A method for separating and determining easily hydrolyzed large polar compounds

Abstract

The present invention discloses a method for separating and measuring easily hydrolyzed polar compounds, comprising the following steps: dissolving acyclovir in a diluent to form a sample solution, and using high performance liquid chromatography to detect the standard solution and the sample solution; liquid chromatography conditions: chromatographic column: hybrid silica gel column; mobile phase A: buffer solution; mobile phase B: acetonitrile; isocratic elution; mobile phase flow rate: 0.4-0.6 ml / min; column temperature: 25-35°C; detection wavelength: UV254nm; injection volume: 20 μl; running time: 50 min. The present invention can inhibit the hydrolysis of easily hydrolyzed polar compound impurities during the injection process, the real reaction impurity detection amount, high precision, room temperature operation, good reproducibility, and can effectively separate and measure the content of easily hydrolyzed polar compounds.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, in particular to a method for separating and determining easily hydrolyzed highly polar compounds, and specifically to a method for separating and determining the impurity content of easily hydrolyzed highly polar compounds in acyclovir. Background Art

[0002] Acyclovir is an antiviral drug.

[0003] Chemical structure:

[0004]

[0005] Acyclovir is a synthetic purine nucleoside analog. It is mainly used for various infections caused by herpes simplex virus, and can be used for primary or recurrent skin, mucosal, external genital infections and HSV infections in immunocompromised people. It is the first choice for the treatment of HSV encephalitis, and is superior to adenosine in reducing morbidity and mortality. It can also be used for infections such as herpes zoster, Epstein-Barr virus, and chickenpox in immunocompromised people. It is only used topically on the skin, and acyclovir is less absorbed by the skin.

[0006] In the quality control of acyclovir, some impurities are extremely unstable and can be rapidly hydrolyzed in water, weak acid or weak base systems. For example, diacetylguanine (impurity A) can be rapidly hydrolyzed in water to remove the acyl group and become monoacetylguanine (impurity B) and guanine; therefore, the content of diacetylguanine, monoacetylguanine and guanine in acyclovir cannot be truly reflected during the detection process.

[0007] Chinese patent CN202010731803.X reports that the ultraviolet dual-wavelength absorbance ratio detection method can be used to quickly and accurately control the reaction endpoint and finished product quality by comparing qualified finished products. The ultraviolet spectrophotometry method has poor specificity and cannot accurately quantify the content of monoacetylguanine in the reaction solution and diacetylguanine.

[0008] Da Yuanfeng et al. reported that the ultra-high pressure liquid chromatography (UPLC) method was used to determine the purity of diacetylguanine (Da Yuanfeng, Lu Chunyan, Mo Wenjuan, Research on the Detection Method of N-2,9-Diacetylguanine, International Medical and Health Guide, Vol. 19, 2013, 1675-1677). Due to the high column efficiency of UPLC, the short chromatographic column and the short analysis time, the retention time of monoacetylguanine and diacetylguanine was only 1.1 minutes and 2.0 minutes, which can achieve better separation. Due to the fast analysis speed of ultra-high pressure liquid chromatography, hydrolysis can be suppressed. At the same time, we also repeated the article using HPLC detection, but the retention time of diacetylguanine was 2 minutes, monoacetylguanine and diacetylguanine co-flowed, and the chromatographic peaks of diacetylguanine and diacetylguanine completely overlapped, and only one chromatographic peak appeared in the liquid chromatogram, indicating that under the conditions of the reference, diacetylguanine and monoacetylguanine were completely separated.

[0009] Therefore, there is an urgent need for a high performance liquid chromatography method for separating and determining impurities in acyclovir that can solve these problems, inhibit the degradation of easily hydrolyzed large polar compound impurities in acyclovir, and completely separate related impurities and has simple operation and good reproducibility. Summary of the invention

[0010] Since ultra-high pressure liquid chromatographs are expensive, most pharmaceutical companies are not equipped with this equipment. Therefore, the method for analyzing easily hydrolyzed polar compounds through ordinary HPLC disclosed in this patent has certain practicality. At the same time, the difference between HILIC chromatographic columns and ordinary C-18 chromatographic columns is cleverly utilized. When the HILIC chromatographic column has a large proportion of organic phase, the hydrolysis of diacetylguanine in the chromatographic analysis process is reduced, and at the same time, the retention of highly polar compounds is strong, which solves the problem of instability and inability to quantify diacetylguanine encountered in the analysis process. This method is innovative.

[0011] This method is also applicable to the detection of EP impurity Q in acyclovir finished products. The European Pharmacopoeia 10.0 (EP10.0) analysis method was used to detect impurity Q in acyclovir finished products. It was found that impurity Q was hydrolyzed under the chromatographic conditions to produce acyclovir. The accuracy in the validation of the analysis method could not meet the regulatory requirements, indicating that the EP10.0 analysis method could not accurately quantify the content of impurity Q in acyclovir finished products. The above analysis method, which can be used to detect a class of easily hydrolyzed large polar compounds, can well suppress the hydrolysis problem of impurity Q in liquid chromatography analysis.

[0012] In order to solve the above technical problems, the present invention provides a method for separating and determining easily hydrolyzed large polar compounds, comprising the following steps:

[0013] The sample containing the easily hydrolyzed polar compound is dissolved in a diluent to form a sample solution, and the standard solution and the sample solution are tested by high performance liquid chromatography;

[0014] Wherein, the diluent is 10% dimethyl sulfoxide acetonitrile solution;

[0015] Liquid chromatography conditions:

[0016] Chromatographic column: hybrid silica gel column; mobile phase A: trifluoroacetic acid solution; mobile phase B: acetonitrile; isocratic elution; mobile phase flow rate: 0.4-0.6 ml / min; column temperature: 25-35°C; detection wavelength: UV254 nm; injection volume: 20 μl; running time: 50 min.

[0017] As a preferred embodiment of the above technical solution, the method for separating and determining easily hydrolyzed large polar compounds provided by the present invention further includes some or all of the following technical features:

[0018] As an improvement of the above technical solution, the sample containing the easily hydrolyzed large polar compound is acyclovir or diacetylguanine.

[0019] As an improvement of the above technical solution, the sample containing the easily hydrolyzed polar compound is acyclovir, which contains impurities A, B and Q; the specific names and chemical structures of the above compounds are as follows:

[0020] Impurity A is diacetylguanine, with the structural formula

[0021] Impurity B is monoacetylguanine, with the structural formula

[0022] Impurity Q is acyclovir EP impurity Q, with the structural formula

[0023] Acyclovir, the structural formula is:

[0024] As an improvement of the above technical solution, in the method for separating and determining easily hydrolyzed large polar compounds, the following solutions need to be prepared:

[0025] Impurity B stock solution: Take 10 mg of impurity B, dissolve it in a 100 ml volumetric flask with 10% dimethyl sulfoxide acetonitrile solution and dilute to the mark, shake well;

[0026] Mixed standard solution: Take 10 mg of diacetylguanine sample and place it in a 100 ml volumetric flask, add 0.5 ml of impurity B stock solution, dissolve it with 10% dimethyl sulfoxide acetonitrile solution and dilute to the scale, shake well;

[0027] Sample solution: Take 10 mg of diacetylguanine sample, put it in a 100 ml volumetric flask, dissolve it with 10% dimethyl sulfoxide acetonitrile solution and dilute to the scale, shake well;

[0028] Blank solution: 10% dimethyl sulfoxide in acetonitrile.

[0029] As an improvement of the above technical solution, the isocratic elution is specifically performed according to the following volume ratio: 96-94% A+4-6% B.

[0030] As an improvement of the above technical solution, the isocratic elution is specifically performed according to the following volume ratio: 95% A+5% B.

[0031] As an improvement of the above technical solution, the hybrid silica gel column is a bonded dihydroxypropyl hybrid silica gel column.

[0032] As an improvement of the above technical solution, the pH value of the buffer solution is 3.0-4.0.

[0033] As an improvement of the above technical solution, the pH value of the buffer solution is adjusted by trifluoroacetic acid.

[0034] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: the method provided by the patent can solve these problems, can inhibit the degradation of easily hydrolyzed large polar compound impurities, truly reflect the content of easily hydrolyzed large polar compounds, and the method is simple, can completely separate acyclovir and impurities, has high precision, operates at room temperature, has good solution reproducibility, and has cheap reagents.

[0035] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented in accordance with the contents of the specification. In order to make the above and other purposes, features and advantages of the present invention more obvious and easy to understand, the following is a detailed description in combination with the preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the drawings of the embodiment are briefly introduced below.

[0037] Figure 1 The liquid chromatogram of the blank solution prepared in Example 1;

[0038] Figure 2 The liquid chromatography analysis spectrum of the mixed standard solution of Example 1;

[0039] Figure 3 The linear spectrum of impurity A of the linear solution prepared in Example 1;

[0040] Figure 4 The linear spectrum of impurity B of the linear solution prepared in Example 1;

[0041] Figure 5 The mixed solution spectrum prepared in Example 2;

[0042] Figure 6 This is the sample solution spectrum prepared in Example 2. DETAILED DESCRIPTION

[0043] The specific implementation modes of the present invention are described in detail below, which is a part of this specification. The principles of the present invention are explained through examples. Other aspects, features and advantages of the present invention will become clear through the detailed description.

[0044] Example 1

[0045] 1) Preparation of solution:

[0046] Blank solution: 10% dimethyl sulfoxide acetonitrile solution;

[0047] Impurity A stock solution: Weigh 20 mg of impurity A reference substance into a 100 ml volumetric flask, accurately weigh, dissolve and dilute to the mark with DMSO, and mix well.

[0048] Impurity B stock solution: Take 10 mg of impurity B, dissolve it in a 100 ml volumetric flask with 10% dimethyl sulfoxide acetonitrile solution and dilute to the mark, shake well;

[0049] Mixed standard solution: Take 10 mg of diacetylguanine sample and place it in a 100 ml volumetric flask, add 0.5 ml of impurity B stock solution, dissolve it with 10% dimethyl sulfoxide acetonitrile solution and dilute to the scale, shake well;

[0050] Sample solution: Take 10 mg of diacetylguanine sample, put it in a 100 ml volumetric flask, dissolve it with 10% dimethyl sulfoxide acetonitrile solution and dilute to the scale, shake well;

[0051] Linear stock solution A: Accurately pipette 14.0 ml of impurity B stock solution to 100 ml, add acetonitrile to dilute to the scale, and shake well.

[0052] Impurity linear 10% solution: Accurately pipette 0.5 ml of linear stock solution A into a 10 ml volumetric flask, add acetonitrile to dilute to the mark, and mix well.

[0053] Impurity linear 20% solution: Accurately pipette 1.0 ml of linear stock solution A into a 10 ml volumetric flask, add acetonitrile to dilute to the mark, and mix well.

[0054] Impurity linear 40% solution: Accurately pipette 2.0 ml of linear stock solution A into a 10 ml volumetric flask, add acetonitrile to dilute to the mark, and mix well.

[0055] Impurity linear 80% solution: Accurately pipette 4.0 ml of linear stock solution A into a 10 ml volumetric flask, add acetonitrile to dilute to the mark, and mix well.

[0056] Impurity linear 100% solution: Accurately pipette 5.0 ml of linear stock solution A into a 10 ml volumetric flask, add acetonitrile to dilute to the mark, and mix well.

[0057] Impurity linear 200% solution: same as linear stock solution A

[0058] Linear stock solution B: Accurately pipette 1.0 ml of impurity A stock solution to 100 ml, add acetonitrile to dilute to the scale, and shake well.

[0059] Diacetylguanine linear LOQ solution: Take the quantitation limit solution.

[0060] Diacetylguanine linear solution-1: Accurately pipette 1.0 ml of linear stock solution B into a 10 ml volumetric flask, add acetonitrile to dilute to the mark, and mix well.

[0061] Diacetylguanine linear solution-2: Accurately pipette 4.0 ml of linear stock solution B into a 10 ml volumetric flask, add acetonitrile to dilute to the mark, and mix well.

[0062] Diacetylguanine linear solution-3: Accurately pipette 5.0 ml of linear stock solution B into a 10 ml volumetric flask, add acetonitrile to dilute to the mark, and mix well.

[0063] Diacetylguanine Linear Solution-4: Isolinear Stock Solution B

[0064] 2) The standard solution and the sample solution were tested by high performance liquid chromatography, and the high performance liquid chromatography conditions were as follows:

[0065] High performance liquid chromatograph: Agilent 1260VWD;

[0066] Chromatographic column: YMC HILIC hybrid silica column (250 mm × 4.6 mm × 5 μm);

[0067] Mobile phase A: trifluoroacetic acid solution (pH = 3.0);

[0068] Mobile phase B: acetonitrile;

[0069] Isocratic elution: A:B = 5:95

[0070] Mobile phase flow rate: 0.5 ml / min;

[0071] Column temperature: 30°C;

[0072] Detection wavelength: UV254nm;

[0073] Injection volume: 20μl.

[0074] Figure 1 The liquid chromatogram of the blank solution prepared in Example 1; Figure 2This is the liquid chromatography analysis spectrum of the mixed standard solution of Example 1.

[0075] A Linearity Test:

[0076] Inject the blank sample and linear solution in sequence.

[0077] Standard curve drawing:

[0078] Under the set chromatographic conditions, the analysis was performed separately. The calibration curve was drawn according to the mass concentration and peak area of ​​the standard series. Linear regression was performed with the ratio of the linear solution as the horizontal axis (%) and the peak area as the vertical axis (Y), and the regression equation and correlation coefficient were obtained. Within the measurement range, each impurity and valacyclovir had a good linear relationship, and the linear correlation coefficient R was not less than 0.9990. The data are shown in Tables 1 and 2. Figure 3 The linear spectrum of impurity A of the linear solution prepared in Example 1; Figure 4 The linear spectrum of impurity B of the linear solution prepared in Example 1;

[0079] Table 1 - Liquid Chromatography Impurity A Chromatographic Peak Analysis Results of Example 1 Linear Solution

[0080]

[0081] Table 2 - Liquid Chromatography Impurity B Chromatographic Peak Analysis Results of Example 1 Linear Solution

[0082]

[0083] Example 2

[0084] 1) Preparation of solution:

[0085] Blank solution: 10% dimethyl sulfoxide acetonitrile solution;

[0086] Impurity Q stock solution: Take 10 mg of impurity Q, dissolve it in a 10 ml volumetric flask with 10% dimethyl sulfoxide acetonitrile solution and dilute to the mark, shake well;

[0087] Acyclovir stock solution: Weigh 10 mg of acyclovir sample into a 10 ml volumetric flask, accurately weigh, dissolve and dilute to the scale with 10% dimethyl sulfoxide acetonitrile solution, and mix well.

[0088] Mixed standard solution: Take 1 ml of impurity Q stock solution and 0.1 ml of acyclovir stock solution in a 10 ml volumetric flask, dissolve and dilute to the mark with 10% dimethyl sulfoxide acetonitrile solution, and shake well;

[0089] Sample solution: Take 10 mg of acyclovir sample, put it in a 10 ml volumetric flask, dissolve it with 10% dimethyl sulfoxide acetonitrile solution and dilute to the scale, shake well;

[0090] 2) The standard solution and the sample solution were tested by high performance liquid chromatography, and the high performance liquid chromatography conditions were as follows:

[0091] High performance liquid chromatograph: Agilent 1260VWD;

[0092] Chromatographic column: YMQ HILIC hybrid silica column (250 mm × 4.6 mm × 5 μm);

[0093] Mobile phase A: trifluoroacetic acid solution (pH = 3.0);

[0094] Mobile phase B: acetonitrile;

[0095] Isocratic elution: A:B = 5:95

[0096] Mobile phase flow rate: 0.5 ml / min;

[0097] Column temperature: 30°C;

[0098] Detection wavelength: UV254nm;

[0099] Injection volume: 20μl.

[0100] Figure 5 The liquid chromatogram of the mixed solution prepared in Example 2; Figure 6 This is the liquid chromatography analysis spectrum of the sample solution in Example 2.

[0101] The mixed solution was injected 6 times continuously, and the peak areas of impurity Q and acyclovir did not change significantly, indicating that impurity Q was not degraded into acyclovir under the chromatographic conditions. The results are as follows:

[0102] Table 3 - Results of the stability study of the mixed solution in Example 2

[0103] Components 1 2 3 4 5 6 average value RSD% Acyclovir 37.962 38.256 38.115 38.194 37.998 38.537 38.177 0.55 Impurity Q 35.404 35.144 34.716 34.397 34.249 34.585 34.749 1.27 .

[0104] The raw materials listed in the present invention, as well as the upper and lower limits and interval values ​​of the raw materials of the present invention, and the upper and lower limits and interval values ​​of the process parameters (such as temperature, time, etc.) can all realize the present invention, and the embodiments are not listed one by one here.

[0105] The above is only a preferred embodiment of the present invention, which certainly cannot be used to limit the scope of rights of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and changes can be made without departing from the principle of the present invention, and these improvements and changes are also regarded as the protection scope of the present invention.

Claims

1. A method for separating and determining easily hydrolyzed polar compounds. It is characterized in that The following steps are included: The sample containing the easily hydrolyzed polar compound is dissolved in a diluent to form a sample solution, and the standard solution and the sample solution are tested by high performance liquid chromatography; Wherein, the diluent is 10% dimethyl sulfoxide acetonitrile solution; Liquid chromatography conditions: Chromatographic column: hybrid silica gel column; mobile phase A: trifluoroacetic acid solution; mobile phase B: acetonitrile; isocratic elution; mobile phase flow rate: 0.4-0.6 ml / min; column temperature: 25-35 °C; detection wavelength: UV254 nm; injection volume: 20 μl; running time: 50 min; The sample containing the easily hydrolyzed polar compound is acyclovir or diacetylguanine; The method is used to separate and determine the impurity Q in the sample acyclovir, or the monoacetylguanine impurity in the sample diacetylguanine; Impurity Q is acyclovir EP impurity Q, with the structural formula The structural formula of diacetylguanine is The structural formula of monoacetylguanine is The structural formula of acyclovir is:

2. The method for separating and measuring easily hydrolyzed large polar compounds as claimed in claim 1, Features: The isocratic elution is specifically performed according to the following volume ratio: 96-94% A+4-6% B.

3. The method for separating and measuring easily hydrolyzed large polar compounds as claimed in claim 1, Features: The isocratic elution is specifically performed according to the following volume ratio: 95% A+5% B.

4. The method for separating and measuring easily hydrolyzed large polar compounds as claimed in claim 1, Features: The hybrid silica gel column is a bonded dihydroxypropyl hybrid silica gel column.

5. The method for separating and measuring easily hydrolyzed large polar compounds as claimed in claim 1, Features: The pH value of the trifluoroacetic acid solution is 3.0-4.

0.

6. The method for separating and measuring easily hydrolyzed large polar compounds as claimed in claim 5, Features: The pH value of the trifluoroacetic acid solution is adjusted by using trifluoroacetic acid.

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