Method for detecting reaction substrate of amoenavir intermediate

Abstract

The invention discloses a reaction substrate detection method of an amonetavir intermediate, which comprises first adding a derivatization reagent to a test solution of the amonetavir intermediate to undergo a derivatization reaction, converting the reaction substrate acyl chloride in the test solution into a corresponding derivative compound, then adding water to terminate the reaction, and calculating the reaction substrate acyl chloride content by detecting the residual carboxylic acid content. The method is simple, efficient, and highly accurate, and will not cause irreversible damage to the detection instrument.

Description

Technical Field

[0001] The present invention relates to the technical field of compound detection, and in particular to a reaction substrate detection method for an amoenavir intermediate. Background Art

[0002] In the prior art, for the reaction monitoring of carboxylic acid to generate acyl chloride, a derivatization method is often used to detect acyl chloride, such as Chinese patent publications CN116718691A, CN107014944A, etc. Since acyl chloride is very active and a chlorinating agent such as thionyl chloride or phosphorus oxychloride is required as a reaction reagent during the reaction, directly analyzing the acyl chloride content by HPLC or GC often produces erroneous results (due to interference from the chlorinating agent), and acyl chloride may also cause irreversible damage to the instrument and chromatographic column, and the acyl chloride compound may react with the stationary phase of the chromatographic column.

[0003] In addition, for the reaction of preparing acyl chloride from carboxylic acid, HPLC method often uses DAD detector to detect the content of acyl chloride by detecting the derivatized acyl chloride. However, the product of the reaction system after derivatization is complex, and the use of DAD detector may not be able to avoid matrix interference of substrate and its synthetic intermediates.

[0004] Amonetvir is an anti-herpes zoster virus drug, and 1,1-dioxo-hexahydro-1L6-thiopyran-4-carboxylic acid is an important intermediate in its synthesis. Since acyl chlorides are more active than corresponding carboxylic acids, reactions using acyl chlorides as raw materials often have higher yields. Therefore, acyl chlorides are often used as raw materials in the process of preparing amides, esters, and anhydrides. In the synthesis of amonetvir, 1,1-dioxo-hexahydro-1L6-thiopyran-4-carboxylic acid is usually reacted with a chlorinating agent to generate 1,1-dioxo-hexahydro-1L6-thiopyran-4-carboxylic acid. Monitoring the consumption of the substrate 1,1-dioxo-hexahydro-1L6-thiopyran-4-carboxylic acid and the amount of the product 1,1-dioxo-hexahydro-1L6-thiopyran-4-carboxylic acid during the reaction is very important for the process control of the reaction. However, there is currently no report on how to accurately detect substrates and products during the reaction. Summary of the invention

[0005] The purpose of the present invention is to provide a method for detecting a reaction substrate of an amonetavir intermediate in view of the above-mentioned deficiencies in the prior art. The detection method is simple, efficient, highly accurate, and will not cause irreversible damage to the detection instrument.

[0006] The technical solution of the present invention is described in detail as follows:

[0007] In the first aspect, the present invention provides a method for detecting a reaction substrate of an amonetavir intermediate, which comprises first adding a derivatization reagent to a test solution of the amonetavir intermediate to carry out a derivatization reaction, converting the reaction substrate acyl chloride in the test solution into a corresponding derivative compound, then adding water to terminate the reaction, and calculating the reaction substrate acyl chloride content by detecting the residual carboxylic acid content.

[0008] The test solution is the sample solution for detection, which contains acyl chloride. The derivatization reagent is any one of aniline, p-methoxyaniline, benzyl alcohol, phenol, and methanol. The derivative compounds after the reaction of different derivatization reagents with acyl chloride are as follows: aniline → 1,1-dioxo-N-phenylthiocyclohexane-3-carboxamide; p-methoxyaniline → 2-(1,1-dioxothiocyclohexane-3-yl)-N-(3-methoxyphenyl)carboxamide; methanol → 1,1-dioxothiocyclohexane-4-carboxylic acid methyl ester; phenol → 1,1-dioxothiocyclohexane-4-carboxylic acid phenyl ester; benzyl alcohol → 1,1-dioxothiocyclohexane-4-carboxylic acid benzyl ester.

[0009] Optionally or preferably, in the above detection method, the detection of the residual carboxylic acid content is completed using a liquid chromatograph, a reference solution is prepared, the reference solution and the test solution are respectively injected into the liquid chromatograph, the chromatogram is recorded, and the carboxylic acid content in the test sample is calculated by an external standard method based on the concentration of the reference solution.

[0010] The reference solution is a solution of known carboxylic acid concentration used to calculate the carboxylic acid content in the test solution. The external standard method is a one-point external standard method, which uses a reference solution of a certain concentration and the test solution to inject multiple times under the same conditions, measure the average value of the peak area, and use these data to calculate the amount of the component to be tested in the sample.

[0011] Optionally or preferably, in the above detection method, the liquid chromatograph is an electrospray liquid chromatograph.

[0012] Optionally or preferably, in the above detection method, the chromatographic conditions of the electrospray liquid chromatograph are:

[0013] Chromatographic column: C18 column;

[0014] Mobile phase A: 0.2% trifluoroacetic acid aqueous solution (volume percentage concentration of trifluoroacetic acid is 0.2%);

[0015] Mobile phase B: acetonitrile

[0016] Gradient elution program:

[0017] Time (min) 0 15 16 26 27 42 Mobile phase A (volume %) 100 100 20 20 100 100 Mobile phase B (volume %) 0 0 80 80 0 0

[0018] Optionally or preferably, in the above detection method, the chromatographic conditions further include:

[0019] Column temperature: 40°C;

[0020] Injection volume: 10 μl;

[0021] Flow rate: 1.0ml / min;

[0022] CAD atomizer temperature: 40℃.

[0023] Optionally or preferably, in the above detection method, the chromatographic column is a YMC Triart C18 chromatographic column with specifications: 4.6×150 mm, 3 μm.

[0024] Optionally or preferably, in the above detection method, the reaction substrate acyl chloride is 1,1-dioxo-hexahydro-1L6-thiopyran-4-carbonyl chloride, and the carboxylic acid is 1,1-dioxo-hexahydro-1L6-thiopyran-4-carboxylic acid.

[0025] Optionally or preferably, in the above detection method, the test solution and the reference solution are both diluted with an acetonitrile aqueous solution. The volume ratio of acetonitrile to water in the acetonitrile aqueous solution is preferably 3:7, which can minimize the solvent effect.

[0026] Optionally or preferably, in the above detection method, the derivatization reagent is aniline, and after adding the derivatization reagent, acetonitrile is also added as a solvent, and then ultrasound is used for dispersion and dissolution, and finally water is added for dilution.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The method of the present invention calculates the acyl chloride in the test sample of the reaction substrate of the intermediate of amonetvir to be tested by detecting carboxylic acid, and the detection object is the carboxylic acid remaining after the test sample is reacted. Compared with the previous detection of derivative products, the detection of carboxylic acid is easier to locate the peak position of carboxylic acid, and there is no need to prepare a reference substance of the derivative separately, and there is no need to calibrate the derivative as an external standard reference substance. The operation is simple and the result is accurate.

[0029] According to the method of the present invention, aniline is added to a test solution to cause a derivatization reaction, so that the acyl chloride in the test solution is converted into an amide compound. The amount of aniline added is usually far more than 10 times the equivalent of the acyl chloride to ensure that no acyl chloride remains. Water is then added to terminate the reaction. Since aniline reacts rapidly with the acyl chloride, adding water will basically not affect the accuracy of the result, so that before injection, the sample becomes a stable system and will not damage the chromatographic column.

[0030] When testing the content of 1,1-dioxo-hexahydro-1L6-thiopyran-4-carbonyl chloride, an intermediate of amonetvir, acetonitrile can be added as a solvent after adding aniline, and ultrasound can be used to fully disperse and dissolve the test sample and aniline to facilitate the full reaction of aniline and acyl chloride. Adding water can also effectively prevent the unreacted carboxylic acid from reacting with thionyl chloride to form acyl chloride during the test, and then reacting with aniline, resulting in the consumption of the remaining carboxylic acid and inaccurate test results. In addition, it can prevent thionyl chloride (which can react quickly with water) from damaging the chromatographic column.

[0031] Compared with the common liquid chromatograph detector DAD (diode array) detector, the present invention uses an electrospray detector (CAD), whose response is not affected by the ultraviolet absorbing group of the compound, and can detect weak ultraviolet absorbing compounds that the DAD detector cannot detect. The method of the present invention uses a CAD detector to avoid the influence of a small amount of strong ultraviolet absorbing products in the reaction system after derivatization, and the detection target object is the reaction substrate carboxylic acid of the acyl chloride. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is the chromatogram of blank solution 1 in Example 1;

[0033] Figure 2 The chromatogram of reference solution 1 in Example 1;

[0034] Figure 3 It is the chromatogram of test solution 1 in Example 1;

[0035] Figure 4 It is the chromatogram of blank solution 2 in Example 2;

[0036] Figure 5 The chromatogram of reference solution 2 in Example 2;

[0037] Figure 6 This is the chromatogram of test solution 2 in Example 2;

[0038] Figure 7 The chromatogram of the test solution 3 diluted with DMF in Example 3;

[0039] Figure 8 The chromatogram of the test solution 3 diluted with DMSO in Example 3;

[0040] Fig. 9 The chromatogram of the test solution 3 diluted with acetonitrile in Example 3;

[0041] Fig.10 The chromatogram of the reference solution 5-aniline in Example 5;

[0042] Fig.11 The chromatogram of the test solution 5-aniline in Example 5;

[0043] Fig.12 The chromatogram of the reference solution 5-p-methoxyaniline in Example 5;

[0044] Fig.13 The chromatogram of the test solution 5-p-methoxyaniline in Example 5;

[0045] Fig.14 The chromatogram of 5-benzyl alcohol solution of the reference substance in Example 5 is shown in FIG.

[0046] Fig.15 The chromatogram of 5-benzyl alcohol in the test solution in Example 5;

[0047] Fig.16 The chromatogram of the reference solution 5-phenol in Example 5 is shown below:

[0048] Fig.17 The chromatogram of 5-phenol of the test solution in Example 5;

[0049] Fig.18 The chromatogram of the reference solution 5-methanol in Example 5;

[0050] Fig.19 This is the chromatogram of the test solution 5-methanol in Example 5;

[0051] Fig. 20 The chromatogram of 6-aniline of the reference substance solution of Example 6 is shown in FIG. DETAILED DESCRIPTION

[0052] In order to enable those skilled in the art to better understand the present application, the present application will be described clearly and completely below in conjunction with the embodiments and drawings. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without creative work should fall within the scope of protection of the present application.

[0053] In the following examples, the equipment used is:

[0054] HPLC-DAD: Manufacturer: Shimadzu, Model: LC-20AD;

[0055] HPLC-CAD: Manufacturer: Thermo, Model: Ultimate 3000.

[0056] Reagents used:

[0057] 1,1-Dioxo-hexahydro-1L6-thiopyran-4-carboxylic acid, manufacturer: Shandong Xuande;

[0058] 1,1-Dioxo-hexahydro-1L6-thiopyran-4-carbonyl chloride solution, manufacturer: homemade.

[0059] p-Anisidine, Manufacturer: Aladdin, Grade / Content: 99%

[0060] Aniline, manufacturer: Chuandong Chemical, grade / content: AR;

[0061] Phenol, manufacturer: Kelon, grade / content: AR;

[0062] Benzyl alcohol, manufacturer: Cologne, grade / content: AR;

[0063] Methanol, manufacturer: Honeywell, grade / content: HPLC;

[0064] Acetonitrile, manufacturer: Honeywell, grade / content: HPLC;

[0065] Example 1 CAD detector can effectively avoid aniline interference

[0066] Detection method: Liquid chromatography

[0067] Detector: CAD detector

[0068] Solution preparation:

[0069] Solvent 1: A mixture of acetonitrile and water in a volume ratio of 3:7.

[0070] Blank solution 1: Pipette 1 ml of aniline into a 10 ml volumetric flask, dilute to the mark with solvent 1, and shake well.

[0071] Reference substance stock solution 1: Accurately weigh 100 mg of reference substance 1,1-dioxo-hexahydro-1L6-thiopyran-4-carboxylic acid, place in a 10 ml volumetric flask, dissolve with solvent 1 by ultrasonication, dilute to scale, shake well, and obtain (containing 10 mg / ml of 1,1-dioxo-hexahydro-1L6-thiopyran-4-carboxylic acid)

[0072] Reference solution 1: Pipette 1 ml of reference stock solution 1 into a 10 ml volumetric flask, add 1 ml of aniline, shake, let stand for 5 minutes, then dilute to the mark with solvent 1, shake well to obtain (containing 1 mg / ml of 1,1-dioxo-hexahydro-1L6-thiopyran-4-carboxylic acid).

[0073] Test solution 1: Take 0.5 ml of 1,1-dioxo-hexahydro-1L6-thiopyran-4-carbonyl chloride solution (concentration is 80 mg / ml, solvent is THF, tetrahydrofuran), add 0.5 ml of aniline, add 3 ml of acetonitrile, ultrasonicate for 5 min, dilute to scale with ultrapure water, and shake well. (Contains 4 mg / ml of 1,1-dioxo-hexahydro-1L6-thiopyran-4-carbonyl chloride).

[0074] Inject blank solution 1, reference solution 1 and test solution 1 into the liquid chromatograph respectively and record the chromatograms.

[0075] Chromatographic conditions:

[0076] project condition Mobile phase 1A 0.2% trifluoroacetic acid aqueous solution (volume percentage concentration) Mobile phase 1B Acetonitrile Flow rate 1.0ml / min Injection volume 10μl Sample tray temperature No temperature control Chromatographic columns YMC Triart C18, 4.6×150mm, 3μm Solvent Effect Eliminator yes Column temperature 40℃ Detector CAD CAD flow rate On CAD atomizer temperature 40℃ Needle wash solution Acetonitrile

[0077] Elution procedure:

[0078] Time (min) 0 15 16 26 27 42 Mobile phase A (%) 100 100 20 20 100 100 Mobile phase B (%) 0 0 80 80 0 0

[0079] The chromatograms of blank solution 1, reference solution 1, and test solution 1 are as follows: Figure 1-3 As shown, Figure 1 The blank solvent 1 only contained aniline, and no peak was observed in the first 15 minutes. Figure 2 It is a reference solution of 1,1-dioxo-hexahydro-1L6-thiopyran-4-carboxylic acid, and its peak time is about 8.8min. Figure 3 The peak at around 8.8min is 1,1-dioxo-hexahydro-1L6-thiopyran-4-carboxylic acid in the test solution after 1,1-dioxo-hexahydro-1L6-thiopyran-4-carboxylic acid chloride was derivatized with aniline. There is no interference from impurities on both sides, indicating that this method has good specificity. Due to the special requirements of the CAD detector, only volatile buffers such as formic acid, acetic acid, ammonium formate, ammonium acetate, and trifluoroacetic acid cannot exceed 0.3%, so the mobile phase 1A cannot use phosphoric acid and ammonia water, and is replaced with trifluoroacetic acid compatible with the CAD detector.

[0080] Example 2 DAD detector cannot avoid aniline interference

[0081] The other conditions are the same as those in Example 1, and a DAD detector is used as a detector.

[0082] Solution preparation:

[0083] Solvent 2: A mixture of acetonitrile and water in a volume ratio of 3:7.

[0084] Blank solution 2: Pipette 1 ml of aniline into a 10 ml volumetric flask, dilute to the mark with solvent 2, and shake well.

[0085] Reference substance stock solution 2: Accurately weigh 100 mg of the reference substance 1,1-dioxo-hexahydro-1L6-thiopyran-4-carboxylic acid, place it in a 10 ml volumetric flask, dissolve it with ultrasonic solvent 2, make up to the scale, shake well, and obtain (containing 10 mg / ml of 1,1-dioxo-hexahydro-1L6-thiopyran-4-carboxylic acid).

[0086] Reference solution 2: Pipette 1 ml of reference stock solution 2 into a 10 ml volumetric flask, add 1 ml of aniline, shake, let stand for 5 minutes, then dilute to the mark with solvent 2, shake well, and obtain (containing 1 mg / ml of 1,1-dioxo-hexahydro-1L6-thiopyran-4-carboxylic acid).

[0087] Test solution 2: Take 0.5 ml of 1,1-dioxo-hexahydro-1L6-thiopyran-4-carbonyl chloride solution (concentration 80 mg / ml, solvent THF, tetrahydrofuran), add 0.5 ml of aniline, add 3 ml of acetonitrile, ultrasonicate for 5 min, dilute to scale with ultrapure water, and shake well. (Contains 4 mg / ml of 1,1-dioxo-hexahydro-1L6-thiopyran-4-carbonyl chloride).

[0088] Inject blank solution 2, reference solution 2 and test solution 2 into the liquid chromatograph respectively and record the chromatograms.

[0089] Chromatographic conditions:

[0090]

[0091]

[0092] Elution procedure:

[0093] Time (min) 0 15 16 26 27 42 Mobile phase A (%) 100 100 20 20 100 100 Mobile phase B (%) 0 0 80 80 0 0

[0094] The chromatograms of blank solution 2, reference solution 2, and test solution 2 are as follows: Figure 4-6 As shown, Figure 4 Blank solution 2 contains only aniline, and there is a large aniline peak in the first 10 min, which will interfere with the detection of carboxylic acid; Figure 5 This is the response of carboxylic acid in blank solution 2. Figure 6 This is the response of carboxylic acid in test solution 2. Figure 5 , 6 can all show that the carboxylic acid peak is at the end of the aniline peak. At this time, due to the matrix effect, the carboxylic acid peak is small, and unknown impurities may appear around the carboxylic acid peak (at 10.628min) to interfere with the determination. Figure 3 and Figure 6 It was found that aniline had little interference with the detection of carboxylic acid in the CAD detector and the method had better specificity without the interference of other strong ultraviolet absorbing substances.

[0095] Example 3 Screening of diluents

[0096] Solution preparation:

[0097] Test solution 3: Take 0.5 ml of 1,1-dioxo-hexahydro-1L6-thiopyran-4-carbonyl chloride solution (concentration is 80 mg / ml, solvent is THF), place it in a 10 ml volumetric flask, add 0.5 ml of aniline, then add different diluents (75% DMF water, 75% DMSO water, 75% acetonitrile water, all by volume) to dissolve and dilute to the scale, ultrasonicate for 5 minutes, and shake well (containing 4 mg / ml of 1,1-dioxo-hexahydro-1L6-thiopyran-4-carbonyl chloride).

[0098] The test sample 3 diluted with the above-mentioned different diluents was injected into the liquid chromatograph and the chromatogram was recorded.

[0099] Chromatographic conditions:

[0100]

[0101]

[0102] Elution procedure:

[0103] Time / min 0 3 23 25 26 36 A(％) 100 100 30 30 100 100 B(％) 0 0 70 70 0 0

[0104] The chromatograms of the test solution 3 diluted with DMF, the test solution 3 diluted with DMSO, and the test solution 3 diluted with acetonitrile are as follows: Figure 7-9 As shown, Figure 7 The test solution 3 is diluted with DMF, Figure 8 , 9 In contrast, DMF as a diluent masks the carboxylic acid peak. Figure 8 The test solution 3 is diluted with DMSO. Fig. 9 Compared with the test solution 3 diluted with acetonitrile, the matrix effect is more obvious. The preferred reaction solvent is acetonitrile, that is, the organic phase in the diluent is acetonitrile.

[0105] Example 4 Recovery rate of the method of the present invention

[0106] Solution preparation:

[0107] Solvent 4: acetonitrile-water (volume ratio 3:1).

[0108] Reference substance stock solution 4: Take 50 mg of the reference substance 1,1-dioxo-hexahydro-1L6-thiopyran-4-carboxylic acid, accurately weigh it, put it in a 10 ml volumetric flask, dilute it to the scale with ultrapure water, shake well, and obtain (containing 5 mg / ml of reference substance).

[0109] Reference solution 4: Take a total of 0.1 ml of reference stock solution 4, place it in a 10 ml volumetric flask, add 0.5 ml of aniline and dilute it with solvent 4 to a 0.05 mg / ml solution.

[0110] Test solution 4: Take 0.5 ml of acyl chloride solution (concentration is 100 mg / ml, solvent is THF), put it in a 10 ml volumetric flask, add 0.5 ml of aniline, shake, react for 5 minutes, add 1 ml of ultrapure water to quench the reaction, dilute to the scale with solvent 4, shake well, and obtain.

[0111] 1% spiked test solution 4: Take 0.5 ml of acyl chloride solution (concentration is 100 mg / ml, solvent is THF), put it in a 10 ml volumetric flask, add 0.5 ml of aniline, shake, react for 5 minutes, add 1 ml of ultrapure water to quench the reaction, add 0.1 ml of reference stock solution, dilute to the scale with solvent 4, shake well, and obtain.

[0112] 5% spiked test solution 4: Take 0.5 ml of acyl chloride solution (concentration is 100 mg / ml, solvent is THF), put it in a 10 ml volumetric flask, add 0.5 ml of aniline, shake, react for 5 minutes, add 1 ml of ultrapure water to quench the reaction, add 0.5 ml of reference stock solution, dilute to the scale with solvent 4, shake well, and obtain.

[0113] 10% spiked test solution 4: Take 0.5 ml of acyl chloride solution (concentration is 100 mg / ml, solvent is THF), put it in a 10 ml volumetric flask, add 0.5 ml of aniline, shake, react for 5 minutes, add 1 ml of ultrapure water to quench the reaction, add 1 ml of reference stock solution, dilute to the scale with solvent 4, shake well, and obtain.

[0114] Chromatographic conditions:

[0115] project condition Mobile phase 1A 0.2% trifluoroacetic acid aqueous solution (volume percentage concentration) Mobile phase 1B Acetonitrile Flow rate 1.0ml / min Injection volume 10μl Sample tray temperature No temperature control Chromatographic columns YMC Triart C18, 4.6×150mm, 3μm Solvent Effect Eliminator yes Column temperature 40℃ Detector CAD CAD flow rate On CAD atomizer temperature 40℃ Needle wash solution Acetonitrile

[0116] Elution procedure:

[0117] Time (min) 0 15 16 26 27 42 Mobile phase A (%) 100 100 20 20 100 100 Mobile phase B (%) 0 0 80 80 0 0

[0118] Recovery rate calculation formula:

[0119]

[0120] M std : The amount of reference substance in the 100% spiked test sample derivative solution (mg); P: The content of the reference substance, calculated as 100%; V std : Dilution multiple of reference substance in 100% spiked test substance derivative solution; A 标 : Average peak area of ​​reference substance in 100% spiked test substance derivatization solution; m 标: The amount of the test sample in the 100% spiked test sample derivatization solution, mg; V 标 : dilution volume of the test sample in the 100% spiked test sample derivatization solution, ml; V s : dilution multiple of the test sample derivative solution; A s : peak area of ​​target peak in the test sample derivatization solution; m s : Test sample weight (mg).

[0121] Calculate the recovery rate by standard addition method:

[0122]

[0123] When the carboxylic acid content in the test sample was between 1% and 10%, the recovery rate ranged from 97% to 105%, and the RSDs from the 1% level to the 10% level were 2.0%, 1.3%, and 1.0%, respectively.

[0124] Example 5 Derivatization Reagent Screening

[0125] Alternative derivatization reagents: aniline, p-methoxyaniline, benzyl alcohol, phenol, methanol.

[0126] Solution preparation:

[0127] Solvent 5: acetonitrile-water (volume ratio 3:1).

[0128] Reference substance stock solution 5: Accurately weigh 200 mg of reference substance 1,1-dioxo-hexahydro-1L6-thiopyran-4-carboxylic acid, place it in a 20 ml volumetric flask, dissolve it with solvent 5, make up to the mark, shake well, and obtain (containing 10 mg / ml of reference substance).

[0129] Reference substance solution 5-aniline: Pipette 1 ml of reference substance stock solution 5 into a 10 ml volumetric flask, add 1 ml of aniline, shake, let stand for 5 minutes, then dilute to the mark with solvent 5, shake well, and you have the solution (containing reference substance 1 mg / ml).

[0130] Reference substance solution 5-benzyl alcohol: Pipette 1 ml of reference substance stock solution 5 into a 10 ml volumetric flask, add 1 ml of benzyl alcohol, shake, let stand for 5 minutes, then dilute to the mark with solvent 5, shake well, and you have the solution (containing 1 mg / ml of reference substance).

[0131] Reference substance solution 5-methanol: Pipette 1 ml of reference substance stock solution 5 into a 10 ml volumetric flask, add 1 ml of methanol, shake, let stand for 5 minutes, then dilute to the mark with solvent 5, shake well, and you have the solution (containing reference substance 1 mg / ml).

[0132] Reference substance solution 5-phenol: Pipette 1 ml of reference substance stock solution 5 into a 10 ml volumetric flask, add 1 g of phenol, shake, let stand for 5 minutes, then dilute to the mark with solvent 5, shake well, and you have the solution (containing reference substance 1 mg / ml).

[0133] Reference substance solution 5-p-methoxyaniline: Pipette 1 ml of reference substance stock solution 5 into a 10 ml volumetric flask, add 1 g of p-methoxyaniline, shake, let stand for 5 minutes, then dilute to the mark with solvent 5, shake well, and you have the solution (containing 1 mg / ml of reference substance).

[0134] Test solution 5: Take 0.5 ml of 1,1-dioxo-hexahydro-1L6-thiopyran-4-carbonyl chloride solution (concentration of 80 mg / ml, solvent is THF), place in a 10 ml volumetric flask, add 0.5 ml of aniline (or 0.5 g of phenol, 0.5 ml of benzyl alcohol, 0.5 g of p-methoxyaniline, 0.5 ml of methanol), add solvent to dissolve and dilute to scale, ultrasonicate for 5 min, shake well (containing 4 mg / ml of 1,1-dioxo-hexahydro-1L6-thiopyran-4-carbonyl chloride). Mark them as test solution 5-aniline, test solution 5-phenol, test solution 5-benzyl alcohol, test solution 5-p-methoxyaniline and test solution 5-methanol respectively.

[0135] Chromatographic conditions:

[0136] project condition Mobile phase 5A Mobile phase 5A: 0.2% trifluoroacetic acid in water (volume percentage concentration) Mobile phase 5B Mobile phase 5B: acetonitrile Flow rate 1.0ml / min Injection volume 10μl Sample tray temperature No temperature control Chromatographic columns YMC Triart C18 4.6×150mm, 3μm Column temperature 40℃ Detector CAD CAD setting temperature 40℃ Needle wash solution Acetonitrile

[0137] Elution procedure:

[0138] Time / min 0 15 16 26 27 42 A / ％ 100 100 20 20 100 100 B / ％ 0 0 80 80 0 0

[0139] The chromatograms of each sample are shown in Figure 10-19 As shown, Figure 10-19 These are the results of derivatization using different derivatization reagents, corresponding to aniline, p-methoxyaniline, benzyl alcohol, phenol, and methanol. A peak of carboxylic acid can be observed around 8 min, with no obvious impurity interference on either side. Using the CAD detector, there are more optional derivatization reagents with less interference.

[0140] Example 6 Detector RID (Differential Refractive Index Detector)

[0141] Solution preparation:

[0142] Solvent 6: acetonitrile-water (volume ratio 3:7).

[0143] Reference substance stock solution 6: Accurately weigh 200 mg of reference substance 1,1-dioxo-hexahydro-1L6-thiopyran-4-carboxylic acid, place it in a 20 ml volumetric flask, dissolve it with solvent 6, make up to the mark, shake well, and obtain (reference substance concentration 10 mg / ml).

[0144] Reference solution 6-aniline: Pipette a total of 1 ml of reference stock solution 6 into a 10 ml volumetric flask, add 1 ml of aniline, shake, let stand for 5 minutes, then dilute to the mark with solvent 6, shake well, and you have (reference concentration 1 mg / ml).

[0145] Chromatographic conditions:

[0146]

[0147] Elution procedure:

[0148] Time / min 0 15 16 26 27 42 A / ％ 100 100 20 20 100 100 B / ％ 0 0 80 80 0 0

[0149] The chromatogram of the reference solution 6-aniline is as follows Fig. 20 As shown, the peak of carboxylic acid is at 12.230 min. There are still interferences from unknown peaks before and after the peak of carboxylic acid in the figure, and the baseline is raised, indicating that the matrix effect brought by the derivatization reagent cannot be avoided under the conditions of the differential detector, which has a certain impact on the detection of carboxylic acid.

[0150] The invention concept is described in detail using specific examples herein, and the description of the above embodiments is only used to help understand the core idea of ​​the invention. It should be pointed out that any obvious modification, equivalent substitution or other improvement made by a person of ordinary skill in the art without departing from the invention concept should be included in the protection scope of the present invention.

Claims

1. A method for detecting a reaction substrate of an amoenavir intermediate, characterized in that: First, a derivatization reagent is added to the test solution of the amonetvir intermediate to carry out a derivatization reaction, wherein the derivatization reagent is aniline or p-methoxyaniline, so that the reaction substrate 1,1-dioxo-hexahydro-1L6-thiopyran-4-carbonyl chloride in the test solution is converted into a corresponding derivative compound, and then water is added to terminate the reaction, and the content of the reaction substrate 1,1-dioxo-hexahydro-1L6-thiopyran-4-carbonyl chloride is calculated by detecting the content of the remaining 1,1-dioxo-hexahydro-1L6-thiopyran-4-carboxylic acid; The test solution and the reference solution were diluted with acetonitrile-water solution; The chromatographic conditions of the electrospray chromatograph were: Chromatographic column: YMC Triart C18 column; Mobile phase A: 0.2% trifluoroacetic acid in water; Mobile phase B: acetonitrile; Gradient elution program:

2. The detection method according to claim 1, characterized in that: The residual 1,1-dioxo-hexahydro-1L6-thiopyran-4-carboxylic acid content was detected using a liquid chromatograph, a reference solution was prepared, the reference solution and the test solution were injected into the liquid chromatograph respectively, the chromatogram was recorded, and the 1,1-dioxo-hexahydro-1L6-thiopyran-4-carboxylic acid content in the test sample was calculated by the external standard method based on the concentration of the reference solution.

3. The detection method according to claim 1, characterized in that: The chromatographic conditions also include: Column temperature: 40°C; Injection volume: 10 μl; Flow rate: 1.0ml / min; CAD atomizer temperature: 40℃.

4. The detection method according to claim 1, characterized in that: The specifications of the chromatographic column are: 4.6×150 mm, 3 μm.

5. The detection method according to claim 1, characterized in that: The derivatization reagent is aniline. After the derivatization reagent is added, acetonitrile is added as a solvent, and then ultrasonic dispersion is used for dissolution, and finally water is added for dilution.

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