A method for detecting residual formic acid solvent in octopaine intermediate products

Abstract

This invention relates to a method for detecting residual formic acid in octopaine intermediates. The method involves first derivatizing the formic acid before a column, followed by detection using gas chromatography with a flame ionization detector. This method overcomes the difficulty of detecting formic acid in octopaine intermediates using ion chromatography or liquid chromatography due to their extremely poor solubility in common solvents. Furthermore, it prevents the degradation of octopaine intermediates into formic acid under high-temperature gas-phase conditions through structural modification. The substance preventing the degradation of octopaine intermediates into formic acid acts as a catalyst, catalyzing a faster and more complete reaction of formic acid and ensuring its complete conversion to ethyl formate. This improves detection efficiency while ensuring the reliability of the octopaine intermediate's quality. This method can quantitatively detect formic acid levels above 0.02% in octopaine intermediates, exhibiting high sensitivity, strong practicality, and a simple and rapid detection process.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analytical chemistry and relates to a method for detecting residual formic acid, a solvent in opipcapone intermediates. Specifically, it relates to a method for detecting residual formic acid, a solvent in opipcapone intermediates, using pre-column derivatized GC-FID headspace separation. Background Technology

[0002] Opicapon is an innovative third-generation peripherally selective catechol-O-methyltransferase (COMT) inhibitor. It was first approved in the European Union in 2016 and by the US FDA and Japan in 2020 for the treatment of Parkinson's disease. As a third-generation COMT inhibitor, compared to the previous two generations (such as entacapone and tocapone), opicapon has a higher enzyme binding affinity and only requires once-daily oral administration, significantly improving patient compliance. As adjunctive therapy to levodopa / carbidopa, opicapon can reduce the "wearing-off" and "on-off" phenomena caused by long-term medication, improve fluctuations in motor symptoms, and help patients better control their motor symptoms.

[0003] The chemical name of octopaine 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 structural formula is:

[0004]

[0005] The synthetic route of octopaine is shown below (octopaine process patent WO2013089573A1):

[0006]

[0007]

[0008] According to the above synthetic route, 2,5-dichloro-3-(5-(4-hydroxy-3-methoxy-5-nitrophenyl)-1,2,4-oxadiazol-3-yl)-4,6-dimethylpyridine-1-oxide is an important intermediate product of ocpicappone, namely chemical structure 7. Chemical structure 6 is reacted with urea peroxide, trifluoroacetic anhydride, and dichloromethane to produce crude chemical structure 7, which is then crystallized from toluene and formic acid to obtain chemical structure 7. This route inevitably contains residual formic acid as a solvent in the ocpicappone intermediate product. The amount of residual formic acid significantly affects subsequent synthetic processes; therefore, strict control of the residual formic acid content in the ocpicappone intermediate product is crucial for the synthesis of the ocpicappone active pharmaceutical ingredient. Based on the current edition of the Chinese Pharmacopoeia and ICH Q3C(R9), the limit of 5000 ppm for Class III residual formic acid is studied and controlled. Summary of the Invention

[0009] Currently, no reports have been found regarding the detection methods for residual formic acid in opipcapone intermediate products.

[0010] Chinese patent CN116500160A discloses a method for determining the content of formic acid and / or acetic acid in ceftazidime; CN112684074A discloses a method for determining the content of calcium formate; literature discloses the determination of formic acid content in secnidazole by HPLC (Du Daoming et al.); literature discloses the determination of formic acid in formate salts by high performance liquid chromatography (Wan Yu et al.); and literature discloses the examination of formic acid content in ticagrelor raw materials. All of these methods use high performance liquid chromatography to detect formic acid content.

[0011] The literature discloses the determination of formic acid content in industrial glacial acetic acid by gas chromatography and the determination of formic acid in cyclohexane by gas chromatography (Li Huawen et al.), both of which use a GC thermal conductivity detector for detection.

[0012] The National Standard of the People's Republic of China GB / T 17068-1997 describes the gas chromatography method for determining formic acid in workshop air. Literature also discloses the gas chromatography method for determining the content of formic acid residues in ceftazidime raw materials (Zhong Baoxin et al.) and the gas chromatography method for determining formic acid in air (Liang Lu et al.). All of these methods involve the esterification reaction of formic acid with ethanol using sulfuric acid as a catalyst to produce ethyl formate, followed by GC-FID headspace sampling for detection. None of these methods investigated the formic acid conversion rate.

[0013] Chinese patent CN117470989A discloses a method for detecting residual formic acid solvent in a pharmaceutical raw material. The method involves reacting formic acid with n-butanol using sulfuric acid as a catalyst to generate n-butyl formate, followed by detection using GC-FID headspace sampling. However, the method does not examine the formic acid conversion rate.

[0014] Because formic acid shows almost no response on a flame ionization detector (FID) under gas chromatography and low sensitivity on a thermal conductivity detector (TCD), and because ocpicarbon intermediates have extremely poor solubility in common solvents (such as water, methanol, ethanol, isopropanol, acetonitrile, tetrahydrofuran, toluene, ethylbenzene, etc.) under liquid chromatography or ion chromatography, it is difficult to detect formic acid content in ocpicarbon intermediates using ion chromatography, liquid chromatography, and gas chromatography. Based on existing derivative technologies, attempts have been made to use sulfuric acid as a catalyst to react with alcohols such as ethanol, isopropanol, or n-propanol under headspace heating to generate the corresponding ester compounds, followed by GC-FID detection. However, due to the extremely poor solubility of ocpicarbon intermediates in common solvents and their easy degradation to formic acid under acidic gas-phase heating, alcohols and water cannot be directly used as diluents for solution preparation. Instead, common solvents such as dimethyl sulfoxide (DMSO) are used. N,N -Dimethylformamide, N,NWhen dimethylacetamide is used as a diluent, it cannot guarantee the complete dissolution of a certain concentration of octopaine intermediate under heating conditions. Furthermore, due to the inherent limitations of their respective processes, they contain 10%–20% formic acid interference. N,N Heating dimethylformamide in the presence of sulfuric acid further hydrolyzes it to release a large amount of formic acid, thus hindering the detection method that relies on the esterification reaction of formic acid with alcohols to form ester compounds. Other methods considered included reacting potassium borohydride and zinc chloride with formic acid to produce methanol, and reacting thionyl chloride with formic acid to produce formyl chloride, which then reacts with ethanol to form ethyl formate. However, experimental results showed that neither method successfully derivatized the compounds.

[0015] Based on the chemical structural formula of the octopaine intermediate, the inventors deduced that its structure originates from the methoxy group, which degrades to formic acid under acidic gas-phase conditions. The carbon-oxygen bond in the octopaine intermediate breaks under high-temperature, strong acid conditions to form methanol. Since the octopaine intermediate itself contains an oxide structure, methanol is further oxidized to formaldehyde or formic acid under these conditions. Therefore, the inventors, in conjunction with the octopaine process route, used aluminum chloride (AlCl3), a strong Lewis acid, to bind with the oxygen atom in the methoxy group (O–CH3), leading to increased polarity and breakage of the methoxy bond. This removes the hydroxyl protecting group from the octopaine intermediate structure, thus preventing further degradation of the methoxy group under high-temperature, acidic conditions to formic acid, which would interfere with detection and accurate quantification. This solves the problem of detecting formic acid content in the octopaine intermediate. Aluminum chloride, the dehydroxyl protecting group reagent, can act as a catalyst to accelerate and complete the reaction between formic acid and ethanol, and also ensure the complete conversion of formic acid to ethyl formate (existing literature and patents have only examined the recovery rate of formic acid, without considering the conversion rate, which is a certain deficiency in the method). While improving detection efficiency, it also ensures the reliability and quality of the octopaine intermediate product. The diluent... N -Methylpyrrolidone can ensure the complete dissolution of a certain concentration of octopaine intermediate under gas-phase heating conditions, and it does not interfere with the detection of formic acid.

[0016] In this invention, the Chinese chemical name of the ocpicabon intermediate is: 2,5-dichloro-3-(5-(4-hydroxy-3-methoxy-5-nitrophenyl)-1,2,4-oxadiazol-3-yl)-4,6-dimethylpyridine-1-oxide, and its chemical structural formula is Chemical Structure 7:

[0017]

[0018] Formic acid, chemical structural formula is:

[0019]

[0020] In an embodiment of the present invention, the formic acid-ethanol derivatization reaction equation is as follows:

[0021]

[0022] In an embodiment of the present invention, the reaction equation for the removal of the hydroxyl protecting group from the octopaben intermediate product structure by the strong Lewis acid aluminum chloride (AlCl3) is as follows:

[0023]

[0024] This invention provides a method for detecting residual formic acid, a solvent, in octopaine intermediates. This method includes at least the following conditions:

[0025] (i) Prepare an aluminum chloride ethanol solution;

[0026] (ii) Formic acid reference solution or ethyl formate reference solution is derivatized with aluminum chloride ethanol solution and sulfuric acid;

[0027] (iii) The octopaine intermediate product test solution was derivatized with aluminum chloride ethanol solution and sulfuric acid;

[0028] (iv) Detection by gas chromatography.

[0029] According to the detection method of the present invention, the detection method includes the following steps:

[0030] (1) Preparation of aluminum chloride ethanol solution: Weigh 1g of aluminum chloride, dissolve and dilute to 10ml with anhydrous ethanol, and shake well;

[0031] (2) Preparation of blank solution: Accurately measure N- Place 2 ml of methylpyrrolidone in an empty bottle, accurately add an appropriate amount of aluminum chloride ethanol solution, shake gently to mix, then add an appropriate amount of sulfuric acid, cap, seal, and shake well.

[0032] (3) Preparation of reference solution: Weigh formic acid reference standard or ethyl formic acid reference standard, place in a volumetric flask, add... N- Dissolve and dilute methylpyrrolidone to prepare a reference solution; accurately measure 2 ml of the reference solution, place it in a headspace bottle, accurately add an appropriate amount of aluminum chloride ethanol solution, shake gently to mix, then add an appropriate amount of sulfuric acid, cap, seal, and shake well.

[0033] (4) Preparation of test solution: Weigh the sample of octopaine intermediate product to be tested, place it in a head bottle, and accurately add N- Dissolve 2 ml of methylpyrrolidone by gentle shaking, then accurately add an appropriate amount of aluminum chloride ethanol solution, gently shake to mix, then add an appropriate amount of sulfuric acid, cap, seal, and shake well.

[0034] (5) Take equal amounts of the blank solution, reference solution, and test solution, respectively, and inject them into the FID gas chromatograph for analysis. Record the chromatograms and calculate the formic acid content by peak area using the external standard method. The molecular weight of formic acid is 46.03, and the molecular weight of ethyl formate is 74.08. If the reference solution is ethyl formate, then the product factor 46.03 / 74.08 needs to be added to the external standard method formula.

[0035] According to the detection method of the present invention, the gas chromatography conditions include:

[0036] Chromatographic column: Capillary column with nitroterephthalic acid modified polyethylene glycol as the stationary phase;

[0037] Sample introduction method: headspace injection;

[0038] Headspace equilibration time: 30 min ± 2 min; Cycle time: 35 min ± 2 min;

[0039] Headspace equilibrium temperature: 70℃±2℃, metering loop temperature: 80℃±2℃, transmission line temperature: 90℃±2℃;

[0040] Headspace oscillation: 5±2;

[0041] Detector: Flame Ionization Detector (FID);

[0042] Column heating program: Initial temperature is 40℃±5℃, maintain for 7 to 10 minutes, increase to 120℃ at a rate of 10℃ per minute, then increase to 240℃ at a rate of 50℃ per minute, and maintain for 5 to 10 minutes.

[0043] Carrier gas nitrogen flow rate: 1.9 ml / min~2.1 ml / min;

[0044] Hydrogen flame ionization detector temperature: 245℃~255℃;

[0045] Inlet temperature: 215℃~225℃;

[0046] Split ratio: 1:1 to 20:1.

[0047] Furthermore, according to the detection method of the present invention, in step (3), the concentration of the reference solution is 10 μg / ml ~ 500 μg / ml (formic acid) or 16.1 μg / ml ~ 804.7 μg / ml (ethyl formate), preferably 100 μg / ml (formic acid) or 160.9 μg / ml (ethyl formate).

[0048] Furthermore, according to the detection method of the present invention, in steps (2), (3), and (4), the amount of aluminum chloride ethanol solution added is 200 μl to 500 μl, preferably 400 μl.

[0049] Furthermore, according to the detection method of the present invention, in steps (2), (3), and (4), the amount of sulfuric acid added is 50 μl to 100 μl, preferably 50 μl.

[0050] Furthermore, according to the detection method of the present invention, in step (4), the concentration of the test sample solution is 2 mg / ml to 40 mg / ml, preferably 20 mg / ml.

[0051] Furthermore, according to the detection method of the present invention, in step (5), the capillary column with nitro-terephthalic acid modified polyethylene glycol as the stationary phase is Perkin Elmer® Elite-FFAP (30m×0.53mm, 1μm).

[0052] Furthermore, according to the detection method of the present invention, in step (5), the gas chromatography conditions are as follows:

[0053] Carrier gas nitrogen flow rate: 2.0 ml / min; and / or

[0054] Hydrogen flame ionization detector temperature: 250℃; and / or

[0055] Inlet temperature: 220℃; and / or

[0056] shunt ratio: 10:1; and / or

[0057] Headspace equilibration time: 30 min, cycle time: 35 min; and / or

[0058] Headspace equilibrium temperature: 70℃, metering loop temperature: 80℃, transfer line temperature: 90℃; and / or

[0059] Head-on oscillation: 5.

[0060] This invention provides a method for detecting residual formic acid, a solvent in octopaine intermediates. By selecting specific chromatographic conditions, diluents, and dehydroxyl protecting reagents, the content of residual formic acid in octopaine intermediates can be separated and detected simply, quickly, and accurately, thereby ensuring the quality control of octopaine raw materials and providing guidance for the development of synthesis processes.

[0061] Obviously, based on the above description of the present invention, and in accordance with 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.

[0062] The following detailed description of specific embodiments further illustrates the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0063] Figure 1 The figure shows the chromatogram of blank solution from the specificity test of the method for detecting the content of residual formic acid in the octopaine intermediate product in Example 1.

[0064] Figure 2 This shows the chromatogram of the method validation specificity test for the detection of residual formic acid content in the octopaine intermediate product in Example 1 - system suitability solution / reference solution / formic acid localization solution.

[0065] Figure 3 This shows the specificity test of the method for detecting the content of residual formic acid in the octopaine intermediate product in Example 1 - chromatogram of ethyl formate localization solution.

[0066] Figure 4 This shows the chromatogram of the test solution in the method validation experiment for the determination of residual formic acid content in the octopaine intermediate product in Example 1.

[0067] Figure 5 The image shows the specificity test of the method for detecting the content of residual formic acid in the octopaine intermediate product in Example 1 - chromatogram of mixed solution.

[0068] Figure 6 This shows the chromatogram of the solution for the determination of residual solvent formic acid content in the octopaine intermediate product in Example 2, which is a validation experiment of the limit of quantitation and limit of detection.

[0069] Figure 7 This shows the chromatogram of the limit of quantitation and limit of detection experiment - limit of quantitation solution for the method validation of the detection method for the content of residual formic acid in the octopaine intermediate product in Example 2.

[0070] Figure 8 The figure shows the linearity and range experiment of the method for detecting the content of residual solvent formic acid in the octopaine intermediate product in Example 3 - C-250% linear solution chromatogram.

[0071] Figure 9The image shows the chromatogram of the solution with 100% recovery rate in the method validation experiment for the detection of residual formic acid content in the octopaine intermediate product in Example 4 - A-100% recovery rate.

[0072] Figure 10 The image shows the chromatogram of the reference solution from the conversion rate experiment of the method validation for the detection of residual formic acid content in the octopaine intermediate product in Example 6.

[0073] Figure 11 This shows the chromatogram of the test sample solution from the conversion rate experiment of the method validation for the detection of residual formic acid content in the octopaine intermediate product in Example 6.

[0074] Figure 12 The image shows the chromatogram of the ethyl formate reference solution (200 µl anhydrous ethanol + 50 µl sulfuric acid) from the experimental study on the addition amount of derivatized reagents in the octopaine intermediate product in Example 7.

[0075] Figure 13 The image shows the chromatogram of the formic acid reference solution (200 µl anhydrous ethanol + 50 µl sulfuric acid) from the experimental study on the addition amount of derivatized reagents in the octopaine intermediate product in Example 7.

[0076] Figure 14 This shows the chromatogram of the derivative reagent addition experiment for the determination of residual solvent formic acid content in octopaine intermediate product in Example 7 - ethyl formate reference solution (50µl of 100mg / ml aluminum chloride ethanol solution + 50µl of sulfuric acid).

[0077] Figure 15 This shows the chromatogram of the formic acid reference solution (50µl of 100mg / ml aluminum chloride ethanol solution + 50µl of sulfuric acid) from the experimental verification of the determination of residual solvent formic acid content in the octopaine intermediate product in Example 7.

[0078] Figure 16 This shows the chromatogram of the derivative reagent addition experiment for the determination of residual solvent formic acid content in octopaine intermediate product in Example 7 - ethyl formate reference solution (200µl of 100mg / ml aluminum chloride ethanol solution + 50µl of sulfuric acid).

[0079] Figure 17 This shows the chromatogram of the formic acid reference solution (200µl of 100mg / ml aluminum chloride ethanol solution + 50µl of sulfuric acid) from the experimental verification of the method for detecting the content of residual solvent formic acid in the octopaine intermediate product in Example 7.

[0080] Figure 18This shows the chromatogram of the test solution (200µl of 100mg / ml aluminum chloride ethanol solution + 50µl of sulfuric acid) from the experimental validation of the method for detecting the content of residual solvent formic acid in the octopaine intermediate product in Example 7.

[0081] Figure 19 This shows the chromatogram of the spiked solution of the test sample (200µl of 100mg / ml aluminum chloride ethanol solution + 50µl of sulfuric acid) from the experimental validation of the method for detecting the content of residual solvent formic acid in the octopaine intermediate product in Example 7.

[0082] Figure 20 This shows the chromatogram of the derivative reagent addition experiment for the determination of residual solvent formic acid content in octopaine intermediate product in Example 7 - ethyl formate reference solution (300µl of 100mg / ml aluminum chloride ethanol solution + 50µl of sulfuric acid).

[0083] Figure 21 This shows the chromatogram of the formic acid reference solution (300µl of 100mg / ml aluminum chloride ethanol solution + 50µl of sulfuric acid) from the experimental verification of the determination of residual solvent formic acid content in the octopaine intermediate product in Example 7.

[0084] Figure 22 This shows the chromatogram of the test solution (300µl of 100mg / ml aluminum chloride ethanol solution + 50µl of sulfuric acid) from the experimental validation of the method for detecting the content of residual solvent formic acid in the octopaine intermediate product in Example 7.

[0085] Figure 23 This shows the chromatogram of the spiked solution of the test sample (300µl of 100mg / ml aluminum chloride ethanol solution + 50µl of sulfuric acid) from the experimental validation of the method for detecting the content of residual solvent formic acid in the octopaine intermediate product in Example 7.

[0086] Figure 24 This shows the chromatogram of the formic acid reference solution (500µl of 100mg / ml aluminum chloride ethanol solution + 0µl of sulfuric acid) from the experimental validation of the method for detecting the content of residual solvent formic acid in the octopaine intermediate product in Example 7.

[0087] Figure 25 This shows the chromatogram of the derivative reagent addition experiment for the determination of residual solvent formic acid content in octopaine intermediate product in Example 7 - ethyl formate reference solution (200µl of 100mg / ml aluminum chloride ethanol solution + 100µl of sulfuric acid).

[0088] Figure 26This shows the chromatogram of the formic acid reference solution (200µl of 100mg / ml aluminum chloride ethanol solution + 100µl of sulfuric acid) from the experimental verification of the method for detecting the content of residual solvent formic acid in the octopaine intermediate product in Example 7.

[0089] Figure 27 The figure shows the chromatogram of blank solution ① of the experimental test for the easy degradation of formic acid in octopaine intermediate product under acidic conditions when aluminum chloride was not added in Comparative Example 1.

[0090] Figure 28 The figure shows the chromatogram of the control solution ① of the experimental test for the easy degradation of formic acid in the octopaine intermediate product under acidic conditions when aluminum chloride was not added in Comparative Example 1.

[0091] Figure 29 The figure shows the chromatogram of the test sample solution ① of the experimental test for the easy degradation of formic acid in octopaine intermediate product under acidic conditions when aluminum chloride was not added in Comparative Example 1.

[0092] Figure 30 This represents the chromatogram of the spiked solution ① of the test sample in Comparative Example 1 when aluminum chloride was not added, showing that the octopaine intermediate product was easily degraded into formic acid under acidic conditions.

[0093] Figure 31 This represents the chromatogram of blank solution ② in the test for the degradation of octopcapone intermediate into formic acid under acidic conditions, without the addition of aluminum chloride in Comparative Example 1. (100 μl anhydrous ethanol + 100 μl sulfuric acid)

[0094] Figure 32 This shows the chromatogram of the control solution ② in the test of the easy degradation of octopaine intermediate to formic acid under acidic conditions in Comparative Example 1 without the addition of aluminum chloride. (100 μl anhydrous ethanol + 100 μl sulfuric acid)

[0095] Figure 33 This represents the chromatogram of test solution ② (100 μl anhydrous ethanol + 100 μl sulfuric acid) in Comparative Example 1 without the addition of aluminum chloride, illustrating the degradation of octopaine intermediate into formic acid under acidic conditions.

[0096] Figure 34 This represents the chromatogram of the spiked solution ② of the octopcapone intermediate product under acidic conditions, as tested in Comparative Example 1 without the addition of aluminum chloride, demonstrating its tendency to degrade into formic acid. (100 μl anhydrous ethanol + 100 μl sulfuric acid)

[0097] Figure 35 This shows the chromatogram of blank solution ① in Comparative Example 2, using isopropanol and n-propanol as derivatizing reagents and sulfuric acid catalysis. (20 μl isopropanol + 20 μl sulfuric acid)

[0098] Figure 36 This shows the chromatogram of Comparative Example 2, using isopropanol and n-propanol as derivatizing reagents and sulfuric acid catalysis – reference solution ①. (20 μl isopropanol + 20 μl sulfuric acid)

[0099] Figure 37 This shows the chromatogram of the blank solution ② in Comparative Example 2, using isopropanol and n-propanol as derivatizing reagents and sulfuric acid catalysis. (20 μl n-propanol + 20 μl sulfuric acid)

[0100] Figure 38 This shows the chromatogram of Comparative Example 2, using isopropanol and n-propanol as derivatizing reagents and sulfuric acid catalysis – reference solution ②. (20 μl n-propanol + 20 μl sulfuric acid) Detailed Implementation

[0101] The present invention will be further illustrated by the following examples, but it should be understood that the following examples are not limited to the scope of the present invention. The reagents used in the following examples are all commercially available.

[0102] (1) Information on reagents, samples and reference standards

[0103] Sample of octopaine intermediate product (Chongqing Liujiang Pharmaceutical Technology Co., Ltd., batch number: C04-E-25031701);

[0104] Sample of octopaine intermediate product (Chongqing Liujiang Pharmaceutical Technology Co., Ltd., batch number: C04-E-25031401B);

[0105] Anhydrous formic acid (Shanghai Maclean Biochemical Technology Co., Ltd., analytical grade, batch number: C15637549, purity: 98%).

[0106] Ethyl formate (Shanghai Aladdin Biochemical Technology Co., Ltd., anhydrous grade, batch number: E1620063, content: 98%)

[0107] Aluminum chloride (Shanghai Maclean Biochemical Technology Co., Ltd., analytical grade, batch number: C14382379, purity: 99%).

[0108] Anhydrous ethanol (Chengdu Nuoershi Technology Co., Ltd., chromatographic grade, batch number: 2023042001, content: ≥99.9%).

[0109] Acetonitrile (Shenzhen Arthur Biotechnology Co., Ltd., chromatographic grade, batch number: A0131250110D, content: ≥99.9%).

[0110] Toluene (Chongqing Chuandong Chemical (Group) Co., Ltd., chromatographic grade, batch number: 20241201, content: ≥99.8%)

[0111] Ethylbenzene (Shanghai Aladdin Biochemical Technology Co., Ltd., spectral grade, batch number: G20R172, content: 99%)

[0112] Sulfuric acid (Chengdu Kelong Chemical Co., Ltd., analytical grade, batch number: 2022050602, content: 95.0-98.0%).

[0113] Dimethyl sulfoxide (Norsch, chromatographic grade, batch number: 2024061101, ≥99.7%).

[0114] N -Methylpyrrolidone (Shanghai Aladdin Biochemical Technology Co., Ltd., GC, batch number: H2315034, content: ≥99.5%)

[0115] Trifluoroacetic acid (Shanghai Aladdin Biochemical Technology Co., Ltd., analytical grade, batch number: B2309632, content: 99.5%).

[0116] Isopropanol (Shenzhen Arthur Biotechnology Co., Ltd., chromatographic grade, batch number: A0530241201D, content: ≥99.9%).

[0117] n-Propanol (GENERAL-REAGENT, analytical grade, batch number: P2672678, purity: ≥99.5%)

[0118] Dichloromethane (Honeywell, GC, lot number: EE763-US, ≥99.9%).

[0119] Potassium borohydride (Comio Reagent, analytical grade, batch number: 20230316, ≥97.0%).

[0120] Zinc chloride (Da Mao Chemical Reagent Factory, analytical grade, batch number: 20190815, content: ≥98.0%).

[0121] Pyridine (Chongqing Chuandong Chemical (Group) Co., Ltd., analytical grade, batch number: 20160601, purity: 99.5%)

[0122] Tetrahydrofuran (Honeywell, GC, lot number: W1FM1H, 99.981%).

[0123] Thionyl chloride (Chongqing Chuandong Chemical (Group) Co., Ltd., analytical grade, batch number: 20220602, content: ≥99.0%).

[0124] (2) Main instruments

[0125] Gas chromatograph: Agilent 7890B+7697A;

[0126] Electronic balance: XSE105DU;

[0127] Water bath constant temperature oscillator: SHZ-B.

[0128] (3) The detection method refers to gas chromatography (General Chapter 0521, Part IV, Chinese Pharmacopoeia 2020 Edition).

[0129] Example 1

[0130] Validation of the method for detecting residual formic acid content in octopaine intermediate products: specificity and system suitability experiments.

[0131] 1. Main technical parameters of gas chromatography

[0132] Gas chromatograph: Agilent 7890B+7697A; Detector: Flame ionization detector (FID);

[0133] Column type: Perkin Elmer® Elite-FFAP (30m × 0.53mm, 1μm); Split ratio: 10:1;

[0134] Sample introduction method: headspace injection; headspace shaking: 5; headspace equilibration time: 30 min, circulation time: 35 min;

[0135] Headspace equilibrium temperature: 70℃, metering loop temperature: 80℃, transfer line temperature: 90℃;

[0136] Carrier gas nitrogen flow rate: 2.0 ml / min; Inlet temperature: 220℃; Detector temperature: 250℃;

[0137] Heating program: Initial temperature is 40℃, maintain for 10 minutes, increase to 120℃ at a rate of 10℃ per minute, then increase to 240℃ at a rate of 50℃ per minute, and maintain for 5 minutes.

[0138] Diluent: N -Methylpyrrolidone.

[0139] 2. Determination method

[0140] (1) Solution preparation

[0141] Aluminum chloride ethanol solution: Accurately weigh 5.0079 g of aluminum chloride, place it in a 50 ml volumetric flask, add anhydrous ethanol to dissolve and dilute to the mark, and shake well.

[0142] Blank solution: Accurately measure N- Place 2 ml of methylpyrrolidone into a 20 ml headspace vial, accurately add 400 μl of aluminum chloride ethanol solution, gently shake to mix, then add 50 μl of sulfuric acid, cap, seal, and shake well.

[0143] System suitability solution ① / Reference solution / Formic acid derivative positioning solution: Accurately weigh 253.0 mg of formic acid reference standard, place it in a 50 ml volumetric flask, and add... N- Dissolve and dilute methylpyrrolidone to the mark, shake well, and prepare formic acid stock solution; accurately measure 1 ml of formic acid stock solution, place it in a 50 ml volumetric flask, and use... N- Dilute methylpyrrolidone to the mark and shake well; accurately measure 2 ml and place it in a 20 ml headspace vial, accurately add 400 μl of aluminum chloride ethanol solution, shake gently to mix, then add 50 μl of sulfuric acid, cap, seal, and shake well.

[0144] System suitability solution ② / ethyl formate positioning solution: Accurately weigh 400.1 mg of ethyl formate reference standard, place it in a 50 ml volumetric flask, and add... N- Dissolve and dilute methylpyrrolidone to the mark, shake well, and prepare ethyl formate stock solution; accurately measure 1 ml of ethyl formate stock solution, place it in a 50 ml volumetric flask, and use... N- Dilute methylpyrrolidone to the mark and shake well; accurately measure 2 ml and place it in a 20 ml headspace vial, accurately add 400 μl of aluminum chloride ethanol solution, shake gently to mix, then add 50 μl of sulfuric acid, cap, seal, and shake well.

[0145] Trifluoroacetic acid positioning solution: Accurately weigh 84.8 mg of trifluoroacetic acid, place it in a 50 ml volumetric flask, and add... N- Dissolve and dilute methylpyrrolidone to the mark, shake well, accurately measure 2 ml, place in a 20 ml headspace vial, accurately add 400 μl of aluminum chloride ethanol solution, shake gently to mix, then add 50 μl of sulfuric acid, cap, seal, and shake well.

[0146] Acetonitrile positioning solution: Accurately weigh 20.3 mg of acetonitrile, place it in a 50 ml volumetric flask, and add... N- Dissolve and dilute methylpyrrolidone to the mark, shake well, accurately measure 2 ml, place in a 20 ml headspace vial, accurately add 400 μl of aluminum chloride ethanol solution, shake gently to mix, then add 50 μl of sulfuric acid, cap, seal, and shake well.

[0147] Toluene positioning solution: Accurately weigh 46.3 mg of toluene and place it in a 50 ml volumetric flask. Add... N- Dissolve and dilute methylpyrrolidone to the mark, shake well, accurately measure 2 ml, place in a 20 ml headspace vial, accurately add 400 μl of aluminum chloride ethanol solution, shake gently to mix, then add 50 μl of sulfuric acid, cap, seal, and shake well.

[0148] Dichloromethane positioning solution: Accurately weigh 32.2 mg of dichloromethane and place it in a 50 ml volumetric flask. Add... N-Dissolve and dilute methylpyrrolidone to the mark, shake well, accurately measure 2 ml, place in a 20 ml headspace vial, accurately add 400 μl of aluminum chloride ethanol solution, shake gently to mix, then add 50 μl of sulfuric acid, cap, seal, and shake well.

[0149] Mixed control solution: Accurately measure 1 ml of the above formic acid stock solution and 1 ml each of trifluoroacetic acid positioning solution, dichloromethane positioning solution, acetonitrile positioning solution, and toluene positioning solution, and place them in the same 50 ml volumetric flask. N- Dilute methylpyrrolidone to the mark and shake well.

[0150] Mixed solution: Accurately weigh 41.0 mg of the test sample (batch number: C04-E-25031401B), place it in a 20 ml headspace vial, accurately add 2 ml of mixed control solution, shake gently to dissolve, then accurately add 400 μl of aluminum chloride ethanol solution, shake gently to mix, then add 50 μl of sulfuric acid, cap, seal, and shake well.

[0151] Test solution: Accurately weigh 39.8 mg of the test sample (batch number: C04-E-25031401B), place it in a 20 ml headspace vial, and accurately add... N- Dissolve 2 ml of methylpyrrolidone by gentle shaking, then precisely add 400 μl of aluminum chloride ethanol solution, gently shake to mix, then add 50 μl of sulfuric acid, cap, seal, and shake well.

[0152] (2) Detection

[0153] Inject the blank solution, each positioning solution, system suitability solution / reference solution / formic acid derivative positioning solution, test solution, and mixed solution into the gas chromatogram headspace and record the chromatograms. A typical chromatogram is shown below. Figures 1-5 The results for each solution are shown in Table 1 and Table 2.

[0154] (3) Results

[0155] Depend on Figure 1 It can be seen that the blank solution does not interfere with the detection of formic acid derivatives;

[0156] Depend on Figure 2 It can be seen that the formic acid derivative eluted at around 6.6 min, with no interfering peaks nearby; from Figure 3 It can be seen that ethyl formate eluted at around 6.6 min, with no interfering peaks nearby; the retention times of the formic acid derivative peaks were consistent with those of the ethyl formate peak.

[0157] Depend on Figure 4 It can be seen that there are no interfering peaks near the elution peak of the formic acid derivative in the test solution;

[0158] Depend on Figure 5 It can be seen that there are no interfering peaks near the elution peak of the formic acid derivative in the mixed solution.

[0159] Table 1. Results of Specificity Experiment in Example 1

[0160]

[0161] Table 2 Results of System Applicability Test in Example 1

[0162]

[0163] (4) Conclusion

[0164] Neither the blank solution nor the test solution interfered with the detection of formic acid derivatives; the minimum resolution between the main peak and each impurity peak in the mixed solution and the formic acid derivative peak was 9.80, which is greater than 1.5. This indicates that the method has good specificity.

[0165] The system suitability of solution ① was good. After six consecutive injections, the retention time RSD was 0.04%, which is less than 1.0%, and the peak area RSD was 3.05%, which is less than 10.0%. The system suitability of solution ② was good. After six consecutive injections, the retention time RSD was 0.03%, which is less than 1.0%, and the peak area RSD was 3.74%, which is less than 10.0%. This indicates that the system suitability of this method is good. Example 2

[0166] Method validation experiments for the determination of residual formic acid content in octopaine intermediate products: Limit of quantitation and limit of detection.

[0167] 1. Main technical parameters of gas chromatography: same as in Example 1.

[0168] 2. Determination method

[0169] (1) Solution preparation

[0170] Aluminum chloride ethanol solution: Accurately weigh 5.0079 g of aluminum chloride, place it in a 50 ml volumetric flask, add anhydrous ethanol to dissolve and dilute to the mark, and shake well.

[0171] Blank solution: Accurately measure N- Place 2 ml of methylpyrrolidone into a 20 ml headspace vial, accurately add 400 μl of aluminum chloride ethanol solution, gently shake to mix, then add 50 μl of sulfuric acid, cap, seal, and shake well.

[0172] Formic acid stock solution: Accurately weigh 253.0 mg of formic acid reference standard, place it in a 50 ml volumetric flask, and add... N- Dissolve and dilute methylpyrrolidone to the mark, then shake well.

[0173] Limit of Quantitation Solution: Accurately measure 0.2 ml of formic acid stock solution and place it in a 50 ml volumetric flask. N-Dilute methylpyrrolidone to the mark and shake well; accurately measure 2 ml and place it in a 20 ml headspace vial, accurately add 400 μl of aluminum chloride ethanol solution, shake gently to mix, then add 50 μl of sulfuric acid, cap, seal, and shake well.

[0174] Detection limit solution: Accurately measure 0.1 ml of formic acid stock solution and place it in a 50 ml volumetric flask. N- Dilute methylpyrrolidone to the mark and shake well; accurately measure 2 ml and place it in a 20 ml headspace vial, accurately add 400 μl of aluminum chloride ethanol solution, shake gently to mix, then add 50 μl of sulfuric acid, cap, seal, and shake well.

[0175] (2) Detection

[0176] Inject the blank solution, limit of quantitation solution, and limit of detection solution into the headspace gas chromatogram and record the chromatograms. A typical chromatogram is shown below. Figures 6-7 The results of the detection limit and quantitation limit are shown in Table 3.

[0177] (3) Results

[0178] Depend on Figure 6 and Figure 7 It can be seen that this method has high detection sensitivity and can quantitatively detect formic acid content higher than 0.02% in octopaine intermediate products.

[0179] Table 3. Results of the detection limit and quantitation limit experiments in Example 2

[0180]

[0181] (4) Conclusion

[0182] The limit of quantitation (LOQ) solution concentration of the formic acid derivative was 19.835 μg / ml, equivalent to 19.8% of the limit concentration and 0.099% of the sample concentration, with a signal-to-noise ratio (S / N) of 96.6, which is greater than 10; the limit of detection (LOD) concentration was 9.918 μg / ml, equivalent to 9.9% of the limit concentration and 0.050% of the sample concentration, with a S / N of 52.4, which is greater than 3.

[0183] This indicates that the method has good sensitivity. Example 3

[0184] Method validation of residual formic acid content detection in octopaine intermediate products: linearity and range experiments

[0185] 1. Main technical parameters of gas chromatography: same as in Example 1.

[0186] 2. Determination method

[0187] (1) Solution preparation

[0188] Aluminum chloride ethanol solution: Accurately weigh 5.0079 g of aluminum chloride, place it in a 50 ml volumetric flask, add anhydrous ethanol to dissolve and dilute to the mark, and shake well.

[0189] Blank solution: Accurately measure N- Place 2 ml of methylpyrrolidone into a 20 ml headspace vial, accurately add 400 μl of aluminum chloride ethanol solution, gently shake to mix, then add 50 μl of sulfuric acid, cap, seal, and shake well.

[0190] Linear stock solution: Accurately weigh 253.0 mg of formic acid reference standard, place it in a 50 ml volumetric flask, and add... N- Dissolve and dilute methylpyrrolidone to the mark, then shake well.

[0191] Linear solution: Accurately measure the linear mother liquor and prepare it with acetonitrile according to the table below.

[0192]

[0193] C-LOQ solution: Take the "Limit of Quantitation Solution" under the "Limit of Quantitation and Limit of Detection" section.

[0194] For each linear solution: accurately measure 2 ml of each linear solution and place them in different headspace vials. Accurately add 400 μl of aluminum chloride ethanol solution to each vial, shake gently to mix, then add 50 μl of sulfuric acid, cap, seal, and shake well.

[0195] (2) Detection

[0196] The blank solution and each linear solution were subjected to headspace gas chromatography (HGC), and the chromatograms were recorded. Typical chromatograms are shown below. Figure 8 The linearity and range experimental results of formic acid derivatives are shown in Table 4.

[0197] (3) Results

[0198] Table 4. Results of Linearity and Range Experiments in Example 3

[0199]

[0200] (4) Conclusion

[0201] Within the concentration range of 19.835 μg / ml to 495.880 μg / ml, the linear regression equation between the concentration of formic acid derivative and the peak area was y = 1.7374x + 12.575, with a correlation coefficient r of 0.9993, which is greater than 0.995; the Y-intercept accounted for 6.7% of the 100% response value, which is less than 25%.

[0202] The results indicate that the formic acid derivative exhibits good linearity within the concentration range of 19.835 μg / ml to 495.880 μg / ml. Example 4

[0203] Accuracy test of the methodology for detecting residual formic acid content in octopaine intermediate products

[0204] 1. Main technical parameters of gas chromatography: Same as in Example 1

[0205] 2. Determination method

[0206] (1) Solution preparation

[0207] Aluminum chloride ethanol solution: Accurately weigh 5.0079 g of aluminum chloride, place it in a 50 ml volumetric flask, add anhydrous ethanol to dissolve and dilute to the mark, and shake well.

[0208] Blank solution: Accurately measure N- Place 2 ml of methylpyrrolidone into a 20 ml headspace vial, accurately add 400 μl of aluminum chloride ethanol solution, gently shake to mix, then add 50 μl of sulfuric acid, cap, seal, and shake well.

[0209] Formic acid reference solution: Accurately weigh 253.0 mg of formic acid reference standard, place it in a 50 ml volumetric flask, and add... N- Dissolve and dilute methylpyrrolidone to the mark, shake well, accurately measure 1 ml, and place in a 50 ml volumetric flask. N- Dilute methylpyrrolidone to the mark and shake well; accurately measure 2 ml and place it in a 20 ml headspace vial, accurately add 400 μl of aluminum chloride ethanol solution, shake gently to mix, then add 50 μl of sulfuric acid, cap, seal, and shake well.

[0210] Ethyl formate reference solution: Accurately weigh 400.1 mg of ethyl formate reference standard, place it in a 50 ml volumetric flask, and add... N- Dissolve and dilute methylpyrrolidone to the mark, shake well, accurately measure 1 ml, and place in a 50 ml volumetric flask. N- Dilute methylpyrrolidone to the mark and shake well; accurately measure 2 ml and place it in a 20 ml headspace vial, accurately add 400 μl of aluminum chloride ethanol solution, shake gently to mix, then add 50 μl of sulfuric acid, cap, seal, and shake well.

[0211] Background solution ①: Accurately weigh 39.8 mg of the test sample (batch number: C04-E-25031701), place it in a 20 ml headspace vial, and accurately add... N- Dissolve 2 ml of methylpyrrolidone by gentle shaking, then precisely add 400 μl of aluminum chloride ethanol solution, gently shake to mix, then add 50 μl of sulfuric acid, cap, seal, and shake well.

[0212] A-20% recovery solution: Accurately weigh 40.7 mg of the test sample (batch number: C04-E-25031701), place it in a 20 ml headspace vial, accurately add 2 ml of "C-20% solution" under "Example 3: Linearity and range experiment of the method for detecting residual solvent formic acid content in octopaine intermediate product", shake gently to dissolve, then accurately add 400 μl of aluminum chloride ethanol solution, shake gently to mix, then add 50 μl of sulfuric acid, cap, seal, and shake well.

[0213] A-50% recovery solution: Accurately weigh 39.6 mg of the test sample (batch number: C04-E-25031701), place it in a 20 ml headspace vial, accurately add 2 ml of "C-50% solution" under "Example 3: Linearity and range experiment of the method for detecting residual solvent formic acid content in octopaine intermediate product", shake gently to dissolve, then accurately add 400 μl of aluminum chloride ethanol solution, shake gently to mix, then add 50 μl of sulfuric acid, cap, seal, and shake well.

[0214] Background solution ②: Accurately weigh 43.8 mg of the test sample (batch number: C04-E-25031401B), place it in a 20 ml headspace vial, and accurately add... N- Dissolve 2 ml of methylpyrrolidone by gentle shaking, then precisely add 400 μl of aluminum chloride ethanol solution, gently shake to mix, then add 50 μl of sulfuric acid, cap, seal, and shake well.

[0215] A-100% recovery solution: Accurately weigh 39.6 mg of the test sample (batch number: C04-E-25031401B), place it in a 20 ml headspace vial, accurately add 2 ml of "C-100% solution" under "Example 3: Linearity and range experiment of the method for detecting residual solvent formic acid content in octopaine intermediate product", shake gently to dissolve, then accurately add 400 μl of aluminum chloride ethanol solution, shake gently to mix, then add 50 μl of sulfuric acid, cap, seal, and shake well.

[0216] A-150% recovery solution: Accurately weigh 38.8 mg of the test sample (batch number: C04-E-25031401B), place it in a 20 ml headspace vial, accurately add 2 ml of "C-150% solution" under "Example 3: Linearity and range experiment of the method for detecting residual solvent formic acid content in octopaine intermediate product", shake gently to dissolve, then accurately add 400 μl of aluminum chloride ethanol solution, shake gently to mix, then add 50 μl of sulfuric acid, cap, seal, and shake well.

[0217] A-250% recovery solution: Accurately weigh 41.6 mg of the test sample (batch number: C04-E-25031401B), place it in a 20 ml headspace vial, accurately add 2 ml of "C-250% solution" under "Example 3: Linearity and range experiment of the method for detecting residual solvent formic acid content in octopaine intermediate product", shake gently to dissolve, then accurately add 400 μl of aluminum chloride ethanol solution, shake gently to mix, then add 50 μl of sulfuric acid, cap, seal, and shake well.

[0218] A-500% recovery solution: Accurately weigh 38.7 mg of the test sample (batch number: C04-E-25031401B), place it in a 20 ml headspace vial, accurately add 2 ml of "C-500% solution" under "Example 3: Linearity and range experiment of the method for detecting residual solvent formic acid content in octopaine intermediate product", shake gently to dissolve, then accurately add 400 μl of aluminum chloride ethanol solution, shake gently to mix, then add 50 μl of sulfuric acid, cap, seal, and shake well.

[0219] (2) Detection

[0220] The blank solution, formic acid reference solution, ethyl formic acid reference solution, background solution, and solutions with various recovery rates were injected into the gas chromatogram headspace, and the chromatograms were recorded. A typical chromatogram is shown below. Figure 9 The accuracy results of recovery calculations using formic acid as a reference standard are shown in Table 5, and the accuracy results of recovery calculations using ethyl formate as a reference standard are shown in Table 6.

[0221] (3) Results

[0222] Table 5. Accuracy test results of using formic acid as a reference standard in Example 4.

[0223]

[0224] Table 6. Accuracy test results of using ethyl formate as a reference standard in Example 4.

[0225]

[0226] (4) Conclusion

[0227] In the accuracy experiment: using formic acid as a reference, the recoveries of A-20%, A-50%, A-100%, and A-150% were calculated in the range of 84.3% to 107.3%, with an RSD of 10.0% for each recovery rate of A-50%, A-100%, A-150%, A-250%, and A-500%. Using ethyl formate as a reference, the recoveries of A-20%, A-50%, A-100%, and A-150% were calculated in the range of 84.9% to 108.1%, with an RSD of 10.0% for each recovery rate of A-50%, A-100%, A-150%, A-250%, and A-500%.

[0228] The above recovery rates all meet the requirements of the analytical method validation guidelines in Section 9101 of the General Chapter of the 2020 edition of the Chinese Pharmacopoeia and the range of 80% to 120% specified in ICH; the accuracy of this method is good.

[0229] Note: The residual formic acid content in octopaine intermediate product C04-E-25031401B is 0.65%, which is relatively high (exceeding the limit of 0.5%). Therefore, using it as the background for calculating the recovery rate of A-100% solution has a significant impact, resulting in a lower result. In contrast, the residual formic acid content in batch C04-E-25031701 is 0.10%, which is relatively low. Therefore, using it as the background for calculating the recovery rate of A-20% and A-50% solutions both meet the range of 90% to 108%. After comprehensive evaluation, octopaine intermediate products have no matrix effect on formic acid and have good accuracy. Example 5

[0230] Method validation for the determination of residual formic acid content in octopaine intermediate products: solution stability test

[0231] 1. Main technical parameters of gas chromatography: Same as in Example 1

[0232] 2. Determination method

[0233] (1) Solution preparation

[0234] Aluminum chloride ethanol solution: Accurately weigh 5.0079 g of aluminum chloride, place it in a 50 ml volumetric flask, add anhydrous ethanol to dissolve and dilute to the mark, and shake well.

[0235] Blank solution: Accurately measure N- Place 2 ml of methylpyrrolidone into a 20 ml headspace vial, accurately add 400 μl of aluminum chloride ethanol solution, gently shake to mix, then add 50 μl of sulfuric acid, cap, seal, and shake well.

[0236] Formic acid reference solution: Accurately weigh 253.0 mg of formic acid reference standard, place it in a 50 ml volumetric flask, and add... N-Dissolve and dilute methylpyrrolidone to the mark, shake well, accurately measure 1 ml, and place in a 50 ml volumetric flask. N- Dilute methylpyrrolidone to the mark and shake well; accurately measure 2 ml and place it in a 20 ml headspace vial, accurately add 400 μl of aluminum chloride ethanol solution, shake gently to mix, then add 50 μl of sulfuric acid, cap, seal, and shake well.

[0237] Ethyl formate reference solution: Accurately weigh 400.1 mg of ethyl formate reference standard, place it in a 50 ml volumetric flask, and add... N- Dissolve and dilute methylpyrrolidone to the mark, shake well, accurately measure 1 ml, and place in a 50 ml volumetric flask. N- Dilute methylpyrrolidone to the mark and shake well; accurately measure 2 ml and place it in a 20 ml headspace vial, accurately add 400 μl of aluminum chloride ethanol solution, shake gently to mix, then add 50 μl of sulfuric acid, cap, seal, and shake well.

[0238] Spiked solution for test sample ①: Accurately weigh 39.6 mg of the test sample (batch number: C04-E-25031401B), place it in a 20 ml headspace vial, accurately add 2 ml of "C-100% solution" under "Example 3: Linearity and range experiment of the method for detecting residual solvent formic acid content in octopaine intermediate product", shake gently to dissolve, then accurately add 400 μl of aluminum chloride ethanol solution, shake gently to mix, then add 50 μl of sulfuric acid, cap, seal, and shake well.

[0239] Spiked solution ②: Accurately weigh 39.5 mg of the test sample (batch number: C04-E-25031401B), place it in a 20 ml headspace vial, accurately add 2 ml of "C-100% solution" under "Example 3: Linearity and range test of method for detecting residual solvent formic acid content in octopaine intermediate product", shake gently to dissolve, then accurately add 400 μl of aluminum chloride ethanol solution, shake gently to mix, then add 50 μl of sulfuric acid, cap, seal, and shake well.

[0240] (2) Detection

[0241] The blank solution, formic acid reference solution, ethyl formic acid reference solution, spiked solution ① and spiked solution ② of the test sample were injected into the gas phase headspace at different time points at room temperature, and the chromatograms were recorded. The results of solution stability are shown in Table 7.

[0242] (3) Results

[0243] Table 7 Results of Solution Stability Experiment in Example 5

[0244]

[0245] (4) Conclusion

[0246] When the formic acid reference solution was placed at room temperature for 18.5 hours, the relative deviation between the peak area of ​​the formic acid derivative and the peak area at 0 hours was 2.19%, which is less than 10.0%, indicating that the formic acid reference solution has good stability at room temperature for 18.5 hours.

[0247] When the ethyl formate reference solution and the test sample spiked solution were placed at room temperature for 5 hours, the relative deviation between the peak area of ​​the formic acid derivative and the peak area at 0 hours was less than 10.0%, indicating that the ethyl formate reference solution and the test sample spiked solution had good stability within 5 hours at room temperature. Example 6

[0248] Method validation and conversion rate experiment for detecting residual formic acid content in octopaine intermediate products

[0249] 1. Main technical parameters of gas chromatography: Same as in Example 1

[0250] 2. Determination method

[0251] (1) Solution preparation

[0252] Aluminum chloride ethanol solution: Accurately weigh 5.0079 g of aluminum chloride, place it in a 50 ml volumetric flask, add anhydrous ethanol to dissolve and dilute to the mark, and shake well.

[0253] Blank solution: Accurately measure N- Place 2 ml of methylpyrrolidone into a 20 ml headspace vial, accurately add 400 μl of aluminum chloride ethanol solution, gently shake to mix, then add 50 μl of sulfuric acid, cap, seal, and shake well.

[0254] Reference solution: Accurately weigh 400.1 mg of ethyl formate reference standard, place it in a 50 ml volumetric flask, and add... N- Dissolve and dilute methylpyrrolidone to the mark, shake well, accurately measure 1 ml, and place in a 50 ml volumetric flask. N- Dilute methylpyrrolidone to the mark and shake well; accurately measure 2 ml and place it in a 20 ml headspace vial, accurately add 400 μl of aluminum chloride ethanol solution, shake gently to mix, then add 50 μl of sulfuric acid, cap, seal, and shake well.

[0255] Test solution: Accurately weigh 253.0 mg of formic acid reference standard, place it in a 50 ml volumetric flask, and add... N- Dissolve and dilute methylpyrrolidone to the mark, shake well, accurately measure 1 ml, and place in a 50 ml volumetric flask. N- Dilute methylpyrrolidone to the mark and shake well; accurately measure 2 ml and place it in a 20 ml headspace vial, accurately add 400 μl of aluminum chloride ethanol solution, shake gently to mix, then add 50 μl of sulfuric acid, cap, seal, and shake well.

[0256] (2) Detection

[0257] Inject the blank solution, reference solution, and test solution into the headspace of a gas chromatograph and record the chromatograms. Typical chromatograms are shown below. Figures 10-11 The conversion results are shown in Table 8. (The molecular weight of formic acid is 46.03, and the molecular weight of ethyl formate is 74.078.)

[0258] (3) Results

[0259] Table 8. Results of Conversion Rate Experiment in Example 6

[0260]

[0261] (4) Conclusion

[0262] Using ethyl formate as a reference standard and formic acid as the test sample, the conversion rate was calculated. The results showed that the conversion rate of formic acid to ethyl formate was 96.8%, which is greater than 90%, indicating that the conversion rate of this method is good. Example 7

[0263] Method validation and derivatization experiments on the amount of derivatizing reagent added for the detection of residual formic acid content in octopaine intermediate products

[0264] 1. Main technical parameters of gas chromatography: Same as in Example 1

[0265] 2. Determination method

[0266] (1) Solution preparation

[0267] Aluminum chloride ethanol solution: Accurately weigh 5.0079 g of aluminum chloride, place it in a 50 ml volumetric flask, add anhydrous ethanol to dissolve and dilute to the mark, and shake well.

[0268] Blank solution: Accurately measure N- Place 2 ml of methylpyrrolidone into a 20 ml headspace vial, accurately add 0-500 μl of aluminum chloride ethanol solution, gently shake to mix, then add 0-100 μl of sulfuric acid, cap, seal, and shake well.

[0269] Ethyl formate reference solution: Accurately weigh 400.1 mg of ethyl formate reference standard, place it in a 50 ml volumetric flask, and add... N- Dissolve and dilute methylpyrrolidone to the mark, shake well, accurately measure 1 ml, and place in a 50 ml volumetric flask. N- Dilute methylpyrrolidone to the mark and shake well; accurately measure 2 ml and place it in a 20 ml headspace vial, accurately add 0-500 μl of aluminum chloride ethanol solution, shake gently to mix well, then add 0-100 μl of sulfuric acid, cap, seal, and shake well.

[0270] Formic acid reference solution: Accurately weigh 253.0 mg of formic acid reference standard, place it in a 50 ml volumetric flask, and add... N- Dissolve and dilute methylpyrrolidone to the mark, shake well, accurately measure 1 ml, and place in a 50 ml volumetric flask. N- Dilute methylpyrrolidone to the mark and shake well; accurately measure 2 ml and place it in a 20 ml headspace vial, accurately add 0-500 μl of aluminum chloride ethanol solution, shake gently to mix well, then add 0-100 μl of sulfuric acid, cap, seal, and shake well.

[0271] Test solution: Accurately weigh 40 mg of the test sample (batch number: C04-E-25031401B), place it in a 20 ml headspace vial, and accurately add... N- Dissolve 2 ml of methylpyrrolidone by gentle shaking, then precisely add 0-500 μl of aluminum chloride ethanol solution, gently shake to mix, then add 0-100 μl of sulfuric acid, cap, seal, and shake well.

[0272] Spiked solution for test sample: Accurately weigh 40 mg of the test sample (batch number: C04-E-25031401B), place it in a 20 ml headspace vial, accurately add 2 ml of formic acid reference solution, shake gently to dissolve, then accurately add 0-500 μl of aluminum chloride ethanol solution, shake gently to mix, then add 0-100 μl of sulfuric acid, cap, seal, and shake well.

[0273] (2) Detection

[0274] The blank solution, reference solution, and test solution were injected into the gas chromatogram headspace, and the chromatograms were recorded. The results of the investigation on the amount of derivatizing reagent added are shown in Table 9.

[0275] (3) Results

[0276] Table 9 Results of the investigation on the amount of derivatizing reagent added in Example 7

[0277]

[0278] (4) Conclusion

[0279] The results show that aluminum chloride can act as a catalyst to accelerate and complete the reaction between formic acid and ethanol, ensuring the complete conversion of formic acid to ethyl formate. Sulfuric acid and aluminum chloride also have a synergistic effect. The optimal conversion rate and recovery rate of formic acid to ethyl formate were achieved when the amount of aluminum chloride-ethanol solution added was 400 μl and the amount of sulfuric acid added was 50 μl. Example 8

[0280] Derivation time study of the methodology for detecting residual formic acid content in octopaine intermediate products

[0281] 1. Main technical parameters of gas chromatography: Same as in Example 1

[0282] 2. Determination method

[0283] (1) Solution preparation

[0284] Aluminum chloride ethanol solution: Accurately weigh 5.0079 g of aluminum chloride, place it in a 50 ml volumetric flask, add anhydrous ethanol to dissolve and dilute to the mark, and shake well.

[0285] Blank solution: Accurately measure N- Place 2 ml of methylpyrrolidone into a 20 ml headspace vial, accurately add 400 μl of aluminum chloride ethanol solution, gently shake to mix, then add 50 μl of sulfuric acid, cap, seal, and shake well.

[0286] Ethyl formate reference solution: Accurately weigh 400.1 mg of ethyl formate reference standard, place it in a 50 ml volumetric flask, and add... N- Dissolve and dilute methylpyrrolidone to the mark, shake well, accurately measure 1 ml, and place in a 50 ml volumetric flask. N- Dilute methylpyrrolidone to the mark and shake well; accurately measure 2 ml and place it in a 20 ml headspace vial, accurately add 400 μl of aluminum chloride ethanol solution, shake gently to mix, then add 50 μl of sulfuric acid, cap, seal, and shake well.

[0287] Formic acid reference solution: Accurately weigh 253.0 mg of formic acid reference standard, place it in a 50 ml volumetric flask, and add... N- Dissolve and dilute methylpyrrolidone to the mark, shake well, accurately measure 1 ml, and place in a 50 ml volumetric flask. N- Dilute methylpyrrolidone to the mark and shake well; accurately measure 2 ml and place it in a 20 ml headspace vial, accurately add 400 μl of aluminum chloride ethanol solution, shake gently to mix, then add 50 μl of sulfuric acid, cap, seal, and shake well.

[0288] Spiked solution for test sample: Accurately weigh 40 mg of the test sample (batch number: C04-E-25031401B), place it in a 20 ml headspace vial, accurately add 2 ml of formic acid reference solution, shake gently to dissolve, then accurately add 400 μl of aluminum chloride ethanol solution, shake gently to mix, then add 50 μl of sulfuric acid, cap, seal, and shake well.

[0289] (2) Detection

[0290] The blank solution, ethyl formate reference solution, formic acid reference solution, and spiked solution of the test sample were placed at room temperature for a certain period of time, then heated in a headspace sampler for 30 min, and then injected using gas chromatography headspace. The chromatograms were recorded. The results of the investigation on the amount of derivatizing reagent added are shown in Table 10.

[0291] (3) Results

[0292] Table 10 Results of the Derivative Time Study in Example 8

[0293]

[0294] (4) Conclusion

[0295] The results showed that the relative deviations between the peak areas of formic acid derivatives obtained by heating the formic acid reference solution in a headspace sampler for 30 min after standing at room temperature for 1 h, 16.5 h, and 18.5 h at room temperature and those obtained by heating the formic acid directly in a headspace sampler for 30 min after standing at room temperature for 0 h at room temperature were all less than 5.0%, and the conversion rates at all four time points were greater than 95.0%, indicating that the derivatization time is simply heating the prepared solution in a headspace sampler for 30 min.

[0296] The relative deviation between the peak area of ​​the formic acid derivative obtained by heating the spiked solution of the test sample in the headspace sampler for 30 min after standing at room temperature for 5 h and the peak area of ​​the formic acid obtained by heating it directly in the headspace sampler for 30 min after standing at room temperature for 0 h is less than 5.0%. Combined with the recovery results in Example 4, it can be concluded that the derivatization time is 30 min after preparing the solution and heating it in the headspace sampler.

[0297] Comparative Example 1:

[0298] Without the addition of aluminum chloride, the octopaine intermediate product is easily degraded to formic acid under acidic conditions (experimental study).

[0299] 1. Main technical parameters of gas chromatography

[0300] Gas chromatograph: Agilent 7890B+7697A; Detector: Flame ionization detector (FID);

[0301] Column type: Perkin Elmer® Elite-FFAP (30m × 0.53mm, 1μm); Split ratio: 3:1;

[0302] Sample introduction method: headspace injection; headspace shaking: 5; headspace equilibration time: 30 min, circulation time: 40 min;

[0303] Headspace equilibrium temperature: 70℃, metering loop temperature: 80℃, transfer line temperature: 90℃;

[0304] Carrier gas nitrogen flow rate: 2.0 ml / min; Inlet temperature: 220℃; Detector temperature: 250℃;

[0305] Heating program: Initial temperature is 60℃, maintain for 7 minutes, increase to 140℃ at a rate of 8℃ per minute, then increase to 240℃ at a rate of 25℃ per minute, and maintain for 10 minutes.

[0306] Diluent: Dimethyl sulfoxide.

[0307] 2. Determination method

[0308] (1) Solution preparation

[0309] Blank solution ①: Accurately measure 1 ml of dimethyl sulfoxide, place it in a 20 ml headspace vial, cap it, and seal it.

[0310] Reference solution ①: Accurately weigh 512.9 mg toluene, 500.5 mg acetonitrile and 500.3 mg formic acid, place them in the same 50 ml volumetric flask, dissolve and dilute to the mark with dimethyl sulfoxide, shake well, accurately measure 1 ml, place it in a 50 ml volumetric flask, dilute to the mark with dimethyl sulfoxide, shake well; accurately measure 1 ml, place it in a 20 ml headspace vial, cap and seal.

[0311] Test solution ①: Accurately weigh 201.8 mg of the test sample (batch number: C04-E-25031401B), place it in a 20 ml headspace vial, accurately add 1 ml of dimethyl sulfoxide, cap, seal, and shake well.

[0312] Spiked solution for test sample ①: Accurately weigh 207.3 mg of the test sample (batch number: C04-E-25031401B), place it in a 20 ml headspace vial, accurately add 1 ml of reference solution, cap, seal, and shake well.

[0313] Blank solution ②: Accurately measure 1 ml of dimethyl sulfoxide and place it in a 20 ml headspace vial. Accurately add 100 μl of anhydrous ethanol and 100 μl of sulfuric acid, cap, seal, and shake well.

[0314] Reference solution ②: Accurately weigh 512.9 mg toluene, 500.5 mg acetonitrile and 500.3 mg formic acid, place them in the same 50 ml volumetric flask, dissolve and dilute to the mark with dimethyl sulfoxide, shake well, accurately measure 1 ml, place it in a 50 ml volumetric flask, dilute to the mark with dimethyl sulfoxide, shake well; accurately measure 1 ml, place it in a 20 ml headspace vial, accurately add 100 μl anhydrous ethanol and 100 μl sulfuric acid, cap, seal, and shake well.

[0315] Test solution ②: Accurately weigh 201.8 mg of the test sample (batch number: C04-E-25031401B), place it in a 20 ml headspace vial, accurately add 1 ml of dimethyl sulfoxide, shake gently, then accurately add 100 μl of anhydrous ethanol and 100 μl of sulfuric acid, cap, seal, and shake well.

[0316] Spiked solution ②: Accurately weigh 207.3 mg of the test sample (batch number: C04-E-25031401B), place it in a 20 ml headspace vial, accurately add 1 ml of the reference solution, shake gently, then accurately add 100 μl of anhydrous ethanol and 100 μl of sulfuric acid, cap, seal, and shake well.

[0317] (2) Detection

[0318] Take the blank solutions ① and ②, reference solutions ① and ②, test solutions ① and ②, and spiked test solutions ① and ② from the above-mentioned blank solutions ① and ②, and inject them into the gas chromatogram headspace. Record the chromatograms. Typical chromatograms are shown below. Figures 27-34 .

[0319] (3) Results

[0320] Formic acid peaks were not identifiable in the chromatograms of reference solution ①, test solution ①, and spiked test solution ①, indicating extremely low sensitivity under FID detection conditions. Formic acid derivative peaks were present at approximately 4.6 min in the chromatograms of reference solution ②, test solution ②, and spiked test solution ②. The peak areas of formic acid derivatives in test solution ② and spiked test solution ② were approximately 50–60 times larger than those in reference solution ②. A rough recovery rate of (94824.46872 - 79867.92479) / 1392.41814 × 100% = 1074.1% indicates that the octopaine intermediate product is easily degraded into formic acid under sulfuric acid heating conditions, resulting in a severely unsatisfactory recovery rate and thus affecting the accurate quantification of formic acid.

[0321] (4) Conclusion: The above results show that octopaine intermediate products are easily degraded into formic acid under acidic heating conditions.

[0322] Comparative Example 2:

[0323] An experiment was conducted using isopropanol and n-propanol as derivatizing reagents and sulfuric acid catalysis.

[0324] 1. Main technical parameters of gas chromatography

[0325] Gas chromatograph: Agilent 7890B+7697A; Detector: Flame ionization detector (FID);

[0326] Column type: Perkin Elmer® Elite-FFAP (30m × 0.53mm, 1μm); Split ratio: 3:1;

[0327] Sample introduction method: headspace injection; headspace shaking: 5; headspace equilibration time: 30 min, circulation time: 40 min;

[0328] Headspace equilibrium temperature: 70℃, metering loop temperature: 80℃, transfer line temperature: 90℃;

[0329] Carrier gas nitrogen flow rate: 2.0 ml / min; Inlet temperature: 220℃; Detector temperature: 250℃;

[0330] Heating program: Initial temperature is 60℃, maintain for 7 minutes, increase to 140℃ at a rate of 8℃ per minute, then increase to 240℃ at a rate of 25℃ per minute, and maintain for 10 minutes.

[0331] Diluent: N -Methylpyrrolidone.

[0332] 2. Determination method

[0333] (1) Solution preparation

[0334] Blank solution ①: Accurately measure N Place 1 ml of methylpyrrolidone into a 20 ml headspace vial, accurately add 20 μl of isopropanol and 20 μl of sulfuric acid, cap, seal, and shake well.

[0335] Reference solution ①: Accurately weigh 504.0 mg toluene, 499.4 mg acetonitrile, and 507.4 mg formic acid, place them in the same 50 ml volumetric flask, and add... N Dissolve and dilute methylpyrrolidone to the mark, shake well, accurately measure 1 ml, and place in a 50 ml volumetric flask. N Dilute methylpyrrolidone to the mark and shake well; accurately measure 1 ml and place it in a 20 ml headspace vial, accurately add 20 μl of isopropanol and 20 μl of sulfuric acid, cap, seal, and shake well.

[0336] Blank solution ②: Accurately measure N Place 1 ml of methylpyrrolidone into a 20 ml headspace vial, accurately add 20 μl of n-propanol and 20 μl of sulfuric acid, cap, seal, and shake well.

[0337] Reference solution ②: Accurately weigh 504.0 mg toluene, 499.4 mg acetonitrile, and 507.4 mg formic acid, place them in the same 50 ml volumetric flask, and add... N Dissolve and dilute methylpyrrolidone to the mark, shake well, accurately measure 1 ml, and place in a 50 ml volumetric flask. N Dilute methylpyrrolidone to the mark and shake well; accurately measure 1 ml and place it in a 20 ml headspace vial, accurately add 20 μl of n-propanol and 20 μl of sulfuric acid, cap, seal, and shake well.

[0338] (2) Detection

[0339] Take blank solutions ① and ②, and reference solutions ① and ② as described above, and perform headspace gas chromatography (GC-COD) to record the chromatograms. Typical chromatograms are shown below. Figures 35-38 .

[0340] (3) Results

[0341] In blank solution ①, an interference peak of isopropyl formate (RT approximately 4.9 min) was observed, accounting for approximately 303.47143 / 1502.61622×100%=20.2%; in blank solution ②, an interference peak of n-propyl formate (RT approximately 6.2 min) was observed, accounting for approximately 227.13468 / 1575.00047×100%=14.4%.

[0342] (4) Conclusion: The above results show that the method of using isopropanol and n-propanol as derivatizing reagents and sulfuric acid catalysis is not suitable for the detection of formic acid content in ocpicaben intermediate products.

Claims

1. A method for detecting residual formic acid in an opicapone intermediate product, characterized in that, The detection method comprises the following steps: (1) Preparation of aluminum chloride ethanol solution: take 1g of aluminum chloride, dissolve and dilute to 10ml with anhydrous ethanol, and shake well; (2) Preparation of blank solution: accurately measure 2ml of N-methyl pyrrolidone, place it in a top empty bottle, accurately add a proper amount of aluminum chloride ethanol solution, shake slightly to mix, then add a proper amount of sulfuric acid, cover, seal, and shake well; (3) Preparation of control solution: weigh the formic acid control or formic acid ethyl ester control, place it in a measuring bottle, dissolve and dilute with N-methyl pyrrolidone to prepare a control solution; accurately measure 2ml of the control solution, place it in a top empty bottle, accurately add a proper amount of aluminum chloride ethanol solution, shake slightly to mix, then add a proper amount of sulfuric acid, cover, seal, and shake well; (4) Preparation of test sample solution: weigh the intermediate product of opicapone to be tested, place it in a top empty bottle, accurately add 2ml of N-methyl pyrrolidone, shake slightly to dissolve, then accurately add a proper amount of aluminum chloride ethanol solution, shake slightly to mix, then add a proper amount of sulfuric acid, cover, seal, and shake well; (5) Respectively, take equal amounts of the above blank solution, control solution and test sample solution, inject into the FID gas chromatograph for determination, record the chromatogram, and calculate the formic acid content by the external standard method with peak area; The chemical name of the intermediate product of opicapone is: 2,5-dichloro-3-(5-(4-hydroxy-3-methoxy-5-nitrophenyl)-1,2,4-oxadiazol-3-yl)-4,6-dimethylpyridine-1-oxide; In step (3), the concentration of the formic acid control solution is 10μg / ml~500μg / ml, or the concentration of the formic acid ethyl ester control solution is 16.1μg / ml~804.7μg / ml; In steps (2), (3) and (4), the amount of aluminum chloride ethanol solution added is 200μl~500μl; In steps (2), (3) and (4), the amount of sulfuric acid added is 50μl~100μl; In step (4), the concentration of the test sample solution is 2mg / ml~40mg / ml.

2. The detection method of claim 1, wherein: The gas chromatography conditions include: Chromatographic column: capillary column with nitroterephthalic acid modified polyethylene glycol as stationary phase; Injection mode: headspace injection; Headspace equilibrium time: 30min±2min; cycle time: 35min±2min; Headspace equilibrium temperature: 70℃±2℃; quantification ring temperature: 80℃±2℃; transmission line temperature: 90℃±2℃; Headspace shaking: 5±2; Detector: hydrogen flame ionization detector (FID); Column temperature program: initial temperature 40℃±5℃, maintain for 7-10 minutes, increase to 120℃ at a rate of 10℃ per minute, then increase to 240℃ at a rate of 50℃ per minute, maintain for 5-10 minutes; Carrier gas nitrogen flow rate: 1.9ml / min~2.1ml / min; Hydrogen flame ionization detector temperature: 245℃~255℃; Injection port temperature: 215℃~225℃; Split ratio: 1:1 to 20:

1.

3. The method of claim 1, wherein: The concentration of the formic acid control solution is 100 μg / ml, or the concentration of the ethyl formate control solution is 160.9 μg / ml.

4. The method of claim 1, wherein: In steps (2), (3) and (4), the amount of the added aluminum chloride ethanol solution is 400 μl.

5. The detection method according to any one of claims 1 to 4, characterized in that: In step (5), in the gas chromatography conditions, The flow rate of the carrier gas nitrogen is 2.0 ml / min; The hydrogen flame ionization detector temperature is 250℃; The injection port temperature is 220℃; The split ratio is 10:1; The headspace equilibrium time is 30 min; the cycle time is 35 min; The headspace equilibrium temperature is 70℃; the quantification ring temperature is 80℃; the transfer line temperature is 90℃; The headspace shaking is 5.

6. The detection method of claim 2, wherein: In step (5), the capillary column with the nitroterephthalic acid modified polyethylene glycol as the stationary phase is Perkin Elmer® Elite-FFAP 30m×0.53mm, 1 μm.

Images