Detection method of eszopiclone related substances

Abstract

The invention discloses a detection method of eszopiclone related substances, which comprises the following steps: respectively taking an eszopiclone reference substance, an impurity A reference substance and an impurity B reference substance, and fixing the volume by using a diluent of which the pH value is adjusted to 2.5 to obtain a system applicable solution; preparing an eszopiclone test solution and an eszopiclone reference solution by adopting the diluting agent; and respectively measuring the system applicability solution, the test solution and the reference solution, respectively testing and recording high performance liquid chromatograms, and calculating according to an external standard method to obtain the content of each related substance in eszopiclone. After the pH value of the diluent is adjusted to 2.5, (1) the solution stability is good, and the diluent does not need to be newly prepared temporarily; (2) the positioning retention time of the impurity A is consistent with the retention time of the impurity A in the test sample labeling solution, and the positioning peak retention time of other known impurities is consistent with the retention time of each impurity peak in the test sample labeling solution. The detection method provided by the invention has better durability and higher accuracy.

Description

Technical Field

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

[0002] Insomnia is a common health problem that affects the quality and quantity of sleep. Long-term insomnia may cause complications such as memory loss, mood swings, and decreased immunity, and even increase the risk of chronic diseases such as cardiovascular disease and diabetes, indirectly affecting the quality of life and life expectancy.

[0003] Drug therapy is a common treatment for insomnia. Eszopiclone is the dextrorotatory single isomer of zopiclone, which is used to treat short-term and chronic insomnia. Compared with traditional barbiturate and benzodiazepine sedative hypnotics, eszopiclone is better in improving sleep quality and shortening the time to fall asleep, has fewer adverse reactions, and has no obvious drug resistance after long-term use. It is an ideal sedative hypnotic.

[0004] The structure of eszopiclone is as follows:

[0005] Related substances are one of the key quality attributes in drug research, and their content is a direct indicator of drug purity. In the current standards, ChP2020 and USP2022 both contain HPLC detection methods for related substances of dexzopiclone. After research, the inventors found that the ChP2020 related substance method for dexzopiclone has the following defects: (1) The test sample is unstable and needs to be freshly prepared before use; (2) The peak shape of impurity I (impurity A) in the system suitability solution, the dexzopiclone peak and the dexzopiclone peak in the test solution are poor and tailing is serious, and the peak shape does not improve significantly after replacing the chromatographic column, which has poor durability for the chromatographic column.

[0006] Compared with the ChP2020 method, the impurity B peak, impurity C peak, impurity D peak, impurity A peak and the impurity A peak in the USP2022 related substance method of eszopiclone can be effectively separated, and the peak shape is good, but there are still some shortcomings: the retention time of impurity A in the impurity A positioning solution (14.227min) is quite different from the retention time of impurity A in the test sample spiked solution (13.072min), which is inconsistent.

[0007] In order to better control the quality of dexzopiclone, it is urgent to develop a universal method for detecting dexzopiclone-related substances. Summary of the invention

[0008] In view of the deficiencies of the prior art, the object of the present invention is to provide a method for detecting related substances of dexzopiclone. Under the chromatographic conditions provided by the present invention, each impurity can be effectively separated from the main peak, and the peak shape is good. The retention time of impurity A is consistent with the retention time of impurity A in the spiked solution of the test sample, and the retention time of the other known impurities is consistent with the retention time of each impurity peak in the spiked solution of the test sample. The method is verified to have good specificity, precision, and accuracy, and can overcome the defects of the detection method of related substances of dexzopiclone in the current standard, and can effectively solve the problems in the background technology.

[0009] The present invention is achieved through the following technical solutions: A method for detecting dexamethasone-related substances comprises the following steps: System suitability solution: Take appropriate amount of dexzopiclone reference substance, impurity A reference substance and impurity B reference substance, dissolve them in diluent to make system suitability solution; Test solution: Take an appropriate amount of dexzopiclone test sample and dissolve it in diluent to make a test solution; Reference solution: Take an appropriate amount of eszopiclone reference substance and dissolve it in a diluent to make a reference solution; Determination method: Accurately measure the system suitability solution, the test solution and the reference solution respectively, inject them into the high performance liquid chromatograph, and record the chromatogram; according to the chromatogram, calculate by the external standard method to obtain the content of each related substance in dexzopiclone; Wherein, the related substances include impurity A, impurity B, impurity C and impurity D, and the structure is as follows: Impurity A ; Impurity B ; Impurity C ; Impurity D ; The diluent is a mixed solution of acetonitrile and sodium dodecyl sulfate-sodium dihydrogen phosphate buffer solution, with a pH value of 2.5; in the diluent, the volume ratio of acetonitrile to buffer solution is 628:1000.

[0010] A further improvement of the present invention is: The system suitability solution preparation method is as follows: take appropriate amounts of dexzopiclone reference substance, impurity A reference substance, and impurity B reference substance, add appropriate amounts of diluent, dissolve by ultrasound, and dissolve to a fixed volume with the diluent to obtain a solution, wherein the concentration of dexzopiclone is 0.04 mg / mL, the concentration of impurity A is 0.04 mg / mL, and the concentration of impurity B is 0.04 mg / mL.

[0011] Furthermore, the test solution preparation method is as follows: take an appropriate amount of dexzopiclone test sample, accurately weigh it, add an appropriate amount of diluent, dissolve it by ultrasound, and dissolve it with the diluent to make a fixed volume to prepare a solution with a concentration of 4 mg / L.

[0012] Furthermore, the reference solution preparation method is as follows: take an appropriate amount of dexzopiclone reference substance, accurately weigh it, add an appropriate amount of diluent, dissolve it by ultrasound, and dissolve it with the diluent to make a solution with a concentration of 0.004 mg / L.

[0013] A further improvement of the present invention is: The chromatographic conditions of the high performance liquid chromatograph are as follows: Column: GL Sciences Inertsil ODS-3V, 250 mm × 4.6 mm, 5 μm; Mobile phase: a mixed solution of acetonitrile and sodium dodecyl sulfate-sodium dihydrogen phosphate buffer, with the pH value adjusted to 4.0 with phosphoric acid; wherein the volume ratio of acetonitrile to buffer was 628:1000; Flow rate: 1.5 mL / min; Detection wavelength: 220nm; Column temperature: 30°C; Sample chamber temperature: 5°C; Injection volume: 20mL.

[0014] A further improvement of the present invention is: In the chromatogram of the system suitability solution, the order of peaks is impurity B peak, impurity A peak and eszopiclone peak, the separation between the impurity A peak and the eszopiclone peak should not be less than 2.0, and the tailing factor of the eszopiclone peak should not be greater than 1.5; in the chromatogram of the reference solution, the RSD of the eszopiclone peak area should not exceed 5.0%.

[0015] Furthermore, the chromatogram of the test solution is recorded to 2.7 times the retention time of the main component right-zopiclone peak.

[0016] Furthermore, in the chromatogram of the test solution, if there are impurity peaks, the content of each impurity shall be calculated using the peak area of ​​eszopiclone according to the external standard method. Impurity A shall not be greater than 0.10%, impurity B shall not be greater than 0.10%, other individual impurities shall not be greater than 0.10%, and the total amount of impurities shall not be greater than 0.3%.

[0017] A further improvement of the present invention is: The sodium dodecyl sulfate-sodium dihydrogen phosphate buffer solution is prepared as follows: appropriate amounts of sodium dodecyl sulfate and sodium dihydrogen phosphate are taken, dissolved in water, and diluted to a fixed volume to obtain a solution; wherein the concentration of sodium dodecyl sulfate is 8.1 g / L, and the concentration of sodium dihydrogen phosphate is 2.1 g / L. Beneficial Effects

[0018] Compared with the prior art, the present invention has the following obvious advantages: After the pH value of the diluent of the present invention is adjusted from pH 4.0 to pH 2.5, (1) the solution has good stability and does not need to be freshly prepared before use; (2) the retention time of impurity A is consistent with the retention time of impurity A in the spiked solution of the test sample, and the retention time of the other known impurities is consistent with the retention time of each impurity peak in the spiked solution of the test sample. This shows that the detection method provided by the present invention has better durability and higher accuracy.

[0019] The method is verified to have good specificity, precision and accuracy in the detection method provided by the present invention, and can overcome the defects of the detection method of dexamethasone-related substances in the current standard.

[0020] The method provided by the present invention can effectively detect dexamethasone-related substances, help evaluate the production process and quality control of dexamethasone, ensure its purity and quality, and thus guarantee its efficacy and safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the blank solvent spectrum; Figure 2 is the system suitability solution spectrum; Figure 3 is the reference solution spectrum; Figure 4 It is the spectrum of the test sample spiked solution; Figure 5 This is the spectrum of the test solution (batch number: 20220901) in Example 1; Figure 6 This is the spectrum of the test solution (batch number: 20221001) in Example 2; Figure 7 This is the spectrum of the test solution (batch number: 20221002) in Example 3; Figure 8 This is the spectrum of the test solution (batch number: 20221201) in Example 4; Fig. 9 This is the spectrum of the test solution (batch number: 20221202) in Example 5; Fig.10 This is the spectrum of the test solution (batch number: 20221203) in Example 6; Fig.11 The standard curve of impurity A in the linearity and range of Example 7; Fig.12 The standard curve diagram of impurity B in the linearity and range of Example 7; Fig.13The standard curve diagram of the impurity C in the linearity and range of Example 7; Fig.14 The standard curve diagram of impurity D in the linearity and range of Example 7; Fig.15 This is a standard curve diagram of the linearity and range of dexamethasone in Example 7. DETAILED DESCRIPTION

[0022] The instruments, chromatographic columns, reagents, reference substances and samples used in the present invention are as follows: Instruments and Columns

[0023] Reagents

[0024] Reference

[0025] sample

[0026] The analysis method provided by the present invention (1) Chromatographic conditions and detection conditions: The chromatographic conditions and detection conditions of the methods provided by the present invention and Comparative Example 1 (Analysis method for related substances of dexzopiclone USP2022) are shown in Table 1 below: Table 1

[0027] (2) Impurity A positioning solution: Take about 10 mg of the impurity A reference substance, accurately weigh it, place it in a 25 mL volumetric flask, dissolve it with a diluent and dilute it to the mark, shake it well, and use it as the impurity A stock solution; accurately measure 2 mL of the impurity A stock solution, place it in a 20 mL volumetric flask, dilute it to the mark with a diluent, shake it well, and use it as the impurity A positioning solution.

[0028] (3) Impurity B locating solution: Take about 10 mg of the Impurity B reference substance, accurately weigh it, place it in a 25 mL volumetric flask, dissolve it with a diluent and dilute it to the mark, shake it well, and use it as the Impurity B stock solution; accurately measure 2 mL of the Impurity B stock solution, place it in a 20 mL volumetric flask, dilute it to the mark with a diluent, shake it well, and use it as the Impurity B locating solution.

[0029] (4) Impurity C locating solution: Take about 2 mg of the impurity C reference substance, accurately weigh it, place it in a 10 mL volumetric flask, dissolve it with a diluent and dilute it to the mark, shake it well, and use it as the impurity C stock solution; accurately measure 2 mL of the impurity C stock solution, place it in a 10 mL volumetric flask, dilute it to the mark with a diluent, shake it well, and use it as the impurity C locating solution.

[0030] (5) Impurity D localizing solution: Take about 10 mg of the impurity D reference substance, accurately weigh it, place it in a 25 mL volumetric flask, dissolve it with a diluent and dilute it to the mark, shake it well, and use it as the impurity D stock solution; accurately measure 2 mL of the impurity D stock solution, place it in a 20 mL volumetric flask, dilute it to the mark with a diluent, shake it well, and use it as the impurity D localizing solution.

[0031] (6) Reference solution: Take about 10 mg of dextropiclone reference substance, accurately weigh it, place it in a 25 mL volumetric flask, dissolve it with a diluent and dilute it to the mark, shake well, and use it as the reference stock solution; accurately measure 1 mL of the reference stock solution, place it in a 100 mL volumetric flask, dilute it to the mark with a diluent, shake well, and use it as the dextropiclone reference solution.

[0032] (7) System suitability solution: Accurately measure 2 mL each of the impurity A stock solution, impurity B stock solution, and reference substance stock solution, place in a 20 mL volumetric flask, dilute to the mark with diluent, and shake well to prepare the system suitability solution.

[0033] (8) Test solution: Take about 40 mg of dextropiclone test sample, accurately weigh it, place it in a 10 mL volumetric flask, dilute it to the mark with diluent, shake well, and use it as the test solution.

[0034] (9) Test sample spike solution: Take about 40 mg of the dexzopiclone test sample, weigh accurately, and place in a 10-mL volumetric flask. Add 1 mL each of the impurity A stock solution, impurity B stock solution, and impurity D stock solution, and 2 mL of the impurity C stock solution. Add diluent and sonicate to dissolve and dilute to the scale. Shake well and use this as the test sample spike solution.

[0035] The system suitability solution, each impurity location solution, and the test sample spiked solution were checked by the methods provided by the present invention and comparative example 1, and the chromatograms were recorded. The results are shown in Tables 2 to 4 as follows: Table 2 System suitability solution test results

[0036] Table 3 Test results of each impurity localization solution

[0037] Table 4 Test results of spiked solution of test sample

[0038] in conclusion: The present invention provides a method in which the retention time of the impurity B, impurity C and impurity D peaks is consistent with the retention time of each impurity peak in a test sample spiked solution, and the retention time of the impurity A is consistent with the retention time of the impurity A in a test sample spiked solution, each impurity can be effectively separated from the other impurities and from the main peak, and the peak shape is good.

[0039] Comparative Example 1 provides a method: the impurity B peak, the impurity C peak, the impurity D peak, the impurity A peak and the peak of eszopiclone can be effectively separated, and the peak shape is good. Among them, the retention time of each positioning peak of impurity B, impurity C, and impurity D is consistent with the retention time of each impurity peak in the test sample spiked solution, but the retention time of impurity A positioning (14.227min) is quite different from the retention time of impurity A in the test sample spiked solution (13.072min).

[0040] In summary, through data comparison, the present invention can overcome the defects of the method provided in Comparative Example 1.

[0041] Example 1-6 Sample Testing 1. Analytical methods: The chromatographic conditions and detection conditions are shown in Table 1.

[0042] Test solution, reference solution, system suitability solution: preparation method see the analysis method provided in the present invention.

[0043] 2. Determination of R-zopiclone API samples: According to the analytical method for related substances of dexzopiclone, the related substances of 6 batches of raw materials were determined and the spectra were recorded, such as Figures 5 to 10 The results are shown in Table 5 below.

[0044] Table 5 Sample measurement results

[0045] The results showed that the determination results of related substances in 6 batches of dexamethasone raw material samples were in compliance with the regulations.

[0046] Example 7 Detection Method Verification 1. Precision (1) Instrument precision Take the reference solution and repeat the measurement 6 times according to the analytical method for related substances, calculate the RSD of the peak area and retention time, and the results are shown in Tables 6 to 9.

[0047] Table 6 Instrument precision measurement results (Instrument No.: JQ1312)

[0048] Table 7 Instrument precision results (Instrument No.: JQ1316-VWD)

[0049] Table 8 Instrument precision results (Instrument No.: JQ1312-PDA)

[0050] Table 9 Instrument precision results (Instrument No.: QC-M-182)

[0051] The results showed that the instrumental precision of the analytical method for dexzopiclone-related substances was good.

[0052] (2) Repeatability Test solution, reference solution, system suitability solution: preparation method see the analysis method provided in the present invention.

[0053] Test sample spiked solution (prepare 6 portions in parallel): the preparation method is as described in the analysis method provided by the present invention.

[0054] According to the analytical method for related substances of eszopiclone, 6 samples of spiked solutions were determined. The recoveries of impurities A and B and the RSDs of other single impurities and total impurities in the 6 samples of spiked solutions were investigated. The results are shown in Table 10.

[0055] Table 10 Repeatability test results

[0056] The results showed that the analytical method for eszopiclone-related substances had good repeatability.

[0057] (3) Intermediate precision Test solution, reference solution, system suitability solution: preparation method see the analysis method provided in the present invention.

[0058] Test sample spiked solution (prepare 6 portions in parallel): the preparation method is as described in the analysis method provided by the present invention.

[0059] Different personnel were investigated, on different dates and using different instruments, to determine the test sample spiked solutions according to the analytical method for related substances of dexamethasone. The recoveries of impurities A and B and the RSDs of other single impurities and total impurities in the test sample spiked solutions (12 portions in total, including 6 portions under the repeatability item) were investigated. The results are shown in Table 11.

[0060] Table 11 Intermediate precision test results

[0061] The results showed that the intermediate precision of the analytical method for dexamethasone-related substances was good.

[0062] 2. Limit of detection and limit of quantification The detection limit and quantification limit were determined by the signal-to-noise ratio method, with the corresponding concentration when the signal-to-noise ratio was approximately 3:1 as the detection limit and the corresponding concentration when the signal-to-noise ratio was approximately 10:1 as the quantification limit.

[0063] (1) Detection limit of impurity A Accurately measure 3 mL of the quantitative limit solution of impurity A, place it in a 10 mL volumetric flask, dilute to the scale with diluent, shake well, and use this as the detection limit solution of impurity A. The concentration is 0.02459 μg / mL, which is 0.61% of the limit.

[0064] (2) Impurity A Quantification Limit Accurately measure 0.1 mL of the stock solution of impurity A under the linearity and range items, place it in a 50 mL volumetric flask, dilute it to the scale with diluent, shake well, accurately measure 1 mL, place it in a 10 mL volumetric flask, dilute it to the scale with diluent, shake well, and use it as the quantitative limit solution of impurity A. The concentration is 0.08198 μg / mL, which is 2.05% of the limit.

[0065] Take the quantitative limit solution of impurity A and repeat the injection 6 times to examine the peak area RSD. The results are shown in Table 12.

[0066] Table 12 Results of determination of the limit of quantification of impurity A

[0067] The results showed that the quantification limit of impurity A had good repeatability.

[0068] (3) Detection limit of impurity B Accurately measure 3 mL of the quantitative limit solution of impurity B, place it in a 10 mL volumetric flask, dilute to the scale with diluent, shake well, and use this as the detection limit solution of impurity B with a concentration of 0.00247 μg / mL, which is 0.062% of the limit.

[0069] (4) Impurity B Quantification Limit Accurately measure 0.1 mL of the stock solution of impurity B under the linearity and range items, place it in a 50 mL volumetric flask, dilute it to the scale with diluent, shake well, accurately measure 1 mL, place it in a 100 mL volumetric flask, dilute it to the scale with diluent, shake well, and use it as the quantitative limit solution of impurity B. The concentration is 0.00823 μg / mL, which is 0.21% of the limit.

[0070] The quantitative limit solution of impurity B was taken and repeated injection was performed 6 times to examine the peak area RSD. The results are shown in Table 13.

[0071] Table 13 Results of determination of the limit of quantification of impurity B

[0072] The results showed that the quantitative limit of impurity B had good repeatability.

[0073] (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, dilute to the scale with diluent, shake well, and use this as the detection limit solution of impurity C with a concentration of 0.00467 μg / mL, which is 0.12% of the limit.

[0074] (6) Impurity C Quantification Limit Accurately measure 0.2 mL of the stock solution of impurity C under the linearity and range items, place it in a 50 mL volumetric flask, dilute it to the scale with diluent, shake well, accurately measure 1 mL, place it in a 50 mL volumetric flask, dilute it to the scale with diluent, shake well, and use it as the quantitative limit solution of impurity C. The concentration is 0.01557 μg / mL, which is 0.39% of the limit.

[0075] The quantitative limit solution of impurity C was taken and repeated injection was performed 6 times to examine the peak area RSD. The results are shown in Table 14.

[0076] Table 14 Results of determination of the limit of quantification of impurity C

[0077] The results showed that the quantitative limit of impurity C had good repeatability.

[0078] (7) Detection limit of impurity D Accurately measure 3 mL of the quantitative limit solution of impurity D, place it in a 10 mL volumetric flask, dilute to the scale with diluent, shake well, and use this as the detection limit solution of impurity D. The concentration is 0.00760 μg / mL, which is 0.19% of the limit.

[0079] (8) Impurity D quantitative limit Accurately measure 0.1 mL of the impurity D stock solution under the linearity and range items, place it in a 50 mL volumetric flask, dilute it to the scale with a diluent, shake well, accurately measure 3 mL of the solution, place it in a 100 mL volumetric flask, dilute it to the scale with a diluent, and use this as the impurity D quantitative limit solution with a concentration of 0.02532 μg / mL, which is 0.63% of the limit.

[0080] Take the quantitative limit solution of impurity D and repeat the injection 6 times to examine the peak area RSD. The results are shown in Table 15.

[0081] Table 15 Results of determination of the limit of quantification of impurity D

[0082] The results showed that the quantitative limit of impurity D had good repeatability.

[0083] (9) Detection limit of eszopiclone Accurately measure 3 mL of the quantification limit solution of dexzopiclone, place it in a 10 mL volumetric flask, dilute to the scale with diluent, shake well, and use this as the detection limit solution of dexzopiclone with a concentration of 0.02462 μg / mL, which is 0.62% of the limit.

[0084] (10) Limit of quantification of eszopiclone Accurately measure 0.1 mL of the dexzopiclone stock solution under the linearity and range items, place it in a 50 mL volumetric flask, dilute it to the scale with diluent, shake well, accurately measure 1 mL, place it in a 10 mL volumetric flask, dilute it to the scale with diluent, shake well, and use this as the dexzopiclone quantitative limit solution with a concentration of 0.08208 μg / mL, which is 2.05% of the limit.

[0085] The quantitative limit solution of dexamethasone was taken and injected 6 times repeatedly to examine the peak area RSD. The results are shown in Table 16.

[0086] Table 16 Determination results of the limit of quantification of eszopiclone

[0087] The results showed that the limit of quantification of eszopiclone had good repeatability.

[0088] 3. Linearity and range Take about 10 mg of impurity A reference substance, accurately weigh it, place it in a 25 mL volumetric flask, dissolve it with diluent and dilute it to the scale, shake it well, and use it as the impurity A stock solution.

[0089] Take about 10 mg of impurity B reference substance, accurately weigh it, place it in a 25 mL volumetric flask, dissolve it with diluent and dilute it to the scale, shake it well, and use it as the impurity B stock solution.

[0090] Take about 2 mg of impurity C reference substance, accurately weigh it, place it in a 10 mL volumetric flask, dissolve it with diluent and dilute it to the scale, shake it well, and use it as the impurity C stock solution.

[0091] Take about 10 mg of impurity D reference substance, accurately weigh it, place it in a 25 mL volumetric flask, dissolve it with diluent and dilute it to the scale, shake it well, and use it as the impurity D stock solution.

[0092] Take about 10 mg of dexzopiclone reference substance, weigh accurately, place in a 25 mL volumetric flask, dissolve and dilute to the scale with diluent, shake well, and use as dexzopiclone stock solution.

[0093] Accurately measure 2 mL each of impurity A stock solution, impurity B stock solution, impurity D stock solution, and dexzopiclone stock solution, and 4 mL of impurity C stock solution, place them in the same 20 mL volumetric flask, dilute to the scale with diluent, shake well, and use as linear stock solution.

[0094] Accurately measure 0.2mL, 0.5mL, 0.8mL, 1.0mL, 1.2mL, and 2.0mL of the linear stock solution, place them in 10mL volumetric flasks respectively, dilute to the scale with diluent, shake well, and use them as linear 1, linear 2, linear 3, linear 4, linear 5, and linear 6 solutions.

[0095] Accurately measure the linear 1 to linear 6 solutions and the quantitative limit solutions of each impurity, inject them into the liquid chromatograph respectively, record the chromatogram, calculate the linear regression equation, and the results are shown in Figures 11 to 15 and Tables 17 to 21.

[0096] Table 17 Impurity A linear determination results

[0097] The results show that the impurity A has a good linear relationship in the concentration range of 0.08198~8.1980μg / mL. Linear regression equation: y=24205x-510.57, r=0.9999.

[0098] Table 18 Impurity B linear relationship determination results

[0099] The results showed that the impurity B had a good linear relationship in the concentration range of 0.00823~8.2320 μg / mL. The linear regression equation was: y=34257x-381.66, r=1.

[0100] Table 19 Impurity C linear relationship determination results

[0101] The results show that the impurity C has a good linear relationship in the concentration range of 0.01557~7.7842 μg / mL. The linear regression equation is: y=31438x-406.84, r=1.

[0102] Table 20 Impurity D linear relationship determination results

[0103] The results showed that the impurity D had a good linear relationship in the concentration range of 0.02532~8.4396 μg / mL. The linear regression equation was: y=23171x-464.95, r=0.9999.

[0104] Table 21 Results of linear relationship determination of eszopiclone

[0105] The results showed that the linear relationship of eszopiclone was good in the concentration range of 0.08208~8.2076 μg / mL. The linear regression equation was: y=27882x-759.43, r=0.9999.

[0106] 4. Accuracy Impurity A stock solution, Impurity B stock solution: See the linearity and range items for the configuration method.

[0107] Impurity stock solution: Accurately measure 5 mL of impurity A stock solution and impurity B stock solution, place them in a 50 mL volumetric flask, dilute to the scale with diluent, and shake well.

[0108] Impurity A quantitative limit stock solution: Accurately measure 0.1 mL of impurity A stock solution, place it in a 50 mL volumetric flask, dilute to the scale with diluent, and shake well.

[0109] Impurity B quantitative limit stock solution: Accurately measure 0.1 mL of impurity B stock solution, place it in a 50 mL volumetric flask, dilute to the scale with diluent, and shake well.

[0110] Test solution: Take about 40 mg of the product, weigh accurately, place in a 10 mL volumetric flask, add diluent, ultrasonically dissolve and dilute to the scale, and shake well.

[0111] Reference substance solution: Take about 10 mg of dextropiclone reference substance, accurately weigh it, place it in a 25 mL volumetric flask, dissolve it with a diluent and dilute it to the mark, shake well, and use it as the reference substance stock solution; accurately measure 1 mL of the reference substance stock solution, place it in a 100 mL volumetric flask, dilute it to the mark with a diluent, and shake well.

[0112] System suitability solution: Accurately measure 2 mL each of impurity A stock solution, impurity B stock solution, and reference substance stock solution, place in a 20 mL volumetric flask, dilute to the scale with diluent, and shake well.

[0113] Quantitation limit test sample spike solution: Take about 40 mg of this product, accurately weigh, place in a 10 mL volumetric flask, accurately add 1 mL of impurity A quantitation limit stock solution, 0.1 mL of impurity B quantitation limit stock solution, add diluent to dissolve and dilute to the scale, shake well, and use as the quantitation limit test sample spike solution (prepare 3 copies in parallel).

[0114] 80% test sample spiked solution: Take about 40 mg of the product, accurately weigh it, put it in a 10 mL volumetric flask, accurately add 0.8 mL of impurity stock solution, add diluent and ultrasonically dissolve it and dilute it to the scale, shake well, and use it as the 80% test sample spiked solution (prepare 3 copies in parallel).

[0115] 100% test sample spiked solution: Take about 40 mg of the product, accurately weigh it, put it in a 10 mL volumetric flask, accurately add 1 mL of the impurity stock solution, add diluent and ultrasonically dissolve it and dilute it to the scale, shake well, and use it as the 100% test sample spiked solution (prepare 3 copies in parallel).

[0116] 120% test sample spiked solution: Take about 40 mg of the product, accurately weigh, place in a 10 mL volumetric flask, accurately add 1.2 mL of impurity stock solution, add diluent and ultrasonically dissolve and dilute to the scale, shake well, and use this as the 120% test sample spiked solution (prepare 3 copies in parallel).

[0117] Accurately measure each of the above solutions and inject them into the liquid chromatograph respectively. Record the chromatogram and calculate the recovery rate and RSD of impurity A and impurity B in the spiked solution of the test sample. The results are shown in Tables 22 to 23.

[0118] Table 22 Accuracy determination results of impurity A

[0119] Table 23 Accuracy determination results of impurity B

[0120] The results showed that the analytical method for eszopiclone-related substances had good accuracy.

[0121] 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 dexamethasone-related substances, characterized in that: The steps include: System suitability solution: Take appropriate amount of dexzopiclone reference substance, impurity A reference substance and impurity B reference substance, dissolve them in diluent to make system suitability solution; Test solution: Take an appropriate amount of dexzopiclone test sample and dissolve it in diluent to make a test solution; Reference solution: Take an appropriate amount of eszopiclone reference substance and dissolve it in a diluent to make a reference solution; Determination method: Accurately measure the system suitability solution, the test solution and the reference solution respectively, inject them into the high performance liquid chromatograph, and record the chromatogram; according to the chromatogram, calculate by the external standard method to obtain the content of each related substance in dexzopiclone; Wherein, the related substances include impurity A, impurity B, impurity C and impurity D, and the structure is as follows: Impurity A ; Impurity B ; Impurity C ; Impurity D ; The diluent is a mixed solution of acetonitrile and sodium dodecyl sulfate-sodium dihydrogen phosphate buffer, with a pH value of 2.5; in the diluent, the volume ratio of acetonitrile to buffer is 628:1000.

2. The method for detecting a dexamethasone-related substance according to claim 1, characterized in that: The system suitability solution preparation method is as follows: take appropriate amounts of dexzopiclone reference substance, impurity A reference substance, and impurity B reference substance, add appropriate amounts of diluent, dissolve by ultrasound, and dissolve and fix the volume with the diluent to obtain a solution; wherein the concentration of dexzopiclone is 0.04 mg / mL, the concentration of impurity A is 0.04 mg / mL, and the concentration of impurity B is 0.04 mg / mL.

3. The method for detecting a dexamethasone-related substance according to claim 1, characterized in that: The test solution preparation method is as follows: take an appropriate amount of dexzopiclone test sample, accurately weigh it, add an appropriate amount of diluent, dissolve it by ultrasound, and dissolve it with the diluent to make a solution with a concentration of 4 mg / L.

4. The method for detecting a dexamethasone-related substance according to claim 1, characterized in that: The preparation method of the reference substance solution is as follows: take an appropriate amount of dexzopiclone reference substance, accurately weigh it, add an appropriate amount of diluent, dissolve it by ultrasonication, and dissolve it with the diluent to make a solution with a concentration of 0.004 mg / L.

5. The method for detecting a dexamethasone-related substance according to claim 1, characterized in that: The chromatographic conditions of the high performance liquid chromatograph are as follows: Column: GL Sciences Inertsil ODS-3V, 250 mm × 4.6 mm, 5 μm; Mobile phase: a mixed solution of acetonitrile and sodium dodecyl sulfate-sodium dihydrogen phosphate buffer, with the pH value adjusted to 4.0 with phosphoric acid; wherein the volume ratio of acetonitrile to buffer was 628:1000; Flow rate: 1.5 mL / min; Detection wavelength: 220nm; Column temperature: 30°C; Sample chamber temperature: 5°C; Injection volume: 20mL.

6. The method for detecting a dexamethasone-related substance according to claim 1, characterized in that: In the chromatogram of the system suitability solution, the order of peaks is impurity B peak, impurity A peak and eszopiclone peak, the separation between the impurity A peak and the eszopiclone peak should not be less than 2.0, and the tailing factor of the eszopiclone peak should not be greater than 1.5; in the chromatogram of the reference solution, the RSD of the eszopiclone peak area should not exceed 5.0%.

7. The method for detecting a dexamethasone-related substance according to claim 1, characterized in that: The chromatogram of the test solution was recorded to 2.7 times the retention time of the main component s-zopiclone peak.

8. The method for detecting a dexazopiclone-related substance according to claim 7, characterized in that: In the chromatogram of the test solution, if there are impurity peaks, the content of each impurity shall be calculated using the peak area of ​​eszopiclone according to the external standard method. Impurity A shall not be greater than 0.10%, impurity B shall not be greater than 0.10%, other individual impurities shall not be greater than 0.10%, and the total amount of impurities shall not be greater than 0.3%.

9. The method for detecting a dexazopiclone-related substance according to claim 1 or 5, characterized in that: The sodium dodecyl sulfate-sodium dihydrogen phosphate buffer solution is prepared as follows: appropriate amounts of sodium dodecyl sulfate and sodium dihydrogen phosphate are taken, dissolved in water, and diluted to a fixed volume to obtain a solution; wherein the concentration of sodium dodecyl sulfate is 8.1 g / L, and the concentration of sodium dihydrogen phosphate is 2.1 g / L.

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