A method for determining the content of DDQ and DDHQ in agomelatine

Abstract

The invention provides a method for determining the content of DDQ and DDHQ in agomelatine, belonging to the technical field of drug detection, wherein a control solution, a test solution and a test spiked solution are subjected to chromatographic analysis by a high performance liquid chromatograph. The invention establishes a method for determining the content of DDQ and DDHQ in agomelatine, and effectively separates and quantitatively detects DDQ and DDHQ that may exist in agomelatine. The invention has strong specificity, high sensitivity, good repeatability and good durability, and is of great significance for the quality control of DDQ and DDHQ in agomelatine.

Description

Technical Field

[0001] The invention relates to the technical field of drug detection, and in particular to a method for determining the contents of DDQ and DDHQ in agomelatine. Background Art

[0002] Agomelatine is a multi-target antidepressant that is a melatonin receptor agonist (MT1 and MT2 receptors) and a 5-HT2C receptor antagonist. It has a good antidepressant effect. Its mechanism of action breaks through the traditional monoamine neurotransmitter system. By activating melatonin receptors MT1 and MT2 and antagonizing 5-HT2C receptors, it has the function of regulating biological rhythms while exerting antidepressant effects. It is the only melatonin receptor agonist used to treat depression. Agomelatine has a positive phase adjustment effect on sleep and induces an advance in sleep phase.

[0003] The chemical name of agomelatine is N-[2-(7-methoxy-1-naphthyl)ethyl]acetamide, and its molecular formula is C 15 H 17 NO2, CAS No. 138112-76-2, chemical structure is as follows:

[0004]

[0005] 2,3-Dichloro-5,6-dicyanobenzoquinone (DDQ for short) is used as a catalyst in the synthesis process of agomelatine, which can be converted into 4,5-dichloro-3,6-dihydroxyphthalonitrile (DDHQ for short) after the reaction. DDQ is widely used as a catalyst in organic synthesis, and both DDQ and DDHQ contain genotoxic warning structural quinones, and the toxicological concern threshold (TTC, 1.5 μg per day) is used to control the risk of medication. Based on the TTC calculation method, the maximum daily dose of agomelatine is 50 mg, and it is calculated that the limits of DDQ and DDHQ should not exceed 30 ppm. However, the current testing methods have limitations such as low detection sensitivity, non-repeatability, and inaccurate detection.

[0006] Therefore, it is very important to provide a safe, reliable, sensitive, accurate and durable method for determining the contents of DDQ and DDHQ in agomelatine. Summary of the invention

[0007] The object of the present invention is to provide a method for determining the contents of DDQ and DDHQ in agomelatine, so as to solve the technical problems of low sensitivity and accuracy, poor durability and poor repeatability in the prior art.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a method for determining the contents of DDQ and DDHQ in agomelatine, comprising the following steps:

[0010] (1) Mixing DDQ reference substance, DDHQ reference substance and methanol to obtain a reference solution;

[0011] The agomelatine test sample and methanol were mixed to obtain a test sample solution;

[0012] The DDQ reference substance, DDHQ reference substance, agomelatine test substance and methanol were mixed to obtain the test sample spiked solution;

[0013] (2) The control solution, the test solution and the test solution spiked with the test sample were subjected to chromatographic detection, and the contents of DDQ and DDHQ in agomelatine were calculated by the external standard method based on the peak area and concentration;

[0014] The mobile phase A of the chromatographic detection is a mixed solution of a buffered saline solution and triethylamine in a volume ratio of 900-1100:1, and the mobile phase B is methanol.

[0015] Further, the concentrations of DDQ and DDHQ in the control solution are independently 0.12 to 0.36 μg / mL;

[0016] The concentration of the test solution is 6-10 mg / mL;

[0017] The concentration of agomelatine in the sample spiked solution was 6-10 mg / mL, the concentration of DDQ was 0.12-0.36 μg / mL, and the concentration of DDHQ was 0.12-0.36 μg / mL.

[0018] Furthermore, the conditions for the chromatographic detection are: flow rate of 0.9-1.1 mL / min, column temperature of 28-32° C., sample chamber temperature of 3-5° C., injection volume of 18-22 μL, detection wavelength of 218-222 nm, and detection time of 18-22 min.

[0019] Furthermore, in step (2), the chromatographic detection uses an octadecylsilane bonded silica gel column with a column length of 100 to 300 mm, an inner diameter of 4 to 5 mm, a filler particle size of 3 to 5 μm, and the model of the chromatographic column is Waters XBridgeC18.

[0020] Furthermore, in step (2), the procedure of gradient elution in chromatographic detection is:

[0021] At 0-6 min, it is 85-45% mobile phase A and 15-55% mobile phase B;

[0022] From 6 to 12 minutes, excluding 6 minutes, it is 45 to 25% mobile phase A and 55 to 75% mobile phase B;

[0023] At 12 to 15 minutes, excluding 12 minutes, 25 to 85% of mobile phase A and 75 to 15% of mobile phase B;

[0024] During 15 to 20 minutes (excluding 15 minutes), the mobile phase A was 85% and the mobile phase B was 15%.

[0025] Furthermore, the buffered saline solution is a potassium dihydrogen phosphate aqueous solution, and the concentration of the potassium dihydrogen phosphate aqueous solution is 1-2 g / L.

[0026] Furthermore, the pH of the mobile phase A is 2-4, and the reagent used to adjust the pH is phosphoric acid.

[0027] Beneficial effects of the present invention:

[0028] (1) The present invention improves the detection sensitivity of DDQ and DDHQ by utilizing high performance liquid chromatography, using a gradient elution program, and combining with a suitable diluent. The detection limit of DDQ is 0.0116 μg / mL, and the quantification limit is 0.0232 μg / mL. The detection limit of DDHQ is 0.0030 μg / mL, and the quantification limit is 0.0060 μg / mL.

[0029] (2) The determination method of the present invention has strong specificity, good repeatability and good durability, which provides a basis for the establishment of the quality standard of agomelatine.

[0030] (3) The determination method of the present invention has mild conditions and has no stringent requirements on high performance liquid chromatographs, chromatographic columns and analytical reagents, and is applicable to conventional liquid chromatographs, chromatographic columns and analytical reagents. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is the detection chromatogram of the blank solution of Example 1;

[0032] Figure 2 is the detection chromatogram of the control solution of Example 1;

[0033] Figure 3 The detection chromatogram of the test solution with batch number SS16-P-2305001 in Example 1;

[0034] Figure 4 is the linearity graph of DDQ in linear solutions with different concentrations;

[0035] Figure 5 The linearity graph of DDHQ in linear solutions with different concentrations. DETAILED DESCRIPTION

[0036] The present invention provides a method for determining the contents of DDQ and DDHQ in agomelatine, comprising the following steps:

[0037] (1) Mixing DDQ reference substance, DDHQ reference substance and methanol to obtain a reference solution;

[0038] The agomelatine test sample and methanol were mixed to obtain a test sample solution;

[0039] The DDQ reference substance, DDHQ reference substance, agomelatine test substance and methanol were mixed to obtain the test sample spiked solution;

[0040] (2) The control solution, the test solution and the test solution spiked with the test sample were subjected to chromatographic detection, and the contents of DDQ and DDHQ in agomelatine were calculated by the external standard method based on the peak area and concentration.

[0041] In the present invention, a blank solution is also included when performing chromatographic detection, and the blank solution is methanol.

[0042] In the present invention, the mobile phase A of the chromatographic detection is a mixed solution of a buffered saline solution and triethylamine, with a volume ratio of 900-1100:1, preferably 950-1050:1, and more preferably 1000:1; the mobile phase B is methanol.

[0043] In the present invention, the concentrations of DDQ and DDHQ in the control solution are independently 0.12 to 0.36 μg / mL, preferably 0.20 to 0.28 μg / mL, and more preferably 0.24 μg / mL;

[0044] The concentration of the test solution is 6 to 10 mg / mL, preferably 7 to 9 mg / mL, and more preferably 8 mg / mL;

[0045] The concentration of agomelatine in the sample spiked solution is 6-10 mg / mL, preferably 7-9 mg / mL, and more preferably 8 mg / mL; the concentration of DDQ is 0.12-0.36 μg / mL, preferably 0.20-0.28 μg / mL, and more preferably 0.24 μg / mL; the concentration of DDHQ is 0.12-0.36 μg / mL, preferably 0.20-0.28 μg / mL, and more preferably 0.24 μg / mL.

[0046] In the present invention, the conditions for the chromatographic detection are:

[0047] The flow rate is 0.9 to 1.1 mL / min, preferably 1 mL / min;

[0048] The column temperature is 28-32°C, preferably 29-31°C, and more preferably 30°C;

[0049] The sample chamber temperature is 3-5°C, preferably 3.5-4.5°C, and more preferably 4°C;

[0050] The injection volume is 18 to 22 μL, preferably 19 to 21 μL, and more preferably 20 μL;

[0051] The detection wavelength is 218-222 nm, preferably 219-221 nm, and more preferably 220 nm;

[0052] The detection time is 18 to 22 minutes, preferably 19 to 21 minutes, and more preferably 20 minutes.

[0053] In the present invention, in the step (2), the chromatographic column used for chromatographic detection is an octadecylsilane bonded silica gel column, with a column length of 100 to 300 mm, preferably 100 to 200 mm, and more preferably 150 mm; an inner diameter of 4 to 5 mm, preferably 4.2 to 4.8 mm, and more preferably 4.6 mm; a filler particle size of 3 to 5 μm, preferably 3.5 to 5 μm, and more preferably 5 μm; and the model of the chromatographic column is preferably Waters XBridge C18.

[0054] In the present invention, in step (2), the procedure of gradient elution in chromatographic detection is:

[0055] At 0-6 min, it is 85-45% mobile phase A and 15-55% mobile phase B;

[0056] From 6 to 12 minutes, excluding 6 minutes, it is 45 to 25% mobile phase A and 55 to 75% mobile phase B;

[0057] At 12 to 15 minutes, excluding 12 minutes, 25 to 85% of mobile phase A and 75 to 15% of mobile phase B;

[0058] During 15 to 20 minutes (excluding 15 minutes), the mobile phase A was 85% and the mobile phase B was 15%.

[0059] In the present invention, the buffered saline solution is a potassium dihydrogen phosphate aqueous solution, and the concentration of the potassium dihydrogen phosphate aqueous solution is 1 to 2 g / L, preferably 1.2 to 1.8 g / L, and more preferably 1.4 to 1.6 g / L.

[0060] In the present invention, the pH of the mobile phase A is 2 to 4, preferably 2.5 to 3.5, and more preferably 3.0; the reagent used to adjust the pH is preferably phosphoric acid.

[0061] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0062] The structural formula of DDQ used in the embodiment of the present invention is The CAS number is 84-58-2, the manufacturer is Heyuan Broad Spectrum Biotechnology Co., Ltd., the batch number is GB126001-01, and the content is 95.56%; the structural formula of DDHQ is CAS No. 4640-41-9, manufacturer Heyuan Broad Spectrum Biotechnology Co., Ltd., batch No. GD351218-01, content 98.51%; agomelatine test sample manufacturer Jiangsu Weiqida Pharmaceutical Co., Ltd., batch No. SS16-P-2305001, AG16-2305002, AG16-2305003 and AG16-2305004; methanol manufacturer Merck k), batch number 20230522-34; the manufacturer of potassium dihydrogen phosphate is Scharlau Co., Ltd. of Spain, batch number 19266901; the manufacturer of triethylamine is Sinopharm Group, batch number 20230706-01, content ≥99.0%; the manufacturer of phosphoric acid is Aladdin Biochemical Technology Co., Ltd. (aladdin), batch number 20230315-01, content is 85-90%.

[0063] The electronic balance used in the embodiments of the present invention is manufactured by Mettler, and the models are XS205, XPR10 and ML204; the pH meter is manufactured by Mettler, and the model is S220; the high performance liquid chromatograph is manufactured by Waters Technology (Shanghai) Co., Ltd. (Waters), and the model is Waters e2695 / UV2489; the chromatographic column is manufactured by Waters, and the model is Waters XBridge C18; the workstation is manufactured by Agilent, and the model is Openlab CDS2.4.

[0064] Example 1

[0065] Methanol was used as blank solution;

[0066] The DDQ reference substance, the DDHQ reference substance and methanol were mixed to obtain a control solution, wherein the concentrations of DDQ and DDHQ in the control solution were independently 0.24 μg / mL;

[0067] Agomelatine test samples with batch numbers SS16-P-2305001, AG16-2305002, AG16-2305003 and AG16-2305004 were mixed with methanol to obtain test solution, and the concentration of the test solution was 8 mg / mL;

[0068] The DDQ reference substance, the DDHQ reference substance, the agomelatine test substance and methanol were mixed to obtain a test sample spiked solution, wherein the concentration of the agomelatine test substance in the test sample spiked solution was 8 mg / mL, the concentration of DDQ was 0.24 μg / mL and the concentration of DDHQ was 0.24 μg / mL;

[0069] The blank solution, control solution, test solution and test spiked solution were subjected to chromatographic detection, and the contents of DDQ and DDHQ in agomelatine were calculated by peak area and concentration using the external standard method;

[0070] The conditions for chromatographic detection were as follows: chromatographic column: Waters XBridge C18, 4.6×150mm×5μm; trapping column: Welch 7.8×50mm; column temperature: 30°C; sample chamber temperature: 4°C; wavelength: 220nm; flow rate: 1.0mL / min; injection volume: 20μL; running time: 20min; mobile phase A: 1.36g / L potassium dihydrogen phosphate aqueous solution plus 0.1% triethylamine, adjusted to pH 3.0 with phosphoric acid; mobile phase B: methanol;

[0071] The gradient elution program was:

[0072] At 0-6 min, it is 85-45% mobile phase A and 15-55% mobile phase B;

[0073] From 6 to 12 minutes, excluding 6 minutes, it is 45 to 25% mobile phase A and 55 to 75% mobile phase B;

[0074] At 12 to 15 minutes, excluding 12 minutes, 25 to 85% of mobile phase A and 75 to 15% of mobile phase B;

[0075] During 15 to 20 minutes (excluding 15 minutes), the mobile phase A was 85% and the mobile phase B was 15%.

[0076] The test solution in Example 1 was tested, and the results of the determination of DDQ and DDHQ contents are shown in Table 1.

[0077] Table 1 Determination results of DDQ and DDHQ contents in test solution

[0078] Test sample batch number DDQ content DDHQ content SS16-P-2305001 Not detected Not detected AG16-2305002 Not detected Not detected AG16-2305003 Not detected Not detected AG16-2305004 Not detected Not detected

[0079] Figure 1 It is the detection chromatogram of the blank solution of Example 1; Figure 2 is the detection chromatogram of the control solution of Example 1; Figure 3 The chromatogram is the test chromatogram of the test solution with batch number SS16-P-2305001 in Example 1. Figures 1 to 3It can be seen that the peak order is DDQ, DDHQ, and the separation degree of DDQ and DDHQ should be no less than 1.5. According to the external standard method, the DDQ in the test solution should not exceed 30ppm, and the DDHQ should not exceed 30ppm. As can be seen from Table 1, DDQ and DDHQ were not detected in the test solution, and the test results met the requirements.

[0080] The determination method of the present invention was tested for specificity: methanol, test solution, test spiked solution, and control solution were tested according to the chromatographic conditions in Example 1, with one injection each. The test results are shown in Table 2.

[0081] Table 2 Specificity test results

[0082]

[0083] As can be seen from Table 2, methanol has no interference with the detection of DDQ and DDHQ, and the separation degree between DDQ and DDHQ is greater than 1.5, indicating that the determination method of the present invention has good specificity.

[0084] The system suitability test of the determination method of the present invention was carried out: blank solution and control solution were tested according to the chromatographic conditions in Example 1, the blank solution was injected into 1 needle, and the control solution was injected into 5 needles. The test results are shown in Table 3.

[0085] Table 3 System suitability test results

[0086]

[0087] As shown in Table 3, the separation degree of DDQ and DDHQ is greater than 1.5, and the RSD of the peak areas of DDQ and DDHQ in the reference solution for 5 consecutive times is less than 10%, indicating that the system applicability of the determination method of the present invention meets the requirements.

[0088] The quantitative limit and detection limit of the determination method of the present invention were tested: the control solution was diluted to a certain concentration, and the concentration when the peak signal-to-noise ratio was about 10:1 was taken as the quantitative limit concentration, and 6 injections of the quantitative limit solution were injected continuously; the concentration when the peak signal-to-noise ratio was about 3:1 was taken as the detection limit concentration, and 2 injections of the detection limit solution were injected continuously. The test results are shown in Table 4.

[0089] Table 4 Quantitation limit and detection limit test results

[0090]

[0091] As shown in Table 4, the detection limit and quantification limit of DDQ and DDHQ are relatively low, indicating that the detection sensitivity of the method is high and can meet the determination requirements.

[0092] The linearity and range of the determination method of the present invention were tested: linear solutions of different concentrations were prepared with reference substances and injected respectively. The concentration range of DDQ in the linear solution was 0.0232-0.3478 μg / mL, and the concentration range of DDHQ was 0.0060-0.3596 μg / mL. Figure 4 is the linearity graph of DDQ in linear solutions with different concentrations; Figure 5 The linearity graph of DDHQ in linear solutions of different concentrations is shown in Table 5. The standard curve is drawn with concentration as the abscissa and peak area as the ordinate. The test results of linearity and range are shown in Table 5.

[0093] Table 5 Linearity and range test results

[0094] name Linear equations Correlation coefficient scope DDQ y=25.373x-0.0091 0.9988 0.0232~0.3478μg / mL DDH y=140.31x-0.0591 0.9999 0.0060~0.3596μg / mL

[0095] Depend on Figures 4-5 As shown in Table 5, using the determination method of the present invention, both DDQ and DDHQ have a good linear relationship.

[0096] The accuracy test of the determination method of the present invention was carried out: the test sample, the mixed solution of DDQ and DDHQ and the diluent were made into recovery solutions, and the mass fractions of the mixed solution of DDQ and DDHQ in the recovery solution were 80%, 100% and 120%, respectively. Three portions of high, medium and low concentrations were prepared in parallel, and the accuracy test results are shown in Table 6.

[0097] Table 6 Accuracy test results

[0098]

[0099] As shown in Table 6, the recoveries of DDQ and DDHQ in the determination method of the present invention are both within the range of 80-120%, and the RSDs of the 9 recoveries are all less than 10%, indicating that the determination method of the present invention has good accuracy.

[0100] The determination method of the present invention was subjected to a precision test: Experimenter A prepared 1 portion of the control solution and 6 portions of the test sample spiked solution in parallel according to the preparation method of the control solution and the test sample spiked solution in Example 1, and tested them separately as the repeatability results. Experimenter B used different instruments and different chromatographic columns on different dates to prepare 1 portion of the control solution and 6 portions of the test sample spiked solution in parallel according to the preparation method of the control solution and the test sample spiked solution in Example 1, and tested them separately as the intermediate precision results, and calculated the RSD of the 12 results. The precision results are shown in Table 7.

[0101] Table 7 Precision test results

[0102]

[0103]

[0104] As shown in Table 7, in the 12 results, the RSDs of the DDQ and DDHQ contents were all less than 10%, indicating that the determination method of the present invention has good precision.

[0105] The solution stability test of the determination method of the present invention was carried out: the control solution and the test solution were prepared according to Example 1, and placed at 4°C for several hours. The separation of DDQ and DDHQ in the control solution at each time point, the RSD of the peak areas of DDQ and DDHQ, and the RSD values ​​of the DDQ and DDHQ contents in the test solution and the content test results were investigated. The solution stability test results are shown in Table 8.

[0106] Table 8 Solution stability test results

[0107]

[0108] As shown in Table 8, the control solution was placed at 4°C for 46 hours, and the separation of DDQ and DDHQ in the control solution at each time point was greater than 1.5, and the RSD of the peak areas of DDQ and DDHQ were less than 10%, indicating that the control solution was stable for 46 hours at 4°C. The test solution was placed at 4°C for 46 hours, and DDQ and DDHQ were not detected in the test solution at each time point, indicating that the test solution was stable for 46 hours at 4°C.

[0109] Example 2

[0110] Compared with Example 1, the only difference is that the flow rate of the chromatographic detection is 0.9 mL / min.

[0111] Example 3

[0112] Compared with Example 1, the only difference is that the flow rate of the chromatographic detection is 1.1 mL / min.

[0113] Example 4

[0114] Compared with Example 1, the only difference is that the injection volume for chromatographic detection is 18 μL.

[0115] Example 5

[0116] Compared with Example 1, the only difference is that the injection volume for chromatographic detection is 22 μL.

[0117] Example 6

[0118] Compared with Example 1, the only difference is that the column temperature of the chromatographic detection is 28°C.

[0119] Example 7

[0120] Compared with Example 1, the only difference is that the column temperature of the chromatographic detection is 32°C.

[0121] Example 8

[0122] Compared with Example 1, the only difference is that the pH value of the mobile phase A for chromatographic detection is 2.9.

[0123] Example 9

[0124] Compared with Example 1, the only difference is that the pH value of the mobile phase A for chromatographic detection is 3.1.

[0125] The durability test was performed on Examples 1 to 9, and the test results of the separation of DDQ and DDHQ in the control solution, the RSD of the peak areas of DDQ and DDHQ in the control solution, the RSD values ​​of DDQ and DDHQ in the test sample spiked solution, and the DDQ and DDHQ content results are shown in Table 9.

[0126] Table 9 Durability test results

[0127]

[0128]

[0129] As shown in Table 8, when the flow rate, injection volume, column temperature, and mobile phase pH were changed, the separation of DDQ and DDHQ in the control solution was greater than 1.5, the RSDs of the peak areas of DDQ and DDHQ were less than 10%, and the RSDs of the contents of DDQ and DDHQ were less than 10%, indicating that the determination method of the present invention has good durability.

[0130] As can be seen from the above embodiments, the present invention provides a method for determining the content of DDQ and DDHQ in agomelatine, which can well detect the content of DDQ and DDHQ under specific chromatographic columns and detection conditions, thereby achieving control of DDQ and DDHQ genotoxic residues in agomelatine. The present invention improves the sensitivity and separation of DDQ and DDHQ by utilizing high performance liquid chromatography, using a gradient elution program, and combining a suitable diluent. The method has strong specificity, good repeatability, and good durability, and can accurately detect DDQ and DDHQ genotoxic residues in products.

[0131] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for determining the contents of DDQ and DDHQ in agomelatine, characterized in that: The following steps are involved: (1) Mix DDQ reference substance, DDHQ reference substance and methanol to obtain a reference solution; The agomelatine test sample and methanol were mixed to obtain a test sample solution; The DDQ reference substance, DDHQ reference substance, agomelatine test substance and methanol were mixed to obtain the test sample spiked solution; (2) The control solution, the test solution and the test solution spiked with the test sample were subjected to chromatographic detection, and the contents of DDQ and DDHQ in agomelatine were calculated by the external standard method based on the peak area and concentration; The mobile phase A of the chromatographic detection is a mixed solution of a buffered saline solution and triethylamine in a volume ratio of 900-1100:1, and the mobile phase B is methanol; The conditions of the chromatographic detection are: flow rate of 0.9~1.1mL / min, column temperature of 28~32°C, sample chamber temperature of 3~5°C, injection volume of 18~22µL, detection wavelength of 218~222nm, and detection time of 18~22min; In the step (2), the chromatographic detection uses an octadecylsilane bonded silica gel column with a column length of 150 mm, an inner diameter of 4.6 mm, and a filler particle size of 5 μm; In the step (2), the procedure of gradient elution in the chromatographic detection is: At 0-6 min, it is 85-45% mobile phase A and 15-55% mobile phase B; From 6 to 12 minutes, excluding 6 minutes, the mobile phase is 45 to 25% A and 55 to 75% B. At 12-15 min (excluding 12 min), 25-85% mobile phase A and 75-15% mobile phase B; From 15 to 20 minutes, excluding 15 minutes, it is 85% mobile phase A and 15% mobile phase B; The buffered saline solution is a potassium dihydrogen phosphate aqueous solution, and the concentration of the potassium dihydrogen phosphate aqueous solution is 1-2 g / L; The pH of the mobile phase A is 2-4, and the reagent used to adjust the pH is phosphoric acid; The structural formula of the DDQ is The structural formula of DDHQ is .

2. The measuring method according to claim 1, characterized in that The concentrations of DDQ and DDHQ in the control solution were independently 0.12 to 0.36 µg / mL; The concentration of the test solution is 6-10 mg / mL; The concentration of agomelatine in the sample spiked solution was 6-10 mg / mL, the concentration of DDQ was 0.12-0.36 µg / mL, and the concentration of DDHQ was 0.12-0.36 µg / mL.

3. The measuring method according to claim 1 or 2, characterized in that In the step (2), the model of the chromatographic column is Waters XBridge C18.

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