A method for simultaneous determination of three related substances in opicapone starting materials

Abstract

The present invention relates to a method for simultaneously detecting three related substances in an opicapone starting material. The method employs gas chromatography with a hydrogen flame ionization detector (FID) to simultaneously detect the three related substances in the opicapone starting material. This method not only addresses the issue of weak and highly variable UV absorption of the three related substances under liquid chromatography conditions, preventing simultaneous detection, but also eliminates the matrix effect produced by the opicapone starting material under gas chromatography conditions and improves the poor resolution between the three related substances. This method improves detection efficiency while also ensuring the authenticity and reliability of the opicapone starting material. This method can simultaneously and quantitatively detect levels of the three related substances exceeding 0.02% in the opicapone starting material, demonstrating high sensitivity, good resolution, and robust practicality, while also simplifying and facilitating a rapid detection process.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical analytical chemistry and relates to a method for simultaneously detecting three related substances in an opicapone starting material, and specifically relates to a method for simultaneously detecting the three related substances morpholine, acetylacetone and cyanoacetamide in an opicapone starting material by using a GC-FID direct sampling method. Background Art

[0002] Opicapone (Ongentys) is a tyrosine hydroxylase (COMT) inhibitor developed by the Portuguese pharmaceutical company Bial, primarily used to treat Parkinson's disease. Opicapone was first approved by the European Medicines Agency (EMA) in June 2016 as an adjunct therapy to levodopa / carbidopa dehydrogenase inhibitors. Opicapone is indicated for Parkinson's patients experiencing "off" episodes (i.e., "off" periods). As an adjunct therapy to levodopa / carbidopa, opicapone, when used in combination with levodopa, can significantly reduce "off" time and prolong "on" time, helping patients better control motor symptoms.

[0003] The Chinese chemical name of opicapone is: 2,5-dichloro-3-(5-(3,4-dihydroxy-5-nitrophenyl)-1,2,4-oxadiazol-3-yl)-4,6-dimethylpyridine 1-oxide, and its chemical structure is:

[0004]

[0005] A synthetic route of opicapone is shown below (opicapone compound patent CN101248064B):

[0006]

[0007]

[0008] According to the above synthesis route, 2,5-dichloro-4,6-dimethylnicotinonitrile is an important starting material for opicapone. The synthesis route of 2,5-dichloro-4,6-dimethylnicotinonitrile, the starting material for opicapone, in the prior art is as follows:

[0009]

[0010] The aforementioned synthetic route for 2,5-dichloro-4,6-dimethylnicotinonitrile shows that acetylacetone and cyanoacetamide, the main raw materials, react with morpholine to produce structure 1. Structure 1 then reacts with thionyl chloride to produce structure 2, which then reacts with phosphorus oxychloride to produce structure 3, the opicapone starting material. The process impurities acetylacetone, cyanoacetamide, and morpholine are unavoidable in the opicapone starting material of this route, and their presence significantly impacts drug safety. During drug development, the impurity content of starting materials is subject to strict limits for drug quality. Therefore, establishing analytical methods for these three related substances in the opicapone starting material is crucial for effectively controlling the quality of the opicapone API and related formulations. Summary of the Invention

[0011] Currently, no reports have been found on the simultaneous detection of three related substances, morpholine, acetylacetone and cyanoacetamide, in the starting materials of opicapone.

[0012] Chinese patent CN113607837A discloses a method for detecting cyanoacetamide and its related substances, CN111413451A discloses a method for detecting cyanoacetamide by reverse phase high performance liquid chromatography, CN103926333A discloses a method for detecting cyanoacetamide by high performance liquid chromatography, CN117388396A discloses a method for analyzing and detecting related substances in cyanoacetamide, and the literature discloses a method for determining cyanoacetamide by normal phase high performance liquid chromatography (Wei Yang. [J]. Modern Pesticides, 2014, 13(3)). All of them use high performance liquid chromatography to detect cyanoacetamide.

[0013] Chinese patent CN116626188A discloses a method for detecting morpholine as an impurity in pegbilprofen. This method extracts morpholine and then uses gas chromatography for detection. CN118169292A discloses a method for simultaneously determining the content of morpholine and pyridine in wastewater. This method uses liquid chromatography-mass spectrometry to detect morpholine. CN115144480A discloses a method for detecting morpholine and / or tetramethylmethanediamine in a rosuvastatin intermediate. This method uses p-toluenesulfonyl chloride to react with morpholine and then uses high-performance liquid chromatography for detection.

[0014] In the group standard T / CSTM00075-2019 Chemical Reagent Acetylacetone Standard issued by the Zhongguancun Materials Testing Technology Alliance, gas chromatography is used to detect the acetylacetone content.

[0015] Because morpholine, acetylacetone, and cyanoacetamide exhibit weak UV absorption, lack characteristic absorption, and exhibit significantly different properties from the opicapone starting material, 2,5-dichloro-4,6-dimethylnicotinonitrile, they cannot be simultaneously detected by HPLC. Therefore, a gas chromatography method was developed and optimized for the detection of morpholine, acetylacetone, and cyanoacetamide in the opicapone starting material. However, the experimental results showed that the opicapone starting material exhibited a matrix effect on acetylacetone, resulting in a poor peak shape for acetylacetone and affecting accurate quantification. This also reduced the resolution between acetylacetone and morpholine, necessitating higher column efficiency requirements. Results were generally consistent across nonpolar, intermediate-polar, and highly polar capillary column conditions.

[0016] Regarding the matrix effect of acetylacetone caused by the opicapone starting material, significant differences in pH between the reference and test solutions were observed. Various reagents were added to mitigate this effect. Results showed that diluents containing 4% ammonia, 8% triethylamine, 0.25%–2% dimethyl sulfoxide, and 1% phosphoric acid failed to improve the peak shape of acetylacetone in the opicapone starting material or enhance the resolution between acetylacetone and morpholine. Furthermore, these diluents resulted in degradation of morpholine and cyanoacetamide. Although a diluent containing 0.1%–1% glacial acetic acid eliminated the matrix effect and improved the peak shape of acetylacetone in the opicapone starting material, it consumed some morpholine, causing instability and degradation. Given these circumstances, simultaneous detection of morpholine, acetylacetone, and cyanoacetamide in the opicapone starting material was difficult.

[0017] Based on the chemical structures of the opicapone starting material, morpholine, acetylacetone, and cyanoacetamide, the inventors used acetic anhydride to react with morpholine to generate N-acetylmorpholine and glacial acetic acid. N-acetylmorpholine has good stability, while glacial acetic acid can improve the matrix effect of the opicapone starting material and does not chemically react with the opicapone starting material, acetylacetone, and cyanoacetamide. This achieves the simultaneous detection of the contents of three related substances, morpholine, acetylacetone, and cyanoacetamide, in the opicapone starting material.

[0018] In the present invention, the Chinese chemical name of the opicapone starting material is: 2,5-dichloro-4,6-dimethylnicotinonitrile, and its chemical structure is Chemical Structure 3:

[0019] .

[0020] Acetylacetone, chemical structure:

[0021] .

[0022] Cyanoacetamide, chemical structure:

[0023] .

[0024] Morpholine, chemical structure:

[0025] .

[0026] In an embodiment of the present invention, the morpholine and acetic anhydride derivatization reaction equation is as follows:

[0027]

[0028] The present invention provides a detection method for simultaneously and accurately determining three related substances, morpholine, acetylacetone, and cyanoacetamide, in an opicapone starting material. The detection method comprises the following steps:

[0029] (1) Prepare a mixed reference solution: weigh the morpholine reference, acetylacetone reference and cyanoacetamide reference, place them in the same volumetric flask, add acetonitrile to dissolve and dilute to prepare a mixed reference solution; alternatively, dissolve and dilute the morpholine reference, acetylacetone reference and cyanoacetamide reference in acetonitrile to obtain a morpholine reference solution, an acetylacetone reference solution and a cyanoacetamide reference solution, and mix them to prepare a mixed reference solution; accurately measure an appropriate amount of the mixed reference solution, place it in an empty bottle, accurately add an appropriate amount of acetic anhydride, cover, seal, mix, let stand in a water bath at 30°C for several minutes, and shake well;

[0030] (2) Prepare the test solution: weigh the opicapone starting material to be tested, place it in a volumetric flask, add acetonitrile to dissolve and dilute to prepare the test solution; accurately measure an appropriate amount of the test solution, place it in an empty bottle, accurately add an appropriate amount of acetic anhydride, cover, seal, mix, let it stand in a water bath at 30°C for several minutes, and shake it evenly;

[0031] (3) Take equal amounts of the mixed reference solution and test solution and inject them into the FID gas chromatograph for measurement;

[0032] According to the detection method of the present invention, the gas chromatography conditions include:

[0033] Chromatographic column: capillary column with bonded cross-linked amine as stationary liquid

[0034] Injection method: direct injection

[0035] Detector: flame ionization detector (FID);

[0036] Column temperature program: initial temperature is 100℃±5℃, maintain for 8-10 minutes, increase the temperature to 160℃ at a rate of 2.5℃ per minute, then increase the temperature to 255℃ at a rate of 50℃ per minute, maintain for 8-10 minutes;

[0037] Carrier gas nitrogen flow rate: 1.1ml / min~1.3ml / min;

[0038] Hydrogen flame ionization detector temperature: 295℃~305℃;

[0039] Inlet temperature: 295℃~305℃;

[0040] Split ratio: 0.5-5:1;

[0041] Injection volume: 0.5μl~2μl.

[0042] Furthermore, according to the detection method of the present invention, in step (1), the concentrations of the mixed reference solution: morpholine, acetylacetone and cyanoacetamide are all 10 μg / ml to 125 μg / ml, preferably all 50 μg / ml.

[0043] Furthermore, according to the detection method of the present invention, in the step (1), the volume ratio of the mixed reference solution added to the headspace bottle to acetic anhydride is 20 to 100:1, preferably 50:1, and more preferably the volume of the mixed reference solution is 2 ml and the volume of acetic anhydride is 40 μl.

[0044] Furthermore, according to the detection method of the present invention, in step (2), the concentration of the test solution is 10 mg / ml to 125 mg / ml, preferably 50 mg / ml.

[0045] Furthermore, according to the detection method of the present invention, in the step (2), the volume ratio of the test solution to acetic anhydride added to the headspace bottle is 20 to 100:1, preferably 50:1, and more preferably the volume of the test solution is 2 ml and the volume of acetic anhydride is 40 μl.

[0046] Furthermore, according to the detection method of the present invention, in the step (3), the capillary column with bonded cross-linked amines as the stationary phase is Agilent CP-Volamine (30m×0.32mm).

[0047] Furthermore, the detection method according to the present invention is characterized in that: in the step (3), the gas chromatography conditions are:

[0048] Carrier gas nitrogen flow rate: 1.2 ml / min; and / or

[0049] Flame ionization detector temperature: 300°C; and / or

[0050] Inlet temperature: 300°C; and / or

[0051] Split ratio: 2:1; and / or

[0052] Injection volume: 1 μl.

[0053] The present invention provides a method for simultaneously detecting three related substances, namely morpholine, acetylacetone and cyanoacetamide, in an opicapone starting material. By selecting specific chromatographic conditions and diluents, the contents of the three related substances, namely morpholine, acetylacetone and cyanoacetamide, in the opicapone starting material can be simply, quickly and accurately separated and detected, thereby ensuring the controllable quality of the opicapone raw material and providing guidance for the development of the synthesis process.

[0054] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0055] The following further details the above content of the present invention through specific embodiments. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 The figure shows the chromatogram of blank solution in the specificity test of the methodological validation for simultaneous detection of three related substances in the opicapone starting material in Example 1.

[0057] Figure 2 The figure shows the chromatogram of the acetylacetone localization solution in the methodological validation specificity experiment for the simultaneous detection of three related substances in the opicapone starting material in Example 1.

[0058] Figure 3 The figure shows the chromatogram of the cyanoacetamide localization solution in the methodological validation specificity experiment for the simultaneous detection of three related substances in the opicapone starting material in Example 1.

[0059] Figure 4 The figure shows the chromatogram of the morpholine derivative localization solution in the methodological validation specificity experiment for the simultaneous detection of three related substances in the opicapone starting material in Example 1.

[0060] Figure 5 The figure shows the chromatogram of the test sample solution in the methodological validation specificity experiment for simultaneous detection of three related substances in the opicapone starting material in Example 1.

[0061] Figure 6 The figure shows the chromatogram of the mixed solution of the methodological validation specificity experiment for simultaneous detection of three related substances in the opicapone starting material in Example 1.

[0062] Figure 7The figure shows the chromatogram of the system suitability solution / reference solution in the methodological validation experiment for simultaneous detection of three related substances in the opicapone starting material in Example 1.

[0063] Figure 8 The figure shows the detection limit solution chromatogram of the methodological validation experiment for simultaneous detection of three related substances in the opicapone starting material in Example 2.

[0064] Figure 9 The figure shows the chromatogram of the quantification limit solution of the experiment for simultaneous detection of three related substances in the opicapone starting material in Example 2.

[0065] Figure 10 The figure shows the linearity and range test for the simultaneous detection of three related substances in the opicapone starting material in Example 3 - C-150% linear solution chromatogram.

[0066] Figure 11 The figure shows the solution chromatogram of the accuracy test of the methodological verification for simultaneous detection of three related substances in the opicapone starting material in Example 4 - A - 100% recovery rate.

[0067] Figure 12 The figure shows the chromatogram of the 4-acetylmorpholine reference solution in the conversion rate verification experiment of the method for simultaneous detection of three related substances in the opicapone starting material in Example 6.

[0068] Figure 13 The figure shows the chromatogram of the morpholine test solution in the conversion rate verification experiment of the simultaneous detection of three related substances in the opicapone starting material in Example 6.

[0069] Figure 14 The figure shows the results of the conversion rate verification experiment of the simultaneous detection of three related substances in the opicapone starting material in Example 6 - LC-MS positive ion full scan mode of the morpholine derivative solution.

[0070] Figure 15 The figure shows the chromatogram of the diluted solution of morpholine derivative in the conversion rate verification experiment of the method for simultaneous detection of three related substances in the starting material of opicapone in Example 6.

[0071] Figure 16 The figure shows the chromatogram of the reference solution of the robustness test for the simultaneous detection of three related substances in the opicapone starting material in Example 7 - robustness condition 1.

[0072] Figure 17 Shown is the chromatogram of the spiked solution of the test sample in the robustness experiment for the simultaneous detection of three related substances in the opicapone starting material, robustness condition 1, in Example 7.

[0073] Figure 18 The figure shows the chromatogram of the reference solution of the robustness test for the simultaneous detection of three related substances in the opicapone starting material in Example 7 - robustness condition 2.

[0074] Figure 19 The figure shows the chromatogram of the spiked solution of the test sample in the robustness test of the methodological validation for the simultaneous detection of three related substances in the opicapone starting material in Example 7 - robustness condition 2.

[0075] Figure 20 The figure shows the chromatogram of the blank solution (non-polar column DB-1) obtained by using the non-polar DB-1 and the medium-polar InertCap 624 columns to investigate the separation between morpholine and acetylacetone in Comparative Example 1.

[0076] Figure 21 The figure shows the separation between morpholine and acetylacetone using the non-polar DB-1 and the medium-polar InertCap 624 columns in Comparative Example 1 - the chromatogram of the acetylacetone positioning solution (non-polar column DB-1).

[0077] Figure 22 The figure shows the chromatogram of the morpholine positioning solution (non-polar column DB-1) used in Comparative Example 1 to investigate the separation between morpholine and acetylacetone using non-polar DB-1 and medium-polar InertCap 624 columns.

[0078] Figure 23 The figure shows the chromatogram of the cyanoacetamide positioning solution (non-polar column DB-1) used in Comparative Example 1 to investigate the separation between morpholine and acetylacetone using non-polar DB-1 and medium-polarity InertCap 624 columns.

[0079] Figure 24 The figure shows the chromatogram of acetylacetone positioning solution (direct injection on amine column) for investigating the feasibility of the method for three related substances in comparative example 2 using direct injection on a bonded and cross-linked amine chromatographic column.

[0080] Figure 25 The figure shows the chromatogram of the morpholine positioning solution (direct injection on amine column) for comparative example 2, which was used to investigate the feasibility of the method for three related substances by direct injection on a bonded and cross-linked amine chromatographic column.

[0081] Figure 26 The figure shows the chromatogram of cyanoacetamide positioning solution (direct injection on amine column) for comparative example 2, which was used to investigate the feasibility of the method for three related substances by direct injection on a bonded and cross-linked amine chromatographic column.

[0082] Figure 27 The figure shows the chromatogram of blank solution (acetonitrile) obtained by direct injection of acetonitrile as diluent for comparative example 3 to investigate the feasibility of the method for three related substances.

[0083] Figure 28 The figure shows the chromatogram of the reference solution (acetonitrile) obtained by direct injection of acetonitrile as a diluent for comparative example 3 to investigate the feasibility of the method for three related substances.

[0084] Figure 29 The figure shows the chromatogram of the spiked solution (acetonitrile) of the test sample in comparative example 3, which was used for direct injection of acetonitrile as the diluent to investigate the feasibility of the method for three related substances.

[0085] Figure 30 The chromatogram of the test sample spiked solution (containing 4% ammonia water) is shown in Comparative Example 4, which uses ammonia water, triethylamine, dimethyl sulfoxide, phosphoric acid, and glacial acetic acid to investigate whether the matrix effect can be eliminated.

[0086] Figure 31 The chromatogram of the test sample spiked solution (containing 8% triethylamine) is shown in Comparative Example 4, which uses ammonia, triethylamine, dimethyl sulfoxide, phosphoric acid, and glacial acetic acid to investigate whether the matrix effect can be eliminated.

[0087] Figure 32 The chromatogram of the test sample spiked solution (containing 0.25% dimethyl sulfoxide) is shown in Comparative Example 4, which uses ammonia, triethylamine, dimethyl sulfoxide, phosphoric acid, and glacial acetic acid to investigate whether the matrix effect can be eliminated.

[0088] Figure 33 The chromatogram of the test sample spiked solution (containing 2% dimethyl sulfoxide) is shown in Comparative Example 4, which uses ammonia, triethylamine, dimethyl sulfoxide, phosphoric acid, and glacial acetic acid to investigate whether the matrix effect can be eliminated.

[0089] Figure 34 The chromatogram of the test sample spiked solution (containing 1% phosphoric acid) is shown in Comparative Example 4, which uses ammonia water, triethylamine, dimethyl sulfoxide, phosphoric acid, and glacial acetic acid to investigate whether the matrix effect can be eliminated.

[0090] Figure 35 The chromatogram of the reference standard solution (containing 0.1% glacial acetic acid) is shown in Comparative Example 4, which uses ammonia water, triethylamine, dimethyl sulfoxide, phosphoric acid, and glacial acetic acid to investigate whether the matrix effect can be eliminated.

[0091] Figure 36 The chromatogram of the test sample spiked solution (containing 1% glacial acetic acid) is shown in Comparative Example 4, which uses ammonia water, triethylamine, dimethyl sulfoxide, phosphoric acid, and glacial acetic acid to investigate whether the matrix effect can be eliminated. DETAILED DESCRIPTION

[0092] The present invention is further described by the following examples, but it should be understood that the following examples are not intended to limit the scope of the present invention. The reagents used in the following examples are all readily available on the market.

[0093] (1) Information on reagents, test drugs, samples and reference substances

[0094] Opicapone starting material sample (Jiangxi Hanhe Pharmaceutical Co., Ltd., batch number: HH023-240701);

[0095] Anhydrous ethanol (Chengdu Norsch Technology Co., Ltd., chromatographically pure, batch number: 2023042001, content: ≥99.9%);

[0096] Acetonitrile (Shenzhen Arthur Biotechnology Co., Ltd., chromatographically pure, batch number: A0131250110D, content: ≥99.9%);

[0097] Ethyl acetate (Chengdu Kelong Chemical Co., Ltd., analytical grade, batch number: 2022120901, content: ≥99.5%);

[0098] n-Heptane (Shenzhen Arthur Biotechnology Co., Ltd., chromatographically pure, batch number: A3330240901D, content: ≥99.0%);

[0099] Morpholine (Chengdu Kelong Chemical Reagent Factory, analytical grade, batch number: 20130320, content: ≥99.0%);

[0100] Acetylacetone (Shanghai Adamas Reagent Co., Ltd., batch number: P3145072, content: 99%);

[0101] Cyanoacetamide (Shanghai Adamas Reagent Co., Ltd., batch number: P2835616, content: 99%);

[0102] Dimethyl sulfoxide (Honeywell, chromatographic grade, batch number: 2024061101, ≥99.7%);

[0103] 4-Acetylmorpholine (Adamas, analytical grade, batch number: P2690826, 98%+)

[0104] Glacial acetic acid (Chengdu Kelong Chemical Co., Ltd., analytical grade, batch number: 2024092501, content: ≥99.5%);

[0105] Acetic anhydride (Chengdu Kelong Chemical Co., Ltd., analytical grade, batch number: 2024080201, content: ≥98.5%);

[0106] Phosphoric acid (Chongqing Chuandong Chemical (Group) Co., Ltd., analytical grade, batch number: 20231201, content: ≥85.0%);

[0107] Ammonia (Chongqing Chuandong Chemical (Group) Co., Ltd., analytical grade, batch number: 20240501, content: 25-28%);

[0108] Triethylamine (Chongqing Chuandong Chemical (Group) Co., Ltd., analytical grade, batch number: 20230901, content: ≥99.0%).

[0109] (2) Main instruments

[0110] Gas chromatograph: Agilent 7890B;

[0111] Electronic balance: XSE105DU;

[0112] Water bath constant temperature oscillator: SHZ-B.

[0113] (3) The detection method refers to the gas chromatography method (General Chapter 0521 of Part IV of the 2020 edition of the Chinese Pharmacopoeia).

[0114] Example 1: Specificity test and system suitability test for simultaneous detection of three related substances in opicapone starting material

[0115] 1. Main technical parameters of gas chromatography

[0116] Gas chromatograph: Agilent 7890B; detector: flame ionization detector (FID); chromatographic column model: Agilent CP-Volamine (30m×0.32mm); flow rate: 1.2ml / min;

[0117] Injection method: direct injection; Injection volume: 1µl;

[0118] Detector temperature: 300°C; Inlet temperature: 300°C; Split ratio: 2:1;

[0119] Heating program: initial temperature 100°C, maintain for 8 minutes, increase to 160°C at a rate of 2.5°C per minute, then increase to 255°C at a rate of 50°C per minute, maintain for 10 minutes;

[0120] Diluent: acetonitrile.

[0121] 2. Determination method

[0122] (1) Solution preparation

[0123] Blank solution: Accurately measure 2 ml of acetonitrile and place it in an empty bottle. Accurately add 40 μl of acetic anhydride, cover, seal, mix well, let it stand in a water bath at 30°C for 30 minutes, and shake well.

[0124] Morpholine Fixing Solution: Accurately weigh 49.8 mg of morpholine reference solution into a 50 ml volumetric flask. Dissolve in acetonitrile and dilute to the mark. Shake well to prepare the morpholine stock solution. Accurately measure 1 ml of the morpholine stock solution into a 20 ml volumetric flask. Dilute to the mark with acetonitrile and shake well. Accurately measure 2 ml into an empty headspace vial. Accurately add 40 μl of acetic anhydride, cap, seal, mix, and incubate in a water bath at 30°C for 30 minutes. Shake well.

[0125] Acetylacetone Fixing Solution: Accurately weigh 29.3 mg of acetylacetone reference solution into a 25 mL volumetric flask. Dissolve in acetonitrile and dilute to the mark. Shake well to prepare the acetylacetone stock solution. Accurately measure 1 mL of the acetylacetone stock solution into a 20 mL volumetric flask. Dilute to the mark with acetonitrile and shake well. Accurately measure 2 mL into an empty headspace vial. Accurately add 40 μL of acetic anhydride, cap, seal, mix, and incubate in a water bath at 30°C for 30 minutes. Shake well.

[0126] Cyanoacetamide Fixing Solution: Accurately weigh 27.3 mg of cyanoacetamide reference solution into a 25 mL volumetric flask. Dissolve in acetonitrile and dilute to the mark. Shake well to prepare the cyanoacetamide stock solution. Accurately measure 1 mL of cyanoacetamide stock solution into a 20 mL volumetric flask. Dilute to the mark with acetonitrile and shake well. Accurately measure 2 mL into an empty headspace vial. Accurately add 40 μL of acetic anhydride, cap, seal, mix, and incubate in a water bath at 30°C for 30 minutes. Shake well.

[0127] Ethanol Fixing Solution: Accurately weigh 250.2 mg of anhydrous ethanol into a 50 mL volumetric flask. Dissolve in acetonitrile and dilute to the mark. Shake well to prepare the ethanol stock solution. Accurately measure 1 mL of the ethanol stock solution into a 20 mL volumetric flask. Dilute to the mark with acetonitrile and shake well. Accurately measure 2 mL into a headspace vial. Accurately add 40 μL of acetic anhydride, cap, seal, mix, and incubate in a water bath at 30°C for 30 minutes. Shake well.

[0128] n-Heptane Fixing Solution: Accurately weigh 249.8 mg of n-heptane into a 50 ml volumetric flask. Dissolve with acetonitrile and dilute to the mark. Shake well to prepare the n-heptane stock solution. Accurately measure 1 ml of the n-heptane stock solution into a 20 ml volumetric flask. Dilute to the mark with acetonitrile and shake well. Accurately measure 2 ml into a headspace vial. Accurately add 40 μl of acetic anhydride, cap, seal, mix, and incubate in a water bath at 30°C for 30 minutes. Shake well.

[0129] Ethyl acetate fixative solution: Accurately weigh 256.9 mg of ethyl acetate into a 50 mL volumetric flask. Dissolve in acetonitrile and dilute to the mark. Shake well to prepare the ethyl acetate stock solution. Accurately measure 1 mL of the ethyl acetate stock solution into a 20 mL volumetric flask. Dilute to the mark with acetonitrile and shake well. Accurately measure 2 mL into an empty headspace vial. Accurately add 40 μL of acetic anhydride, cap, seal, mix, and incubate in a water bath at 30°C for 30 minutes. Shake well.

[0130] Acetonitrile positioning solution: Take acetonitrile and you will get it.

[0131] System Suitability Solution / Reference Solution: Accurately measure 1 mL each of the morpholine stock solution, acetylacetone stock solution, and cyanoacetamide stock solution into a 20 mL volumetric flask. Dilute to the mark with acetonitrile and shake well. Accurately measure 2 mL and place into an empty headspace vial. Accurately add 40 μL of acetic anhydride, cap, seal, mix, and incubate in a water bath at 30°C for 30 minutes. Shake well.

[0132] Test solution: Accurately weigh 997.6 mg of the test sample into a 20 ml volumetric flask. Dissolve in acetonitrile and dilute to the mark. Shake well. Accurately measure 2 ml into an empty headspace vial. Accurately add 40 μl of acetic anhydride. Cover, seal, mix, and incubate in a water bath at 30°C for 30 minutes. Shake well.

[0133] Mixed solution: Accurately weigh 999.9 mg of the test sample into a 20 ml volumetric flask. Accurately add 1 ml of each of the above stock solutions, dissolve in acetonitrile, dilute to volume, and shake well. Accurately measure 2 ml and place in an empty headspace vial. Accurately add 40 μl of acetic anhydride, cap, seal, mix, and allow to stand in a water bath at 30°C for 30 minutes. Shake well.

[0134] (2) Detection

[0135] Take the blank solution, each positioning solution, system suitability solution / reference solution, test solution and mixed solution and inject them directly, and record the chromatogram. Typical chromatograms are shown in Figures 1 to 7 The results of each solution are shown in Table 1 and Table 2.

[0136] (3) Results

[0137] Depend on Figure 1 It can be seen that the blank solutions did not interfere with the detection of acetylacetone, cyanoacetamide and morpholine derivatives;

[0138] Depend on Figure 2 It can be seen that acetylacetone peaks at around 11.4 min, and there are no interfering peaks nearby. Figure 3 It can be seen that cyanoacetamide peaks at around 21.2 min, and there are no interfering peaks nearby. Figure 4 It can be seen that the morpholine derivative peaks at around 33.5 min, and there are no interfering peaks nearby;

[0139] Depend on Figure 5 It can be seen that there are no interfering peaks near the peaks of acetylacetone, cyanoacetamide and morpholine derivatives in the test solution;

[0140] Depend on Figure 6 It can be seen that there are no interfering peaks near the peaks of acetylacetone, cyanoacetamide and morpholine derivatives in the mixed solution.

[0141] Table 1 Specificity experimental results of Example 1

[0142]

[0143] Table 2 System applicability test results of Example 1

[0144]

[0145] (4) Conclusion

[0146] Neither the blank solution nor the test solution interfered with the detection of acetylacetone, cyanoacetamide, and morpholine derivatives. The minimum resolution between acetylacetone, cyanoacetamide, and morpholine derivatives and other adjacent impurities in the mixed solution was 3.02, greater than 1.5. This indicates that the method has good specificity.

[0147] The system suitability solution was injected six times continuously. The RSD of the retention time of acetylacetone was 0.03%, less than 1.0%, and the RSD of the peak area was 1.27%, less than 10.0%; the RSD of the retention time of cyanoacetamide was 0.02%, less than 1.0%, and the RSD of the peak area was 1.18%, less than 10.0%; the RSD of the retention time of morpholine derivatives was 0.01%, less than 1.0%, and the RSD of the peak area was 0.77%, less than 10.0%, indicating that the system applicability of this method is good.

[0148] Example 2: Simultaneous Detection of Three Related Substances in Opicapone Starting Material by Limit of Quantitation and Limit of Detection

[0149] 1. Main technical parameters of gas chromatography: same as in Example 1.

[0150] 2. Determination method

[0151] (1) Solution preparation

[0152] Blank solution: Accurately measure 2 ml of acetonitrile and place it in an empty bottle. Accurately add 40 μl of acetic anhydride, cover, seal, mix well, let it stand in a water bath at 30°C for 30 minutes, and shake well.

[0153] Mixed stock solution: Accurately weigh 51.7 mg of acetylacetone reference substance, 52.2 mg of cyanoacetamide reference substance and 51.2 mg of morpholine reference substance, place them in the same 50 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and obtain.

[0154] Quantitation limit solution: Accurately measure 0.2 ml of the mixed stock solution into a 20 ml volumetric flask, dilute to the mark with acetonitrile, and shake well. Accurately measure 2 ml into an empty headspace vial, accurately add 40 μl of acetic anhydride, cap, seal, mix, and let stand in a water bath at 30°C for 30 minutes, then shake well.

[0155] Detection limit solution: Accurately measure 0.1 ml of the mixed stock solution into a 20 ml volumetric flask, dilute to the mark with acetonitrile, and shake well. Accurately measure 2 ml into an empty headspace vial, accurately add 40 μl of acetic anhydride, cap, seal, mix well, and let stand in a water bath at 30°C for 30 minutes, then shake well.

[0156] (2) Detection

[0157] Take the blank solution, quantitative limit solution and detection limit solution and inject them directly, and record the chromatogram. Typical chromatograms are shown in Figures 8 and 9 The detection limit and quantification limit results are shown in Table 3.

[0158] (3) Results

[0159] Depend on Figure 8 and Figure 9 It can be seen that this method has high detection sensitivity and can quantitatively detect three related substances, acetylacetone, cyanoacetamide and morpholine derivatives, in the starting material of opicapone at a concentration higher than 0.02%.

[0160] Table 3 Detection limit and quantification limit experimental results of Example 2

[0161]

[0162] (4) Conclusion

[0163] The quantification limit of acetylacetone solution concentration is 10.237 μg / ml, equivalent to 20.5% of the limit concentration and 0.020% of the test sample concentration, with a signal-to-noise ratio (S / N) of 18.9, greater than 10; the detection limit concentration is 5.118 μg / ml, equivalent to 10.2% of the limit concentration and 0.010% of the test sample concentration, with a signal-to-noise ratio (S / N) of 10.1, greater than 3;

[0164] The quantification limit solution concentration of cyanoacetamide was 10.336 μg / ml, equivalent to 20.7% of the limit concentration and 0.021% of the test sample concentration, with a signal-to-noise ratio (S / N) of 19.1, which was greater than 10. The detection limit concentration was 5.168 μg / ml, equivalent to 10.3% of the limit concentration and 0.010% of the test sample concentration, with a signal-to-noise ratio (S / N) of 10.1, which was greater than 3.

[0165] The quantification limit of the morpholine derivative solution concentration was 10.138 μg / ml, equivalent to 20.3% of the limit concentration and 0.020% of the test sample concentration, with a signal-to-noise ratio (S / N) of 99.1, greater than 10. The detection limit was 5.069 μg / ml, equivalent to 10.1% of the limit concentration and 0.010% of the test sample concentration, with a signal-to-noise ratio (S / N) of 53.9, greater than 3.

[0166] This shows that this method has good sensitivity.

[0167] Example 3: Linearity and range test for simultaneous detection of three related substances in opicapone starting material

[0168] 1. Main technical parameters of gas chromatography: same as in Example 1.

[0169] 2. Determination method

[0170] (1) Solution preparation

[0171] Blank solution: Accurately measure 2 ml of acetonitrile and place it in an empty bottle. Accurately add 40 μl of acetic anhydride, cover, seal, mix well, let it stand in a water bath at 30°C for 30 minutes, and shake well.

[0172] Linear mother solution: Accurately weigh 51.7 mg of acetylacetone reference substance, 52.2 mg of cyanoacetamide reference substance and 51.2 mg of morpholine reference substance, place them in the same 50 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and obtain.

[0173] Linear solution: Accurately measure the linear stock solution and prepare it with acetonitrile according to the table below.

[0174]

[0175] LOQ solution: Take the LOQ solution under “Limit of Quantitation and Limit of Detection”.

[0176] Each linear solution: Accurately measure 2 ml of each linear solution and place it in different headspace bottles. Accurately add 40 μl of acetic anhydride to each bottle, cover, seal, mix well, let it stand in a water bath at 30°C for 30 minutes, and shake well.

[0177] (2) Detection

[0178] Take the blank solution and each linear solution and inject them directly, and record the chromatogram. Typical chromatograms are shown in Figure 10 The linearity and range test results of acetylacetone are shown in Table 4, the linearity and range test results of cyanoacetamide are shown in Table 5, and the linearity and range test results of morpholine derivatives are shown in Table 6.

[0179] (3) Results

[0180] Table 4 Example 3 Acetylacetone linearity and range test results

[0181]

[0182] Table 5 Example 3 Cyanoacetamide linearity and range test results

[0183]

[0184] Table 6 Example 3 Morpholine derivative linearity and range test results

[0185]

[0186] (4) Conclusion

[0187] In the concentration range of 10.237 μg / ml to 127.958 μg / ml, the linear regression equation of acetylacetone concentration and peak area was y = 2.0356x + 1.7819, and the correlation coefficient r was 0.9999, which was greater than 0.995. The Y-axis intercept accounted for 1.7% of the 100% response value, which was less than 25%.

[0188] In the concentration range of 10.336 μg / ml to 129.195 μg / ml, the linear regression equation between cyanoacetamide concentration and peak area was y = 1.0952x-0.7567, with a correlation coefficient r of 0.9998, which was greater than 0.995; the Y-axis intercept accounted for 1.4% of the 100% response value, which was less than 25%;

[0189] In the concentration range of 10.138 μg / ml to 126.720 μg / ml, the linear regression equation between the concentration of morpholine derivatives and the peak area was y = 2.2571x-0.2886, with a correlation coefficient r of 0.9999, which was greater than 0.995; the Y-axis intercept accounted for 0.26% of the 100% response value, which was less than 25%;

[0190] It shows that acetylacetone has good linearity in the concentration range of 10.237μg / ml~127.958μg / ml, cyanoacetamide has good linearity in the concentration range of 10.336μg / ml~129.195μg / ml, and morpholine derivatives have good linearity in the concentration range of 10.138μg / ml~126.720μg / ml.

[0191] Example 4: Accuracy test for simultaneous detection of three related substances in opicapone starting material

[0192] 1. Main technical parameters of gas chromatography: same as in Example 1

[0193] 2. Determination method

[0194] (1) Solution preparation

[0195] Blank solution: Accurately measure 2 ml of acetonitrile and place it in an empty bottle. Accurately add 40 μl of acetic anhydride, cover, seal, mix well, let it stand in a water bath at 30°C for 30 minutes, and shake well.

[0196] Mixed stock solution: Accurately weigh 51.7 mg of acetylacetone reference substance, 52.2 mg of cyanoacetamide reference substance and 51.2 mg of morpholine reference substance, place them in the same 50 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and obtain.

[0197] Reference solution: Accurately measure 1 ml of the mixed stock solution into a 20 ml volumetric flask, dilute to the mark with acetonitrile, and shake well. Accurately measure 2 ml into an empty headspace vial, accurately add 40 μl of acetic anhydride, cap, seal, mix, and let stand in a water bath at 30°C for 30 minutes, then shake well.

[0198] Background solution: Accurately weigh 997.6 mg of the test sample into a 20 ml volumetric flask. Dissolve in acetonitrile and dilute to the mark. Shake well. Accurately measure 2 ml and place in an empty headspace vial. Accurately add 40 μl of acetic anhydride. Cover, seal, mix, and incubate in a water bath at 30°C for 30 minutes. Shake well.

[0199] A-20% Recovery Solution: Accurately weigh 995.8 mg of the test sample into a 20 ml volumetric flask. Accurately add 0.2 ml of each of the above stock solutions, dissolve in acetonitrile, dilute to volume, and shake well. Accurately measure 2 ml and place in an empty headspace vial. Accurately add 40 μl of acetic anhydride, cap, seal, mix, and incubate in a water bath at 30°C for 30 minutes. Shake well.

[0200] A-50% Recovery Solution: Accurately weigh 1001.3 mg of the test sample into a 20 ml volumetric flask. Accurately add 0.5 ml of each of the above stock solutions, dissolve in acetonitrile, dilute to volume, and shake well. Accurately measure 2 ml and place in an empty headspace vial. Accurately add 40 μl of acetic anhydride, cap, seal, mix, and incubate in a water bath at 30°C for 30 minutes. Shake well.

[0201] A-100% Recovery Solution: Accurately weigh 1003.4 mg of the test sample into a 20 ml volumetric flask. Accurately add 1 ml of each of the above stock solutions, dissolve in acetonitrile, dilute to volume, and shake well. Accurately measure 2 ml and place in an empty headspace vial. Accurately add 40 μl of acetic anhydride, cap, seal, mix, and incubate in a water bath at 30°C for 30 minutes. Shake well.

[0202] A-150% Recovery Solution: Accurately weigh 1001.2 mg of the test sample into a 20 ml volumetric flask. Accurately add 1.5 ml of each of the above stock solutions, dissolve in acetonitrile, dilute to volume, and shake well. Accurately measure 2 ml and place in an empty headspace vial. Accurately add 40 μl of acetic anhydride, cap, seal, mix, and incubate in a water bath at 30°C for 30 minutes. Shake well.

[0203] (2) Detection

[0204] Take the blank solution, reference solution, background solution and each recovery solution and inject them directly, and record the chromatogram. Figure 11 The accuracy results of acetylacetone are shown in Table 7, the accuracy results of cyanoacetamide are shown in Table 8, and the accuracy results of morpholine derivatives are shown in Table 9.

[0205] (3) Results

[0206] Table 7 Example 4 Acetylacetone Accuracy Test Results

[0207]

[0208] Table 8 Example 4 Cyanoacetamide Accuracy Test Results

[0209]

[0210] Table 9 Example 4 Morpholine derivative accuracy test results

[0211]

[0212] (4) Conclusion

[0213] In the accuracy experiment, the recoveries of acetylacetone A-20%, A-50%, A-100%, and A-150% were in the range of 95.2% to 104.7%, and the RSD of the recoveries of A-50%, A-100%, and A-150% were 5.01%, less than 10.0%.

[0214] The recoveries of cyanoacetamide A-20%, A-50%, A-100%, and A-150% were in the range of 96.7% to 103.0%, and the RSD of each of the recoveries of A-50%, A-100%, and A-150% was 3.56%, which was less than 10.0%;

[0215] The recoveries of morpholine derivatives A-20%, A-50%, A-100%, and A-150% were in the range of 95.4% to 102.6%, and the RSD of the recoveries of A-50%, A-100%, and A-150% was 4.03%, which was less than 10.0%;

[0216] The recoveries of acetylacetone, cyanoacetamide, and morpholine derivatives all meet the requirements of the 9101 Validation Guidelines for Analytical Methods in the 2020 edition of the Chinese Pharmacopoeia and the range of 90% to 108% specified in the ICH.

[0217] This method has good accuracy.

[0218] Example 5: Simultaneous Stability Test of Three Related Substances in Opicapone Starting Material

[0219] 1. Main technical parameters of gas chromatography: same as in Example 1

[0220] 2. Determination method

[0221] (1) Solution preparation

[0222] Blank solution: Accurately measure 2 ml of acetonitrile and place it in an empty bottle. Accurately add 40 μl of acetic anhydride, cover, seal, mix well, let it stand in a water bath at 30°C for 30 minutes, and shake well.

[0223] Mixed stock solution: Accurately weigh 51.7 mg of acetylacetone reference substance, 52.2 mg of cyanoacetamide reference substance and 51.2 mg of morpholine reference substance, place them in the same 50 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and obtain.

[0224] Reference solution: Accurately measure 1 ml of the mixed stock solution into a 20 ml volumetric flask, dilute to the mark with acetonitrile, and shake well. Accurately measure 2 ml into an empty headspace vial, accurately add 40 μl of acetic anhydride, cap, seal, mix, and let stand in a water bath at 30°C for 30 minutes, then shake well.

[0225] Test sample spike solution: Accurately weigh 1003.4 mg of the test sample into a 20 mL volumetric flask. Accurately add 1 mL of each of the above stock solutions, dissolve in acetonitrile, dilute to volume, and shake well. Accurately measure 2 mL and place into an empty headspace vial. Accurately add 40 μL of acetic anhydride, cap, seal, mix, and incubate in a water bath at 30°C for 30 minutes. Shake well.

[0226] (2) Detection

[0227] Directly inject the blank solution, reference solution, and test sample spiked solution, and record the chromatograms. The stability results of the reference solution are shown in Table 10, and the stability results of the test sample spiked solution are shown in Table 11.

[0228] (3) Results

[0229] Table 10 Example 5 Reference solution stability test results

[0230]

[0231] Table 11 Example 5 Test sample spiked solution stability test results

[0232]

[0233] (4) Conclusion

[0234] When the reference solution and the test sample spiked solution were placed at room temperature for 22.5 hours, the relative deviations between the peak areas of acetylacetone, cyanoacetamide and morpholine derivatives and the peak areas at 0 hours were all less than 10.0%, indicating that both the reference solution and the test sample spiked solution were stable at room temperature within 22.5 hours.

[0235] Example 6: Simultaneous detection of the conversion rate of three related substances in the starting material of opicapone

[0236] 1. Main technical parameters of gas chromatography: same as in Example 1

[0237] 2. Determination method

[0238] (1) Solution preparation

[0239] Blank solution: Accurately measure 2 ml of acetonitrile and place it in an empty bottle. Accurately add 40 μl of acetic anhydride, cover, seal, mix well, let it stand in a water bath at 30°C for 30 minutes, and shake well.

[0240] 4-Acetylmorpholine Reference Solution: Accurately weigh 79.1 mg of 4-acetylmorpholine into a 50 ml volumetric flask. Dissolve in acetonitrile and dilute to the mark. Shake well. Accurately measure 1 ml into a 20 ml volumetric flask, dilute to the mark with acetonitrile, and shake well. Accurately measure 2 ml into a headspace vial. Accurately add 40 μl of acetic anhydride, cap, seal, mix, and incubate in a water bath at 30°C for 30 minutes. Shake well.

[0241] Morpholine Test Solution: Prepare 6 replicates of the "System Suitability Solution / Reference Solution" under "Example 1." Accurately weigh 49.8 mg of morpholine into a 50 ml volumetric flask. Dissolve in acetonitrile and dilute to volume. Shake well. Accurately measure 1 ml into a 20 ml volumetric flask, dilute to volume with acetonitrile, and shake well. Accurately measure 2 ml into a headspace vial. Accurately add 40 μl of acetic anhydride, cap, seal, mix, and incubate in a water bath at 30°C for 30 minutes. Shake well.

[0242] Morpholine derivative solution: Place 0.2 ml of morpholine, 0.215 ml of acetic anhydride, and 5 ml of acetonitrile in a headspace vial. Cover, seal, mix thoroughly, and incubate at room temperature for 2 hours. (Morpholine and acetic anhydride are added in a 1:1 molar ratio. Based on theoretical analysis, ignoring the amount of water in the acetonitrile that hydrolyzes the acetic anhydride, the resulting morpholine derivative concentration is approximately 54.90 mg / ml.)

[0243] Morpholine derivative diluted solution: Accurately measure 1 ml of morpholine derivative solution and place it in a 20 ml headspace bottle. Add 13.8 ml of acetonitrile, cover, seal, shake well, remove the cap, accurately measure 1 ml and place it in a 50 ml volumetric flask. Dilute to the mark with acetonitrile and shake well. (Theoretical concentration is approximately 74.19 μg / ml)

[0244] (2) Detection

[0245] Take the blank solution, 4-acetylmorpholine reference solution, morpholine test solution and morpholine derivative diluted solution and inject them directly into gas chromatography. At the same time, perform LC-MS qualitative detection on the morpholine derivative solution and record the chromatogram. Typical chromatograms are shown in Figures 12 to 15 ; The morpholine conversion results are shown in Table 12.

[0246] (3) Results

[0247] Table 12 Example 6 Morpholine conversion rate experimental results

[0248]

[0249] (4) Conclusion

[0250] The final derivative product generated by morpholine and acetic anhydride is 4-acetylmorpholine or N-acetylmorpholine (molecular formula: C6H 11 NO2, molecular weight: 129.157). LC-MS qualitative analysis revealed a molecular weight of 129 for the derivative product, consistent with that of 4-acetylmorpholine. Purity determination revealed a purity of 93.2% for the morpholine derivative product (the water content in acetonitrile consumes some acetic anhydride, resulting in 6.8% remaining unreacted after a 1:1 molar ratio of morpholine to acetic anhydride), exceeding 90.0%.

[0251] The conversion rate was calculated using 4-acetylmorpholine as a reference substance and morpholine as a test substance. The results showed that the conversion rate of morpholine to 4-acetylmorpholine was 97.0%, which was greater than 90%, indicating that this method had a good conversion rate.

[0252] Example 7: Durability test for simultaneous detection of three related substances in opicapone starting material

[0253] 1. Main technical parameters of gas chromatography:

[0254] Durability condition 1: Except for the injection port temperature of 295°C, the column initial temperature of 95°C, the FID detector temperature of 295°C and the nitrogen flow rate of 1.1 ml / min, the other chromatographic conditions were the same as those in Example 1.

[0255] Durability Condition 2: Except for the injection port temperature of 305°C, the column initial temperature of 105°C, the FID detector temperature of 305°C and the nitrogen flow rate of 1.3 ml / min, the other chromatographic conditions were the same as those in Example 1.

[0256] 2. Determination method

[0257] (1) Solution preparation

[0258] Blank solution: Accurately measure 2 ml of acetonitrile and place it in an empty bottle. Accurately add 40 μl of acetic anhydride, cover, seal, mix well, let it stand in a water bath at 30°C for 30 minutes, and shake well.

[0259] Morpholine stock solution: Accurately weigh 49.8 mg of morpholine reference substance, place it in a 50 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and obtain.

[0260] Acetylacetone stock solution: Accurately weigh 29.3 mg of acetylacetone reference substance, place it in a 25 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and obtain.

[0261] Cyanoacetamide stock solution: Accurately weigh 27.3 mg of cyanoacetamide reference substance, place it in a 25 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and obtain.

[0262] Reference solution: Accurately measure 1 ml each of morpholine stock solution, acetylacetone stock solution, and cyanoacetamide stock solution into a 20 ml volumetric flask. Dilute to the mark with acetonitrile and shake well. Accurately measure 2 ml and place into an empty headspace vial. Accurately add 40 μl of acetic anhydride, cap, seal, mix, and allow to stand in a water bath at 30°C for 30 minutes. Shake well.

[0263] Test solution: Accurately weigh 997.6 mg of the test sample into a 20 ml volumetric flask. Dissolve in acetonitrile and dilute to the mark. Shake well. Accurately measure 2 ml into an empty headspace vial. Accurately add 40 μl of acetic anhydride. Cover, seal, mix, and incubate in a water bath at 30°C for 30 minutes. Shake well.

[0264] Test sample spike solution: Accurately weigh 1005.8 mg of the test sample into a 20 mL volumetric flask. Accurately add 1 mL each of the morpholine stock solution, acetylacetone stock solution, and cyanoacetamide stock solution. Dissolve in acetonitrile and dilute to the mark. Shake well. Accurately measure 2 mL and place into an empty headspace vial. Accurately add 40 μL of acetic anhydride. Cover, seal, mix, and incubate in a water bath at 30°C for 30 minutes. Shake well.

[0265] (2) Detection

[0266] Take the blank solution, reference solution, test solution and test solution spiked with the above solution and inject directly, and record the chromatogram. Typical chromatograms are shown in Figures 16 to 19 ; The durability condition results are shown in Table 13.

[0267] (3) Results

[0268] Table 13 Durability test results of Example 7

[0269]

[0270] (4) Conclusion

[0271] Under normal conditions, durability condition 1, and durability condition 2, the blank solution and the test solution did not interfere with the detection of acetylacetone, cyanoacetamide, and morpholine derivatives, and the recoveries were all in the range of 90.0% to 108.0%, indicating good durability. That is, slight changes in the injection port temperature, column initial temperature, FID detector temperature, and nitrogen flow rate did not affect the detection of acetylacetone, cyanoacetamide, and morpholine derivatives.

[0272] Example 8: Simultaneous Detection of Three Related Substances in Opicapone Starting Material by Derivatization Reagent Amounts

[0273] 1. Main technical parameters of gas chromatography: same as in Example 1

[0274] 2. Determination method

[0275] (1) Solution preparation

[0276] Blank solution: Accurately measure 1 ml of acetonitrile and place it in an empty bottle. Take 5 parallel portions and accurately add 10 μl, 20 μl, 30 μl, 40 μl and 50 μl of acetic anhydride respectively. Cover, seal, mix well, let stand at room temperature for 30 minutes, and shake well.

[0277] Mixed stock solution: Accurately weigh 51.7 mg of acetylacetone reference substance, 52.2 mg of cyanoacetamide reference substance and 51.2 mg of morpholine reference substance, place them in the same 50 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and obtain.

[0278] Reference solution: Accurately measure 1 ml of the mixed stock solution into a 20 ml volumetric flask, dilute to the mark with acetonitrile, and shake well. Accurately measure 1 ml into an empty headspace vial. Take five parallel aliquots and accurately add 10 μl, 20 μl, 30 μl, 40 μl, and 50 μl of acetic anhydride, respectively. Cover, seal, mix, and let stand at room temperature for 30 minutes. Shake well.

[0279] Test sample spike solution: Accurately weigh 1002.8 mg of the test sample into a 20 ml volumetric flask. Accurately add 1 ml of the mixed stock solution above, dissolve with acetonitrile, dilute to the mark, and shake well. Accurately measure 1 ml and place in an empty headspace vial. Take five parallel aliquots and accurately add 10 μl, 20 μl, 30 μl, 40 μl, and 50 μl of acetic anhydride, respectively. Cover, seal, mix, and let stand at room temperature for 30 minutes. Shake well.

[0280] (2) Detection

[0281] Directly inject the blank solution, reference solution, and test sample spiked solution, and record the chromatograms. The results of the investigation of the amount of derivatization reagent added to the reference solution are shown in Table 14, and the results of the investigation of the amount of derivatization reagent added to the test sample spiked solution are shown in Table 15.

[0282] (3) Results

[0283] Table 14 The results of the investigation on the amount of derivatization reagent added to the reference solution of Example 8 are shown in Table

[0284]

[0285] Table 15 Experimental results of the amount of derivatization reagent added to the spiked solution of the test sample in Example 8

[0286]

[0287] (4) Conclusion

[0288] The above results show that the peak areas of acetylacetone, cyanoacetamide, and morpholine derivatives are essentially identical when acetic anhydride is added in amounts of 10 μl, 20 μl, 30 μl, 40 μl, and 50 μl to the reference and test solutions. The relative deviations of the peak areas of acetylacetone, cyanoacetamide, and morpholine derivatives at 20 μl, 30 μl, 40 μl, and 50 μl of acetic anhydride compared to those at 10 μl are all less than 5.0%, indicating that morpholine is essentially reacted at a 10 μl acetic anhydride addition. The peak areas of acetylacetone and cyanoacetamide are unaffected by the amount of acetic anhydride added. After comprehensive evaluation, the amount of acetic anhydride added was determined to be 20 μl or 40 μl (the amount added to the blank, reference, and test solutions was 1 ml or 2 ml), or equivalent.

[0289] Example 9: Simultaneous Detection of Three Related Substances in Opicapone Starting Material and Derivatization Temperature and Time Investigation Experiment

[0290] 1. Main technical parameters of gas chromatography: same as in Example 1

[0291] 2. Determination method

[0292] (1) Solution preparation

[0293] Blank solution: Accurately measure 2 ml of acetonitrile and place it in an empty bottle. Accurately add 40 μl of acetic anhydride, cover, seal, mix, let stand for several minutes at room temperature or in a water bath at 30°C or 40°C, and shake well.

[0294] Mixed stock solution: Accurately weigh 51.7 mg of acetylacetone reference substance, 52.2 mg of cyanoacetamide reference substance and 51.2 mg of morpholine reference substance, place them in the same 50 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and obtain.

[0295] Reference solution: Accurately measure 1 ml of the mixed stock solution into a 20 ml volumetric flask, dilute to the mark with acetonitrile, and shake well. Accurately measure 2 ml into an empty headspace vial, accurately add 40 μl of acetic anhydride, cap, seal, mix, and let stand for several minutes at room temperature or in a water bath at 30°C or 40°C, then shake well.

[0296] Test sample spike solution: Accurately weigh 1003.4 mg of the test sample into a 20 mL volumetric flask. Accurately add 1 mL of each of the above stock solutions, dissolve in acetonitrile, and dilute to the mark. Shake well. Accurately measure 2 mL and place into an empty headspace vial. Accurately add 40 μL of acetic anhydride. Cover, seal, and mix. Let stand for several minutes at room temperature or in a water bath at 30°C or 40°C. Shake well.

[0297] (2) Detection

[0298] Directly inject the blank solution, reference solution, and test sample spiked solution, and record the chromatograms. Derivatization temperature and time results for the reference solution are shown in Tables 16 and 17, and for the test sample spiked solution are shown in Tables 18 and 19.

[0299] (3) Results

[0300] Table 16 Experimental results of derivatization temperature (30°C) and time of reference solution in Example 9

[0301]

[0302] Table 17 Experimental results of derivatization temperature (40°C) and time of reference solution in Example 9

[0303]

[0304] Table 18 Experimental results of the temperature (30°C) and time of the spiked solution of the test sample in Example 9

[0305]

[0306] Table 19 Experimental results of the temperature (40°C) and time of the spiked solution of the test sample in Example 9

[0307]

[0308] (4) Conclusion

[0309] The relative deviations of the peak areas of acetylacetone, cyanoacetamide and morpholine derivatives of the reference solution and the test solution at 30°C for 0.5h and 1h and those at room temperature for 0h were less than 5.0%;

[0310] The relative deviations of the peak areas of acetylacetone, cyanoacetamide and morpholine derivatives of the reference solution and the test solution at 40°C for 0.5 h and at room temperature for 0 h were less than 5.0%;

[0311] This indicates that morpholine reacts rapidly with acetic anhydride in both the reference solution and the test solution, achieving complete reaction within a short time at room temperature. Furthermore, the peak areas of acetylacetone and cyanoacetamide were unaffected by the derivatization temperature and time. After comprehensive evaluation, to ensure complete reaction even in the lower winter temperatures, the derivatization temperature and time were set at 30°C in a water bath for 30 minutes.

[0312] Comparative Example 1: Separation of Morpholine and Acetylacetone Using Non-Polar DB-1 and Medium-Polar InertCap 624 Columns

[0313] 1. Main technical parameters of gas chromatography

[0314] (1) Gas chromatograph: Agilent 7890B+7697A; Detector: Flame ionization detector (FID);

[0315] Chromatographic column model: DB-1 (30m×0.530mm, 5.00μm); Carrier gas nitrogen flow rate: 1.5ml / min;

[0316] Injection method: headspace injection; headspace equilibrium temperature: 100°C, quantitative loop temperature: 110°C, transfer line temperature: 120°C;

[0317] Detector temperature: 250°C; Inlet temperature: 220°C; Split ratio: 5:1 or 10:1;

[0318] Headspace equilibration time: 30 min, cycle time: 40 min; Headspace shaking: 3;

[0319] Heating program 1: Initial temperature is 40°C, maintained for 10 minutes, then heated to 120°C at a rate of 8°C per minute, maintained for 2 minutes, then heated to 230°C at a rate of 25°C per minute, maintained for 4 minutes;

[0320] Heating program 2: Initial temperature is 40°C, maintain for 10 minutes, increase the temperature to 120°C at a rate of 8°C per minute, then increase the temperature to 160°C at a rate of 12°C per minute, and then increase the temperature to 230°C at a rate of 30°C per minute and maintain for 5 minutes;

[0321] Diluent: dimethyl sulfoxide.

[0322] (2) Gas chromatograph: Agilent 7890B+7697A; Detector: Flame ionization detector (FID);

[0323] Chromatographic column model: InertCap 624 (30 m × 0.530 mm, 3.00 μm); carrier gas nitrogen flow rate: 1.5 ml / min;

[0324] Injection method: headspace injection; headspace equilibrium temperature: 140°C, quantitative loop temperature: 150°C, transfer line temperature: 160°C;

[0325] Detector temperature: 250°C; Inlet temperature: 220°C; Split ratio: 5:1 or 10:1;

[0326] Headspace equilibration time: 30 min, cycle time: 40 min; Headspace shaking: 3;

[0327] Heating program: initial temperature is 40°C, maintain for 10 minutes, increase to 120°C at a rate of 8°C per minute, then increase to 160°C at a rate of 12°C per minute, then increase to 230°C at a rate of 30°C per minute, maintain for 5 minutes;

[0328] Diluent: dimethyl sulfoxide.

[0329] 2. Determination method

[0330] (1) Solution preparation

[0331] Blank solution: Accurately measure 5 ml of dimethyl sulfoxide, place it in a 20 ml headspace bottle, cover and seal.

[0332] Acetylacetone Fixing Solution: Accurately weigh 124.6 mg of acetylacetone into a 50 ml volumetric flask. Dissolve in dimethyl sulfoxide and dilute to volume. Shake well. Accurately measure 5 ml into a 20 ml headspace vial, cap, and seal.

[0333] Morpholine targeting solution: Accurately weigh 126.1 mg of morpholine into a 50 ml volumetric flask. Dissolve in dimethyl sulfoxide and dilute to volume. Shake well. Accurately measure 5 ml into a 20 ml headspace vial, cap, and seal.

[0334] Cyanoacetamide Fixing Solution: Accurately weigh 122.1 mg of cyanoacetamide into a 50 ml volumetric flask. Dissolve in dimethyl sulfoxide and dilute to volume. Shake well. Accurately measure 5 ml into a 20 ml headspace vial, cap, and seal.

[0335] (2) Detection

[0336] Take the blank solution and each positioning solution and inject them into the headspace, and record the chromatogram. Figures 20 to 23 .

[0337] (3) Results

[0338] Under the conditions of the non-polar DB-1 and moderately polar InertCap 624 columns, both acetylacetone and morpholine are significantly interfered with by the dimethyl sulfoxide blank solution and are prone to residue in the system. Cyanoacetamide, due to its high boiling point, has low sensitivity and no peak in the headspace. Furthermore, the separation between acetylacetone and morpholine is poor, requiring high column efficiency.

[0339] (4) Conclusion: The above results indicate that the non-polar DB-1 and medium-polarity InertCap 624 columns and the diluent dimethyl sulfoxide are not suitable for the detection of acetylacetone, morpholine, and cyanoacetamide using headspace injection.

[0340] Comparative Example 2: Investigating the feasibility of the three related substance methods using direct injection of bonded cross-linked amine chromatographic columns

[0341] 1. Main technical parameters of gas chromatography

[0342] Gas chromatograph: Agilent 7890B+7697A; detector: flame ionization detector (FID); chromatographic column model: Agilent CP-Volamine (30m×0.32mm); flow rate: 1.5ml / min;

[0343] Injection method: direct injection; Injection volume: 1µl;

[0344] Detector temperature: 300°C; Inlet temperature: 300°C; Split ratio: 5:1

[0345] Heating program: initial temperature is 100 °C, maintained for 8 minutes, heated to 200 °C at a rate of 8 °C per minute, then heated to 250 °C at a rate of 50 °C per minute, and maintained for 5 minutes.

[0346] Diluent: dimethyl sulfoxide.

[0347] 2. Determination method

[0348] (1) Solution preparation

[0349] Blank solution: dimethyl sulfoxide

[0350] Acetylacetone fixing solution: Accurately weigh 124.6 mg of acetylacetone, place it in a 50 ml volumetric flask, add dimethyl sulfoxide to dissolve and dilute to the scale, shake well, and obtain.

[0351] Morpholine positioning solution: Accurately weigh 126.1 mg of morpholine, place it in a 50 ml volumetric flask, add dimethyl sulfoxide to dissolve and dilute to the scale, shake well, and obtain.

[0352] Cyanoacetamide fixing solution: Accurately weigh 122.1 mg of cyanoacetamide, place it in a 50 ml volumetric flask, add dimethyl sulfoxide to dissolve and dilute to the scale, shake well, and obtain the solution.

[0353] (2) Detection

[0354] Take the blank solution and each positioning solution and inject them directly, and record the chromatogram. Typical chromatograms are shown in Figures 24 to 26 .

[0355] (3) Results

[0356] When dimethyl sulfoxide was used as the diluent for direct injection, the retention times of acetylacetone and morpholine were close to each other and both were interfered by the dimethyl sulfoxide blank solution. However, the peak shape of cyanoacetamide was good and was not interfered by the dimethyl sulfoxide blank solution.

[0357] (4) Conclusion: The above results show that direct injection using a bonded cross-linked amine column can detect cyanoacetamide, but using dimethyl sulfoxide as a diluent will interfere with the detection of acetylacetone and morpholine.

[0358] Comparative Example 3: Investigating the feasibility of three related substance methods using acetonitrile as a diluent and direct injection

[0359] 1. Main technical parameters of gas chromatography

[0360] Gas chromatograph: Agilent 7890B+7697A; detector: flame ionization detector (FID); chromatographic column model: Agilent CP-Volamine (30m×0.32mm); flow rate: 1.5ml / min or 1.2ml / min;

[0361] Injection method: direct injection; Injection volume: 1µl;

[0362] Detector temperature: 300°C; Inlet temperature: 300°C; Split ratio: 3:1 or 5:1

[0363] Heating program 1: Initial temperature is 100°C, maintained for 8 minutes, heated to 200°C at a rate of 5°C per minute, then heated to 250°C at a rate of 50°C per minute, and maintained for 5 minutes.

[0364] Heating program 2: Initial temperature is 100°C, maintained for 8 minutes, then heated to 160°C at a rate of 2.5°C per minute, then heated to 250°C at a rate of 50°C per minute, and maintained for 6 minutes.

[0365] Diluent: acetonitrile.

[0366] 2. Determination method

[0367] (1) Solution preparation

[0368] Blank solution: acetonitrile

[0369] Mix mother liquor: Accurately weigh 51.7 mg of acetylacetone, 51.2 mg of morpholine and 52.2 mg of cyanoacetamide, place them in the same 50 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and obtain.

[0370] Reference solution: Accurately measure 1 ml of the mixed mother solution, place it in a 10 ml volumetric flask, dilute to the mark with acetonitrile, and shake well.

[0371] Test solution: Accurately weigh 1000.3 mg of the test sample, place it in a 10 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and obtain the solution.

[0372] Test sample spiked solution: Accurately weigh 1004.2 mg of the test sample and place it in a 10 ml volumetric flask. Accurately add 1 ml of the mixed mother liquor, dissolve it in acetonitrile and dilute to the scale. Shake well to obtain the solution.

[0373] (2) Detection

[0374] Take the blank solution, reference solution, test solution and test solution spiked with the above solution and inject directly, and record the chromatogram. Typical chromatograms are shown in Figures 27 to 29 .

[0375] (3) Results

[0376] When acetonitrile was used as the diluent for direct injection, the peak shapes and sensitivity of acetylacetone, morpholine, and cyanoacetamide in the reference solution were good. However, the peak shape of acetylacetone in the test sample spiked solution deteriorated, resulting in no baseline separation between it and morpholine. The peak shape and sensitivity of cyanoacetamide were good.

[0377] (4) Conclusion: The above results indicate that the matrix effect of acetylacetone in the test sample will affect the accurate quantification of acetylacetone and reduce the separation between acetylacetone and morpholine, thus affecting the accurate quantification of morpholine.

[0378] Comparative Example 4: Using ammonia, triethylamine, dimethyl sulfoxide, phosphoric acid, and glacial acetic acid to investigate whether the matrix effect can be eliminated

[0379] 1. Main technical parameters of gas chromatography: Except for the temperature rising program, the rest are the same as those in Example 1.

[0380] Heating program 1: Initial temperature is 100°C, maintained for 8 minutes, heated to 160°C at a rate of 2.5°C per minute, then heated to 250°C at a rate of 50°C per minute, and maintained for 6 minutes.

[0381] Heating program 2: Initial temperature is 100°C, maintained for 8 minutes, heated to 160°C at a rate of 2.5°C per minute, then heated to 255°C at a rate of 50°C per minute, and maintained for 10 minutes.

[0382] Diluent: acetonitrile.

[0383] 2. Determination method

[0384] (1) Solution preparation

[0385] Blank solution: acetonitrile

[0386] Mix mother liquor: Accurately weigh 51.7 mg of acetylacetone, 51.2 mg of morpholine and 52.2 mg of cyanoacetamide, place them in the same 50 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and obtain.

[0387] Reference solution: Accurately measure 1 ml of the mixed mother solution and place it in a 10 ml volumetric flask. Dilute to the mark with acetonitrile and shake well. Accurately measure 2 ml of the reference solution and place it in a headspace vial. Accurately add 2 μl of glacial acetic acid, cover, seal, mix well, and let it stand in a water bath at 30°C for 30 min. Shake well.

[0388] Test solution: Accurately weigh 1000.3 mg of the test sample, place it in a 10 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and obtain the solution.

[0389] Test sample spike solution: Accurately weigh 1004.2 mg of the test sample and place it in a 10 ml volumetric flask. Accurately add 1 ml of the mixed mother liquor, dissolve it in acetonitrile and dilute to the mark, shake well, and obtain the solution. ① Accurately measure 1 ml of the test sample spike solution and place it in an empty bottle at the top. Accurately add 40 μl of ammonia water, cover, seal, mix well, and let it stand in a water bath at 30°C for 30 min, and shake well. ② Accurately measure 1 ml of the test sample spike solution and place it in an empty bottle at the top. Accurately add 80 μl of triethylamine, cover, seal, mix well, and let it stand in a water bath at 30°C for 30 min, and shake well. ③ Accurately measure 2 ml of the test sample spike solution and place it in an empty bottle at the top. Accurately add 5 μl of dimethyl sulfoxide, cover, seal, mix well, and let it stand in a water bath at 30°C for 30 min, and shake well. ④ Accurately measure 2 ml of the sample spiked solution, place it in an empty vial, accurately add 40 μl of dimethyl sulfoxide, cover, seal, mix, let it stand in a water bath at 30°C for 30 min, and shake well. ⑤ Accurately measure 2 ml of the sample spiked solution, place it in an empty vial, accurately add 10 μl of phosphoric acid, cover, seal, mix, let it stand in a water bath at 30°C for 30 min, and shake well. ⑥ Accurately measure 2 ml of the sample spiked solution, place it in an empty vial, accurately add 2 μl of glacial acetic acid, cover, seal, mix, let it stand in a water bath at 30°C for 30 min, and shake well. ⑦ Accurately measure 2 ml of the sample spiked solution, place it in an empty vial, accurately add 10 μl of glacial acetic acid, cover, seal, mix, let it stand in a water bath at 30°C for 30 min, and shake well.

[0390] (2) Detection

[0391] Take the blank solution, reference solution, test solution and test solution spiked with the above solution and inject directly, and record the chromatogram. Typical chromatograms are shown in Figures 30 to 36 .

[0392] (3) Results

[0393] Diluents containing 4% ammonia, 8% triethylamine, 0.25%–2% dimethyl sulfoxide, and 1% phosphoric acid failed to improve the peak shape of acetylacetone in the opicapone starting material or enhance the resolution between acetylacetone and morpholine, and all resulted in degradation of morpholine and cyanoacetamide. Although a diluent containing 0.1%–1% glacial acetic acid eliminated matrix effects in the opicapone starting material and improved the acetylacetone peak shape, it consumed some morpholine, causing instability and degradation, and also resulted in peak tailing.

[0394] (4) Conclusion: The above results indicate that ammonia, triethylamine, dimethyl sulfoxide, phosphoric acid, and glacial acetic acid are not feasible for eliminating the matrix effect of opicapone starting material on acetylacetone.

Claims

1. A method for simultaneously detecting three related substances in the starting material of opicapone, wherein: The chemical name of the opicapone starting material is 2,5-dichloro-4,6-dimethylnicotinonitrile; the three related substances are morpholine, acetylacetone and cyanoacetamide; the detection method comprises the following steps: (1) Prepare a mixed reference solution: weigh the morpholine reference, acetylacetone reference and cyanoacetamide reference, place them in the same volumetric flask, add acetonitrile to dissolve and dilute to prepare a mixed reference solution; alternatively, dissolve and dilute the morpholine reference, acetylacetone reference and cyanoacetamide reference in acetonitrile to obtain a morpholine reference solution, an acetylacetone reference solution and a cyanoacetamide reference solution, and mix them to prepare a mixed reference solution; accurately measure an appropriate amount of the mixed reference solution, place it in an empty bottle, accurately add an appropriate amount of acetic anhydride, cover, seal, mix, let stand in a water bath at 30°C for several minutes, and shake well; (2) Prepare the test solution: weigh the opicapone starting material to be tested, place it in a volumetric flask, add acetonitrile to dissolve and dilute to prepare the test solution; accurately measure an appropriate amount of the test solution, place it in an empty bottle, accurately add an appropriate amount of acetic anhydride, cover, seal, mix, let it stand in a water bath at 30°C for several minutes, and shake it evenly; (3) Take equal amounts of the mixed reference solution and test solution and inject them into the FID gas chromatograph for measurement; Among them, gas chromatography conditions include: Chromatographic column: capillary column with bonded cross-linked amine as stationary liquid Injection method: direct injection Detector: flame ionization detector (FID); Column temperature program: initial temperature is 100℃±5℃, maintain for 8-10 minutes, increase the temperature to 160℃ at a rate of 2.5℃ per minute, then increase the temperature to 255℃ at a rate of 50℃ per minute, maintain for 8-10 minutes; Carrier gas nitrogen flow rate: 1.1ml / min~1.3ml / min; Hydrogen flame ionization detector temperature: 295℃~305℃; Inlet temperature: 295℃~305℃; Split ratio: 0.5-5:1; Injection volume: 0.5μl~2μl.

2. The detection method according to claim 1, wherein: In the step (1), the concentrations of the mixed reference solution: morpholine, acetylacetone and cyanoacetamide are all 10 μg / ml to 125 μg / ml.

3. The detection method according to claim 2, wherein: In the step (1), the concentrations of the mixed reference solution: morpholine, acetylacetone and cyanoacetamide are all 50 μg / ml.

4. The detection method according to claim 1, wherein: In the step (1), the volume ratio of the mixed reference solution added to the headspace bottle to acetic anhydride is 20 to 100:

1.

5. The detection method according to claim 4, wherein: In the step (1), the volume ratio of the mixed reference solution added to the headspace bottle to acetic anhydride is 50:

1.

6. The detection method according to claim 5, characterized in that: The volume of the mixed reference solution was 2 ml, and the volume of acetic anhydride was 40 μl.

7. The detection method according to claim 1, wherein: In the step (2), the concentration of the test solution is 10 mg / ml to 125 mg / ml.

8. The detection method according to claim 7, wherein: In the step (2), the concentration of the test solution is 50 mg / ml.

9. The detection method according to claim 1, wherein: In the step (2), the volume ratio of the test solution added to the headspace bottle to acetic anhydride is 20 to 100:

1.

10. The detection method according to claim 9, characterized in that: In the step (2), the volume ratio of the test solution added to the headspace bottle to acetic anhydride is 50:

1.

11. The detection method according to claim 10, characterized in that: The volume of the test solution is 2 ml, and the volume of acetic anhydride is 40 μl.

12. The detection method according to any one of claims 1 to 11, characterized in that: In the steps (1) and (2), the standing time in the water bath at 30° C. is 30 minutes.

13. The detection method according to any one of claims 1 to 11, characterized in that: In the step (3), the gas chromatography conditions are: Carrier gas nitrogen flow rate: 1.2 ml / min; and / or Flame ionization detector temperature: 300°C; and / or Inlet temperature: 300°C; and / or Split ratio: 2:1; and / or Injection volume: 1 μl.

14. The detection method according to any one of claims 1 to 11, characterized in that: In the step (3), the capillary column with bonded cross-linked amines as the stationary phase is Agilent CP-Volamine 30m×0.32mm.

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