CN112730659B

CN112730659B - Method for detecting related substances of Reidesciclovir intermediate

Info

Publication number: CN112730659B
Application number: CN202011516941.2A
Authority: CN
Inventors: 马燕; 罗锋林
Original assignee: Jiangsu Zenji Pharmaceuticals Ltd
Current assignee: Jiangsu Zenji Pharmaceuticals Ltd
Priority date: 2020-12-21
Filing date: 2020-12-21
Publication date: 2022-05-27
Anticipated expiration: 2040-12-21
Also published as: CN112730659A

Abstract

The invention discloses a method for detecting related substances of a Reidesciclovir intermediate, wherein the Reidesciclovir intermediate has a structure shown in a formula I, and the method comprises the following steps: (1) preparing a reference substance and a test solution; (2) and (3) detection: adopting high performance liquid chromatography, wherein the mobile phase A is 0.05-0.5% of acid buffer solution, the mobile phase B is organic solvent, the detection wavelength is 220-240 nm, and the elution gradient mobile phase A is calculated by volume ratio for 0 min: 100% → 90%, 0-15 min: 95% → 50%, 15 to 30 minutes: 50% → 0%, 30 to 50 minutes: 100% → 90%; (3) and (3) calculating the content of the related substances in the detection map in the step (2). The detection method establishes a specific detection method of the Rudexiluwei intermediate, can accurately detect the content of each related substance in the Rudexiluwei intermediate, and has good specificity, sensitivity, repeatability and stability.

Description

Method for detecting related substances of Reidesciclovir intermediate

Technical Field

The invention relates to a detection method of related substances of a Reidesciclovir intermediate, in particular to a detection method of related substances of a Reidesciclovir intermediate, which can accurately detect the content of each related substance in the Reidesciclovir intermediate.

Background

Reddesivir (Remdesivir) was developed by Gilidd science, a nucleoside analog with antiviral activity, EC against SARS-CoV and MERS-CoV in HAE cells₅₀Value of 74nM, EC against murine hepatitis virus in delayed brain tumor cells₅₀The value was 30 nM. The chemical name of the compound is (S) -2- (((S) - ((((2R, 3S,4R,5R) -5- (4-aminopyrrolo [2, 1-f)][1,2,4]Triazin-7-yl) -5-cyano-3,4-dihydroxytetrahydrofuran-2-yl) methoxy) (phenoxy) phosphorusAcyl) amino) propionic acid 2-ethylbutyl ester, known as (S) -2-ethylbutylyl-2- (((S) - ((2R,3S,4R,5R) -5- (4-aminopyrorrolo- [2, 1-f)][1,2,4]-triazin-7-yl) -5-cyanoo-3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) (phenoxy) phosphoryl) amino) propanoate having the CAS number: 1809249-37-3, molecular formula C₂₇H₃₅N₆O₈P, molecular weight: 602.58, the chemical structure is:

the united states biopharmaceutical company girelide board president and president performed official Daniel europay (Daniel O' Day) on 9/10/2020, and data from a three-phase trial of the drug rdsievir against the new corona virus has been published in the well-known medical journal new england. Phase three clinical data is described from a biorandomized, double blind placebo-controlled phase three study conducted by the national institute for allergy and infectious disease, covering approximately 1060 hospitalized patients worldwide. The clinical data of the three-phase Reidesciclovir shows that the average recovery time of the patients receiving Reidesciclovir treatment is 5 days faster than that of the patients treated by other medicines, and the recovery time of the patients with serious illness accounting for 85 percent of the total study number is 7 days faster; in addition, Reidesciclovir reduces the likelihood of a patient developing a more severe stage of the disease, and the largest group of patients in clinical trials, patients who use low flow oxygen due to severe illness, have reduced mortality rates after Reidesciclovir.

The chemical name of the Ruidesacvir intermediate is (2R,3R,4S,5R) -2- (4-aminopyrrole- [1, 2-f)][1,2,4]-triazin-7-yl) -3, 4-dihydroxy-5- (methoxy) tetrahydrofuran-2-carbonitrile having the CAS number: 1191237-69-0, formula: c₁₂H₁₃N₅O₄Molecular weight: 291.27, the chemical structure is:

as a key intermediate for synthesizing the raw material of the Reidesvir, impurities contained in the Reidesvir inevitably can be brought into a Reidesvir product in a synthesis reaction, and the quality, safety and effectiveness of the Reidesvir product are directly influenced. Therefore, if impurities in the starting material of the ridciclovir can be detected or controlled, the preparation of the ridciclovir is more beneficial. At present, no method for detecting substances related to the intermediates is available.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to provide a detection method of related substances of a Redcisvir intermediate, which has good specificity, sensitivity, repeatability and stability.

The technical scheme is as follows: the detection method of the related substances of the Reidesciclovir intermediate comprises the following steps:

(1) preparing a reference substance and a test solution;

(2) and (3) detection: adopting high performance liquid chromatography, wherein the mobile phase A is 0.05-0.5% of acid buffer solution, the mobile phase B is organic solvent, the detection wavelength is 220-240 nm, and the volume ratio of the mobile phase A in the elution gradient is 0 min: 100% → 90%, 0-15 min: 95% → 50%, 15 to 30 minutes: 50% → 0%, 30 to 50 minutes: 100% → 90%;

(3) and (3) calculating the content of the related substances in the detection map in the step (2).

The high performance liquid detection method adopts a diode array detector, and the used reference substances are as follows:

the preparation method of the test solution comprises the following steps:

precisely weighing a proper amount of a test sample, placing the test sample into a measuring flask, precisely adding 10% of methanol in the final volume, adding 85% of phosphoric acid in the volume of 5% of methanol, dissolving, diluting to scale with the mobile phase A, and shaking up to prepare a test sample solution of 0.25 mg/ml.

The preparation method of each reference substance solution comprises the following steps:

(1) intermediate control solution

The preparation method of the test solution is the same as that of the test solution.

(2) Reference substance solution of each related substance

Precisely weighing a proper amount of a sample, placing the sample in a measuring flask, precisely adding 10% methanol in final volume, adding 85% phosphoric acid in 5% methanol volume, dissolving, diluting with mobile phase A to scale, and shaking up to obtain 0.25mg/ml impurity stock solutions; precisely transferring each impurity stock solution, placing into a measuring flask, adding diluent to dilute to scale, shaking up, and making into 6 μ g/ml impurity reference substance solution.

The preparation method of the system applicability solution comprises the following steps:

precisely transferring the impurity stock solutions, diluting with the mobile phase A to a constant volume to scale, shaking up, and obtaining a system applicability solution of 6 mu g/ml.

Preferably, the concentration of the test solution in the step (1) is 0.1-1.0 mg/ml.

Preferably, the detection wavelength in the step (2) is 220-238 nm.

Preferably, in the step (2), the mobile phase A is 0.1-0.5% of acid buffer solution, and the mobile phase B is methanol. Further preferably, the mobile phase a in step (2) is one or more of aqueous trifluoroacetic acid solution, aqueous formic acid solution, aqueous phosphoric acid solution and aqueous perchloric acid solution. More preferably, the mobile phase A in the step (2) is 0.1-0.5% formic acid solution.

Preferably, in the elution gradient in step (2), the ratio of mobile phase a to mobile phase a is measured by volume ratio, 0 min: 95%, 0-15 min: 95% → 50%, 15 to 30 minutes: 50% → 5%, 30 to 40 minutes: 95 percent.

Preferably, the chromatographic column in the step (2) is an alkylsilane bonded silica chromatographic column, and the flow rate is 0.8-1.2 ml/min, preferably 1.0 ml/min; the column temperature is 30-35 ℃; the concentration of the sample is preferably 0.25mg/ml, and the sample amount of the sample is preferably 5. mu.l. More preferably, the chromatographic column is a C18 chromatographic column, a C8 chromatographic column, a phenyl column, a cyano column or an amino column, and the length of the column is 150-250 mm, more preferably 250 mm; the granularity of the chromatographic column packing is 3.0-5.0 mu m, and more preferably 5.0 mu m; the diameter of the chromatographic column is 2.0-4.6 mm, and the preferred diameter is 4.6 mm.

Preferably, the content of the related substances in the step (3) is calculated according to an area normalization method.

Has the advantages that: compared with the prior art, the invention has the following remarkable advantages:

(1) a detection method for related substances related to the Reidesciclovir intermediate is established, and the quality condition of the Reidesciclovir intermediate can be accurately and effectively reflected, so that the quality of the subsequently prepared Reidesciclovir bulk drug can be effectively controlled;

(2) the method is simple and convenient to operate, can accurately detect the content of each impurity in the Rudexiliwei intermediate, and has good specificity (the separation degree between each impurity and a main component is not less than 1.5), sensitivity (the detection line sensitivity reaches 0.02 percent, the quantitative limit sensitivity reaches 0.05 percent), repeatability (the content of each impurity RSD which is less than 0.5 percent is not more than 10.0 percent, the content of each impurity RSD which is 0.5 to 2.0 percent is not more than 5.0 percent, the purity and the total impurity RSD are not more than 2.0 percent) and stability (a sample is stable within 24 hours at room temperature);

(3) the method has strong adaptability, can be used for quality control of the whole preparation process of the RedeWest and can also be used for quality control of the RedeWest bulk drug.

Drawings

FIG. 1 is an HPLC chromatogram of the applicability of the system of the present application;

FIG. 2 is an HPLC chromatogram of a test article of the present application;

the individual labeled chromatographic peaks in FIG. 1: 1. impurity a, 2, impurity b, 3, Reidcisvir intermediate, 4, impurity c, 5, impurity d, 6, impurity e, 7, impurity f, 8, impurity g, 9 and impurity h.

Detailed Description

The technical solution of the present invention is further explained below with reference to the examples and the accompanying drawings.

Example 1: specificity test

1. Detecting chromatographic conditions

A chromatographic column: c18(250 mm. times.4.6 mm, 5 μm);

a detector: a Diode Array Detector (DAD);

detection wavelength: 238 nm;

flow rate: 1.0 ml/min;

column temperature: 30 ℃;

diluent agent: 0.1% phosphoric acid water-methanol (95: 5);

sample introduction volume: 5 μ l.

2. Preparation of reference substance and test solution

The specificity test requires verification that the blank solution is non-interfering at the time of retention of the main peak in the test and control solutions, and that the degree of separation between impurities and the main component.

The preparation method of each impurity and main component is as follows:

impurity a localization solution: accurately weighing 15mg of the impurity a reference substance, putting the reference substance into a 50ml measuring flask, adding 5ml of methanol, adding 0.25ml of 85% phosphoric acid, performing ultrasonic dissolution, diluting with a mobile phase A to a constant volume to scale, and shaking up to obtain an impurity a stock solution; precisely transferring 1.0ml of the impurity a stock solution, putting the stock solution into a 50ml measuring flask, adding a diluent to dilute the stock solution to a scale, and shaking up the stock solution to be used as an impurity a positioning solution.

Impurity b localization solution: accurately weighing 15mg of the reference substance of the impurity b, putting the reference substance into a 50ml measuring flask, adding 5ml of methanol, adding 0.25ml of 85% phosphoric acid, performing ultrasonic dissolution, diluting with the mobile phase A to a constant volume to scale, and shaking up to obtain a stock solution of the impurity b; precisely transferring 1.0ml of the impurity b stock solution, putting the impurity b stock solution into a 50ml measuring flask, adding a diluent to dilute the impurity b stock solution to a scale, and shaking up the impurity b stock solution to be used as an impurity b positioning solution.

Intermediate positioning solution: accurately weighing 12.5mg of intermediate reference substance, placing in a 50ml measuring flask, adding 5ml of methanol, adding 0.25ml of 85% phosphoric acid, dissolving by ultrasonic treatment, diluting with mobile phase A to constant volume, and shaking to obtain intermediate positioning solution.

Impurity c localization solution: accurately weighing 15mg of the impurity c reference substance, putting the reference substance in a 50ml measuring flask, adding 5ml of methanol, adding 0.25ml of 85% phosphoric acid, performing ultrasonic dissolution, diluting with the mobile phase A to a constant volume to scale, and shaking up to obtain an impurity c stock solution; precisely transferring 1.0ml of impurity c stock solution, placing the stock solution in a 50ml measuring flask, adding a diluent to dilute the stock solution to a scale, and shaking up the stock solution to be used as impurity c positioning solution.

Impurity d localization solution: accurately weighing 15mg of the impurity d reference substance, putting the reference substance in a 50ml measuring flask, adding 5ml of methanol, adding 0.25ml of 85% phosphoric acid, performing ultrasonic dissolution, diluting with the mobile phase A to a constant volume to scale, and shaking up to obtain an impurity d stock solution; precisely transferring 1.0ml of the impurity d stock solution, putting the stock solution into a 50ml measuring flask, adding a diluent to dilute the stock solution to a scale, and shaking the stock solution uniformly to obtain an impurity d positioning solution.

Impurity e localization solution: accurately weighing 15mg of an impurity e reference substance, putting the reference substance into a 50ml measuring flask, adding 5ml of methanol, adding 0.25ml of 85% phosphoric acid, performing ultrasonic dissolution, diluting with a mobile phase A to a constant volume to scale, and shaking up to obtain an impurity e stock solution; precisely transferring 1.0ml of impurity e stock solution, placing the stock solution in a 50ml measuring flask, adding a diluent to dilute the stock solution to a scale, and shaking up the stock solution to be used as impurity e positioning solution.

Impurity f localization solution: precisely weighing 15mg of the impurity f reference substance, putting the reference substance in a 50ml measuring flask, adding 5ml of methanol, adding 0.25ml of 85% phosphoric acid, dissolving by ultrasonic, diluting with the mobile phase A to a constant volume to scale, and shaking up to obtain an impurity f stock solution; precisely transferring 1.0ml of the impurity f stock solution, putting the impurity f stock solution into a 50ml measuring flask, adding a diluent to dilute the impurity f stock solution to a scale, and shaking up the impurity f stock solution to be used as an impurity f positioning solution.

Impurity g localization solution: accurately weighing 15mg of an impurity g reference substance, putting the reference substance in a 50ml measuring flask, adding 5ml of methanol, adding 0.25ml of 85% phosphoric acid, performing ultrasonic dissolution, diluting with a mobile phase A to a constant volume to scale, and shaking up to obtain an impurity g stock solution; precisely transferring 1.0ml of the impurity g stock solution, putting the impurity g stock solution into a 50ml measuring flask, adding a diluent to dilute the impurity g stock solution to a scale, and shaking the impurity g stock solution uniformly to serve as an impurity g positioning solution.

Impurity h positioning solution: accurately weighing 15mg of impurity h reference substance, placing in a 50ml measuring flask, adding 5ml of methanol, adding 0.25ml of 85% phosphoric acid, diluting with methanol to constant volume, shaking up to obtain impurity h stock solution; precisely transferring 1.0ml of the impurity h stock solution, placing the impurity h stock solution in a 50ml measuring flask, adding a diluent to dilute the impurity h stock solution to a scale, and shaking up to obtain an impurity h positioning solution.

Blank solution: precisely measuring 5ml of methanol, placing the methanol into a 50ml measuring flask, adding 0.25ml of 85% phosphoric acid, diluting to scale with the mobile phase A, and shaking up.

System applicability solution: accurately weighing 12.5mg of a Reidesciclovir intermediate reference substance, putting the reference substance into a 50ml measuring flask, adding 5ml of methanol, adding 0.25ml of 85% phosphoric acid, dissolving by ultrasonic, accurately transferring 1ml of each impurity stock solution, diluting with a mobile phase A to a constant volume to scale, shaking up, and using the solution as a system applicability solution.

Test solution: weighing the product about 12.5mg, placing the product in a 50ml measuring flask, firstly adding 5ml of methanol and then 0.25ml of 85% phosphoric acid, carrying out ultrasonic dissolution, diluting with the mobile phase A to a constant volume to scale, and shaking up.

With reference to the detection chromatographic conditions, precisely measuring 5 μ l of each solution, injecting into a liquid chromatograph, and recording chromatogram, the results are shown in table 1 and fig. 1-2.

TABLE 1 results of the specificity test

As can be seen from Table 1 and FIGS. 1 to 2, the blank solution was not interfered at the retention time of the main peak in the test and control solutions; the separation degree between the impurities and the main components is more than or equal to 1.5, which indicates that the specificity of the detection method meets the quality control requirement.

Example 2: sensitivity test

Taking the system applicability solution prepared in the embodiment 1, gradually diluting to a proper multiple, and taking the solution with the signal-to-noise ratio of more than or equal to 10: 1 as a quantitative limiting solution; taking the solution with the signal-to-noise ratio of more than or equal to 3: 1 as the detection limit solution.

With reference to the detection chromatographic conditions of example 1, 5. mu.l of each of the above solutions was precisely measured, and the solutions were injected into a liquid chromatograph, and a sample of a quantitative limiting solution was continuously injected into 6 needles, and a sample of a detection limiting solution was injected into 1 needle, and chromatograms were recorded, and the results are shown in tables 2 to 3.

TABLE 2 quantitative Limit verification results

Name (R)	Concentration of μ g/ml	S/N	Sensitivity (%)
				Impurity a	0.11708	24.68	0.05
Impurity b	0.11746	38.48	0.05
				Intermediate product	0.12240	21.07	0.05
Impurity c	0.11109	15.98	0.05
				Impurity d	0.11211	27.9	0.05
Impurity e	0.11940	14.0	0.05
				Impurity f	0.11341	17.0	0.05
Impurity g	0.12099	11.6	0.05

TABLE 3 detection Limit verification results

Name (R)	Concentration of μ g/ml	S/N	Sensitivity (%)
				Impurity a	0.05854	14.2	0.02
Impurity b	0.05873	22.9	0.02
				Intermediates	0.06120	13.0	0.02
Impurity c	0.05554	9.9	0.02
				Impurity d	0.05606	24.8	0.02
Impurity e	0.05970	7.5	0.02
				Impurity f	0.05671	10.5	0.02
Impurity g	0.06050	6.4	0.02

As can be seen from tables 2 to 3, the detection limit sensitivity of the detection method of the present application is 0.02%, and the quantitative limit sensitivity is 0.05%, which indicates that the specificity of the detection method of the present application meets the quality control requirements.

Example 3: repeatability test

Test solution: weighing about 12.5mg of a sample, placing the sample in a 50ml measuring flask, adding 5ml of methanol, adding 0.25ml of 85% phosphoric acid, performing ultrasonic dissolution, diluting with a mobile phase A to a constant volume to scale, shaking up, and preparing 6 parts in parallel.

With reference to the conditions of detection chromatography in example 1, 5. mu.l each of the above solutions was measured precisely, and the solution was injected into a liquid chromatograph, and the chromatogram was recorded, and the results are shown in Table 4.

TABLE 4 results of repeated investigation

Note: "ND" means not detected.

As can be seen from Table 4, the number of impurity peaks above the quantitative limit is kept unchanged, RSD of single impurity content and purity both meet the requirements (RSD of each impurity with content less than 0.5% is not more than 10.0%, RSD of each impurity with content 0.5% -2.0% is not more than 5.0%, purity and total RSD are not more than 2.0%), which indicates that the repeatability of the detection method meets the quality control requirement.

Example 4: stability test of solution

The system suitability solution and the sample solution prepared in example 1 were measured by a precision measuring method under the conditions of the detection chromatography in example 1, and the measured solutions were injected into a liquid chromatograph, followed by recording the chromatogram, and the results are shown in Table 5.

TABLE 5 test article solution stability investigation results

Note: "ND" means not detected.

As can be seen from Table 5, the ratio of the peak area of each impurity to the peak area at 0 hour at different time points in 24 hours of the test solution at room temperature is 80.0-120.0%, which indicates that the stability of the detection method of the present application meets the quality control requirements.

Claims

1. A method for detecting related substances of a Reidesciclovir intermediate is characterized in that the method is used for detecting the following Reidesciclovir intermediate and impurities,

the detection method comprises the following steps:

(1) preparing a reference substance and a test solution;

(2) and (3) detection: adopting high performance liquid chromatography, wherein the chromatographic column is a C18 chromatographic column, the mobile phase A is 0.1-0.5% formic acid solution, the mobile phase B is methanol, the detection wavelength is 220-240 nm, and the mobile phase A in the elution gradient is counted by volume ratio for 0 min: 95%, 0-15 min: 95% → 50%, 15 to 30 minutes: 50% → 5%, 30 to 40 minutes: 95 percent;

2. The detection method according to claim 1, wherein the concentration of the sample solution in the step (1) is 0.1 to 1.0 mg/ml.

3. The detection method according to claim 1, wherein the detection wavelength in the step (2) is 220 to 238 nm.

4. The detection method according to claim 1, wherein the flow rate of the mobile phase detected in the step (2) is 0.8-1.2 mL/min, and the column temperature is 30-35 ℃.

5. The detection method according to claim 1, wherein the length of the chromatographic column in the step (2) is 150-250 mm, the particle size of the chromatographic column packing is 3.0-5.0 μm, and the diameter of the chromatographic column is 2.0-4.6 mm.

6. The detection method according to claim 1, wherein the content of the substance of interest in the step (3) is calculated by an area normalization method.