A method for detecting diastereoisomer impurities of peramivir

Abstract

The present invention relates to the technical field of analysis and detection, and in particular to a method for detecting diastereoisomer impurities of peramivir. The present invention performs high performance liquid chromatography detection on a sample liquid to be tested to obtain the impurity content in the sample liquid to be tested, and the detection conditions of high performance liquid chromatography include: a mobile phase system includes mobile phase A and mobile phase B, wherein the mobile phase A is a mixture of a weak acid buffer salt and a weak acid, and the mobile phase B is methanol; the flow rate of the mobile phase system is 0.8 mL / min; the elution mode is isocratic elution; the volume ratio of the mobile phase A to the mobile phase B is 80:20, and the detection method provided by the present invention meets the detection requirements of diastereoisomer impurities of peramivir.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, and in particular to a method for detecting diastereoisomer impurities of Peramivir. Background Art

[0002] Peramivir trihydrate has neuraminidase (NA) inhibitory effect, shows strong antiviral activity against influenza A and B viruses, and is effective against influenza A and B virus infections.

[0003] Peramivir has five chiral centers, involving multiple enantiomers and diastereomers, and the control of isomers is the key to product quality control. Domestic and foreign literature pays more attention to the control of enantiomers, and there are relatively few discussions on the detection methods of diastereoisomer impurities. Diastereoisomers (structural formulas see Formula I and Formula II) are degradation impurities and need to be studied and controlled in Peramivir injection. However, since these two impurities are difficult to separate, there are no relevant reports on the detection and quantification methods of these two impurities at home and abroad. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a method for detecting diastereomeric impurities of Peramivir. The detection method provided by the present invention can simultaneously detect diastereomeric impurities having the structures of Formula I and Formula II.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides a method for detecting diastereoisomer impurities of Peramivir, comprising the following steps:

[0006] The sample solution to be tested is subjected to high performance liquid chromatography detection, and the mass percentage of diastereoisomer impurities in the sample solution to be tested is obtained by self-control method according to the detection peak area;

[0007] The detection conditions of high performance liquid chromatography include: the mobile phase system includes mobile phase A and mobile phase B, the mobile phase A is a mixture of weak acid buffer salt and weak acid, and the mobile phase B is methanol;

[0008] The flow rate of the mobile phase system is 0.8 mL / min;

[0009] The elution mode is isocratic elution; the volume ratio of the mobile phase A to the mobile phase B is 80:20;

[0010] The peramivir diastereoisomer impurities have the structures of Formula I and Formula II:

[0011]

[0012] Preferably, the chromatographic column of the high performance liquid chromatography is an octylsilane bonded silica gel chromatographic column; the column temperature is 30° C.; the injection volume is 10 μL; and the detection wavelength is 205 nm.

[0013] Preferably, the weak acid buffer salt in the mobile phase A is phosphate, acetate or carbonate.

[0014] Preferably, the weak acid in the mobile phase A is trifluoroacetic acid, phosphoric acid, formic acid or acetic acid.

[0015] Preferably, the concentration of the weak acid buffer salt in the mobile phase A is 0.01 mol / L.

[0016] Preferably, the volume concentration of the weak acid in the mobile phase A is 0.1%.

[0017] Preferably, the self-control method comprises the following steps:

[0018] The sample solution to be tested and the sample dilution to be tested are subjected to high performance liquid chromatography detection to obtain the peak area of ​​the impurity to be tested in the sample solution to be tested and the peak area of ​​peramivir in the sample dilution to be tested, respectively;

[0019] The mass percentage of diastereoisomer impurities in peramivir is calculated by formula a;

[0020]

[0021] In the formula a, Ai is the peak area of ​​the impurity to be detected in the sample solution to be detected;

[0022] A S is the peak area of ​​peramivir in the diluent of the sample to be tested;

[0023] n is the dilution factor of the sample solution to be tested.

[0024] Preferably, the value range of n is 400-600.

[0025] The present invention provides a method for detecting diastereomeric impurities of peramivir, comprising the following steps: performing high performance liquid chromatography detection on a sample liquid to be tested, adopting a self-control method according to the detection peak area, and obtaining the mass percentage of diastereomeric impurities in the sample liquid to be tested in peramivir; the detection conditions of the high performance liquid chromatography include: the mobile phase system includes mobile phase A and mobile phase B, the mobile phase A is a mixed solution of a weak acid buffer salt and a weak acid, and the mobile phase B is methanol; the chromatographic column is an octylsilane bonded silica gel chromatographic column; the flow rate of the mobile phase system is 0.8 mL / min; the elution mode is isocratic elution; the volume ratio of the mobile phase A to the mobile phase B is 80:20. The present invention separates the diastereomeric impurities having the structures of formula I and formula II by limiting the detection conditions, and then obtains the impurity content by using the self-control method. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is the chromatogram of blank solvent;

[0027] Figure 2 is the chromatogram of blank excipient;

[0028] Figure 3 is the chromatogram of the test solution;

[0029] Figure 4 is the chromatogram of the test sample spiked solution;

[0030] Figure 5 is the chromatogram of the sample solution to be tested;

[0031] Figure 6 is the chromatogram of the control solution. DETAILED DESCRIPTION

[0032] The present invention provides a method for detecting diastereoisomer impurities of peramivir, comprising the following steps: performing high performance liquid chromatography detection on a sample liquid to be detected, and using a self-control method according to the detection peak area to obtain the mass percentage of diastereoisomer impurities in the sample liquid to be detected in peramivir;

[0033] The detection conditions of high performance liquid chromatography include: the mobile phase system includes mobile phase A and mobile phase B, the mobile phase A is a mixture of weak acid buffer salt and weak acid, and the mobile phase B is methanol; the chromatographic column is an octylsilane bonded silica gel chromatographic column;

[0034] The flow rate of the mobile phase system is 0.8 mL / min;

[0035] The elution mode is isocratic elution; the volume ratio of the mobile phase A to the mobile phase B is 80:20;

[0036] The peramivir diastereoisomer impurities have the structures of Formula I and Formula II:

[0037]

[0038] In the present invention, unless otherwise specified, the reagents used are commercially available products well known to those skilled in the art.

[0039] In the present invention, the conditions for the high performance liquid chromatography detection include: the mobile phase system includes mobile phase A and mobile phase B. In the present invention, the mobile phase A is a mixture of weak acid buffer salt and weak acid. In the present invention, the mobile phase B is methanol; the methanol is preferably chromatographic grade methanol; the weak acid buffer salt in the mobile phase A is preferably phosphate, acetate or carbonate, more preferably phosphate; the phosphate is preferably potassium dihydrogen phosphate; the concentration of the weak acid buffer salt in the mobile phase A is preferably 0.01 mol / L; in the present invention, the weak acid in the mobile phase A is preferably trifluoroacetic acid, phosphoric acid, formic acid or acetic acid, more preferably trifluoroacetic acid, and the volume concentration of the weak acid in the mobile phase A is preferably 0.1%; the chromatographic column is preferably an octylsilane bonded silica gel chromatographic column, more preferably an Agilent ZORBAX RX-C8 chromatographic column.

[0040] In the present invention, the flow rate of the mobile phase system is 0.8 mL / min.

[0041] In the present invention, the elution mode is isocratic elution.

[0042] In the present invention, the column temperature of the HPLC detection is preferably 30°C; and the injection volume is preferably 10 μL.

[0043] In the present invention, the self-control method comprises the following steps:

[0044] The sample solution to be tested and the sample dilution to be tested are subjected to high performance liquid chromatography detection to obtain the peak area of ​​the impurity to be tested in the sample solution to be tested and the peak area of ​​peramivir in the sample dilution to be tested, respectively;

[0045] The mass percentage of diastereoisomer impurities in peramivir is calculated by formula a.

[0046]

[0047] Wherein, Ai is the peak area of ​​the impurity to be detected in the sample solution to be detected; A S is the peak area of ​​peramivir in the diluent of the sample to be tested; n is the dilution multiple of the sample to be tested, and the value range of n is preferably 400-600, more preferably 500.

[0048] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0049] In an embodiment of the present invention, the conditions for high performance liquid chromatography detection include: the mobile phase system includes mobile phase A and mobile phase B (the volume ratio of mobile phase A to mobile phase B is 80:20), the mobile phase A is a mixture of potassium dihydrogen phosphate and trifluoroacetic acid, the concentration of potassium dihydrogen phosphate is 0.01 mol / L, and the volume concentration of trifluoroacetic acid is 0.1%; the mobile phase B is methanol; the chromatographic column is an Agilent ZORBAX RX-C8 chromatographic column; the column temperature is 30°C; the injection volume is 10 μL; and the flow rate of the mobile phase system is 0.8 mL / min.

[0050] Example 1

[0051] Specificity test

[0052] 1. Preparation of samples and working solutions

[0053] 1. Impurity with structure of formula I (named as impurity B) localization solution

[0054] Take an appropriate amount of impurity B reference substance, dissolve it in water and dilute it to make a solution containing about 40 μg per 1 mL.

[0055] 2. Impurity with structure of formula II (named as impurity C) positioning solution

[0056] Take an appropriate amount of impurity C reference substance, dissolve it in water and dilute it to make a solution containing about 40 μg per 1 mL.

[0057] 3. Test sample spike solution

[0058] Accurately measure 10 mL of Peramivir Injection and place it in a 25 mL volumetric flask. Add 2.5 mL each of Impurity B Location Solution and Impurity C Location Solution, dilute to the mark with water, and shake well.

[0059] 4. Test solution

[0060] Accurately measure 10 mL of Peramivir Injection, place it in a 25 mL volumetric flask, dilute to the mark with water, and shake well.

[0061] 5. Blank excipient solution: Accurately measure 4 mL of excipient solution (excluding impurities B and C), place in a 10 mL volumetric flask, dilute to the scale with solvent, and shake well.

[0062] 6. Blank solvent: water

[0063] 2. Detection

[0064] The blank solvent, blank excipient solution, test sample solution and test sample spiked solution were tested according to the “HPLC test conditions”. The test results are shown in Figure 1 to Figure 4 and Table 1, Figure 1 is the chromatogram of blank solvent; Figure 2is the chromatogram of the blank excipient. Figure 1-2 It can be seen that solvents and excipients do not interfere with the detection of impurities. Figure 3 is the chromatogram of the test solution. Figure 3 It can be seen that impurity B is contained in the peramivir injection. Figure 4 is the chromatogram of the test sample spiked solution. Figure 4 It can be seen that impurity B and impurity C can be well separated, and the separation degree is greater than 1.5.

[0065] Table 1 Test results of blank solvent, blank excipient solution, test solution and test spiked solution

[0066]

[0067]

[0068] Example 2

[0069] Take an appropriate amount of the test solution, add 25%, 100%, 150% limit concentration (limit concentration 0.2%: impurity B or impurity C in the mass percentage of peramivir) of impurity B and impurity C reference substances to obtain the test solution, and prepare 3 parallel copies of each.

[0070] Acceptable standard (set with reference to the Chinese Pharmacopoeia): The recovery rate of 25% limit concentration is between 85% and 110%, the recovery rates of 100% and 150% limit concentrations are both between 90% and 108%, and the RSD value is no more than 10%.

[0071] The test liquid was tested according to the “High Performance Liquid Chromatography Test Conditions”, and the test results are shown in Table 2.

[0072] Table 2 Test results of accuracy test

[0073]

[0074]

[0075] As can be seen from Table 2, the recovery rate of 25% limit concentration in the present invention is between 85% and 110%, and the recovery rates of 100% and 150% limit concentrations are both between 90% and 108%, and the RSD value is less than 10%, and the accuracy of this method is good. After method verification, the method has good specificity and meets the requirements for accuracy, and can be used for the detection of impurities B and impurities C.

[0076] Example 3

[0077] Solution preparation:

[0078] Sample solution to be tested: Take an appropriate amount of peramivir injection and quantitatively dilute it with water to make a solution containing approximately 2 mg of peramivir per 1 mL.

[0079] Control solution (test sample diluent): Accurately measure an appropriate amount of the test sample solution and quantitatively dilute it with water to prepare a solution containing approximately 4 μg of peramivir per 1 mL.

[0080] The sample solution to be tested and the control solution were tested according to the "High Performance Liquid Chromatography Test Conditions". According to the self-control method, the content of impurities in the sample was calculated according to formula a, where n is 500.

[0081] The test results of the sample solution and the control solution are shown in Figure 5-6 ,in Figure 5 The spectrum information of is shown in Tables 3 and 4. Figures 5-6 As shown in Tables 3 and 4, the test results show that the impurity B accounts for 0.05% of the mass percentage of peramivir in the test sample solution, and the impurity C is not detected.

[0082] Table 3 Figure 5 Detection of chromatogram information

[0083]

[0084]

[0085] Table 4 Figure 6 Detection of chromatogram information

[0086]

[0087] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for detecting diastereoisomer impurities of Peramivir, characterized in that: The following steps are involved: The sample solution to be tested is subjected to high performance liquid chromatography detection, and the mass percentage of diastereoisomer impurities in the sample solution to be tested is obtained by self-control method according to the detection peak area; The detection conditions of high performance liquid chromatography include: the mobile phase system includes mobile phase A and mobile phase B, the mobile phase A is a mixture of potassium dihydrogen phosphate and trifluoroacetic acid, the concentration of potassium dihydrogen phosphate is 0.01 mol / L, and the volume concentration of trifluoroacetic acid is 0.1%; The mobile phase B is methanol; the chromatographic column is an octylsilane bonded silica gel chromatographic column; The flow rate of the mobile phase system is 0.8 mL / min; The elution mode is isocratic elution; the volume ratio of the mobile phase A to the mobile phase B is 80:20; The peramivir diastereoisomer impurities have the structures of Formula I and Formula II: Formula I; Formula II.

2. The detection method according to claim 1, characterized in that: The column temperature of the HPLC was 30°C; the injection volume was 10 μL; and the detection wavelength was 205 nm.

3. The detection method according to claim 1, characterized in that: The self-control method comprises the following steps: The sample solution to be tested and the sample dilution to be tested are subjected to high performance liquid chromatography detection to obtain the peak area of ​​the impurity to be tested in the sample solution to be tested and the peak area of ​​peramivir in the sample dilution to be tested, respectively; The mass percentage of diastereoisomer impurities in peramivir is calculated by formula a; Impurities (%) = Formula a In the formula a, Ai is the peak area of ​​the impurity to be detected in the sample solution to be detected; A S is the peak area of ​​peramivir in the diluent of the sample to be tested; n is the dilution factor of the sample solution to be tested.

4. The detection method according to claim 3, characterized in that: The value range of n is 400~600.

Images