Shield RP18 (150mm*4.6mm, 3.5um) were purchased from Waters Technology (Shanghai) Co., Ltd., the chromatographic column ShimPack GIST C18-AQ (150mm*4.6mm, 3um) was purchased from Shimadzu Enterprise Management (China) Co., Ltd., and the chromatographic column YMC Triart C18 ExRS (150mm*4.6mm, 3um) was purchased from Shanghai Hanyao Instrument Equipment Co., Ltd.

[0047] Example 1 Screening of Chromatographic Columns and Mobile Phase A - Using different chromatographic columns in different weak elution mobile phases to analyze the intermediate NEL-SIP-02 and its related impurities, the steps are as follows:

[0048] Solution preparation:

[0049] Diluent: Take 200 mL of acetonitrile, 800 mL of ultrapure water, and 0.8 mL of commercially available 85 wt% phosphoric acid and mix them. That is, acetonitrile: 0.1 v / v% phosphoric acid = 20:80, v / v.

[0050] NEL-SI-01 stock solution: weigh 23.93 mg into a 10 mL volumetric flask, add diluent to dissolve, dilute to volume, and shake well.

[0051] NEL-SIP-02-IMP-01 stock solution: weigh 2.73 mg into a 10 mL volumetric flask, add diluent to dissolve, dilute to volume, and shake well.

[0052] NEL-SIP-02-IMP-15 stock solution: weigh 2.50 mg into a 10 mL volumetric flask, add diluent to dissolve, dilute to volume, and shake well.

[0053] NEL-SIP-02-IMP-02 stock solution: weigh 24.04 mg into a 10 mL volumetric flask, add diluent to dissolve, dilute to volume, and shake well.

[0054] NEL-SIP-02-IMP-03 stock solution: weigh 30.32 mg into a 10 mL volumetric flask, add diluent to dissolve, dilute to volume, and shake well.

[0055] NEL-SIP-02-IMP-13 stock solution: weigh 24.61 mg into a 10 mL volumetric flask, add diluent to dissolve, dilute to volume, and shake well.

[0056] NEL-SIP-02-IMP-05 stock solution: weigh 24.49 mg into a 10 mL volumetric flask, add diluent to dissolve, dilute to volume, and shake well.

[0057] ADR-3-IMP-08 stock solution: weigh 24.75 mg into a 10 mL volumetric flask, add diluent to dissolve, dilute to volume, and shake well.

[0058] Preparation of test sample spiked solution: weigh 30.28 mg of NEL-SIP-02 into a 50 mL volumetric flask, add 0.25 mL each of NEL-SI-01, NEL-SIP-02-IMP-02, NEL-SIP-02-IMP-13, NEL-SIP-02-IMP-05, and ADR-3-IMP-08 stock solutions; 2.5 mL of NEL-SIP-02-IMP-01 stock solution; 0.5 mL of NEL-SIP-02-IMP-15 stock solution; 0.5 mL of NEL-SIP-02-IMP-03 stock solution, dilute with diluent and make up to volume, shake well.

[0059] Chromatographic columns: Waters Atlantis T3 (150mm*4.6mm, 3um), YMC Triart C18 ExRS (150mm*4.6mm, 3um), ShimPack GIST C18-AQ (150mm*4.6mm, 3um), Waters Shield RP18 (150mm*4.6mm, 3.5um)

[0060] Chromatographic instrument: Thermo U3000 high performance liquid chromatograph

[0061] Injection volume: 10 μL.

[0062] Phase B is acetonitrile; detection wavelength is 210nm; flow rate: 1.0ml / min; column temperature: 35℃; gradient:

[0063] Time / min MPA% MPB% 0 92 8 35 20 80 38 20 80 38.1 92 8 45 92 8

[0064] Note: MPA refers to phase A, i.e. weak elution mobile phase (aqueous phase); MPB refers to phase B, i.e. strong elution mobile phase (organic phase).

[0065] Considering that the intermediate NEL-SIP-02 is an alkaline compound, there is a tailing phenomenon when analyzing under acidic conditions. Adding chaotropic agents such as sodium perchlorate and sodium hexafluorophosphate to the mobile phase can improve the peak shape of alkaline compounds. And the improvement effect increases with the increase of the concentration of the chaotropic agent. In addition, chromatographic columns with different filler types will also improve the peak shape of alkaline compounds to a certain extent. Therefore, the two factors of chromatographic column and sodium perchlorate concentration are investigated together. The peak shape of NEL-SIP-02 under different chromatographic columns and different sodium perchlorate concentrations is studied.

[0066] The results are shown in Table 2 below:

[0067] Table 2 Peak shape of NEL-SIP-02 in different chromatographic columns and different sodium perchlorate concentrations

[0068]

[0069] Note: Phosphoric acid was used to adjust the pH of phase A to 2.1. The concentrations of potassium dihydrogen phosphate and sodium perchlorate refer to those in phase A.

[0070] As can be seen from Table 2 above, when using a Waters Shield RP18 (150mm*4.6mm, 3.5um) column and the sodium perchlorate concentration is 100mM, the asymmetry of NEL-SIP-02 is the smallest, reaching 1.31, between 0.8 and 1.5, and the peak height is relatively high, at 3277.54mAU.

[0071] Example 2 Screening of mobile phase B - using phase B with different ratios of methanol and acetonitrile to analyze the intermediate NEL-SIP-02 and its related impurities, the steps are as follows:

[0072] Solution preparation: Use the test solution in Example 1.

[0073] Chromatographic column: Waters Shield RP18 (150mm*4.6, 3.5um)

[0074] Chromatographic instrument: Thermo U3000 high performance liquid chromatograph

[0075] Injection volume: 10 μL.

[0076] Phase A is 10mM potassium dihydrogen phosphate + 100mM sodium perchlorate, pH 2.1; detection wavelength 210nm; flow rate: 1.0ml / min; column temperature: 30℃; gradient:

[0077] Time / min MPA% MPB% 0 95 5 40 20 80 40.1 95 5 48 95 5

[0078] The volume ratios of phase B were acetonitrile:methanol = 50:50, acetonitrile:methanol = 75:25, acetonitrile:methanol = 80:20, and acetonitrile:methanol = 100:0, respectively, to study the separation of NEL-SIP-02 and its related impurities at different organic phase ratios.

[0079] When phase B uses acetonitrile:methanol = 50:50, v / v, the separation results of NEL-SIP-02 and its related impurities are as follows Figure 1 As shown:

[0080] Figure 1The impurity NEL-SIP-02-IMP-15 at 28.043min is impurity NEL-SIP-02-IMP-15. Impurity NEL-SIP-02-IMP-15 and impurity NEL-SIP-02-IMP-01 are a pair of positional isomers on the benzene ring. When acetonitrile:methanol 50:50 is used, the separation between these two impurities is 2.71. The separation between NEL-SIP-02-IMP-02 and the unknown impurity at 31.347min is only 0.50, which cannot meet the separation requirements.

[0081] Phase B uses acetonitrile:methanol = 75:25, v / v, and the separation results of NEL-SIP-02 and its related impurities are as follows Figure 2 As shown:

[0082] Figure 2 The impurity NEL-SIP-02-IMP-15 at 25.730min is impurity NEL-SIP-02-IMP-15. Impurity NEL-SIP-02-IMP-15 and impurity NEL-SIP-02-IMP-01 are a pair of positional isomers on the benzene ring. When acetonitrile:methanol 75:25 is used, the separation between these two impurities is 2.01. The separation between NEL-SIP-02-IMP-02 and the unknown impurity at 29.140min is only 1.16, which cannot meet the separation requirements.

[0083] The separation results of NEL-SIP-02 and its related impurities were as follows: Figure 3 As shown:

[0084] Figure 3 The impurity NEL-SIP-02-IMP-15 at 25.464min is impurity NEL-SIP-02-IMP-15. Impurity NEL-SIP-02-IMP-15 and impurity NEL-SIP-02-IMP-01 are a pair of positional isomers on the benzene ring. When using acetonitrile:methanol 80:20, the separation between these two impurities is 1.96. The separation between NEL-SIP-02-IMP-02 and the unknown impurity at 28.913min is 1.38. Considering that the separation between known impurities and unknown impurities is greater than 1.2, the separation requirements can be met.

[0085] When phase B uses acetonitrile:methanol=100:0, v / v, the separation results of NEL-SIP-02 and its related impurities are as follows Figure 4 As shown:

[0086] Figure 4The impurity NEL-SIP-02-IMP-15 at 23.653min is impurity NEL-SIP-02-IMP-15. Impurity NEL-SIP-02-IMP-15 and impurity NEL-SIP-02-IMP-01 are a pair of positional isomers on the benzene ring. When using acetonitrile:methanol 100:0, the resolution between these two impurities is 1.39. The resolution between NEL-SIP-02-IMP-02 and the unknown impurity at 26.973min is 1.38.

[0087] The separation of NEL-SIP-02 and its related impurities when the volume ratio of phase B was acetonitrile:methanol = 50:50, acetonitrile:methanol = 75:25, acetonitrile:methanol = 80:20, and acetonitrile:methanol = 100:0 was investigated. Under these four conditions, the impurities NEL-SIP-02-IMP-13, ADR-3-IMP-08, NEL-SI-01, NEL-SIP-02-IMP-03 and the main peak NEL-SIP-02 can meet the separation requirements. The difficulty of separation is concentrated on the two pairs of compounds of impurities NEL-SIP-02-IMP-01 and NEL-SIP-02-IMP-15, and NEL-SIP-02-IMP-02 and unknown impurities. The specific results are summarized in Table 3 below:

[0088] Table 3 Peaks of impurities NEL-SIP-02-IMP-01, NEL-SIP-02-IMP-15, NEL-SIP-02-IMP-02 and unknown impurities

[0089]

[0090] As can be seen from Table 3 above, as the proportion of acetonitrile in phase B increases, the elution capacity increases and the retention time of the same compound decreases; the separation of impurities NEL-SIP-02-IMP-01 and NEL-SIP-02-IMP-15 decreases as the proportion of acetonitrile increases, and the separation of NEL-SIP-02-IMP-02 and unknown impurities increases as the proportion of acetonitrile increases. When the proportion of acetonitrile reaches 80%, the separation between NEL-SIP-02-IMP-02 and unknown impurities reaches a maximum of 1.38, and even if the proportion of acetonitrile is increased to 100%, the separation between the two is still 1.38. Therefore, considering the separation between the two pairs of compounds, the proportion of acetonitrile NEL-SIP-02-IMP-01 and NEL-SIP-02-IMP-15, and NEL-SIP-02-IMP-02 and unknown impurities, the acetonitrile ratio is selected to be 80%, that is, acetonitrile: methanol 80:20.

[0091] Example 3: Analysis of impurities related to the intermediate NEL-SIP-02 at different column temperatures, the steps are as follows:

[0092] Solution preparation: Use the test solution in Example 1.

[0093] Chromatographic column: Waters Shield RP18 (150mm*4.6mm, 3.5um)

[0094] Chromatographic instrument: Thermo U3000 high performance liquid chromatograph

[0095] Injection volume: 10 μL.

[0096] Phase A is 10 mM potassium dihydrogen phosphate + 100 mM sodium perchlorate, pH 2.1; Phase B is acetonitrile: methanol = 80:20, v / v; Detection wavelength 210 nmnm; Flow rate: 1.0 ml / min; Gradient:

[0097] Time / min MPA% MPB% 0 95 5 40 35 65 40.1 95 5 48 95 5

[0098] The column temperatures were selected as follows: 20℃; 25℃; 30℃; 35℃; 40℃; 45℃, and the separation of NEL-SIP-02 and its related impurities at different column temperatures was studied.

[0099] When the column temperature is 20℃, the separation results of NEL-SIP-02 and its related impurities are as follows: Figure 5 As shown;

[0100] When the column temperature is 25°C, the separation results of NEL-SIP-02 and its related impurities are as follows: Figure 6 As shown;

[0101] When the column temperature is 30℃, the separation results of NEL-SIP-02 and its related impurities are as follows: Figure 7 As shown;

[0102] When the column temperature is 35°C, the separation results of NEL-SIP-02 and its related impurities are as follows: Figure 8 As shown;

[0103] When the column temperature is 40℃, the separation results of NEL-SIP-02 and its related impurities are as follows: Fig. 9 As shown;

[0104] When the column temperature is 45°C, the separation results of NEL-SIP-02 and its related impurities are as follows: Fig.10 shown.

[0105] Similar to the results of phase B in Example 2, when the column temperatures were 20°C, 25°C, 30°C, 35°C, 40°C, and 45°C, the impurities NEL-SIP-02-IMP-13, ADR-3-IMP-08, NEL-SI-01, NEL-SIP-02-IMP-03 and the main peak NEL-SIP-02 could meet the separation requirements under these 6 conditions. The difficulty of separation was concentrated on the 5 compounds of post-main peak impurities NEL-SIP-02-IMP-01, NEL-SIP-02-IMP-15, NEL-SIP-02-IMP-05, NEL-SIP-02-IMP-02 and unknown impurities. The specific results are summarized in Table 4 below:

[0106] Table 4 Peaks of impurities NEL-SIP-02-IMP-01, NEL-SIP-02-IMP-15, NEL-SIP-02-IMP-05, NEL-SIP-02-IMP-02 and unknown impurities

[0107]

[0108] It can be seen from Table 4 above that the separation of impurities NEL-SIP-02-IMP-01 and NEL-SIP-02-IMP-15, and impurity NEL-SIP-02-IMP-02 and unknown impurities deteriorates with increasing column temperature; except that the separation degree between NEL-SIP-02-IMP-15 and NEL-SIP-02-IMP-05 is less than 1.5 when the column temperature is 20°C, the separation degrees between the two are greater than 1.5 under the other column temperature conditions; therefore, considering the separation between these three pairs of compounds, the column temperature is selected to be 30°C, which can fluctuate by ±5°C, that is, 25°C to 35°C, which can satisfy the separation degree between known impurities greater than 1.5, and the separation degree between known impurities and unknown impurities greater than 1.2.

Claims

1. A high performance liquid chromatography detection method for a norepinephrine bitartrate intermediate NEL-SIP-02 and related impurities thereof, wherein the impurities include 4-(chloroacetyl)catechol, 2-(dibenzylamino)-1-(3-(2-(3,4-dihydroxyphenyl)-2-oxoethoxy)-4-hydroxyphenyl)ethane-1-one, 2-(dibenzylamino)-1-(4-(2-(3,4-dihydroxyphenyl)-2-oxoethoxy)-3-hydroxyphenyl)ethane-1-one, 2-(dibenzylamino)-1-(4-hydroxy-3-(2-(4-hydroxy-3-methoxyphenyl)-2-oxoethoxy)phenyl)ethane-1-one, dibenzylamine hydrochloride, 3,4-dihydroxybenzoic acid, N,N-diphenylformamide and 1-(2,4-dihydroxyphenyl)ethanone, characterized in that: A reverse phase chromatographic column with octadecylsilane chemically bonded porous silica gel with carbamate groups embedded in the ligand as filler was used; the weak elution mobile phase was a mixture of 10 mM potassium dihydrogen phosphate and 100 mM sodium perchlorate, pH = 2.1; the strong elution mobile phase was methanol: acetonitrile with a volume ratio of 20:80; the gradient elution program: 0-40 min, the weak elution mobile phase was from 95% to 35%; The following chromatographic conditions were used for the detection: The flow rate of the mobile phase is 0.8~1.2mL / min; The column temperature is 25~35℃; The detector used is an ultraviolet detector with a detection wavelength of 200~230nm.

2. The detection method according to claim 1, characterized in that: The chromatographic column is Waters Shield RP18.

3. The detection method according to claim 2, characterized in that: The specifications of the chromatographic column are: 150mm*4.6mm, 3.5um.

4. The detection method according to claim 1 or 2, characterized in that: The temperature of the chromatographic column is 30°C.

5. The detection method according to claim 1 or 2, characterized in that: The flow rate of the mobile phase is 1.0 mL / min.

6. The detection method according to claim 1 or 2, characterized in that: The detection wavelength is 210 nm.

7. The detection method according to claim 1 or 2, characterized in that: The injection volume was 10 μL.

8. The detection method according to claim 1 or 2, characterized in that: The detection method comprises the following steps: 1.1 Prepare the sample solution with a diluent, wherein the diluent is acetonitrile: 0.1 v / v% phosphoric acid = 20:80, v / v; 1.2 Inject the sample solution obtained in step 1.1 into the chromatographic column of the high performance liquid chromatograph for detection.

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