A method for detecting related substances of eszopiclone intermediate YZ2

Abstract

This invention belongs to the field of drug detection technology, specifically disclosing a method for detecting related substances in eszopiclone intermediate YZ2, comprising the following steps: Test solution: Take an appropriate amount of the eszopiclone intermediate test sample and dissolve and dilute to volume with acetonitrile; Impurity localization solutions: Take appropriate amounts of impurity C, H, and N reference standards and dissolve and dilute to volume with acetonitrile respectively. Determination method: Accurately measure the test solution and the three impurity localization solutions, inject them into a high-performance liquid chromatograph, and record the chromatograms; based on the chromatograms of the impurity localization solutions, locate the peaks of the three impurities, analyze the chromatogram of the test solution, and calculate the content of each related substance in the eszopiclone intermediate using the peak area normalization method. This invention uses high-performance liquid chromatography to monitor the purity of the eszopiclone intermediate by analyzing the content of active ingredients and impurities, ensuring quality and evaluating the production process.

Description

Technical Field

[0001] This invention belongs to the field of drug detection technology, and specifically relates to a method for detecting related substances of eszopiclone intermediate YZ2. Background Technology

[0002] Eszopiclone, chemically known as 6-(5-chloro-2-pyridinyl)-7-[(4-methylpiperazin-1-yl)carboxyoxy]-5,6-dihydropyrrolo[3,4-b]pyrazin-5-one, is a rapid-acting, short-acting non-benzodiazepine sedative-hypnotic. It is the dextrorotatory monoisomer of zopiclone and possesses sedative, anxiolytic, anticonvulsant, and muscle relaxant effects. Studies have shown that eszopiclone is similar to long-acting benzodiazepines in improving sleep quality and similar to short-acting benzodiazepines in shortening sleep onset time. It exhibits fewer residual effects and hangovers than benzodiazepines, and shows no significant drug tolerance with long-term use, making it an ideal sedative-hypnotic drug.

[0003] In the preparation of eszopiclone, a key intermediate is involved, chemically named 6-(5-chloro-2-pyridinyl)-6,7-dihydro-7-hydroxy-5H-pyrrolo[3,4-b]pyrazin-5-one (YZ2), with the following structural formula:

[0004]

[0005] Three related substances are present in the synthesis of zezopiclone intermediate YZ2:

[0006] (1) Impurity C (generated by reduction and hydrogenation of eszopiclone intermediate YZ2);

[0007] (2) Impurity H (starting material for the synthesis of eszopiclone intermediate YZ2);

[0008] (3) Impurity N (generated by hydrolysis of eszopiclone YZ2).

[0009] The structure is as follows:

[0010] Impurity C Impurity H Impurity N .

[0011] Currently, there are few reports in the literature on the detection methods of related substances of eszopiclone intermediate YZ2. In order to strengthen the quality control of eszopiclone intermediate YZ2 and meet the current needs, it is urgent to develop a detection method for related substances of eszopiclone intermediate YZ2. Summary of the Invention

[0012] To address the shortcomings of existing technologies, the present invention aims to provide a method for detecting related substances of eszopiclone intermediate YZ2, which employs high-performance liquid chromatography (HPLC). This method offers high sensitivity, fast analysis speed, and widespread availability of equipment, making it easy to promote and effectively solve the problems in the prior art.

[0013] This invention is achieved through the following technical solution:

[0014] A method for detecting related substances of eszopiclone intermediate 6-(5-chloro-2-pyridyl)-6,7-dihydro-7-hydroxy-5H-pyrrolo[3,4-b]pyrazin-5-one, comprising the following steps:

[0015] Test solution: Take an appropriate amount of eszopiclone intermediate test sample, dissolve and dilute with acetonitrile to prepare the test solution.

[0016] Impurity C positioning solution: Take an appropriate amount of impurity C reference standard, dissolve it in acetonitrile and make up to volume to prepare impurity C positioning solution.

[0017] Impurity H positioning solution: Take an appropriate amount of impurity H reference standard, dissolve and dilute with acetonitrile to prepare impurity H positioning solution.

[0018] Impurity N positioning solution: Take an appropriate amount of impurity N reference standard, dissolve it in acetonitrile and make up to volume to prepare impurity N positioning solution.

[0019] Determination method: Accurately measure the test solution, impurity C positioning solution, impurity H positioning solution, and impurity N positioning solution, respectively, and inject them into the high performance liquid chromatograph and record the chromatograms; locate the peaks of the three impurities according to the chromatograms of the impurity positioning solutions, analyze the chromatogram of the test solution, and calculate the content of each related substance in the eszopiclone intermediate by peak area normalization method.

[0020] The structure of the zopiclone intermediate is as follows:

[0021]

[0022] The relevant substances include impurity C, impurity H, and impurity N, with the following structure:

[0023] Impurity C Impurity H Impurity N .

[0024] A further improvement to the present invention is as follows:

[0025] The test solution is prepared as follows: Take an appropriate amount of the eszopiclone intermediate test sample, dissolve it in acetonitrile and dilute to a final volume to prepare a solution with a concentration of 0.4 mg / mL.

[0026] Furthermore, the method for preparing the impurity C positioning solution is as follows: Take an appropriate amount of impurity C reference standard, dissolve it in acetonitrile, and make up to a volume of 0.4 μg / mL to prepare a solution.

[0027] Furthermore, the method for preparing the impurity H positioning solution is as follows: Take an appropriate amount of impurity H reference standard, dissolve it in acetonitrile, and make up to a volume of 0.4 μg / mL to prepare a solution.

[0028] Furthermore, the method for preparing the impurity N positioning solution is as follows: Take an appropriate amount of impurity N reference standard, dissolve it in acetonitrile, and dilute to a final volume to prepare a solution with a concentration of 8 μg / mL.

[0029] A further improvement to the present invention is as follows:

[0030] The chromatographic conditions of the high-performance liquid chromatograph are as follows:

[0031] Column: Merck Purospher STAR RP-C18, 250 mm × 4.6 mm, 5 μm;

[0032] Mobile phases: Mobile phase A: aqueous solution of perchloric acid and trifluoroacetic acid; Mobile phase B: acetonitrile;

[0033] gradient:

[0034]

[0035] Flow rate: 1.0 mL / min;

[0036] Detection wavelength: 303nm;

[0037] Column temperature: 30℃;

[0038] Injection volume: 20 μL.

[0039] Furthermore, the method for preparing 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, place them in 1000 mL of water, and mix well.

[0040] A further improvement to the present invention is as follows:

[0041] In the chromatogram of the test sample solution, the content of eszopiclone intermediate calculated by peak area normalization shall not be less than 96%.

[0042] 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 exceed 4%, other individual impurities shall not exceed 0.10%, and the total amount of impurities shall not exceed 4%. Beneficial effects

[0043] Compared with the prior art, the present invention has the following obvious advantages:

[0044] I. This invention provides a method for detecting related substances of eszopiclone intermediate YZ2, which uses high performance liquid chromatography, has high sensitivity, fast analysis speed, and is widely available and easy to promote.

[0045] Second, the addition of perchloric acid and trifluoroacetic acid to mobile phase A in this invention can improve peak shape and overcome peak broadening and tailing problems. Under the chromatographic conditions provided by this invention, impurities can be effectively separated from each other and from the main peak, with good peak shape, which facilitates subsequent analysis of the chromatogram.

[0046] Third, the detection method provided by this invention has good specificity, precision, accuracy, robustness and stability. By analyzing the content of active ingredients and impurities in eszopiclone intermediate YZ2, the purity of eszopiclone intermediate YZ2 can be monitored to ensure its quality, and the production process of eszopiclone intermediate YZ2 can be evaluated. Attached Figure Description

[0047] Figure 1 This is a blank solvent spectrum;

[0048] Figure 2 The chromatogram of the solution for locating impurity C;

[0049] Figure 3 The solution spectrum for locating impurity H;

[0050] Figure 4 The solution spectrum for locating impurity N;

[0051] Figure 5 The chromatogram of the spiked solution of the test sample;

[0052] Figure 6 The spectrum of the test solution (batch number: YZ220601B) in Example 1;

[0053] Figure 7 The spectrum of the test solution (batch number: YZ221001B) in Example 2;

[0054] Figure 8 The spectrum of the test solution (batch number: YZ221002B) in Example 3;

[0055] Figure 9 The spectrum of the test solution (batch number: YZ221101B) in Example 4;

[0056] Figure 10 The spectrum of the test solution (batch number: YZ221102B) in Example 5;

[0057] Figure 11The spectrum of the test solution (batch number: YZ221103B) in Example 6;

[0058] Figure 12 This is a standard curve of linearity versus impurity N in Example 7;

[0059] Figure 13 This is a standard curve of linearity versus impurity H in the range of Example 7;

[0060] Figure 14 This is a standard curve of linearity versus impurity C in the range of Example 7;

[0061] Figure 15 This is a standard curve of the linear and range-bound expression of the zopiclone intermediate YZ2 in Example 7. Detailed Implementation

[0062] The instruments, chromatographic columns, reagents, reference standards, and samples used in this invention are as follows:

[0063] Instruments and Columns

[0064]

[0065] reagents

[0066]

[0067] Reference

[0068]

[0069] sample

[0070]

[0071] The present invention will now be described in detail with reference to the embodiments.

[0072] Examples 1-6 Sample Detection

[0073] 1. Analytical Methods

[0074] (1) Test solution: Take an appropriate amount of eszopiclone intermediate YZ2 test sample, accurately weigh it, add an appropriate amount of acetonitrile, sonicate to dissolve, and dilute to volume with acetonitrile to prepare a solution with a concentration of 0.4 mg / mL.

[0075] (2) Impurity C positioning solution: Take an appropriate amount of impurity C reference standard, dissolve it in acetonitrile and make up to volume to prepare a solution with a concentration of 0.4 μg / mL.

[0076] (3) Impurity H positioning solution: Take an appropriate amount of impurity H reference standard, dissolve it in acetonitrile and make up to volume to prepare a solution with a concentration of 0.4 μg / mL.

[0077] (4) Impurity N positioning solution: Take an appropriate amount of impurity N reference standard, dissolve it in acetonitrile and make up to volume to prepare a solution with a concentration of 8 μg / mL.

[0078] (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 the content of each related substance in the eszopiclone intermediate by peak area normalization method.

[0079] (6) Chromatographic conditions:

[0080] Column: Merck Purospher STAR RP-C18, 250 mm × 4.6 mm, 5 μm;

[0081] Mobile phases: Mobile phase A: aqueous solution of perchloric acid and trifluoroacetic acid; Mobile phase B: acetonitrile;

[0082] gradient:

[0083]

[0084] Flow rate: 1.0 mL / min;

[0085] Detection wavelength: 303nm;

[0086] Column temperature: 30℃;

[0087] Injection volume: 20 μL.

[0088] Furthermore, the method for preparing 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, place them in 1000 mL of water, and mix well.

[0089] (7) Limits: If there are impurity peaks in the chromatogram of the test solution, the impurity N shall not exceed 4% according to the peak area normalization method, the other individual impurities shall not exceed 0.10%, and the total amount of impurities shall not exceed 4%.

[0090] (8) Content: The content of eszopiclone intermediate YZ2 in the chromatogram of the test sample solution, calculated by the peak area normalization method, shall not be less than 96%.

[0091] 2. Sample determination

[0092] According to the analytical method for related substances and content determination of eszopiclone intermediate YZ2, 6 batches of YZ2 samples were analyzed, such as... Figures 6 to 11 As shown, the results are presented in Table 1.

[0093] Table 1 Sample Measurement Results

[0094]

[0095] The normalized content results of impurities H and C are shown in Table 2 below:

[0096] Table 2. Normalized content results of impurities H and C

[0097]

[0098] The results showed that the determination results of related substances and contents of the six batches of samples all met the requirements, and the impurities H and C did not exceed 0.03%.

[0099] Example 7 Validation of the detection method

[0100] 1. Exclusivity

[0101] (1) Blank solvent: acetonitrile.

[0102] (2) Location of impurity H: Weigh approximately 2 mg of impurity H reference standard accurately, place it in a 100 mL volumetric flask, dissolve and dilute to the mark with acetonitrile, shake well, and use it as 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.

[0103] (3) Location of impurity N: Weigh approximately 1.6 mg of impurity N reference standard accurately, place it in a 20 mL volumetric flask, dissolve and dilute to the mark with acetonitrile, shake well, and use it as 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.

[0104] (4) Location of impurity C: Weigh approximately 2 mg of impurity C reference standard accurately, place it in a 100 mL volumetric flask, dissolve and dilute to the mark with acetonitrile, shake well, and use it as impurity C stock solution; accurately measure 1 mL of impurity C stock solution, place it in a 50 mL volumetric flask, dilute to the mark with acetonitrile, and shake well.

[0105] (5) Test solution: Take an appropriate amount of eszopiclone intermediate YZ2 test sample, accurately weigh it, add an appropriate amount of acetonitrile, sonicate to dissolve it, and quantitatively dilute it with acetonitrile to prepare a solution containing about 0.4 mg per 1 mL.

[0106] (6) Spiked solution for test sample: Weigh about 20 mg of eszopiclone intermediate YZ2 test sample accurately, place it in a 50 mL volumetric flask, add an appropriate amount of acetonitrile, sonicate to dissolve, accurately add 1 mL of impurity H stock solution, 1 mL of impurity C stock solution and 5 mL of impurity N stock solution, dilute to the mark with acetonitrile and shake well.

[0107] Accurately measure each of the above solutions and inject them into the liquid chromatograph. Record the chromatograms and investigate the interference of the blank solvent on the determination of related substances and contents of eszopiclone intermediate YZ2 and the separation between peaks. The results are shown in Table 3.

[0108] Table 3 Specificity test results

[0109]

[0110] The results showed that in the spiked chromatogram of the test sample, the elution order was YZ2, impurity H, impurity N, and impurity C. The blank solvent did not interfere with the determination of YZ2, and each impurity peak could be effectively separated from the YZ2 peak. The analytical method for the determination of related substances and content of eszopiclone intermediate YZ2 showed good specificity.

[0111] 2. Precision

[0112] (1) Instrument precision

[0113] Accurately measure the impurity N localization solution and repeat the injection 6 times according to the analytical method for determination of related substances and content of eszopiclone intermediate YZ2. Examine the RSD of the peak area and retention time of impurity N in the impurity N localization solution. The results are shown in Tables 4 to 6.

[0114] Table 4 Results of instrument precision measurement (JQ1322)

[0115]

[0116] Table 5 Results of instrument precision measurement (JQ1316)

[0117]

[0118] Table 6 Results of instrument precision measurement (QC-M-181)

[0119]

[0120] The results showed that the analytical method for determining the related substances and content of eszopiclone intermediate YZ2 had good instrument precision.

[0121] (2) Repeatability

[0122] Impurity localization solution: Take an appropriate amount of impurity N reference standard, dissolve and dilute it in acetonitrile to prepare a solution containing approximately 8 μg per 1 mL.

[0123] Test solution: Accurately weigh an appropriate amount of eszopiclone intermediate YZ2, add an appropriate amount of acetonitrile, sonicate to dissolve, and quantitatively dilute with acetonitrile to prepare a solution containing approximately 0.4 mg per mL. (Prepare 6 parallel solutions)

[0124] Six test solutions were analyzed according to the related substances and content determination method for eszopiclone intermediate YZ2. The RSDs of impurity N, other individual impurities, total impurities, and content in the six test solutions were investigated, and the results are shown in Table 7.

[0125] Table 7 Repeatability Test Results

[0126]

[0127] The results showed that the analytical method for determining the related substances and content of eszopiclone intermediate YZ2 had good repeatability.

[0128] (3) Intermediate precision

[0129] Impurity localization solution: Take an appropriate amount of impurity N reference standard, dissolve and dilute it in acetonitrile to prepare a solution containing approximately 8 μg per 1 mL.

[0130] Test solution: Accurately weigh an appropriate amount of eszopiclone intermediate YZ2, add an appropriate amount of acetonitrile, sonicate to dissolve, and quantitatively dilute with acetonitrile to prepare a solution containing approximately 0.4 mg per mL. (Prepare 6 parallel solutions)

[0131] Different personnel, on different dates, and using different instruments, analyzed the test solution according to the related substances and content determination method for eszopiclone intermediate YZ2. The RSD of impurity N, other individual impurities, total impurities, and content in the test solution (a total of 12 samples, including 6 samples under the repeatability item) was investigated. The results are shown in Table 8.

[0132] Table 8 Results of intermediate precision determination

[0133]

[0134] The results showed that the intermediate precision of the analytical method for determining the related substances and content of eszopiclone intermediate YZ2 was good.

[0135] 3. Limit of detection and limit of quantitation

[0136] The detection limit and quantitation limit were determined using the signal-to-noise ratio method, with the corresponding concentration at a signal-to-noise ratio of approximately 3:1 as the detection limit and the corresponding concentration at a signal-to-noise ratio of approximately 10:1 as the quantitation limit.

[0137] (1) Detection limit of impurity N

[0138] Accurately measure 3 mL of the impurity N quantitation limit solution and place it in a 10 mL volumetric flask. Dilute to the mark with acetonitrile and shake well. This solution serves as the impurity N detection limit solution. The detection limit concentration is 0.0024 μg / mL, which is 0.03% of the limit.

[0139] (2) Limit of quantification for impurity N

[0140] For the preparation method of linear solution of impurity N, please refer to the section on linearity and range.

[0141] Accurately measure 0.5 mL of the impurity N linear 1 solution and place it in a 100 mL volumetric flask. Dilute to the mark with acetonitrile and shake well. This solution serves as the limit of quantitation (LOQ) solution for impurity N. The LOQ concentration is 0.0081 μg / mL, which is 0.10% of the limit.

[0142] Take the limit of quantitation solution of impurity N, repeat the injection 6 times, and examine the peak area RSD. The results are shown in Table 9.

[0143] Table 9 Results of Impurity N Quantitation Limit Solution Determination

[0144]

[0145] The results showed that the solution with the limit of quantification for impurity N had good precision.

[0146] (3) Detection limit of impurity H

[0147] Accurately measure 3 mL of the quantitation limit solution for impurity H, place it in a 10 mL volumetric flask, dilute to the mark with acetonitrile, and shake well. This solution serves as the detection limit solution for impurity H, with a detection limit concentration of 0.0059 μg / mL, which is 1.48% of the limit.

[0148] (4) Limit of quantification for impurity H

[0149] For the preparation method of linear solution of impurity H, please refer to the section on linearity and range.

[0150] Accurately measure 2.5 mL of the linear 1 solution of impurity H and place it in a 10 mL volumetric flask. Dilute to the mark with acetonitrile and shake well. This solution is used as the limit of quantitation solution for impurity H. The limit of quantitation concentration is 0.0195 μg / mL, which is 4.88% of the limit.

[0151] Take the limit of quantitation solution of impurity H, and repeat the injection 6 times to examine the peak area RSD. The results are shown in Table 10.

[0152] Table 10 Results of Impurity H Quantitation Limit Solution Determination

[0153]

[0154] The results showed that the solution with the limit of quantification for impurity H had good precision.

[0155] (5) Detection limit of impurity C

[0156] Accurately measure 3 mL of the impurity C quantitation limit solution and place it in a 10 mL volumetric flask. Dilute to the mark with acetonitrile and shake well. This solution serves as the impurity C detection limit solution. The detection limit concentration is 0.0048 μg / mL, which is 1.20% of the limit.

[0157] (6) Limit of quantification for impurity C

[0158] For the preparation method of linear solution of impurity C, please refer to the section on linearity and range.

[0159] Accurately measure 2 mL of the linear 1 solution of impurity C and place it in a 10 mL volumetric flask. Dilute to the mark with acetonitrile and shake well. This solution serves as the limit of quantitation (LOQ) solution for impurity C. The LOQ concentration is 0.0160 μg / mL, which is 4.00% of the limit.

[0160] Take the limit of quantitation solution of impurity C, repeat the injection 6 times, and examine the peak area RSD. The results are shown in Table 11.

[0161] Table 11 Results of Impurity C Quantitation Limit Solution Determination

[0162]

[0163] The results showed that the solution with the limit of quantification for impurity C had good precision.

[0164] 4. Linearity and Range

[0165] (1) Impurity N

[0166] Weigh approximately 1.6 mg of impurity N reference standard accurately, place it in a 20 mL volumetric flask, dissolve and dilute to the mark with acetonitrile, and shake well to prepare impurity N stock solution.

[0167] Accurately measure 0.2 mL, 0.5 mL, 0.8 mL, 1.0 mL, 1.2 mL, and 2.0 mL of impurity N stock solution, place them in 10 mL volumetric flasks, dilute to the mark with acetonitrile, and shake well. These are used as linear solutions 1, 2, 3, 4, 5, and 6 for impurity N.

[0168] Accurately measure out solutions of impurity N with linearity 1 to 6 and solutions at the limit of quantitation, inject them separately into the liquid chromatograph, record the chromatograms, calculate the linear regression equation, and see the results. Figure 12 And Table 12.

[0169] Table 12 Results of linear determination of impurity N

[0170]

[0171] The results showed that impurity N exhibited good linearity in the concentration range of 0.0081 μg / mL to 16.2065 μg / mL, with a linear regression equation of y = 67223x - 1824.5 and r = 0.9999.

[0172] (2) Impurity H

[0173] Weigh approximately 2 mg of impurity H reference standard accurately, place it in a 100 mL volumetric flask, dissolve and dilute to the mark with acetonitrile, and shake well to prepare impurity H stock solution.

[0174] Accurately measure 0.2 mL, 0.5 mL, 0.8 mL, 1.0 mL, 1.2 mL, and 2.0 mL of impurity H stock solution, place them in 50 mL volumetric flasks, dilute to the mark with acetonitrile, and shake well to obtain impurity H linear 1, linear 2, linear 3, linear 4, linear 5, and linear 6 solutions.

[0175] Accurately measure out solutions of impurity H with linearity 1 to 6 and solutions at the limit of quantitation, inject them separately into the liquid chromatograph, record the chromatograms, calculate the linear regression equation, and see the results. Figure 13 And Table 13.

[0176] Table 13 Results of linear determination of impurity H

[0177]

[0178] The results showed that impurity H exhibited good linearity in the concentration range of 0.0195 μg / mL to 0.7810 μg / mL, with the linear regression equation being y = 22900x + 248.22 and r = 0.9994.

[0179] (3) Impurity C

[0180] Weigh approximately 2 mg of impurity C reference standard accurately, place it in a 100 mL volumetric flask, dissolve and dilute to the mark with acetonitrile, and shake well to prepare impurity C stock solution.

[0181] Accurately measure 0.2 mL, 0.5 mL, 0.8 mL, 1.0 mL, 1.2 mL, and 2.0 mL of impurity C stock solution, place them in 50 mL volumetric flasks, dilute to the mark with acetonitrile, and shake well. These are used as linear solutions 1, 2, 3, 4, 5, and 6 of impurity C.

[0182] Accurately measure out solutions of impurity C with linearity 1 to 6 and solutions at the limit of quantitation, inject them separately into the liquid chromatograph, record the chromatograms, calculate the linear regression equation, and see the results. Figure 14 And Table 14.

[0183] Table 14 Results of linear determination of impurity C

[0184]

[0185] The results showed that impurity C exhibited good linearity in the concentration range of 0.02318 μg / mL to 38.6280 μg / mL, with the linear regression equation being y = 53866x - 16918 and r = 0.9998.

[0186] (4) Dexzopiclone intermediate YZ2

[0187] Weigh approximately 40 mg of YZ2 accurately, place it in a 50 mL volumetric flask, dissolve and dilute to the mark with acetonitrile, and shake well to prepare the YZ2 stock solution.

[0188] Accurately measure 1.0 mL, 2.5 mL, 4.0 mL, 5.0 mL, and 6.0 mL of YZ2 stock solution and place them in 10 mL volumetric flasks respectively. Dilute to the mark with acetonitrile and shake well to obtain YZ2 linear solutions 1, 2, 3, 4, and 5.

[0189] Accurately measure YZ2 solutions with linearity 1 to 5, inject them into the liquid chromatograph, record the chromatograms, calculate the linear regression equations, and see the results. Figure 15 And Table 15.

[0190] Table 15 Results of YZ2 linearity determination

[0191]

[0192] The results showed that YZ2 exhibited good linearity in the concentration range of 79.7524 μg / mL to 478.5144 μg / mL, with the linear regression equation being y = 67465x + 521443 and r = 0.9996.

[0193] 5. Durability

[0194] Impurity localization solution: Take an appropriate amount of impurity N reference standard, dissolve and dilute it in acetonitrile to prepare a solution of about 8 μg per 1 mL.

[0195] Test solution: Take an appropriate amount of eszopiclone intermediate YZ2 test sample, accurately weigh it, add an appropriate amount of acetonitrile, sonicate to dissolve, and quantitatively dilute with acetonitrile to prepare a solution containing approximately 0.4 mg per 1 mL.

[0196] Following the analytical method for related substances and content determination of eszopiclone intermediate YZ2, the RSD of impurity N, other individual impurities, total impurities and content in the test solution were investigated under different wavelengths (303±2nm), different column temperatures (30±2℃) and different column conditions. The results are shown in Table 16.

[0197] Table 16 Durability Test Results

[0198]

[0199] The results showed that the analytical method for determining the related substances and content of eszopiclone intermediate YZ2 was robust.

[0200] 6. Solution stability

[0201] Impurity localization solution: Take an appropriate amount of impurity N reference standard, dissolve and dilute it in acetonitrile to prepare a solution of about 8 μg per 1 mL.

[0202] Test solution: Take an appropriate amount of eszopiclone intermediate YZ2 test sample, accurately weigh it, add an appropriate amount of acetonitrile, sonicate to dissolve, and quantitatively dilute with acetonitrile to prepare a solution containing approximately 0.4 mg per 1 mL.

[0203] Following the analytical method for related substances and content determination of eszopiclone intermediate YZ2, the RSD of impurity N, other individual impurities, total impurities and content in the test solution within 24 hours was investigated. The results are shown in Table 17.

[0204] Table 17 Results of solution stability test

[0205]

[0206] The results showed that the spiked solution of the test sample had good stability within 24 hours.

[0207] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent transformations or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for detecting related substances of eszopiclone intermediate 6-(5-chloro-2-pyridyl)-6,7-dihydro-7-hydroxy-5H-pyrrolo[3,4-b]pyrazin-5-one, characterized in that, Includes the following steps: Test solution: Take an appropriate amount of eszopiclone intermediate test sample, dissolve and dilute with acetonitrile to prepare test solution; Impurity C positioning solution: Take an appropriate amount of impurity C reference standard, dissolve it in acetonitrile and make up to volume to prepare impurity C positioning solution; Impurity H localization solution: Take an appropriate amount of impurity H reference standard, dissolve it in acetonitrile and make up to volume to prepare impurity H localization solution; Impurity N locating solution: Take an appropriate amount of impurity N reference standard, dissolve it in acetonitrile and make up to volume to prepare impurity N locating 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 chromatograms; locate the peaks of the three impurities according to the chromatograms of the impurity positioning solutions, analyze the chromatogram of the test solution, and calculate the content of each related substance in the eszopiclone intermediate by peak area normalization method. The structure of the zopiclone intermediate is as follows: ； The related substances include impurity C, impurity H, and impurity N, with the following structure: Impurity C Impurity H Impurity N ; The chromatographic conditions of the high-performance liquid chromatograph are as follows: Column: Merck Purospher STAR RP-C18, 250 mm × 4.6 mm, 5 μm; Mobile phases: Mobile phase A: aqueous solution of perchloric acid and trifluoroacetic acid; Mobile phase B: acetonitrile; gradient: Flow rate: 1.0 mL / min; Detection wavelength: 303nm; Column temperature: 30℃; Injection volume: 20 μL.

2. The method for detecting related substances of the eszopiclone 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 is prepared as follows: Take an appropriate amount of the eszopiclone intermediate test sample, dissolve it in acetonitrile and dilute to a final volume to prepare a solution with a concentration of 0.4 mg / mL.

3. The method for detecting related substances of the eszopiclone 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 positioning solution is as follows: Take an appropriate amount of impurity C reference standard, dissolve it in acetonitrile, and make up to a volume of 0.4 μg / mL.

4. The method for detecting related substances of the eszopiclone 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 positioning solution is as follows: Take an appropriate amount of impurity H reference standard, dissolve it in acetonitrile and make up to a volume of 0.4 μg / mL.

5. The method for detecting related substances of the eszopiclone 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 standard, dissolve it in acetonitrile, and make up to a volume of 8 μg / mL to prepare a solution.

6. The method for detecting related substances of the eszopiclone 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 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, place them in 1000 mL of water, and mix well.

7. The method for detecting related substances of the eszopiclone 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 eszopiclone intermediate calculated by peak area normalization shall not be less than 96%.

8. The method for detecting related substances of the eszopiclone 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 solution, if there are impurity peaks, the impurity N shall not exceed 4% according to the peak area normalization method, other individual impurities shall not exceed 0.10%, and the total amount of impurities shall not exceed 4%.

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