Method for detecting related substances of eszopiclone intermediate YZ2

Abstract

The invention belongs to the technical field of medicine detection, and particularly discloses a method for detecting related substances of an eszopiclone intermediate YZ2, which comprises the following steps of: preparing a test solution: taking a proper amount of eszopiclone intermediate test, and dissolving the eszopiclone intermediate test with acetonitrile to a constant volume; an impurity positioning solution: taking proper amounts of reference substances of impurities C, H and N, and respectively dissolving the reference substances with acetonitrile to a constant volume; the determination method comprises the following steps: respectively and precisely measuring the test solution and the three impurity positioning solutions, injecting into a high performance liquid chromatograph, and recording chromatograms; and carrying out peak positioning on the three impurities according to the chromatogram of the impurity positioning solution, analyzing the chromatogram of the test solution, and calculating according to a peak area normalization method to obtain the content of each related substance in the eszopiclone intermediate. According to the present invention, the high performance liquid chromatography is adopted, and the contents of the effective components and the impurities in the eszopiclone intermediate are analyzed, such that the purity of the eszopiclone intermediate can be monitored, the quality can be ensured, and the production process can be evaluated.

Description

Technical Field

[0001] The invention belongs to the technical field of drug detection, and particularly relates to a method for detecting related substances of a dexzopiclone intermediate YZ2. Background Art

[0002] Eszopiclone, chemical name: 6-(5-chloro-2-pyridyl)-7-[(4-methylpiperazine-1-yl)carboxyloxy]-5,6-dihydropyrrolo[3,4-b]pyrazine-5-one. Eszopiclone is a fast-acting, short-acting non-benzodiazepine sedative hypnotic. It is the right-rotating single isomer of zopiclone and has sedative, anxiolytic, anticonvulsant and muscle relaxant effects. Studies have shown that eszopiclone is similar to long-acting benzodiazepines in improving sleep quality and is similar to short-acting benzodiazepines in shortening the time to fall asleep. The residual effect and hangover phenomenon after taking the drug are less than those of benzodiazepines. There is no obvious drug resistance after long-term use. It is an ideal sedative hypnotic drug.

[0003] In the preparation process of eszopiclone, a key intermediate is involved, the chemical name is: 6-(5-chloro-2-pyridyl)-6,7-dihydro-7-hydroxy-5H-pyrrolo[3,4-b]pyrazin-5-one (YZ2), the structural formula is as follows:

[0004] In the synthesis of the dexamethasone intermediate YZ2, there are three related substances: (1) Impurity C (generated by reduction and hydrogenation of the dexamethasone intermediate YZ2); (2) Impurity H (the starting material for the synthesis of eszopiclone intermediate YZ2); (3) Impurity N (generated by hydrolysis of dexamethasone YZ2).

[0005] The structure is as follows: Impurity C ; Impurity H ; Impurity N .

[0006] At present, there are few literature reports on the detection method of related substances of dexzopiclone intermediate YZ2. In order to strengthen the quality control of dexzopiclone intermediate YZ2 and meet existing needs, it is urgent to develop a detection method for related substances of dexzopiclone intermediate YZ2. Summary of the invention

[0007] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a method for detecting related substances of the dexazopiclone intermediate YZ2, which adopts high performance liquid chromatography, has high sensitivity, fast analysis speed, high equipment penetration rate, is easy to promote, and can effectively solve the problems in the background technology.

[0008] The present invention is achieved through the following technical solutions: A method for detecting related substances of 6-(5-chloro-2-pyridyl)-6,7-dihydro-7-hydroxy-5H-pyrrolo[3,4-b]pyrazin-5-one, an intermediate of dexzopiclone, comprises the following steps: Test solution: Take an appropriate amount of the dextrozopiclone intermediate test sample and dissolve it in acetonitrile to make the test solution.

[0009] Impurity C localization solution: Take an appropriate amount of impurity C reference substance and dissolve it in acetonitrile to make the impurity C localization solution.

[0010] Impurity H localization solution: Take an appropriate amount of impurity H reference substance and dissolve it in acetonitrile to make the impurity H localization solution.

[0011] Impurity N localization solution: Take an appropriate amount of impurity N reference substance and dissolve it in acetonitrile to make the impurity N localization solution.

[0012] Determination method: Accurately measure the test solution, impurity C positioning solution, impurity H positioning solution, and impurity N positioning solution, respectively, inject them into the high performance liquid chromatograph, and record the chromatogram; locate the peaks of the three impurities according to the chromatogram of the impurity positioning solution, analyze the chromatogram of the test solution, and calculate by the peak area normalization method to obtain the content of each related substance in the dexzopiclone intermediate.

[0013] Wherein, the structure of the dexazopiclone intermediate is as follows:

[0014] The related substances include impurity C, impurity H, and impurity N, and the structure is as follows: Impurity C ; Impurity H ; Impurity N .

[0015] A further improvement of the present invention is: The test solution preparation method is as follows: take an appropriate amount of the dexzopiclone intermediate test sample, dissolve it in acetonitrile to make a solution with a concentration of 0.4 mg / mL.

[0016] Furthermore, the method for preparing the impurity C localization solution is as follows: take an appropriate amount of impurity C reference substance, dissolve it in acetonitrile to make a solution with a concentration of 0.4 μg / mL.

[0017] Furthermore, the impurity H localization solution is prepared as follows: take an appropriate amount of impurity H reference substance, dissolve it in acetonitrile to make a solution with a concentration of 0.4 μg / mL.

[0018] Furthermore, the method for preparing the impurity N localization solution is as follows: take an appropriate amount of impurity N reference substance, dissolve it in acetonitrile to make a solution with a concentration of 8 μg / mL.

[0019] A further improvement of the present invention is: The chromatographic conditions of the high performance liquid chromatograph are as follows: Chromatographic column: Merck Purospher STAR RP-C18, 250 mm × 4.6 mm, 5 μm; Mobile phase: Mobile phase A: perchloric acid and trifluoroacetic acid aqueous solution; Mobile phase B: acetonitrile; gradient:

[0020] Flow rate: 1.0 mL / min; Detection wavelength: 303nm; Column temperature: 30°C; Injection volume: 20 μL.

[0021] Furthermore, the preparation method of the perchloric acid-trifluoroacetic acid aqueous solution is as follows: take 0.43 mL of perchloric acid with a content of 70%-72% and 1 mL of trifluoroacetic acid, put them into 1000 mL of water, and mix them evenly.

[0022] A further improvement of the present invention is: In the chromatogram of the test sample solution, the content of the dexamethasone intermediate calculated by the peak area normalization method shall not be less than 96%.

[0023] Furthermore, in the chromatogram of the test solution, if there are impurity peaks, calculated by the peak area normalization method, the impurity N shall not be greater than 4%, other single impurities shall not be greater than 0.10%, and the total amount of impurities shall not be greater than 4%. Beneficial Effects

[0024] Compared with the prior art, the present invention has the following obvious advantages: 1. The present invention provides a method for detecting related substances of the dexzopiclone intermediate YZ2, which adopts high performance liquid chromatography, has high sensitivity, fast analysis speed, high equipment penetration rate, and is easy to promote.

[0025] 2. The present invention adds perchloric acid and trifluoroacetic acid to the mobile phase A to improve the peak shape and overcome the problems of peak broadening and tailing; under the chromatographic conditions provided by the present invention, the impurities can be effectively separated from each other and from the main peak, and the peak shape is good, which is convenient for subsequent analysis of the chromatogram.

[0026] 3. The method has been verified to have good specificity, precision, accuracy, durability and stability. By analyzing the contents of active ingredients and impurities in the dexzopiclone intermediate YZ2, the purity of the dexzopiclone intermediate YZ2 can be monitored to ensure its quality, and the production process of the dexzopiclone intermediate YZ2 can be evaluated. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the blank solvent spectrum; Figure 2 Locate the solution spectrum for impurity C; Figure 3 The solution map for impurity H positioning; Figure 4 The solution map for locating impurity N; Figure 5 It is the spectrum of the test sample spiked solution; Figure 6 This is the spectrum of the test solution (batch number: YZ220601B) in Example 1; Figure 7 This is the spectrum of the test solution (batch number: YZ221001B) in Example 2; Figure 8 This is the spectrum of the test solution (batch number: YZ221002B) in Example 3; Fig. 9 This is the spectrum of the test solution (batch number: YZ221101B) in Example 4; Fig.10 This is the spectrum of the test solution (batch number: YZ221102B) in Example 5; Fig.11 This is the spectrum of the test solution (batch number: YZ221103B) in Example 6; Fig.12 The standard curve diagram of impurity N in the linearity and range of Example 7; Fig.13 The standard curve of impurity H in the linearity and range of Example 7; Fig.14 The standard curve diagram of the impurity C in the linearity and range of Example 7; Fig.15 This is a standard curve diagram of the linearity and range of the dexazopiclone intermediate YZ2 in Example 7. DETAILED DESCRIPTION

[0028] The instruments, chromatographic columns, reagents, reference substances and samples used in the present invention are as follows: Instruments and Columns

[0029] Reagents

[0030] Reference

[0031] sample

[0032] The present invention is described in detail below in conjunction with embodiments.

[0033] Example 1-6 Sample Testing 1. Analytical methods (1) Test solution: Take an appropriate amount of the test sample of the intermediate YZ2 of right-zopiclone, accurately weigh it, add an appropriate amount of acetonitrile, dissolve it by ultrasonication, and then dissolve it with acetonitrile to make a solution with a concentration of 0.4 mg / mL.

[0034] (2) Impurity C localization solution: Take an appropriate amount of impurity C reference substance and dissolve it in acetonitrile to make a solution with a concentration of 0.4 μg / mL.

[0035] (3) Impurity H localization solution: Take an appropriate amount of impurity H reference substance and dissolve it in acetonitrile to make a solution with a concentration of 0.4 μg / mL.

[0036] (4) Impurity N localization solution: Take an appropriate amount of impurity N reference substance and dissolve it in acetonitrile to make a solution with a concentration of 8 μg / mL.

[0037] (5) Determination method: Accurately measure the test solution, impurity C positioning solution, impurity H positioning solution, and impurity N positioning solution, respectively, inject them into the high performance liquid chromatograph, and record the chromatogram; locate the peaks of the three impurities according to the chromatogram of the impurity positioning solution, analyze the chromatogram of the test solution, and calculate by the peak area normalization method to obtain the content of each related substance in the dexzopiclone intermediate.

[0038] (6) Chromatographic conditions: Chromatographic column: Merck Purospher STAR RP-C18, 250 mm × 4.6 mm, 5 μm; Mobile phase: Mobile phase A: perchloric acid and trifluoroacetic acid aqueous solution; Mobile phase B: acetonitrile; gradient:

[0039] Flow rate: 1.0 mL / min; Detection wavelength: 303nm; Column temperature: 30°C; Injection volume: 20 μL.

[0040] Furthermore, the preparation method of the perchloric acid-trifluoroacetic acid aqueous solution is as follows: take 0.43 mL of perchloric acid with a content of 70%-72% and 1 mL of trifluoroacetic acid, put them into 1000 mL of water, and mix them evenly.

[0041] (7) Limits: If there are impurity peaks in the chromatogram of the test solution, the impurity N shall not be greater than 4%, other individual impurities shall not be greater than 0.10%, and the total amount of impurities shall not be greater than 4%, calculated by the peak area normalization method.

[0042] (8) Content: In the chromatogram of the test sample solution, the content of the intermediate YZ2 of right-zopiclone calculated by the peak area normalization method shall not be less than 96%.

[0043] 2. Sample determination According to the analytical method for the determination of related substances and content of the dexzopiclone intermediate YZ2, six batches of YZ2 samples were determined, such as Figures 6 to 11 The results are shown in Table 1.

[0044] Table 1 Sample measurement results

[0045] Among them: The normalized content results of impurity H and impurity C are shown in Table 2 below: Table 2 Normalized content results of impurity H and impurity C

[0046] The results showed that the relevant substances and content determination results of the 6 batches of samples were in compliance with the regulations, among which impurity H and impurity C did not exceed 0.03%.

[0047] Example 7 Detection Method Verification 1. Exclusivity (1) Blank solvent: acetonitrile.

[0048] (2) Location of impurity H: Take about 2 mg of impurity H reference substance, accurately weigh it, place it in a 100 mL volumetric flask, add acetonitrile to dissolve and dilute to the mark, shake well, and use it as the impurity H stock solution; accurately measure 1 mL of impurity H stock solution, place it in a 50 mL volumetric flask, dilute to the mark with acetonitrile, and shake well.

[0049] (3) Location of Impurity N: Take about 1.6 mg of Impurity N reference substance, accurately weigh it, place it in a 20 mL volumetric flask, add acetonitrile to dissolve and dilute to the mark, shake well, and use it as the Impurity N stock solution; accurately measure 1 mL of Impurity N stock solution, place it in a 10 mL volumetric flask, dilute to the mark with acetonitrile, and shake well.

[0050] (4) Location of Impurity C: Take about 2 mg of the Impurity C reference substance, accurately weigh it, place it in a 100 mL volumetric flask, add acetonitrile to dissolve and dilute to the mark, shake well, and use it as the Impurity C stock solution; accurately measure 1 mL of the Impurity C stock solution, place it in a 50 mL volumetric flask, dilute to the mark with acetonitrile, and shake well.

[0051] (5) Test solution: Take an appropriate amount of the test sample of the intermediate YZ2 of right-zopiclone, accurately weigh it, add an appropriate amount of acetonitrile, sonicate to dissolve it, and quantitatively dilute it with acetonitrile to make a solution containing approximately 0.4 mg per 1 mL.

[0052] (6) Test sample spiked solution: Take about 20 mg of the test sample of the intermediate YZ2 of right-zopiclone, accurately weigh it, place it in a 50-mL volumetric flask, add an appropriate amount of acetonitrile, sonicate to dissolve, accurately add 1 mL of the impurity H stock solution, 1 mL of the impurity C stock solution, and 5 mL of the impurity N stock solution, dilute to the scale with acetonitrile, and shake well.

[0053] Accurately measure each of the above solutions, inject them into the liquid chromatograph respectively, record the chromatogram, and investigate the interference of the blank solvent on the related substances and content determination of the dexzopiclone intermediate YZ2 and the separation between the peaks. The results are shown in Table 3.

[0054] Table 3 Specificity determination results

[0055] The results showed that in the spiked chromatogram of the test sample, the peak order was YZ2, impurity H, impurity N, and impurity C; the blank solvent did not interfere with the determination of YZ2, and the impurity peaks could be effectively separated from the YZ2 peak. The analytical method for the determination of related substances and content of the dexzopiclone intermediate YZ2 had good specificity.

[0056] 2. Precision (1) Instrument precision The impurity N positioning solution was accurately measured and the sample was repeatedly injected 6 times according to the analytical method for related substances and content determination of the right-zopiclone intermediate YZ2. The RSD of the impurity N peak area and retention time in the impurity N positioning solution was investigated. The results are shown in Tables 4 to 6.

[0057] Table 4 Instrument precision measurement results (JQ1322)

[0058] Table 5 Instrument precision measurement results (JQ1316)

[0059] Table 6 Instrument precision measurement results (QC-M-181)

[0060] The results showed that the instrumental precision of the analytical method for the determination of related substances and content of the intermediate YZ2 of dexzopiclone was good.

[0061] (2) Repeatability Impurity localization solution: Take an appropriate amount of impurity N reference substance, dissolve it in acetonitrile and dilute it to make a solution containing about 8 μg per 1 mL.

[0062] Test solution: Take an appropriate amount of the test sample of the intermediate YZ2 of right-zopiclone, weigh it accurately, add an appropriate amount of acetonitrile, dissolve it by ultrasonication, and quantitatively dilute it with acetonitrile to make a solution containing about 0.4 mg per 1 mL. (Prepare 6 portions in parallel) According to the analytical method for related substances and content determination of the intermediate YZ2 of right zopiclone, 6 test sample solutions were determined. The RSD of impurity N, other single impurities, total impurities and content in the 6 test sample solutions were investigated, and the results are shown in Table 7.

[0063] Table 7 Repeatability test results

[0064] The results showed that the analytical method for the determination of related substances and content of the dexzopiclone intermediate YZ2 had good repeatability.

[0065] (3) Intermediate precision Impurity localization solution: Take an appropriate amount of impurity N reference substance, dissolve it in acetonitrile and dilute it to make a solution containing about 8 μg per 1 mL.

[0066] Test solution: Take an appropriate amount of the test sample of the intermediate YZ2 of right-zopiclone, weigh it accurately, add an appropriate amount of acetonitrile, dissolve it by ultrasonication, and quantitatively dilute it with acetonitrile to make a solution containing about 0.4 mg per 1 mL. (Prepare 6 portions in parallel) Different personnel were investigated on different dates and using different instruments to determine the test solution according to the analytical method for related substances and content determination of the intermediate YZ2 of right-zopiclone. The RSD of impurity N, other single impurities, total impurities and content in the test solution (a total of 12 portions, including 6 portions under the repeatability item) were investigated. The results are shown in Table 8.

[0067] Table 8 Intermediate precision test results

[0068] The results showed that the intermediate precision of the analytical method for the determination of related substances and content of the dexzopiclone intermediate YZ2 was good.

[0069] 3. Limit of detection and limit of quantification The detection limit and quantification limit were determined by the signal-to-noise ratio method. The corresponding concentration when the signal-to-noise ratio was about 3:1 was taken as the detection limit, and the corresponding concentration when the signal-to-noise ratio was about 10:1 was taken as the quantification limit.

[0070] (1) Impurity N detection limit Accurately measure 3 mL of the impurity N quantitative limit solution, place it in a 10 mL volumetric flask, add acetonitrile to dilute to the scale, shake well, and use it as the impurity N detection limit solution. The detection limit concentration is 0.0024 μg / mL, which is 0.03% of the limit.

[0071] (2) Impurity N quantitative limit For the preparation method of impurity N linear 1 solution, please refer to the linearity and range section.

[0072] Accurately measure 0.5 mL of impurity N linear 1 solution, place it in a 100 mL volumetric flask, add acetonitrile to dilute to the scale, shake well, and use this as the impurity N quantitative limit solution. The quantitative limit concentration is 0.0081 μg / mL, which is 0.10% of the limit.

[0073] Take the impurity N quantitative limit solution, repeat the injection 6 times, and examine the peak area RSD. The results are shown in Table 9.

[0074] Table 9 Determination results of impurity N quantitative limit solution

[0075] The results showed that the precision of the quantitative limit solution of impurity N was good.

[0076] (3) Detection limit of impurity H Accurately measure 3 mL of the impurity H quantitative limit solution, place it in a 10 mL volumetric flask, add acetonitrile to dilute to the scale, shake well, and use it as the impurity H detection limit solution. The detection limit concentration is 0.0059 μg / mL, which is 1.48% of the limit.

[0077] (4) Impurity H Quantification Limit For the preparation method of impurity H linear 1 solution, please refer to the linearity and range section.

[0078] Accurately measure 2.5 mL of impurity H linear 1 solution, place it in a 10 mL volumetric flask, add acetonitrile to dilute to the scale, shake well, and use this as the impurity H quantitative limit solution. The quantitative limit concentration is 0.0195 μg / mL, which is 4.88% of the limit.

[0079] The quantitative limit solution of impurity H was taken and repeated injection was performed 6 times to examine the peak area RSD. The results are shown in Table 10.

[0080] Table 10 Determination results of impurity H quantitative limit solution

[0081] The results showed that the precision of the quantitative limit solution of impurity H was good.

[0082] (5) Detection limit of impurity C Accurately measure 3 mL of the quantitative limit solution of impurity C, place it in a 10 mL volumetric flask, add acetonitrile to dilute to the scale, shake well, and use it as the detection limit solution of impurity C. The detection limit concentration is 0.0048 μg / mL, which is 1.20% of the limit.

[0083] (6) Impurity C Quantification Limit For the preparation method of impurity C linear 1 solution, please refer to the linearity and range section.

[0084] Accurately measure 2 mL of impurity C linear 1 solution, place it in a 10 mL volumetric flask, add acetonitrile to dilute to the scale, shake well, and use this as the impurity C quantitative limit solution. The quantitative limit concentration is 0.0160 μg / mL, which is 4.00% of the limit.

[0085] The quantitative limit solution of impurity C was taken and injected 6 times repeatedly to examine the peak area RSD. The results are shown in Table 11.

[0086] Table 11 Determination results of impurity C quantitative limit solution

[0087] The results showed that the precision of the quantitative limit solution of impurity C was good.

[0088] 4. Linearity and range (1) Impurity N Take about 1.6 mg of impurity N reference substance, accurately weigh it, put it in a 20 mL volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and use it as the impurity N stock solution.

[0089] Accurately measure 0.2mL, 0.5mL, 0.8mL, 1.0mL, 1.2mL, and 2.0mL of impurity N stock solution, place them in 10mL volumetric flasks respectively, dilute to the scale with acetonitrile, shake well, and use them as impurity N linear 1, linear 2, linear 3, linear 4, linear 5, and linear 6 solutions.

[0090] Accurately measure the impurity N linear 1 to linear 6 solutions and the quantitative limit solution, inject them into the liquid chromatograph respectively, record the chromatogram, calculate the linear regression equation, and the results are shown in Fig.12 And Table 12.

[0091] Table 12 Impurity N linear determination results

[0092] The results showed that the impurity N had a good linear relationship in the concentration range of 0.0081 μg / mL to 16.2065 μg / mL, and the linear regression equation was: y=67223x-1824.5, r=0.9999.

[0093] (2) Impurity H Take about 2 mg of impurity H reference substance, weigh accurately, place in a 100 mL volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and use as the impurity H stock solution.

[0094] Accurately measure 0.2mL, 0.5mL, 0.8mL, 1.0mL, 1.2mL, and 2.0mL of the impurity H stock solution, place them in 50mL volumetric flasks respectively, dilute to the scale with acetonitrile, and shake well to serve as impurity H linear 1, linear 2, linear 3, linear 4, linear 5, and linear 6 solutions.

[0095] Accurately measure the impurity H linear 1 to linear 6 solutions and the quantitative limit solution, inject them into the liquid chromatograph respectively, record the chromatogram, calculate the linear regression equation, and the results are shown in Fig.13 And Table 13.

[0096] Table 13 Impurity H linear determination results

[0097] The results showed that the impurity H had a good linear relationship in the concentration range of 0.0195 μg / mL to 0.7810 μg / mL, and the linear regression equation was: y=22900x+248.22, r=0.9994.

[0098] (3) Impurity C Take about 2 mg of impurity C reference substance, accurately weigh it, place it in a 100 mL volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and use it as the impurity C stock solution.

[0099] Accurately measure 0.2mL, 0.5mL, 0.8mL, 1.0mL, 1.2mL, and 2.0mL of the impurity C stock solution, place them in 50mL volumetric flasks respectively, dilute to the scale with acetonitrile, and shake well to serve as impurity C linear 1, linear 2, linear 3, linear 4, linear 5, and linear 6 solutions.

[0100] Accurately measure the impurity C linear 1 to linear 6 solutions and the quantitative limit solution, inject them into the liquid chromatograph respectively, record the chromatogram, calculate the linear regression equation, and the results are shown in Fig.14 And Table 14.

[0101] Table 14 Impurity C linear determination results

[0102] The results showed that the impurity C had a good linear relationship in the concentration range of 0.02318 μg / mL to 38.6280 μg / mL, and the linear regression equation was: y=53866x-16918, r=0.9998.

[0103] (4) YZ2, an intermediate of eszopiclone Take about 40 mg of YZ2, weigh it accurately, put it in a 50 mL volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and use it as the YZ2 stock solution.

[0104] Accurately measure 1.0mL, 2.5mL, 4.0mL, 5.0mL, and 6.0mL of YZ2 stock solution, place them in 10mL volumetric flasks respectively, dilute to the scale with acetonitrile, and shake well to make YZ2 linear 1, linear 2, linear 3, linear 4, and linear 5 solutions.

[0105] Accurately measure YZ2 linear 1 to linear 5 solutions, inject them into the liquid chromatograph respectively, record the chromatogram, calculate the linear regression equation, and the results are shown in Fig.15 And Table 15.

[0106] Table 15 YZ2 linear measurement results

[0107] The results showed that YZ2 had a good linear relationship in the concentration range of 79.7524μg / mL to 478.5144μg / mL, and the linear regression equation was: y=67465x+521443, r=0.9996.

[0108] 5. Durability Impurity localization solution: Take an appropriate amount of impurity N reference substance, dissolve it in acetonitrile and dilute it to make a solution of about 8 μg per 1 mL.

[0109] Test solution: Take an appropriate amount of the test sample of the intermediate YZ2 of right-zopiclone, accurately weigh it, add an appropriate amount of acetonitrile, sonicate to dissolve it, and quantitatively dilute it with acetonitrile to make a solution containing about 0.4 mg per 1 mL.

[0110] According to the analytical method for the determination of related substances and contents of the intermediate YZ2 of right-zopiclone, the RSD of impurity N, other single impurities, total impurities and content in the test solution were investigated under different wavelengths (303±2nm), different column temperatures (30±2℃) and different chromatographic column conditions. The results are shown in Table 16.

[0111] Table 16 Durability test results

[0112] The results showed that the analytical method for the determination of related substances and content of the dexzopiclone intermediate YZ2 had good durability.

[0113] 6. Solution stability Impurity localization solution: Take an appropriate amount of impurity N reference substance, dissolve it in acetonitrile and dilute it to make a solution of about 8 μg per 1 mL.

[0114] Test solution: Take an appropriate amount of the test sample of the intermediate YZ2 of right-zopiclone, accurately weigh it, add an appropriate amount of acetonitrile, sonicate to dissolve it, and quantitatively dilute it with acetonitrile to make a solution containing about 0.4 mg per 1 mL.

[0115] According to the analytical method for the determination of related substances and contents of the intermediate YZ2 of right-zopiclone, the RSD of impurity N, other single impurities, the total amount of impurities and the content in the test solution within 24 hours were investigated. The results are shown in Table 17.

[0116] Table 17 Solution stability test results

[0117] The results showed that the spiked solution of the test sample had good stability within 24 hours.

[0118] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent transformation or modification made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting related substances of 6-(5-chloro-2-pyridyl)-6,7-dihydro-7-hydroxy-5H-pyrrolo[3,4-b]pyrazin-5-one, an intermediate of dexzopiclone, characterized in that: The steps include: Test solution: Take an appropriate amount of the intermediate of dexzopiclone and dissolve it in acetonitrile to make a test solution; Impurity C localization solution: Take an appropriate amount of impurity C reference substance and dissolve it in acetonitrile to make an impurity C localization solution; Impurity H localization solution: Take an appropriate amount of impurity H reference substance and dissolve it in acetonitrile to make an impurity H localization solution; Impurity N localization solution: Take an appropriate amount of impurity N reference substance and dissolve it in acetonitrile to make an impurity N localization solution; Determination method: accurately measure the test solution, impurity C positioning solution, impurity H positioning solution, and impurity N positioning solution respectively, inject them into the high performance liquid chromatograph, and record the chromatogram; locate the peaks of the three impurities according to the chromatogram of the impurity positioning solution, analyze the chromatogram of the test solution, and calculate by the peak area normalization method to obtain the content of each related substance in the dexzopiclone intermediate; Wherein, the structure of the dexazopiclone intermediate is as follows: The related substances include impurity C, impurity H, and impurity N, and the structure is as follows: Impurity C ; Impurity H ; Impurity N .

2. The method for detecting related substances of a dexazopiclone intermediate 6-(5-chloro-2-pyridyl)-6,7-dihydro-7-hydroxy-5H-pyrrolo[3,4-b]pyrazin-5-one according to claim 1, characterized in that: The test solution preparation method is as follows: take an appropriate amount of the dexzopiclone intermediate test sample, dissolve it in acetonitrile to make a solution with a concentration of 0.4 mg / mL.

3. The method for detecting related substances of a dexazopiclone intermediate 6-(5-chloro-2-pyridyl)-6,7-dihydro-7-hydroxy-5H-pyrrolo[3,4-b]pyrazin-5-one according to claim 1, characterized in that: The method for preparing the impurity C localization solution is as follows: take an appropriate amount of impurity C reference substance, dissolve it in acetonitrile to make a solution with a concentration of 0.4 μg / mL.

4. The method for detecting related substances of a dexazopiclone intermediate 6-(5-chloro-2-pyridyl)-6,7-dihydro-7-hydroxy-5H-pyrrolo[3,4-b]pyrazin-5-one according to claim 1, characterized in that: The method for preparing the impurity H localization solution is as follows: take an appropriate amount of impurity H reference substance, dissolve it in acetonitrile to make a solution with a concentration of 0.4 μg / mL.

5. The method for detecting related substances of a dexazopiclone intermediate 6-(5-chloro-2-pyridyl)-6,7-dihydro-7-hydroxy-5H-pyrrolo[3,4-b]pyrazin-5-one according to claim 1, characterized in that: The method for preparing the impurity N localization solution is as follows: take an appropriate amount of impurity N reference substance, dissolve it in acetonitrile to make a solution with a concentration of 8 μg / mL.

6. The method for detecting related substances of a dexzopiclone intermediate 6-(5-chloro-2-pyridyl)-6,7-dihydro-7-hydroxy-5H-pyrrolo[3,4-b]pyrazin-5-one according to claim 1, characterized in that: The chromatographic conditions of the high performance liquid chromatograph are as follows: Chromatographic column: Merck Purospher STAR RP-C18, 250 mm × 4.6 mm, 5 μm; Mobile phase: Mobile phase A: perchloric acid and trifluoroacetic acid aqueous solution; Mobile phase B: acetonitrile; gradient: ； Flow rate: 1.0 mL / min; Detection wavelength: 303nm; Column temperature: 30°C; Injection volume: 20 μL.

7. The method for detecting related substances of a dexazopiclone intermediate 6-(5-chloro-2-pyridyl)-6,7-dihydro-7-hydroxy-5H-pyrrolo[3,4-b]pyrazin-5-one according to claim 6, characterized in that: The preparation method of the perchloric acid-trifluoroacetic acid aqueous solution is as follows: take 0.43 mL of perchloric acid with a content of 70%-72% and 1 mL of trifluoroacetic acid, put them into 1000 mL of water, and mix them evenly.

8. The method for detecting related substances of a dexazopiclone intermediate 6-(5-chloro-2-pyridyl)-6,7-dihydro-7-hydroxy-5H-pyrrolo[3,4-b]pyrazin-5-one according to claim 1, characterized in that: In the chromatogram of the test sample solution, the content of the dexamethasone intermediate calculated by the peak area normalization method shall not be less than 96%.

9. The method for detecting related substances of a dexzopiclone intermediate 6-(5-chloro-2-pyridyl)-6,7-dihydro-7-hydroxy-5H-pyrrolo[3,4-b]pyrazin-5-one according to claim 8, characterized in that: In the chromatogram of the test solution, if there are impurity peaks, calculated by the peak area normalization method, the impurity N shall not be greater than 4%, other individual impurities shall not be greater than 0.10%, and the total amount of impurities shall not be greater than 4%.

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