A method for detecting the content of zopiclone impurities

Abstract

This invention relates to a method for detecting zopiclone impurities, the method comprising the following steps: Step A: preparation of diluent; Step B: preparation of test solution: take zopiclone, add diluent, and dilute to prepare a test solution; Step C: preparation of reference solution: take impurity 6-(5-chloro-2-pyridinyl)-7-ethoxy-5,6-dihydropyrrolo[3,4-b]pyrazin-5-one reference standard and zopiclone reference standard, add diluent, sonicate to dissolve, and prepare a reference solution; Step D: determination method: take the test solution and reference solution respectively, inject them into a high-performance liquid chromatograph, record the chromatogram, and calculate the impurity content; wherein, the chromatographic conditions are: using octadecylsilane-bonded silica gel as the packing material; using acetonitrile-buffered saline solution as the mobile phase; flow rate of 0.5 mL to 2.5 mL per minute; detection wavelength of 280 nm to 320 nm; column temperature of 25 °C to 35 °C; sample chamber temperature of 0 °C to 15 °C; injection volume of 10 to 30 μl.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis, specifically relating to a method for detecting the content of zopiclone impurities. Background Technology

[0002] Zopiclone, chemically known as 6-(5-chloro-2-pyridinyl)-7-[(4-methylpiperazin-1-yl)formyloxy]-5,6-dihydropyrrolo[3,4-b]pyrazin-5-one, has the following chemical structure:

[0003]

[0004] Zopiclone is a medication used to treat insomnia, also known as a non-benzodiazepine. Zopiclone, a type of sedative-hypnotic drug, primarily works by selectively binding to benzodiazepine receptors in the central nervous system, enhancing the central inhibitory effect of GABA receptors. It possesses sedative, hypnotic, anti-anxiety, anticonvulsant, and muscle relaxant effects. Clinically, zopiclone is mainly used to treat sleep disorders. It has a rapid onset of action, a long duration of action, and few adverse reactions. It significantly shortens the sleep latency, prolongs sleep time, and improves sleep quality, with a stronger effect than benzodiazepines such as diazepam, flurazepam, clozapine, oxazepam, and triazolam. It has mild respiratory depression, low residual effect the next day, and no significant dependence, making it suitable for various types of insomnia.

[0005] 6-(5-chloro-2-pyridinyl)-7-ethoxy-5,6-dihydropyrrolo[3,4-b]pyrazin-5-one, with the following structure:

[0006]

[0007] For zopiclone active pharmaceutical ingredient (API), the content of related substances must be controlled to ensure that the finished zopiclone API is a qualified drug for production and sale. However, the existing technology does not disclose the detection method for the impurity 6-(5-chloro-2-pyridinyl)-7-ethoxy-5,6-dihydropyrrolo[3,4-b]pyrazin-5-one in zopiclone.

[0008] Existing technologies often employ the self-comparison method for detecting zopiclone impurities. While this method is simple and rapid, it has limitations due to factors such as the response factors of each impurity and the principal component not necessarily being the same, the impurity amount and the principal component amount not necessarily being within the same linear range, and the instrument's integration precision and accuracy differing for trace impurities and major principal components. The external standard method, however, provides more accurate quantification. Therefore, there is an urgent need for a method to detect the impurity 6-(5-chloro-2-pyridinyl)-7-ethoxy-5,6-dihydropyrrolo[3,4-b]pyrazin-5-one in zopiclone. Summary of the Invention

[0009] In view of this, the object of the present invention is to provide a method for detecting the content of zopiclone impurity 6-(5-chloro-2-pyridinyl)-7-ethoxy-5,6-dihydropyrrolo[3,4-b]pyrazin-5-one.

[0010] The method includes the following steps:

[0011] Step A: Preparation of diluent;

[0012] Step B: Preparation of test solution: Take zopiclone, add diluent, and dilute to prepare test solution;

[0013] Step C: Preparation of reference solution: Take impurity 6-(5-chloro-2-pyridinyl)-7-ethoxy-5,6-dihydropyrrolo[3,4-b]pyrazin-5-one reference standard and zopiclone reference standard, add diluent, and sonicate to dissolve to obtain the reference solution.

[0014] Step D: Determination method: Take the test solution and the reference solution respectively, inject them into the high performance liquid chromatograph, record the chromatogram, and calculate the impurity content;

[0015] The chromatographic conditions are as follows: octadecylsilane-bonded silica gel as the packing material; acetonitrile-buffered saline solution as the mobile phase; flow rate of 0.5 mL to 2.5 mL per minute; detection wavelength of 280 nm to 320 nm; column temperature of 25 °C to 35 °C; sample chamber temperature of 0 °C to 15 °C; and injection volume of 10 to 30 μL.

[0016] In the method of the present invention, wherein step D preferably has the following parameters: flow rate of 1.5 mL per minute; detection wavelength of 303 nm; column temperature of 30 °C; sample chamber temperature of 5 °C; and injection volume of 20 μl.

[0017] In the method of the present invention, wherein in step D, the volume ratio of acetonitrile to buffer salt solution is preferably 538:1000, and the buffer salt solution is prepared as follows: take 8.1g of sodium dodecyl sulfate and 1.6g of sodium dihydrogen phosphate, dissolve them in water and dilute to 1000ml, and adjust the pH value to 4.0 with 10% phosphoric acid solution.

[0018] In the method of the present invention, preferably in step A, the diluent is an acetonitrile-buffered salt, and the pH value is adjusted to 2.5±0.05 with 10% phosphoric acid solution; the volume ratio of the acetonitrile-buffered salt is 37:63; the buffered salt is prepared as follows: take 8.1g of sodium dodecyl sulfate and 6.9g of sodium dihydrogen phosphate into 1000ml of water, sonicate for no less than 15 minutes, and filter to obtain the buffered salt.

[0019] In the method of the present invention, preferably in step B, the test solution contains approximately 4 mg of test sample per 1 mL.

[0020] In the method of the present invention, wherein in step C, preferably, the reference solution contains approximately 0.004 mg of reference standard per 1 mL.

[0021] The method of the present invention, wherein the impurity 6-(5-chloro-2-pyridinyl)-7-ethoxy-5,6-dihydropyrrolo[3,4-b]pyrazin-5-one is prepared by the following method: 6-(5-chloro-2-pyridinyl)-5H-pyrrolo[3,4-b]pyrazin-5,7(6H)-dione is reacted with hydrochloric acid ethanol solution in the presence of an organic solvent and a catalyst.

[0022] The organic solvent is selected from one or two of ethanol, methanol, ethyl acetate, tetrahydrofuran, acetone, and diethyl ether.

[0023] The catalyst is selected from one or two of sodium borohydride, potassium borohydride, lithium borohydride, lithium aluminum tetrahydrogen, and sodium hydride;

[0024] The catalyst to the raw material weight ratio is 0.1-0.5:1; the concentration of the hydrochloric acid ethanol solution is 0.6-1.8 mol / L;

[0025] The weight ratio of the hydrochloric acid ethanol solution to 6-(5-chloro-2-pyridinyl)-5H-pyrrolo[3,4-b]pyrazine-5,7(6H)-dione is 2-8:1;

[0026] The reaction temperature is -10℃ to 30℃;

[0027] The reaction time is 2h-10h.

[0028] Preferably, the organic solvent is selected from ethanol, and / or the catalyst is selected from potassium borohydride; and / or the weight ratio of the catalyst to the raw material is 0.2:1, and / or the concentration of the hydrochloric acid ethanol solution is 1.25 mol / L; and / or the weight ratio of the hydrochloric acid ethanol solution to 6-(5-chloro-2-pyridinyl)-5H-pyrrolo[3,4-b]pyrazine-5,7(6H)-dione is 5:1; and / or the reaction temperature is 0℃-10℃; and / or the reaction time is 4h-8h.

[0029] Alternatively, it can be prepared by the following method: 50g of crude zopiclone is stirred and refluxed in 550g of ethanol for 6h, then cooled to 3°C and crystallized overnight. The reflux and crystallization process is repeated twice. The filtrate is filtered and about 100mL is collected. The target impurity 6-(5-chloro-2-pyridinyl)-7-ethoxy-5,6-dihydropyrrolo[3,4-b]pyrazin-5-one is obtained by column chromatography (Rf=0.2, DCM / MeOH=20 / 1).

[0030]

[0031] The inventors discovered that the impurities described in this invention are undetectable in crude zopiclone prepared using conventional techniques. However, if the remaining reaction liquid in the reactor is not filtered promptly or repeatedly refluxed for crystallization during production, the impurities of this invention will appear. For example, the study showed that the crude zopiclone contained no target impurities. After three cycles of reflux and cooling for crystallization, the impurity purity of the filtrate was 44.4%, and the impurity purity of the filter cake was 4.41%. Further analysis of the filtrate was conducted. TCL analysis indicated that the point with Rf = 0.2 was likely the target impurity point. The relative retention time after scraping the sample matched that of the target impurity 6-(5-chloro-2-pyridinyl)-7-ethoxy-5,6-dihydropyrrolo[3,4-b]pyrazin-5-one, thus confirming the TCL position of the target impurity point. The target impurity was subsequently obtained by column chromatography, yielding 0.3 g of a yellowish-brown solid with a purity of 97.7%. Therefore, detecting the impurities of this invention and controlling the preparation process are essential.

[0032] The following is an explanation and description of the terminology used in this invention:

[0033] In the detection method of this invention, the 6-(5-chloro-2-pyridinyl)-7-ethoxy-5,6-dihydropyrrolo[3,4-b]pyrazin-5-one reference standard is obtained by the synthesis method of analogs in the prior art (Heterocycles, 1983, Vol 20, No 6, 985-990), and is purified to the purity of the reference standard by recrystallization or other known purification methods.

[0034] In the detection method of this invention, the zopiclone reference standard is derived from commercially available sources or synthesized according to publicly available literature.

[0035] "Precise weighing" means that the weight should be accurate to one-thousandth of the weight taken;

[0036] "Weighing" means that the weight should be accurate to one-hundredth of the weight taken;

[0037] "Precise measurement" means that the accuracy of the volume measurement should meet the precision requirements of the pipette of that volume in the national standard.

[0038] "Measurement" refers to the use of a graduated cylinder or the selection of a measuring tool according to the significant figures of the volume being measured;

[0039] When the amount to be taken is “about”, it means that the amount to be taken shall not exceed ±10% of the prescribed amount.

[0040] The beneficial technical effects of this invention are as follows:

[0041] (1) The diluent of the present invention uses a low pH, which ensures the stability of the sample, and the determination method is accurate and efficient.

[0042] (2) In the synthesis described in this invention, the impurity synthesis route is simple, the raw material source is sufficient, and the purity of the prepared impurities is high. Regarding solvent selection, anhydrous ethanol solution is preferred as it does not introduce additional side reactions, thereby improving product quality and purity. For the feeding method, the hydrochloric acid ethanol solution should be added to the reaction system dropwise, and the temperature should be controlled below 10°C. Excessive temperature will lead to side reactions and impurity generation, while temperatures below 0°C will slow down the reaction, increasing production time costs. Regarding catalyst selection, this invention has found that sodium borohydride and lithium borohydride can also participate in the reaction normally. Considering catalyst cost, potassium borohydride is selected as the preferred catalyst in this invention. The synthesis route is simple and efficient, and the raw materials used are all common chemical raw materials. This invention has high yield, good purity, and no additional impurities are generated in the reaction. It is easy to perform common detection methods such as HPLC, TLC, and HNMR, filling the gap in the synthesis of this impurity compound.

[0043] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention. Attached Figure Description

[0044] Figure 1 : 1H NMR spectrum of 6-(5-chloro-2-pyridinyl)-7-ethoxy-5,6-dihydropyrrolo[3,4-b]pyrazin-5-one prepared in Example 1;

[0045] Figure 2 CNMR spectrum of 6-(5-chloro-2-pyridinyl)-7-ethoxy-5,6-dihydropyrrolo[3,4-b]pyrazin-5-one prepared in Example 1;

[0046] Figure 3 LCMS spectrum of 6-(5-chloro-2-pyridinyl)-7-ethoxy-5,6-dihydropyrrolo[3,4-b]pyrazin-5-one prepared in Example 1;

[0047] Figure 4 HPLC chromatogram of the impurity reference standard in Example 2;

[0048] Figure 5 HPLC chromatogram of the zopiclone sample in Example 2. Detailed Implementation

[0049] The present invention is further illustrated by the following examples, but these are not intended to limit the invention.

[0050] Example 1

[0051] Preparation of 6-(5-chloro-2-pyridinyl)-7-ethoxy-5,6-dihydropyrrolo[3,4-b]pyrazin-5-one

[0052]

[0053] In a clean 250 mL three-necked flask equipped with a stirrer and thermometer, add 126 mL of anhydrous ethanol, 10.0 g of 6-(5-chloro-2-pyridinyl)-5H-pyrrolo[3,4-b]pyrazine-5,7(6H)-dione, and 0.2 g of potassium borohydride. Turn on the stirrer and place the flask in an ice-water bath. When the internal temperature reaches 0–5 °C, add 50 g of 1.25 mol / L hydrochloric acid ethanol solution dropwise under controlled temperature. After the addition is complete, stir at 0–10 °C for 4 h. Stop the reaction when no raw material remains as detected by TLC. The pH was adjusted to 8-9 with 10% potassium hydroxide aqueous solution. The sample was extracted twice with 200 mL of dichloromethane. The organic phases were combined and distilled under reduced pressure to obtain crude 6-(5-chloro-2-pyridinyl)-7-ethoxy-5,6-dihydropyrrolo[3,4-b]pyrazin-5-one. The crude product was dried under vacuum at 40-50 °C for 4 h to obtain 8.7 g of a pale yellow solid, with a yield of 87.5% and a purity of 99.2%. The ¹H NMR spectrum is shown in the attached figure. Figure 1 The CNMR spectrum is shown in the attached figure. Figure 2 The LCMS spectrum is shown in the attached figure. Figure 3 .

[0054] Example 2: Detection of Zopiclone Related Substances

[0055] Preparation of diluent: Acetonitrile-buffered salt (take 8.1g sodium dodecyl sulfate and 6.9g sodium dihydrogen phosphate into 1000ml water, sonicate for no less than 15 minutes, and filter) (37:63), adjust the pH value to 2.5±0.05 with 10% phosphoric acid solution.

[0056] Preparation of the test solution: Take the zopiclone sample, weigh it accurately, add diluent, sonicate to dissolve, and dilute quantitatively with diluent to prepare a solution containing about 4 mg per ml.

[0057] Reference solution: Take the impurity reference standard 6-(5-chloro-2-pyridinyl)-7-ethoxy-5,6-dihydropyrrolo[3,4-b]pyrazin-5-one, add diluent, sonicate to dissolve, and dilute with diluent to prepare a solution containing about 0.004 mg per ml.

[0058] Chromatographic conditions: Octadecylsilane-bonded silica gel (Agilent Extend-C18, 250 mm × 4.6 mm, 5 μm) was used as the stationary phase; [acetonitrile-buffered saline (8.1 g sodium dodecyl sulfate (a reagent for ion-pair chromatography) and 1.6 g sodium dihydrogen phosphate, dissolved in water and diluted to 1000 ml) (538:1000), pH adjusted to 4.0 with 10% phosphoric acid solution] was used as the mobile phase; the flow rate was 1.5 ml / min; the detection wavelength was 303 nm; the column temperature was 30 °C; the sample chamber temperature was 5 °C; and the injection volume was 20 μl. The retention time of zopiclone was approximately 51.6 min, while the retention time of impurities was approximately 14 min. The resolution of the zopiclone and impurity peaks was greater than 3. The content of the impurity 6-(5-chloro-2-pyridyl)-7-ethoxy-5,6-dihydropyrrolo[3,4-b]pyrazin-5-one in zopiclone was calculated to be 0.20% using the external standard method. The chromatogram of the reference solution is shown below. Figure 4 See Table 1 for the chromatogram of the zopiclone sample. Figure 5 And Table 2.

[0059] Table 1. Chromatographic data of the reference solution

[0060]

[0061]

[0062] Table 2 Chromatographic data of zopiclone samples

[0063]

[0064] Example 3: Study on Linear Relationships

[0065] Accurately weigh approximately 37.5 mg of the impurity reference standard 6-(5-chloro-2-pyridinyl)-7-ethoxy-5,6-dihydropyrrolo[3,4-b]pyrazin-5-one, place it in a 50 mL volumetric flask, add mobile phase solution, shake to dissolve and dilute to the mark, and mix well. Accurately measure 2.5, 5.0, 10.0, 15.0, and 20.0 mL of the above reference standard solution, place them in 100 mL volumetric flasks respectively, and dilute to the mark with mobile phase. Inject the sample three times under the above chromatographic conditions, record the chromatograms, and calculate the linear regression equation: y = 45228x + 858.34, r = 1. The results show that the impurity exhibits good linearity in the concentration range of 0.0164 μg / mL to 8.2163 μg / mL.

[0066] Example 4 Detection Limit Experiment

[0067] The reference solution from Example 2 was injected six times under the same chromatographic conditions as in Example 2, and the chromatographic data were recorded. The limit of detection was 0.0049 μg / ml, which is 0.12% of the limit; the limit of quantitation was 0.0164 μg / ml, which is 0.41% of the limit; and the peak area RSD was 1.99%. The results indicate that the impurity quantitation limit solution has good precision.

[0068] Example 5 Stability Experiment

[0069] The reference solution, which had been stored at room temperature in the dark for 24 hours, was subjected to HPLC analysis every 2 hours under the chromatographic conditions of Example 2. The peak area remained essentially unchanged, indicating that the RSD was 2.40%. The solution showed good stability and met the determination requirements.

[0070] Example 6

[0071] In a clean 250 mL three-necked flask equipped with a stirrer and thermometer, add 126 mL of anhydrous ethanol, 10.0 g of 6-(5-chloro-2-pyridinyl)-5H-pyrrolo[3,4-b]pyrazine-5,7(6H)-dione, and 0.2 g of sodium borohydride. Turn on the stirrer and place the flask in an ice-water bath. When the internal temperature reaches 0–5 °C, add 50 g of 1.25 mol / L hydrochloric acid ethanol solution dropwise under controlled temperature. After the addition is complete, stir at 0–10 °C for 4 h. Stop the reaction when no raw material remains as detected by TLC. The pH was adjusted to 8-9 with 10% sodium hydroxide aqueous solution, and the sample was extracted twice with 200 mL of dichloromethane. The organic phases were combined and distilled under reduced pressure to obtain crude 6-(5-chloro-2-pyridinyl)-7-ethoxy-5,6-dihydropyrrolo[3,4-b]pyrazin-5-one. This crude product was dried under vacuum at 40-50 °C for 4 h to give 8.7 g of a pale yellow solid, with a yield of 86.7% and a purity of 98.8%.

[0072] Example 7

[0073] In a clean 250 mL three-necked flask equipped with a stirrer and thermometer, add 126 mL of anhydrous ethanol, 10.0 g of 6-(5-chloro-2-pyridinyl)-5H-pyrrolo[3,4-b]pyrazine-5,7(6H)-dione, and 0.5 g of potassium borohydride. Turn on the stirrer and place the flask in an ice-water bath. When the internal temperature reaches 0–5 °C, add 50 g of 1.25 mol / L hydrochloric acid-ethanol solution dropwise under controlled temperature. After the addition is complete, stir at 0–10 °C for 4 h. Stop the reaction when no raw material remains as detected by TLC. The pH was adjusted to 8-9 with 10% potassium hydroxide aqueous solution, and the mixture was extracted twice with 200 mL of dichloromethane. The organic phases were combined and distilled under reduced pressure to obtain crude 6-(5-chloro-2-pyridinyl)-7-ethoxy-5,6-dihydropyrrolo[3,4-b]pyrazin-5-one. This crude product was dried under vacuum at 40-50 °C for 4 h to give 8.8 g of a pale yellow solid, with a yield of 87.8% and a purity of 99.3%.

[0074] Example 8

[0075] In a clean 250 mL three-necked flask equipped with a stirrer and thermometer, add 126 mL of anhydrous ethanol, 10.0 g of 6-(5-chloro-2-pyridinyl)-5H-pyrrolo[3,4-b]pyrazine-5,7(6H)-dione, and 0.2 g of potassium borohydride. Turn on the stirrer and place the flask in an ice-water bath. When the internal temperature reaches 10–15 °C, add 50 g of 1.25 mol / L hydrochloric acid ethanol solution dropwise under controlled temperature. After the addition is complete, stir at 10–15 °C for 4 h. Stop the reaction when no raw material remains as detected by TLC. The pH was adjusted to 8-9 with 10% potassium hydroxide aqueous solution, and the sample was extracted twice with 200 mL of dichloromethane. The organic phases were combined and distilled under reduced pressure to obtain crude 6-(5-chloro-2-pyridinyl)-7-ethoxy-5,6-dihydropyrrolo[3,4-b]pyrazin-5-one. The crude product was dried under vacuum at 40-50 °C for 4 h to give 7.0 g of a pale yellow solid, with a yield of 70.1% and a purity of 85.7%.

[0076] Example 9

[0077] In a clean 250 mL three-necked flask equipped with a stirrer and thermometer, add 126 mL of anhydrous ethanol, 10.0 g of 6-(5-chloro-2-pyridinyl)-5H-pyrrolo[3,4-b]pyrazine-5,7(6H)-dione, and 0.2 g of potassium borohydride. Turn on the stirrer and place the flask in an ice-salt bath. When the internal temperature reaches -10 to 0 °C, add 50 g of 1.25 mol / L hydrochloric acid ethanol solution dropwise under controlled temperature. After the addition is complete, stir at -10 to 0 °C for 6 h. Stop the reaction when no raw material remains as detected by TLC. The pH was adjusted to 8-9 by adding 10% potassium hydroxide aqueous solution, and the mixture was extracted twice with 200 mL of dichloromethane. The organic phases were combined and distilled under reduced pressure to obtain crude 6-(5-chloro-2-pyridinyl)-7-ethoxy-5,6-dihydropyrrolo[3,4-b]pyrazin-5-one. The crude product was dried under vacuum at 40-50 °C for 4 h to obtain 8.5 g of pale yellow solid, with a yield of 85.2% and a purity of 98.7%.

[0078] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for detecting zopiclone impurities, comprising the following steps: Step A: Preparation of diluent; Step B: Preparation of test solution: Take zopiclone, add diluent, and dilute to prepare test solution; Step C: Preparation of reference solution: Take impurity 6-(5-chloro-2-pyridinyl)-7-ethoxy-5,6-dihydropyrrolo[3,4-b]pyrazin-5-one reference standard and zopiclone reference standard, add diluent, and sonicate to dissolve to obtain the reference solution. Step D: Determination method: Take the test solution and the reference solution respectively, inject them into the high performance liquid chromatograph, record the chromatogram, and calculate the impurity content; The chromatographic conditions are as follows: octadecylsilane-bonded silica gel as the packing material; acetonitrile-buffered saline solution as the mobile phase; flow rate of 0.5 mL to 2.5 mL per minute; detection wavelength of 280 nm to 320 nm; column temperature of 25 °C to 35 °C; sample chamber temperature of 0 °C to 15 °C; and injection volume of 10 to 30 μL.

2. The method according to claim 1, characterized in that: In step D, the flow rate is 1.5 mL per minute; the detection wavelength is 303 nm; the column temperature is 30 °C; the sample chamber temperature is 5 °C; and the injection volume is 20 μl.

3. The method according to any one of claims 1-2, characterized in that: In step D, the volume ratio of acetonitrile to buffer salt solution is 538:1000. The buffer salt solution is prepared as follows: take 8.1g of sodium dodecyl sulfate and 1.6g of sodium dihydrogen phosphate, dissolve them in water and dilute to 1000ml, and adjust the pH value to 4.0 with 10% phosphoric acid solution.

4. The method according to claim 1, characterized in that: In step A, the diluent is an acetonitrile-buffered salt, and the pH value is adjusted to 2.5±0.05 with 10% phosphoric acid solution; the volume ratio of the acetonitrile-buffered salt is 37:63; the buffered salt is prepared as follows: take 8.1g of sodium dodecyl sulfate and 6.9g of sodium dihydrogen phosphate into 1000ml of water, sonicate for no less than 15 minutes, and filter to obtain the buffered salt.

5. The method according to claim 1, characterized in that: The test solution contains approximately 4 mg of the test sample per 1 mL.

6. The method according to claim 1, characterized in that: The reference solution contains approximately 0.004 mg of reference standard per 1 mL.

7. The method according to any one of claims 1-2, characterized in that: The impurity 6-(5-chloro-2-pyridinyl)-7-ethoxy-5,6-dihydropyrrolo[3,4-b]pyrazin-5-one was prepared by the following method: 6-(5-chloro-2-pyridinyl)-5H-pyrrolo[3,4-b]pyrazin-5,7(6H)-dione was reacted with hydrochloric acid ethanol solution in the presence of an organic solvent and a catalyst.

8. The method according to claim 7, characterized in that: The organic solvent is selected from one or two of ethanol, methanol, ethyl acetate, tetrahydrofuran, acetone, and diethyl ether; And / or, the catalyst is selected from one or two of sodium borohydride, potassium borohydride, lithium borohydride, lithium aluminum tetrahydrogen, and sodium hydride; And / or, the weight ratio of the catalyst to the raw material is 0.1-0.5:1; And / or, the concentration of the hydrochloric acid ethanol solution is 0.6-1.8 mol / L; And / or, the weight ratio of the hydrochloric acid ethanol solution to 6-(5-chloro-2-pyridinyl)-5H-pyrrolo[3,4-b]pyrazine-5,7(6H)-dione is 2-8:1; And / or, the reaction temperature is -10℃ to 30℃; And / or, the reaction time is 2h-10h.

9. The method according to claim 7, characterized in that: wherein, The organic solvent is selected from ethanol, and / or the catalyst is selected from potassium borohydride; and / or the weight ratio of the catalyst to the raw material is 0.2:1, and / or the concentration of the hydrochloric acid ethanol solution is 1.25 mol / L; and / or the weight ratio of the hydrochloric acid ethanol solution to 6-(5-chloro-2-pyridinyl)-5H-pyrrolo[3,4-b]pyrazine-5,7(6H)-dione is 5:1; and / or the reaction temperature is 0℃-10℃; and / or the reaction time is 4h-8h.

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