Novel impurity compound, preparation method, content determination method and application thereof

Abstract

The invention discloses a novel impurity compound with a structure as shown in a formula (I), a preparation method and a content determination method thereof, and application of the compound as an impurity reference substance in medicine quality control. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a new impurity compound, in particular to a new hydroxyoxalamide impurity compound and a preparation method and a content determination method thereof, as well as application of the compound in drug quality control. Background Art

[0002] API N 1 -(3,5)-dichloro-4-(3-(4-fluorobenzyl)-4-hydroxyphenoxy)phenyl)-N 2 -Hydroxyoxamide (hereinafter referred to as CU-40101) can be used to treat androgenic alopecia in a targeted manner. This product has not yet been included in any national pharmacopoeia or drug standard. Chinese invention patent CN104193699A introduces the synthesis method and application of this product.

[0003]

[0004] Molecular formula: C 21 H 15 Cl2FN2O5

[0005] Molecular weight: 465.26

[0006] According to the synthesis process and formulation composition of CU-40101, the sources of organic impurities in this preparation are the process impurities of the raw material drug and the degradation impurities in the raw material drug and the preparation. The impurity information is shown in Table 1:

[0007] Table 1: List of impurities in CU-40101

[0008]

[0009] Summary of the invention

[0010] Technical Problems to be Solved by the Invention

[0011] During the development and research of the CU-40101 preparation product, the inventors found that in addition to the above-mentioned process impurities and degradation impurities, a new unknown impurity was produced in the CU-40101 preparation during the stability study. The unknown impurity was produced and continued to increase under accelerated conditions (25°C). After storage at room temperature for 18 months, the content reached 1.6%. According to the ICH Q3B (R2) impurity guidelines for new drug preparations, the identification limit and definition limit of the impurity of this product are both 1.0%. Therefore, it is necessary to conduct further research such as preparation and structural analysis of the impurity, strictly control the content of the impurity, and further control the quality of the drug.

[0012] Since there are few literatures and reports on the raw material CU-40101, no reports on the above-mentioned unknown impurities have been found in the prior art, and there are no standard products (reference products) on sale, it is impossible to conduct comprehensive and effective quality control of CU-40101.

[0013] In view of the above technical issues, the purpose of the present invention is to separate and identify the structure of the unknown impurity and establish a method for determining the content of the unknown impurity to achieve quality control of the raw material CU-40101.

[0014] Technical means to solve technical problems

[0015] The present invention provides an impurity compound represented by structural formula (I):

[0016]

[0017] The present invention also provides a method for preparing the impurity compound represented by the above structural formula (I), comprising:

[0018] 1) Take the raw material N 1 -(3,5)-dichloro-4-(3-(4-fluorobenzyl)-4-hydroxyphenoxy)phenyl)-N 2 -Hydroxyoxamide is dissolved in ethanol;

[0019] 2) heating the ethanol solution of the raw material drug obtained in step (1) to degrade it;

[0020] 3) separating the degraded API ethanol solution using a preparative liquid chromatograph, and collecting the solution by threshold collection, wherein the threshold is preferably 20;

[0021] 4) drying the collected fraction containing the impurity compound under reduced pressure;

[0022] Wherein, the liquid chromatography conditions in step (3) are as follows:

[0023] Preparative column: C18

[0024] Mobile phase A: 0.08-0.12% (v / v) formic acid-water solution

[0025] Mobile phase B: 0.08-0.12% (v / v) formic acid-acetonitrile solution

[0026] The gradient elution program was:

[0027]

[0028]

[0029] Detector: UV-visible detector,

[0030] Detection wavelength: 248±3nm.

[0031] In step (2) of the preparation method of the present invention, the heating temperature is above 100°C, and / or

[0032] The heating time is more than 24 hours.

[0033] In step (2) of the preparation method of the present invention, the ethanol solution of the raw material drug is heated under pressurized conditions.

[0034] In step (2) of the preparation method of the present invention, the pressurization is to place the ethanol solution of the raw material drug in a pressurized test tube.

[0035] In step (2) of the preparation method of the present invention, the heating method is selected from oven heating, electric heating, microwave heating, and steam heating.

[0036] The present invention also provides a method for determining the content of the impurity compound represented by structural formula (I), wherein the liquid chromatography method is as follows:

[0037] Chromatographic column: C18 column

[0038] Mobile phase A: TFA aqueous solution with a TFA volume percentage of 0.03%-0.07%

[0039] Mobile phase B: TFA acetonitrile solution with a TFA volume percentage of 0.03%-0.07%

[0040] Detector: UV-visible detector

[0041] Detection wavelength: 220±3nm

[0042] Gradient elution program:

[0043]

[0044] In the content determination method of the present invention, the flow rate of the mobile phase is: 0.3±0.1mL / min, and / or

[0045] Sample chamber temperature: 4 ± 1 °C, and / or

[0046] Column temperature: 40℃±3℃.

[0047] In the content determination method of the present invention, the impurity content is calculated by the peak area normalization method according to the following formula:

[0048]

[0049] Among them, A S : Target impurity peak area in the test solution

[0050] AT : The sum of the chromatographic peak areas in the test solution.

[0051] The present invention also provides the use of the impurity compound represented by structural formula (I) as an impurity reference substance in drug quality control, wherein the drug contains N 1 -(3,5)-dichloro-4-(3-(4-fluorobenzyl)-4-hydroxyphenoxy)phenyl)-N 2 -Hydroxyoxamide.

[0052] Beneficial technical effects

[0053] Beneficial effects of the present invention:

[0054] 1. The present invention uses high temperature and pressure to destroy the CU-40101 raw material drug, and uses a preparative liquid chromatograph to achieve CU-40101-IPR01 enrichment and purification.

[0055] 2. Use UPLC-Q Exactive Plus high-resolution liquid chromatography-mass spectrometry system to accurately determine the molecular weight (four decimal places) of unknown degradation impurities, and infer their molecular structures from the mass spectrometry fragment ion peaks.

[0056] 3. The structure of CU-40101-IPR01 is identified by comprehensive use of infrared spectroscopy, ultraviolet spectroscopy, and multiple nuclear magnetic resonance spectra, and the causes of impurities are determined. This can be used for impurity analysis and control of CU-40101-IPR01-related preparations to ensure quality and drug safety.

[0057] 4. This method requires a small amount of sample and is suitable for the qualitative analysis of unknown chemical components that are difficult to obtain. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 :Mass spectrum of target impurity

[0059] Figure 2 : Secondary mass spectrum of target impurity

[0060] Figure 3 : Infrared spectrum of target impurities

[0061] Figure 4 :UV spectrum of target impurity

[0062] Figure 5 : Impurity CU-40101-IPR01 H NMR spectrum ( 1 H NMR)

[0063] Figure 6 :Impurity CU-40101-IPR01 C NMR spectrum ( 13 C NMR), DEPT-135 spectrum

[0064] Figure 7 :Impurity CU-40101-IPR01 NMR F spectrum

[0065] Figure 8 :Impact CU-40101-IPR01 NMR HSQC spectrum

[0066] Fig. 9 :Impurity CU-40101-IPR01 NMR HMBC spectrum

[0067] Fig.10 : Impurity CU-40101-IPR01 NMR 1 H- 1 H COSY spectrum

[0068] Fig.11 : Impurity CU-40101-IPR01 NMR 1 H- 1 H ROESY spectrum DETAILED DESCRIPTION

[0069] definition

[0070] It should be understood that as used herein, the singular forms "a," "an," "an," and "the" include plural forms unless the context clearly dictates otherwise.

[0071] The terms "above", "below", "within", and "at least" herein include the present number unless the context clearly dictates otherwise.

[0072] When the embodiments are described herein as "comprising", "containing", or "including", other similar embodiments described as "consisting of" and / or "consisting essentially of" are also provided. It should also be understood that when the embodiments are described herein as "consisting essentially of", other similar embodiments described as "consisting of" are also provided.

[0073] The term "and / or" as used herein in phrases such as "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0074] The "±" used in phrases such as "A±B" in this document represents all values ​​within the range from the value of AB to the value of A+B. For example, 248±3 represents all values ​​from 245-251, such as 245, 246, 247, 248, 249, 250, 251, and values ​​within the range of 245-251 after rounding.

[0075] Example

[0076] The present invention is described in detail below with reference to the following examples. The examples provide detailed implementation methods and processes to fully disclose and demonstrate that the invention can be implemented. The examples do not represent limitations on the invention, and the protection scope of the present invention is not limited to the following examples.

[0077] The instruments, reagents, materials, experimental animals, etc. whose sources are not specified in the examples of the specification are purchased through general commercial channels. The experimental methods without specific conditions in the examples of the specification are usually carried out according to the conventional conditions in the field or the conditions recommended by the manufacturer.

[0078] The purpose of the present invention is to provide a method for preparing and structurally identifying unknown impurities of a small molecule compound that promotes hair growth.

[0079] The specific steps are as follows:

[0080] 1. Sample preparation

[0081] 1) Dissolve the raw material CU-40101 in ethanol to prepare a solution of about 6 mg / mL;

[0082] 2) Place the CU-40101 ethanol solution in a pressurized test tube, place it in a 105°C oven, and take it out after 72 hours;

[0083] 3) The degraded CU-40101 sample ethanol solution was separated and eluted by preparative liquid chromatography, and the fraction of the unknown impurity was collected. The instrument information and chromatographic conditions were as follows:

[0084] Table 2

[0085]

[0086] 4) Collect the fractions above the collection threshold, and dry the collected fractions of the unknown impurity under reduced pressure to obtain the unknown impurity (purity 95%).

[0087] 2. Structural Identification

[0088] The method for identifying the structure of unknown impurities in a small molecule compound that promotes hair growth provided by the present invention comprises analyzing the above-mentioned unknown impurities by LC-MS, obtaining the accurate molecular weight of the impurity by high-resolution mass spectrometry, inferring the molecular structure by fragment ions, and confirming the impurity structure by nuclear magnetic resonance spectra such as infrared spectroscopy, ultraviolet spectroscopy, hydrogen spectrum, and carbon spectrum.

[0089] LC-MS analysis

[0090] 1) Chromatographic conditions

[0091] Table 3

[0092]

[0093]

[0094] 2) Mass spectrometry conditions

[0095] Table 4

[0096]

[0097]

[0098] 3) Experimental results

[0099] The primary mass spectrum of the target impurity is as follows Figure 1 , detected [M+H] + Quasi-molecular ion peak m / z =

[0100] 493.0726. According to the test results, the target impurity is speculated to be a CU-40101-related impurity with a molecular weight of 492, 28 more than CU-40101. Its molecular formula is speculated to be C 23 H 19 C l2 FN2O5, which contains more C2H4 than CU-40101, may be produced by the dehydration reaction of CU-40101 and ethanol.

[0101] The secondary mass spectrum of the target impurity is as follows Figure 2 The main fragment ions include m / z 109, 176, 246, 282, 308, 325, 351 and 397.

[0102] 4) Structural speculation

[0103] The unknown impurity may be generated by the dehydration reaction of CU-40101 and ethanol. Referring to the structural formula of CU-40101, the dehydration reaction may occur on the amide hydroxyl group to generate structure 1:

[0104]

[0105] Or ethanol and CU-40101 phenolic hydroxyl group form ether, see structure 2:

[0106]

[0107] If the target impurity is structure 1, the possible fragments are as follows:

[0108]

[0109] By analyzing the secondary mass spectrum of the target impurity, possible fragments of structure 1 can be found, but possible fragments of structure 2 are not found.

[0110] In summary, the target impurity result is structure 1. The unknown impurity is produced by the dehydration condensation of the amide hydroxyl group in the CU-40101 structure and ethanol under high temperature conditions. The impurity is named CU-40101IPR01, and its structure is identified by infrared spectroscopy, ultraviolet spectroscopy, and nuclear magnetic resonance spectroscopy.

[0111]

[0112] Molecular formula: C 23 H 19 Cl2FN2O5

[0113] Exact mass: 492.0655

[0114] 1. Infrared spectroscopy

[0115] 1) Test method

[0116] Table 5

[0117] Instrument Model Perkin Elmer, Spectrum Two Fourier Transform Infrared Spectrometer Test conditions Attenuated Total Reflectance Method Scan range <![CDATA[4000cm -1 -400cm -1 ]]> Resolution <![CDATA[4cm -1 ]]> Scan times 3

[0118] 2) Test results see Figure 3 , infrared spectrum data list is shown in Table 6:

[0119] Table 6: List of characteristic absorption peaks of infrared spectrum

[0120]

[0121] 3) Analysis

[0122] 3588.93, 3473.18, 3280.95, 3215.81cm -1 These are the stretching vibration absorption peaks of NH and OH. 3162.82, 3112.36, 3071.19, 3037.99, 3007.45, 2935.74, 2833.49, 2805.60cm -1 It is the stretching vibration absorption peak of CH. 1715.74, 1661.70cm -1It is the stretching vibration absorption peak of C=O. 1584.45, 1544.96, 1516.42, 1505.54cm -1 It is the C=C stretching vibration absorption peak of the benzene ring.

[0123] 2. Ultraviolet spectrum

[0124] 1) Test method

[0125] Table 7

[0126] Instrument Model Perkin Elmer Lambda 25 UV-Vis Spectrophotometer Test conditions Scanning range: 190nm-800nm Solvents Methanol Sample concentration 8.46μg / ml

[0127] 2) Test results see Figure 4 The UV spectrum shows that the sample has two large absorption peaks at 203.22nm and 266.85nm. 203.22nm is attributed to the K band absorption of unsaturated conjugated olefins, and 266.85nm is attributed to the B band absorption of benzene rings.

[0128] 3. Nuclear Magnetic Resonance Spectroscopy

[0129] 1) Test method

[0130]

[0131] 2) Test results

[0132] The test results are as follows Figure 5-11 shown.

[0133] 3) Analysis

[0134]

[0135] Molecular formula: C 23 H 19 Cl2FN2O5

[0136] Molecular weight:493.31

[0137] Table 2: H NMR, 1H-1H COSY, ROESY data list

[0138] No. <![CDATA[ d H (ppm)]]> Type J(Hz) COSY ROESY 1 7.22,1H t 6.9 H-2 H-2,7 2 7.07,1H t 8.8 H-1 H-1 4 7.07,1H t 8.8 H-5 H-5 5 7.22,1H t 6.9 H-4 H-4,7 7 3.81,2H s - - - 9 6.60,1H d 3.0 H-11,12 H-1,5,7,11 11 6.41,1H d 8.9 H-9,12 H-9,12 12 6.73,1H d 8.7 H-9,11 H-11,23-OH 16 7.80,1H s - - 24-NH 18 7.80,1H s - - 24-NH 23-OH 9.21,1H s - - H-7,12 24-NH 9.98,1H s - - H-16,18,25-NH 25-NH 10.51,1H s - 24-NH 26 4.19,2H q 7.1 27-CH3 27-CH3 27 1.25,3H t 7.2 26-CH2 26-CH2

[0139] Note: s = singlet, d = doublet, m = multiplet, t = triplet

[0140] This product has 2 saturated methylene groups. 1 H- 1 H ROESY and chemical shift rules, δ H

[0141] The single peak at 3.82 ppm is attributed to the saturated methylene at position 7, δ HThe triplet peak at 1.25 ppm was assigned to the saturated methylene group at position 27.

[0142] This product has 9 unsaturated methine groups. 1 H- 1 H COSY, ROESY and chemical shift rules, δ H The triplet peak at 7.22 ppm was attributed to the unsaturated methine groups at positions 1 and 5; H The triplet peak at 7.07 ppm was attributed to the unsaturated methine groups at positions 2 and 4; H The doublet peak at 6.60 ppm is attributed to the unsaturated methyl group at position 9; H The doublet peak at 6.41 ppm was attributed to the 11-position unsaturated methine; H The doublet peak at 6.73 ppm was attributed to the 12-position unsaturated methine; H The single peak at 7.80 ppm was attributed to the unsaturated methine groups at the 16- and 18-positions.

[0143] This product has one saturated methyl group. 1 H- 1 H COSY and chemical shift rules, δ H The triplet peak at 1.25 ppm was assigned to the saturated methyl group at position 27.

[0144] Table 3: NMR carbon spectrum, DEPT135, HSQC, HMBC spectrum data list

[0145]

[0146]

[0147]

[0148] DEPT135 spectrum shows that there are two methylene groups in this product. According to the chemical shift rule and HSQC, δ C

[0149] 34.42ppm is attributed to carbon 7, δ C 61.8 ppm was assigned to carbon 26.

[0150] The DEPT135 spectrum shows that there are 9 unsaturated methines in this product. According to the chemical shift rule and HSQC, the attribution is as follows: the carbon at position 1 is symmetrical with the carbon at position 5, the chemical environment and magnetic environment are the same, the carbon signals are the same, and the fluorine splitting δ C 130.30ppm and 130.25ppm are assigned to carbon 1 and carbon 5; carbon 2 and carbon 4 are symmetrical, have the same chemical and magnetic environments, and have the same carbon signals, which are affected by fluorine splitting δ C114.88ppm and 114.74ppm are attributed to carbon 2 and carbon 4; δ C 116.60ppm is attributed to carbon 9; δ C

[0151] 112.61ppm is attributed to carbon 11; δ C 115.56ppm is assigned to carbon at position 12; carbon at positions 16 and 18 are symmetrical, have the same chemical and magnetic environments, and have the same carbon signals. C 120.15 ppm was assigned to carbon 16 and carbon 18.

[0152] DEPT135 spectrum shows that δ C 161.37ppm and 159.77ppm are quaternary carbons. The HMBC spectrum shows that 1-CH, 2-CH, 4-CH, and 5-CH are remotely correlated with them and are attributed to the 3-position carbon.

[0153] DEPT135 spectrum shows that δ C 136.90ppm and 136.88ppm are quaternary carbons. The HMBC spectrum shows that 2-CH, 4-CH and 7-CH2 are remotely related to them, which are attributed to the 6-position carbon.

[0154] DEPT135 spectrum shows that δ C 128.64ppm is a quaternary carbon. The HMBC spectrum shows that 7-CH2, 12-CH and 23-OH are remotely correlated with it and are attributed to the 8-carbon.

[0155] DEPT135 spectrum shows that δ C The quaternary carbon is at 149.25 ppm. The HMBC spectrum shows that 9-CH and 12-CH are remotely correlated with it and it is attributed to the 10-position carbon.

[0156] DEPT135 spectrum shows that δ C 150.23ppm is a quaternary carbon. The HMBC spectrum shows that 7-CH2, 9-CH, 11-CH, 12-CH and 23-OH are remotely related to it and are attributed to the 13th carbon.

[0157] DEPT135 spectrum shows that δ C 142.14ppm is a quaternary carbon. The HMBC spectrum shows that 16-CH and 18-CH are remotely correlated with it, which is attributed to the 14-position carbon.

[0158] DEPT135 spectrum shows that δ C 128.61ppm is a quaternary carbon. The HMBC spectrum shows that 16-CH is remotely correlated with it and is assigned to the 15-carbon.

[0159] DEPT135 spectrum shows that δ C136.15ppm is a quaternary carbon. The HMBC spectrum shows that 16-CH, 18-CH and 24-NH are remotely related to it and are assigned to the 17th carbon.

[0160] DEPT135 spectrum shows that δ C 128.61ppm is a quaternary carbon. The HMBC spectrum shows that 18-CH is remotely correlated with it and is attributed to the 19th carbon.

[0161] DEPT135 spectrum shows that δ C 150.57ppm and δ C 154.22ppm is quaternary carbon, and the HMBC spectrum does not show any correlation with δ C Any correlation point at 150.57 ppm, HMBC spectrum shows that 26-CH C 154.22ppm long-range correlation, attributed to δ C 150.57ppm is carbon at position 20, δ C 154.22ppm is carbon at position 21 (HMBC four-bond long-range correlation).

[0162] There is an absorption peak in the fluorine spectrum of this sample, located at -117.68 ppm, indicating that the compound contains a F atom.

[0163] By comparing the carbon spectrum of impurity CU-40101-IPR01 with CU-40101, it was found that the C-20 (δ C 150.57ppm), C-21(δ C 154.22ppm) and C-20 (δ C 156.26ppm), C-21(δ C 158.74ppm) has undergone a large shift, indicating that it is the site of ethyl substitution. The two-dimensional spectrum shows that 23-OH has HMBC remote correlation with C-8, C-12, and C-13, and ROESY spatial correlation with 7-CH2 and H-12. 24-NH has HMBC remote correlation with C-16, 17, and 18, and REOSY spatial correlation with H-16, H-18, and 25-NH. 26-CH2 has HMBC remote correlation with C-21 and COSY correlation with 27-CH3. In summary, 23-OH, 24-NH and 25-NH in the impurity are not substituted, and the substitution position is on 22-OH. The impurity CU-40101-IPR01 is the product of the dehydration of 22-OH of CU-40101 with ethanol to form an ether. The structure is as follows:

[0164]

[0165] 3. Content Determination

[0166] In order to further investigate the 1 -(3,5)-dichloro-4-(3-(4-fluorobenzyl)-4-hydroxyphenoxy)phenyl)-N 2 -hydroxyoxamide for quality control, the present invention also establishes a method for determining the content of the impurity compound represented by formula (I). The specific steps are as follows:

[0167] (1) Preparation of reference solution: Take an appropriate amount of the impurity compound represented by the reference formula (I), add diluent ethanol, mix well, and prepare a reference solution with a concentration of about 0.01 mg / ml;

[0168] (2) Preparation of test solution: Take the raw material N 1 -(3,5)-dichloro-4-(3-(4-fluorobenzyl)-4-hydroxyphenoxy)phenyl)-N 2 -Hydroxyoxamide 0.2 mg, add diluent ethanol 0.8 mL, mix well, and obtain.

[0169] (3) Chromatographic conditions

[0170] Chromatographic column: C18 column

[0171] Mobile phase A: 0.05% TFA (trifluoroacetic acid) in water

[0172] Mobile phase B: 0.05% TFA (trifluoroacetic acid) acetonitrile solution

[0173] Detector: UV-visible detector

[0174] Detection wavelength: 220nm

[0175] Column temperature: 40°C

[0176] Sample chamber temperature: 4°C

[0177] Injection volume: 10uL

[0178] Gradient elution program:

[0179]

[0180] (3) Content calculation

[0181] Calculate by peak area normalization method according to the following formula:

[0182]

[0183] Among them: A S : Target impurity peak area in the test solution

[0184] A T : The sum of the chromatographic peak areas in the test solution.

[0185] It should be noted that the present invention is not limited to the above implementation modes and examples. The above implementation modes and examples are only examples, and implementation modes that have substantially the same structure as the technical idea and exert the same effect within the scope of the technical solution of the present application are all included in the technical scope of the present application. In addition, within the scope of the subject matter of the present application, various modifications and substitutions of the implementation modes / examples that can be thought of by those skilled in the art, and other methods formed by combining some of the constituent elements in the implementation modes are also included in the scope of the present application, and the scope of protection of the present invention is defined by the attached claims.

Claims

1. Impurity compounds represented by structural formula (I):

2. The method for preparing the impurity compound according to claim 1, include: 1) Take the raw material N 1 -(3,5)-dichloro-4-(3-(4-fluorobenzyl)-4-hydroxyphenoxy)phenyl)-N 2 -Hydroxyoxamide is dissolved in ethanol; 2) heating the ethanol solution of the raw material drug obtained in step (1) to degrade it; 3) separating the degraded API ethanol solution using a preparative liquid chromatograph, using a threshold collection method; 4) drying the collected fraction containing the impurity compound under reduced pressure; Wherein, the liquid chromatography conditions in step (3) are as follows: Preparative column: C18 Mobile phase A: 0.08-0.12% (v / v) formic acid-water solution Mobile phase B: 0.08-0.12% (v / v) formic acid-acetonitrile solution The gradient elution program was: Detector: UV-visible detector Detection wavelength: 248±3nm.

3. The preparation method according to claim 2, wherein in step (2), the heating temperature is above 100°C, and / or The heating time is more than 24 hours, and / or In step (3), the threshold is 20.

4. The preparation method according to claim 2, wherein in step (2), the ethanol solution of the raw material drug is heated under pressurized conditions.

5. The preparation method according to claim 4, wherein the pressurization is performed by placing the API ethanol solution in a pressurized test tube.

6. The preparation method according to claim 2, wherein the heating method is selected from oven heating, electric heating, microwave heating, and steam heating.

7. A method for determining the N content of a raw material drug 1 -(3,5)-dichloro-4-(3-(4-fluorobenzyl)-4-hydroxyphenoxy)phenyl)-N 2 -A method for determining the content of the impurity compound represented by formula (I) in hydroxyoxamide, the liquid chromatography method is as follows: Chromatographic column: C18 column Mobile phase A: TFA aqueous solution with a TFA volume percentage of 0.03%-0.07% Mobile phase B: TFA acetonitrile solution with a TFA volume percentage of 0.03%-0.07% Detector: UV-visible detector Detection wavelength: 220±3nm Gradient elution program:

8. The method of claim 7, wherein the flow rate of the mobile phase is 0.3±0.1 mL / min, and / or Sample chamber temperature: 4 ± 1 °C, and / or Column temperature: 40℃±3℃.

9. The method according to claim 7, wherein the impurity content is calculated by peak area normalization method according to the following formula: in, A S : Target impurity peak area in the test solution A T : The sum of the chromatographic peak areas in the test solution.

10. Use of the impurity compound of claim 1 as an impurity reference substance in drug quality control, wherein the drug contains N 1 -(3,5)-dichloro-4-(3-(4-fluorobenzyl)-4-hydroxyphenoxy)phenyl)-N 2 -Hydroxyoxamide.

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