A method for detecting isomers of isavuconazole sulfate

Abstract

The present invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for detecting the enantiomers of isavuconazole sulfate. In the present invention, the sample to be tested, isavuconazole sulfate and its enantiomers, are hydrolyzed and converted into isavuconazole and its enantiomers under the catalytic action of an enzyme, and isavuconazole and its enantiomers are separated and detected by liquid chromatography, so as to detect the content of the enantiomers of isavuconazole sulfate, and further control the impurity content of the enantiomers in the isavuconazole sulfate raw material or its preparation, ensuring the quality controllability of the isavuconazole sulfate raw material and its preparation. The problem of difficult detection of the enantiomers of isavuconazole sulfate in the quality control of the isavuconazole sulfate raw material and its preparation is solved.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for detecting the enantiomers of isavuconazole sulfate. Background Art

[0002] Isavuconazole sulfate is a new broad-spectrum triazole antifungal drug, first developed by Basilea Pharmaceutica. In 2015, the capsule and lyophilized powder for injection of isavuconazole sulfate were approved by the US FDA for marketing. The chemical name of isavuconazole sulfate is 1-{ (2R,3R)-3-[4-(4-cyanophenyl)-1,3-thiazol-2-yl]-2-(2,5-difluorophenyl)-2-hydroxybutyl}-4-[(1RS)-1-({methyl[3-({[(methylamino)acetyl]oxy}methyl)pyridin-2-yl]carbamoyloxy}ethyl]-1H-1,2,4-triazol-4-ium monosulfate, and its structural formula is as follows:

[0003] 。

[0004] Isavuconazole sulfate is a prodrug of isavuconazole. Under the catalysis of enzymes in vivo, it can be rapidly hydrolyzed into the active part isavuconazole and the inactive dissociation part. Isavuconazole sulfate has 3 chiral centers. The two centers C7 and C8 in the isavuconazole part are both in the R configuration. The third chiral center C29 is not in the isavuconazole part and has both R and S configurations, which is a racemate. Isavuconazole sulfate is a mixture of two epimers of C29. The enantiomers of isavuconazole sulfate have the structures of C7(S), C8(S) and C29(R / S). Since the reference substances of the isavuconazole sulfate enantiomers are very expensive and difficult to prepare, the detection of the isavuconazole sulfate enantiomers in the quality control of isavuconazole sulfate has become a technical problem in this field. The structural formula of the isavuconazole sulfate enantiomers is as follows:

[0005] 。

[0006] The active part of isavuconazole sulfate, isavuconazole, has the chemical name of 4-[2-[(2R,3R)-3-(2,5-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-1,3-thiazol-4-yl]benzonitrile, and its structure is as follows:

[0007] 。

[0008] The enantiomers of isavuconazole have the chemical name of 4-[2-[(2S,3S)-3-(2,5-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butane-2-yl]-1,3-thiazol-4-yl]benzonitrile, and its structure is as follows:

[0009] 。

[0010] In the prior art, a method for detecting related substances in isavuconazole sulfate is disclosed in the patent with the application number CN202311619785.6. In this method, high performance liquid chromatography is used for detection. In the chromatographic detection, an octadecylsilane bonded silica gel is used as the chromatographic column, an aqueous solution of trifluoroacetic acid is used as mobile phase A, and a mixed solution of trifluoroacetic acid methanol - acetonitrile is used as mobile phase B to detect the related substances in isavuconazole sulfate. However, this method can only separate and detect the epimers in isavuconazole sulfate, and cannot separate and detect the enantiomers of isavuconazole sulfate.

[0011] The patent with the application number CN202311185496.X discloses a method and application for detecting isavuconazole by UPLC - MS / MS. In this method, ultra - high performance liquid chromatography - mass spectrometry is used. A C18 column filled with ethylene bridged hybrid particles is used as the chromatographic column, an aqueous solution of formic acid is used as mobile phase A, and an acetonitrile solution of formic acid is used as mobile phase B to separate and detect isavuconazole. However, this method cannot separate and detect the enantiomers of isavuconazole sulfate.

[0012] Therefore, there is an urgent need to establish an accurate and feasible method for detecting the enantiomers of isavuconazole sulfate to achieve the quality control of isavuconazole sulfate and its preparations. Summary of the Invention

[0013] To better control the quality of isavuconazole sulfate drugs and fill the technical gap in detecting the enantiomers of isavuconazole sulfate in the prior art, the present invention provides a method for detecting the enantiomers of isavuconazole sulfate, which at least includes the following steps:

[0014] (1) Preparation of the system suitability solution: Appropriate amounts of isavuconazole and its enantiomers are taken, dissolved and diluted with a first solvent to prepare the system suitability solution;

[0015] (2) Preparation of the test solution: An appropriate amount of the test sample of isavuconazole sulfate is taken, the hydrolysis of isavuconazole sulfate and its enantiomers is accelerated by adding an enzyme test solution, then dissolved and diluted with a second solvent, and finally an enzyme reaction termination solution is added to prepare the test solution;

[0016] (3) Chromatographic test: Appropriate amounts of the system suitability solution and the test solution are taken, injected into the liquid chromatograph, the chromatogram is recorded, and the amount of the enantiomers of isavuconazole sulfate is calculated by the area normalization method. As a preferred technical solution, the detection conditions of the liquid chromatograph are as follows:

[0017] Detector: Ultraviolet detector;

[0018] Chromatographic column: a chiral chromatographic column packed with cellulose tris(3,5-dichlorophenylcarbamate) bonded to the silica surface;

[0019] Mobile phase A: ammonium acetate solution;

[0020] Mobile phase B: a mixed solution of ammonium acetate solution and acetonitrile;

[0021] Flow rate: 0.5 - 1.0 ml / min;

[0022] Column temperature: 25 - 35 °C;

[0023] Detection wavelength: 284 nm;

[0024] Injection volume: 10 μl;

[0025] The mobile phase gradient elution program is as follows:

[0026] 。

[0027] In the present invention, the enzyme test solution is used to accelerate the hydrolysis of isavuconazole sulfate into isavuconazole, and at the same time accelerate the hydrolysis of the isavuconazole sulfate enantiomer into the isavuconazole enantiomer. Since the physicochemical properties of isavuconazole sulfate and its enantiomer are the same, and the hydrolysis rates are the same under the action of the same enzyme, the amount of the isavuconazole sulfate enantiomer can be obtained by detecting the amount of the isavuconazole enantiomer. The hydrolysis conditions of the present invention are relatively mild and will not cause the inversion of the chiral configuration. It can be proved by the enzymatic conversion rate test combined with the accuracy test that the isavuconazole sulfate enantiomer can be completely converted into the isavuconazole enantiomer after enzymatic hydrolysis, and the configuration will not change during the conversion process. The hydrolysis rate of isavuconazole sulfate is very slow without adding catalytic enzyme and more other impurities will be produced.

[0028] As a preferred technical solution, the enzyme test solution is prepared from a buffer solution and an enzyme, and the enzyme is selected from one of trypsin or butyrylcholinesterase.

[0029] As a preferred technical solution, the buffer solution is selected from one of phosphate buffer solution, acetate buffer solution, and citrate buffer solution, and the buffer solution adjusts the pH value in the enzyme test solution to 7 - 9.

[0030] As a more preferred technical solution, the buffer solution is phosphate buffer solution.

[0031] As a more preferred technical solution, the preparation method of the enzyme test solution is: take an appropriate amount of trypsin and make a solution with a trypsin content of 1 wt% using phosphate buffer solution with a pH of 8.0.

[0032] As a preferred technical solution, the enzyme reaction termination solution is prepared from a hydrochloric acid solution and a first solvent, and the concentration of hydrochloric acid in the enzyme reaction termination solution is ≥ 0.1 mol / L.

[0033] As a more preferred technical solution, the concentration of hydrochloric acid in the enzyme reaction termination solution includes but is not limited to 0.10 mol / L, 0.15 mol / L, 0.50 mol / L, 1.0 mol / L, 2.0 mol / L, 3.0 mol / L, 4.0 mol / L, 5.0 mol / L.

[0034] As a more preferred technical solution, the concentration of hydrochloric acid in the enzyme reaction termination solution is 0.10 mol / L.

[0035] As a preferred technical solution, the first solvent is selected from at least one of acetonitrile or its aqueous solution, methanol or its aqueous solution, ethanol or its aqueous solution.

[0036] As a preferred technical solution, the first solvent is an acetonitrile aqueous solution, and the volume percentage of acetonitrile in the acetonitrile aqueous solution is ≥ 30%.

[0037] As a preferred technical solution, the volume percentage of acetonitrile in the first solvent acetonitrile aqueous solution includes but is not limited to 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%.

[0038] As a more preferred technical solution, the volume percentage of acetonitrile in the first solvent acetonitrile aqueous solution is 50%.

[0039] As a preferred technical solution, the first solvent in the enzyme reaction termination solution is an acetonitrile aqueous solution, in which the volume percentage of acetonitrile is 50%.

[0040] As a preferred technical solution, the second solvent is selected from at least one of acetonitrile or its aqueous solution, methanol or its aqueous solution, ethanol or its aqueous solution.

[0041] As a more preferred technical solution, the second solvent is acetonitrile.

[0042] As a more preferred technical solution, the detection conditions of the liquid chromatograph are as follows:

[0043] Detector: ultraviolet detector;

[0044] Chromatographic column: a chiral chromatographic column filled with cellulose tris(3,5-dichlorophenylcarbamate) bonded to the surface of silica gel;

[0045] Mobile phase A: ammonium acetate solution;

[0046] Mobile phase B: A mixed solution of ammonium acetate solution and acetonitrile;

[0047] Flow rate: 0.7 ml / min;

[0048] Column temperature: 30 °C;

[0049] Detection wavelength: 284 nm;

[0050] Injection volume: 10 µl;

[0051] The mobile phase gradient elution program is as follows:

[0052] 。

[0053] As a preferred technical solution, the volume ratio of ammonium acetate solution to acetonitrile in the mobile phase B is (15 - 25):(75 - 85).

[0054] As a preferred technical solution, the pH of the ammonium acetate solution in the mobile phase A and the mobile phase B is 4.5 - 5.5.

[0055] As a more preferred technical solution, the pH of the ammonium acetate solution in the mobile phase A and the mobile phase B is 4.8 - 5.2.

[0056] As a preferred technical solution, the concentration of the ammonium acetate solution in the mobile phase A is 0.01 mol / L.

[0057] As a preferred technical solution, the concentration of the ammonium acetate solution in the mobile phase B is 0.1 mol / L.

[0058] As a preferred technical solution, the pH of the ammonium acetate solution in the mobile phase A is adjusted with glacial acetic acid.

[0059] As a preferred technical solution, the pH of the ammonium acetate solution in the mobile phase B is adjusted with glacial acetic acid.

[0060] As a preferred technical solution, the chiral chromatographic column with silica gel surface bonded with cellulose tris(3,5-dichlorophenylcarbamate) as the filler, model CHIRALPAK IC-3, 4.6 mm × 250 mm, 3 µm.

[0061] The detection method of isavuconazole sulfate enantiomers in the present invention has good applicability, can be applied to the detection of isavuconazole sulfate enantiomers in isavuconazole sulfate raw materials and its preparations, and realizes the quality control of isavuconazole sulfate and its preparations.

[0062] Beneficial effects

[0063] In the present invention, the sample to be tested, isavuconazole sulfate, is hydrolyzed under the action of an enzyme to obtain isavuconazole and its enantiomers. Since the physicochemical properties of isavuconazole sulfate and its enantiomers are the same, they will be equivalently converted into isavuconazole and its enantiomers under the catalysis of the enzyme. Therefore, isavuconazole and its enantiomers are separated and detected by liquid chromatography, so as to realize the detection of the content of isavuconazole sulfate enantiomers, and further control the impurity content of enantiomers in the isavuconazole sulfate raw material medicine or its preparation, ensuring the quality control of the isavuconazole sulfate raw material medicine and its preparation. The problem of difficult detection of isavuconazole sulfate enantiomers in the quality control of isavuconazole sulfate raw material medicine and its preparation is solved. Description of the Drawings

[0064] Figure 1 It is the liquid chromatogram of the test system suitability solution in Example 5.

[0065] Figure 2 It is the liquid chromatogram of the test sample solution in Example 5.

[0066] Figure 3 It is the liquid chromatogram of the system suitability solution under different pH values of the mobile phase in Example 6.

[0067] Figure 4 It is the liquid chromatogram of the system suitability solution under different ratios of acetonitrile in mobile phase B in Example 6.

[0068] Figure 5 It is the liquid chromatogram of different recovery solutions in Example 9.

[0069] Figure 6 It is the liquid chromatogram in the precision test in Example 10.

[0070] Figure 7 It is the linear graph of the isavuconazole reference substance in Example 11.

[0071] Figure 8 It is the linear graph of the isavuconazole enantiomer in Example 11. Detailed Description of the Embodiments

[0072] To facilitate the understanding of the content of the present invention by those skilled in the art, the specific embodiments of the present invention are described in detail below. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as a limitation to the present invention.

[0073] Example 1 Selection of Solvent

[0074] Appropriate amounts of isavuconazole sulfate and isavuconazole were taken respectively, and isavuconazole sulfate and isavuconazole were dissolved with acetonitrile aqueous solutions with different volume ratios of acetonitrile, and the dissolution situation was observed by visual method, as shown in Table 1 below.

[0075] Table 1 - Dissolution of Different Solvents

[0076]

[0077] From the dissolution results, it can be seen that several solvents have good solubility for isavuconazole sulfate, while the acetonitrile aqueous solution (volume ratio of acetonitrile to water is 10:90) has poor solubility for isavuconazole, and the remaining solvents have good solubility for isavuconazole.

[0078] Selection of Chromatographic Conditions in Example 2

[0079] System suitability solution: Take appropriate amounts of isavuconazole and its enantiomer, dissolve and dilute with acetonitrile - aqueous solution (volume ratio of acetonitrile to water is 50:50) to prepare a solution containing 0.6 mg of isavuconazole and 0.6 μg of isavuconazole enantiomer per 1 ml.

[0080] High performance liquid chromatography detection conditions:

[0081] Flow rate: 0.7 ml / min;

[0082] Column temperature: 30 °C;

[0083] Detection wavelength: 284 nm;

[0084] Injection volume: 10 μl;

[0085] Mobile phase and elution gradient: See Test Conditions 1 - 7 in Table 2;

[0086] Chromatographic column: See Test Conditions 1 - 7 in Table 2.

[0087] Screen the liquid chromatography conditions in Test Conditions 1 - 7 in Table 2 respectively with the system suitability solution, record and observe the separation of the two peaks of isavuconazole and isavuconazole enantiomer in the liquid chromatography diagram. The specific screening conditions and results of the chromatography are shown in Table 2.

[0088] Table 2 - Chromatographic Screening Conditions and Results

[0089]

[0090] From the above results, it can be seen that when the chromatographic column packing is cellulose tris(3,5 - dichlorophenylcarbamate) bonded to the silica surface, mobile phase A is 0.01 mol / L ammonium acetate solution (adjust the pH value to 5.0 with glacial acetic acid), mobile phase B is 0.1 mol / L ammonium acetate solution (adjust the pH value to 5.0 with glacial acetic acid) - acetonitrile (volume ratio between ammonium acetate solution and acetonitrile is 20:80) as mobile phase B, and combined with the following elution gradient, the detection peaks of isavuconazole and isavuconazole enantiomer can be separated.

[0091]

[0092] The above is the elution gradient.

[0093] Example 3 Selection of Enzyme Conditions

[0094] Pepsin, trypsin, and butyrylcholinesterase were used as hydrolysis enzymes respectively to explore the enzymatic hydrolysis conditions.

[0095] Isecazolesulfate was enzymatically hydrolyzed with pepsin, trypsin, and butyrylcholinesterase respectively (the specific preparation method is as follows: take an appropriate amount of isecazolesulfate, dissolve and dilute it with the corresponding enzyme solution to make a solution with a concentration of 4 mg / ml of isecazolesulfate. After reacting according to the enzymatic hydrolysis conditions in Table 3, add an equal volume of acetonitrile precisely to dissolve it, shake well, precisely measure 1 ml, and mix it with 1 ml of the enzyme reaction termination solution (the enzyme reaction termination solution is prepared by mixing hydrochloric acid solution and acetonitrile aqueous solution (the volume ratio of acetonitrile to water is 50:50) to make a hydrochloric acid concentration of 0.1 mol / L). The amount of isecazole converted was detected by the external standard method of isecazole reference substance (the chromatographic detection conditions were the test conditions 7 in Example 2, and compared with the theoretical amount of isecazolesulfate converted to isecazole, the enzymatic hydrolysis conversion rate was calculated (the actual amount of isecazolesulfate converted to isecazole / the theoretical amount of isecazolesulfate converted to isecazole = conversion rate), and the results are shown in Table 3.

[0096] Table 3 - Enzymatic Hydrolysis Conditions and Results

[0097]

[0098] Preparation of phosphate buffer solution: Take 6.8 g of potassium dihydrogen phosphate, add 1000 ml of water to dissolve it, and adjust the pH value to the corresponding target pH value with 1 mol / L sodium hydroxide solution.

[0099] From the screening results of the enzymatic hydrolysis conditions in Table 3, it can be seen that trypsin and butyrylcholinesterase have better enzymatic hydrolysis effects. Considering the comprehensive cost, 1 wt% trypsin in pH 8.0 phosphate buffer solution was selected as the enzyme test solution.

[0100] Preparation method of 1 wt% trypsin in pH 8.0 phosphate buffer solution (i.e., trypsin test solution with a trypsin activity of 2500 units / ml): Take 5 g of trypsin (porcine source, enzyme activity 1:250 (1 mg has 250 activity units)), precisely weigh it, add 500 ml of phosphate buffer solution with a pH of 8 to dissolve it, and mix well to obtain it. The preparation methods of the enzyme test solutions in the other test conditions in this example are the same as that of 1 wt% trypsin in pH 8.0 phosphate buffer solution.

[0101] Isavuconazole sulfate is converted into isavuconazole under the action of an enzyme under relatively mild conditions, and no configuration change occurs during the conversion process.

[0102] Example 4 Selection of Conditions for Enzyme Reaction Termination Solution

[0103] Preparation of enzyme reaction termination solution: It is prepared from a hydrochloric acid solution and an acetonitrile solution (the volume ratio between acetonitrile and water in the acetonitrile solution is 50:50). Investigation of hydrochloric acid dosage: After the enzymatic hydrolysis of isavuconazole sulfate in a 37°C water bath, acid needs to be added to terminate the enzymatic reaction activity, which is beneficial to the extraction of the target isavuconazole. Therefore, the hydrochloric acid dosage was investigated. Take 1 ml of the solution after enzymatic hydrolysis of isavuconazole sulfate obtained under Test Condition 4 in Example 3, mix it with 1 ml of hydrochloric acid extraction solutions with different concentrations, and then inject the mixture into a liquid chromatograph (the liquid chromatographic conditions are Test Condition 7 in Example 2) for testing. By detecting the content of isavuconazole and comparing it with the theoretical amount, the conversion rate is calculated (the amount of actual isavuconazole sulfate converted into isavuconazole / the amount of theoretical isavuconazole sulfate converted into isavuconazole = conversion rate), as shown in Table 4 specifically.

[0104] Table 4 - Experimental Results

[0105]

[0106] The results show that when adding an enzyme reaction termination solution with a hydrochloric acid concentration of 0.1 mol / L, the conversion and extraction rate is over 95%. Therefore, it is beneficial for conversion and extraction to inject the solution after enzymatic hydrolysis of the sample in equal proportion with an enzyme reaction termination solution with a hydrochloric acid concentration of 0.1 mol / L.

[0107] Example 5 Specificity Test

[0108] In this example, Test Condition 7 in Example 2 was used as the liquid chromatographic detection condition.

[0109] Preparation of system suitability solution: Take appropriate amounts of isavuconazole and its enantiomer, dissolve and dilute them with an acetonitrile - water solution (the volume ratio of acetonitrile to water is 50:50) to prepare a solution containing 0.6 mg of isavuconazole and 0.6 μg of isavuconazole enantiomer per 1 ml.

[0110] Preparation of sensitivity solution: Take an appropriate amount of isavuconazole enantiomer reference substance, accurately weigh it, dissolve and quantitatively dilute it with an acetonitrile - water solution (the volume ratio of acetonitrile to water is 50:50) to prepare a solution containing approximately 0.3 μg per 1 ml.

[0111] Preparation of the test solution: Weigh accurately 44 mg of the test sample of isavuconazole sulfate, place it in a reaction tube, accurately add 10 ml of trypsin test solution containing 1 wt% of trypsin [Take an appropriate amount of trypsin (porcine source, enzyme activity 1:250 (1 mg has 250 activity units)), add phosphate buffer solution with pH 8.0 (0.05 mol / L potassium dihydrogen phosphate solution, adjust the pH value to 8.0 with 1 mol / L sodium hydroxide solution) to prepare a solution containing 2500 units of trypsin per 1 ml], shake well, seal, and place at 37 °C for 8 hours. Take it out, accurately add 10 ml of acetonitrile to dissolve it, shake well, accurately measure 1 ml, and mix it with 1 ml of hydrochloric acid extraction solution (Take 10 ml of 1 mol / L hydrochloric acid solution, dilute it to 100 ml with acetonitrile - aqueous solution (volume ratio between acetonitrile and water is 50:50)).

[0112] Accurately measure 10 μl of the system suitability solution and inject it into the liquid chromatograph. The chromatographic conditions of the liquid chromatograph are the same as those in Test Condition 7 of Example 2. Record the chromatogram. The test results are shown in Table 4 and Figure 1 , in the chromatogram of the system suitability solution, the isavuconazole enantiomers and isavuconazole elute successively, and the resolution between the two is greater than 1.5, with good separation; the theoretical plate number of the main peak of isavuconazole is greater than 5000, and the peak shape is good.

[0113] Accurately measure 10 µl of the sensitivity solution and inject it into the liquid chromatograph. Record the chromatogram. The results are shown in Table 5. The signal - to - noise ratio of the peak height of the isavuconazole enantiomer peak is 175, which is greater than 10.

[0114] Accurately measure 10 µl of the test solution and inject it into the liquid chromatograph. Record the chromatogram. The results are shown in Table 5 and Figure 2 , no enantiomers are detected in the chromatogram of the test solution, meeting the requirements.

[0115] Table 5 Detection results of the specificity test

[0116]

[0117] It can be seen from the above detection results that it has good sensitivity.

[0118] Example 6 Robustness test

[0119] In this example, Test Condition 7 in Example 2 is used as the liquid chromatography detection condition.

[0120] Preparation of the system suitability solution: The same as in Example 5.

[0121] The durability of the high performance liquid chromatography (HPLC) detection method was investigated by changing the mobile phase ratio and the pH value of the mobile phase. Only one parameter was changed in each test, and the results were compared with those obtained without changing the parameters. Compared with the chromatographic conditions in Test Condition 7 of Example 2, in this example, the acetonitrile volume ratio in Mobile Phase B was changed within the range of 78% - 83%, and the pH values of the buffer solutions in Mobile Phase A and Mobile Phase B were simultaneously changed by ±0.2. 10 μl of the system suitability solution was respectively taken and injected into the liquid chromatograph according to the standard chromatographic conditions, Chromatographic Condition 1, Chromatographic Condition 2, Chromatographic Condition 3, and Chromatographic Condition 4 in Table 5, and the chromatograms were recorded. The chromatographic conditions and test results are shown in Table 6.

[0122] Table 6 Results of the durability test

[0123]

[0124] The test results showed that under the standard chromatographic conditions, Chromatographic Condition 1, Chromatographic Condition 2, Chromatographic Condition 3, and Chromatographic Condition 4, that is, when the pH of the ammonium acetate solution in Mobile Phase A and Mobile Phase B was within the range of 4.8 - 5.2, or when the volume ratio of acetonitrile in Mobile Phase B was within the range of 78% - 83%, the resolution between the chromatographic peaks of isavuconazole and isavuconazole enantiomers was greater than 1.5, meeting all the requirements of the system suitability test, indicating that the method had good durability. The results are shown in Figure 3 and Figure 4 。

[0125] Example 7 Solution stability test

[0126] In this example, Test Condition 7 in Example 2 was used as the liquid chromatography detection condition.

[0127] An isavuconazole enantiomer reference solution was prepared (3 mg of isavuconazole enantiomer reference substance was dissolved with an acetonitrile - aqueous solution (the volume ratio of acetonitrile to water was 50:50) and quantitatively diluted to a solution of 0.6 μg / ml), and it was placed at 5 ± 3°C. Liquid chromatography injection tests were carried out at 0, 16, and 24 hours respectively. Compared with the 0 - hour result, the change value of the peak area was calculated, and the change value of the peak area was not greater than 20%. The isavuconazole enantiomer reference solution had good stability within 24 hours. The results are shown in Table 7.

[0128] Table 7 Results of the reference solution stability test

[0129]

[0130] Prepare the test solution. In this example, the preparation method of the test solution is the same as that in Example 5. The prepared test solution is placed under the condition of 5±3°C, and samples are injected for detection at 0, 8, 12, and 24 hours respectively. Compared with 0 hour, calculate the change value of the amount of enantiomers. The change value of the peak area is not more than 20%. It is determined that the test solution has good stability within 24 hours. The results are shown in Table 8.

[0131] Table 8 Results of the stability test of the test solution

[0132]

[0133] Example 8 Quantitative limit and detection limit test

[0134] In this example, Test Condition 7 in Example 2 is used as the liquid chromatography detection condition.

[0135] Take 17.6 mg of isavuconazole and 3 mg of isavuconazole enantiomer, dissolve them separately with an acetonitrile - aqueous solution (the volume ratio of acetonitrile to water is 50:50) solvent and gradually dilute, shake well, accurately measure 10 μl and inject it into the liquid chromatograph until the response values of the peaks of each component are about 10 times and 3 times higher than the noise signal, which are the quantitative limit and detection limit of the corresponding component respectively. The results are shown in Table 9.

[0136] Table 9 Test results of quantitative limit and detection limit

[0137]

[0138] The test results show that the quantitative limit of isavuconazole is 0.052 μg / ml, the quantitative limit of isavuconazole enantiomer is 0.060 μg / ml, the detection limit of isavuconazole is 0.016 μg / ml, and the detection limit of isavuconazole enantiomer is 0.018 μg / ml.

[0139] Example 9 Accuracy test

[0140] In this example, Test Condition 7 in Example 2 is used as the liquid chromatography detection condition.

[0141] The accuracy of the method is achieved by examining the accuracy of liquid chromatography tests after adding different amounts of isavuconazole sulfate enantiomers to the sample. Examine the amounts of isavuconazole sulfate enantiomers added to the test samples of isavuconazole sulfate as 0.01 wt%, 0.1 wt%, and 0.15 wt% (take the isavuconazole sulfate enantiomer reference substance and make a 0.44 mg / ml solution with acetonitrile-aqueous solution (where the volume ratio of acetonitrile to water is 50:50) as the stock solution. After weighing the test sample, add 10 μl, 100 μl, and 150 μl of the stock solution respectively, and the other preparations are the same as the preparation of the test solution in Example 5), and examine the accuracy of liquid chromatography tests. Use the test condition 7 in Example 2 as the liquid chromatography detection condition to detect the recovery blank solution (prepared in the same way as the test solution in Example 5), 0.01 wt% isavuconazole sulfate enantiomer solution (i.e., the recovery quantitation limit solution), 0.1 wt% isavuconazole sulfate enantiomer solution (i.e., the 100% recovery solution), and 0.15 wt% isavuconazole sulfate enantiomer solution (i.e., the 150% recovery solution). Each concentration is repeated 3 times, and the average recovery rate meets 100±20%, and the relative standard deviation ≤10%. The method has good accuracy, and the results are shown in Table 10 and Figure 5 . The accuracy is calculated as measured amount / added amount × 100%.

[0142] Table 10 Results of accuracy test

[0143]

[0144] From the above test results, it can be seen that the accuracies of the measured isavuconazole enantiomers are all in the range of 80% - 120%. The liquid phase analysis method has good accuracy. From the relatively high detection accuracy, it can be known that after the enzymatic hydrolysis process, the isavuconazole sulfate enantiomers can be completely converted into isavuconazole enantiomers, and no configuration change occurs during the conversion process.

[0145] Example 10 Precision test

[0146] In this example, the test condition 7 in Example 2 is used as the liquid chromatography detection condition.

[0147] The precision test was carried out by means of method repeatability and intermediate precision. Method repeatability was achieved by repeatedly preparing the same batch of test samples 6 times (after preparing the test sample solution as in Example 5, adding isavuconazole enantiomer at a quantity of 0.10 wt%). Calculate the detected amount of enantiomers and the relative standard deviation of the detected amount in each test sample solution, and the RSD does not exceed 10%; Intermediate precision was verified by repeating the "method repeatability" by different analysts, on different dates, and using different instruments, and the RSD does not exceed 10%; And the relative deviation of the results of method repeatability and intermediate precision does not exceed 20%, meeting the requirements. The results are shown in Table 11 and Figure 6 。

[0148] Table 11 Results of precision test

[0149]

[0150] Example 11 Linear Test

[0151] In this example, Test Condition 7 in Example 2 was used as the liquid chromatography detection condition.

[0152] The linearity of the method was achieved by examining the linearity of solutions with different concentrations (Take isavuconazole reference substance and dissolve and dilute it with acetonitrile-aqueous solution (the volume ratio of acetonitrile to water is 50:50) to prepare a series of concentration solutions of 0.05, 0.52, 173.91, 347.81, 521.72, 695.62 μg / ml; Take isavuconazole enantiomer reference substance and dissolve and dilute it with acetonitrile-water (the volume ratio of acetonitrile to water is 50:50) to prepare a series of concentration solutions of 0.06, 0.30, 0.60, 0.90, 1.20 μg / ml). Plot the measured peak area against the concentration to obtain a curve, and calculate the regression equation, correlation coefficient and intercept by the least squares method. The method has good linearity. The results are shown in Table 12 and Figure 7 。

[0153] Table 12 Results of linearity test

[0154]

[0155] From the above results, it can be seen that the linear equations of isavuconazole reference substance and isavuconazole enantiomer have good linearity.

Claims

1. A method for detecting isavuconazole sulfate enantiomers, characterized in that: At least the following steps are included: (1) Preparation of system suitability solution: taking appropriate amounts of isavuconazole and isavuconazole enantiomers, adding a first solvent to dissolve and dilute them to prepare a system suitability solution; (2) Preparation of the test solution: Take an appropriate amount of isavuconazole sulfate test sample, add an enzyme test solution to accelerate the hydrolysis of isavuconazole sulfate and isavuconazole sulfate enantiomers, then add a second solvent to dissolve and dilute, and finally add an enzyme reaction termination solution to prepare a test solution; (3) Chromatographic test: Take appropriate amounts of the system suitability solution and the test solution, inject them into a liquid chromatograph, record the chromatogram, and calculate the amount of isavuconazole sulfate enantiomers by the area normalization method. The liquid chromatograph detection conditions are as follows: Detector: UV detector; Chromatographic column: a chiral chromatographic column with cellulose-tris(3,5-dichlorophenylcarbamate) bonded to the surface of silica gel as filler; Mobile phase A: ammonium acetate solution; Mobile phase B: a mixed solution of ammonium acetate solution and acetonitrile, with a volume ratio of ammonium acetate solution to acetonitrile of 20:80; Flow rate: 0.5~1.0ml / min; Column temperature: 25-35°C; Detection wavelength: 284nm; Injection volume: 10 μl; The mobile phase gradient elution program is as follows: The enzyme test solution is prepared from a buffer solution and an enzyme, wherein the enzyme is selected from trypsin, and 1wt% trypsin pH8.0 phosphate buffer is used as the enzyme test solution. The enzyme reaction termination solution is prepared from a hydrochloric acid solution and a first solvent, and the concentration of the hydrochloric acid in the enzyme reaction termination solution is ≥0.1mol / L.

2. The method for detecting isavuconazole sulfate enantiomers according to claim 1, wherein the buffer solution is selected from a phosphate buffer, an acetate buffer, and a citrate buffer, and the pH value of the enzyme test solution is adjusted to 7 to 9 by the buffer solution.

3. The method for detecting isavuconazole sulfate enantiomers according to claim 1, wherein the first solvent is selected from at least one of acetonitrile or an aqueous solution thereof, methanol or an aqueous solution thereof, and ethanol or an aqueous solution thereof.

4. The method for detecting isavuconazole sulfate enantiomers according to claim 3, wherein the first solvent is an acetonitrile aqueous solution, and the volume proportion of acetonitrile in the acetonitrile aqueous solution is ≥30%.

5. The method for detecting isavuconazole sulfate enantiomers according to claim 1, wherein the second solvent is selected from at least one of acetonitrile or an aqueous solution thereof, methanol or an aqueous solution thereof, and ethanol or an aqueous solution thereof.

6. The method for detecting isavuconazole sulfate enantiomers according to claim 1, wherein the pH of the ammonium acetate solution in the mobile phase A and the mobile phase B is between 4.5 and 5.

5.

7. The method for detecting isavuconazole sulfate enantiomers according to claim 6, wherein the pH of the ammonium acetate solution in the mobile phase A and the mobile phase B is between 4.8 and 5.2.

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