Detection method of isavuconazole sulfate enantiomer

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for detecting an isavuconazole sulfate enantiomer. According to the method, the to-be-detected sample isavuconazole sulfate and the enantiomer thereof are accelerated to be hydrolyzed and converted into isavuconazole and the enantiomer thereof under the catalytic action of enzyme, and the isavuconazole and the enantiomer thereof are separated and detected through liquid chromatography, so that the content of the isavuconazole sulfate enantiomer is detected. Further, the impurity content of enantiomers in the isavuconazole sulfate raw material medicine or the preparation thereof is controlled, and the quality controllability of the isavuconazole sulfate raw material medicine and the preparation thereof is ensured. The problem that the isavuconazole sulfate enantiomer is difficult to detect in the quality control of the isavuconazole sulfate raw material medicine and the preparation thereof is solved.

Description

Technical Field

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

[0002] Isavuconazole sulfate is a new triazole broad-spectrum antifungal drug first developed by Basilea Pharmaceuticals. In 2015, isavuconazole sulfate capsules and powder for injection 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]carbamoyl}oxy)ethyl]-1H-1,2,4-triazol-4-ium monosulfate, and the structural formula is as follows:

[0003] .

[0004] Isavuconazole sulfate is a prodrug of isavuconazole. Under the catalytic action of enzymes in the body, it can be rapidly hydrolyzed into the active part isavuconazole and the inactive dissociated part. Isavuconazole sulfate has three chiral centers. The two centers C7 and C8 of the isavuconazole part are both R configurations. The third chiral center C29 is not on the isavuconazole part and has both R and S configurations. It is a racemate. Isavuconazole sulfate is a mixture of two diastereomers of C29. The enantiomers of isavuconazole sulfate are C7 (S), C8 (S) and C29 (R / S) structures. Since the reference substance of isavuconazole sulfate enantiomers is very expensive and difficult to prepare, the detection of isavuconazole sulfate enantiomers in the quality control of isavuconazole sulfate has become a technical problem in this field. The structural formula of isavuconazole sulfate enantiomers is as follows:

[0005] .

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

[0007] .

[0008] The chemical name of the isavuconazole enantiomer is 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] Prior art, such as the patent with application number CN202311619785.6, discloses a method for detecting related substances in isavuconazole sulfate. In this method, high performance liquid chromatography is used for detection. In the chromatographic detection, octadecylsilane bonded silica gel is used as a chromatographic column, trifluoroacetic acid aqueous solution is used as mobile phase A, and a mixed solution of trifluoroacetic acid methanol-acetonitrile is used as mobile phase B to detect related substances in isavuconazole sulfate. However, this method can only separate and detect diastereomers in isavuconazole sulfate, and cannot separate and detect enantiomers of isavuconazole sulfate.

[0011] The patent with application number CN202311185496.X discloses a method and application of UPLC-MS / MS combined detection of isavuconazole. In this method, ultra-high performance liquid chromatography-mass spectrometry is used, C18 with ethylene bridge hybrid particles as filler is used as a 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, but this method cannot separate and detect the enantiomers of isavuconazole sulfate.

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

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

[0014] (1) Preparation of system suitability solution: Take appropriate amount of isavuconazole and isavuconazole enantiomers, add the first solvent to dissolve and dilute, and prepare the system suitability solution;

[0015] (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;

[0016] (3) Chromatographic test: Take appropriate amounts of system suitability solution and test solution, inject them into a liquid chromatograph, record the chromatogram, and calculate the amount of isavuconazole sulfate enantiomers by area normalization method. As a preferred technical solution, the liquid chromatograph detection conditions are as follows:

[0017] Detector: UV detector;

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

[0019] Mobile phase A: ammonium acetate solution;

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

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

[0022] Column temperature: 25-35°C;

[0023] Detection wavelength: 284nm;

[0024] Injection volume: 10 µl;

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

[0026] .

[0027] In the present invention, the hydrolysis of isavuconazole sulfate into isavuconazole is accelerated by enzyme test solution, and the hydrolysis of isavuconazole sulfate enantiomers into isavuconazole enantiomers is accelerated. Since isavuconazole sulfate and isavuconazole sulfate enantiomers have the same physicochemical properties, the hydrolysis rate is the same under the action of the same enzyme, and the amount of isavuconazole sulfate enantiomers can be obtained by detecting the amount of isavuconazole enantiomers. The hydrolysis conditions of the present invention are relatively mild and will not cause the flipping of the chiral configuration. The enzymatic conversion rate test combined with the accuracy test can prove that the isavuconazole sulfate enantiomers can be completely converted into the isavuconazole enantiomers after enzymatic hydrolysis, and the configuration will not change during the conversion process. The hydrolysis rate of isavuconazole sulfate is very slow without the addition of a catalytic enzyme and more other impurities will be generated.

[0028] As a preferred technical solution, the enzyme test solution is prepared from a buffer solution and an enzyme, wherein 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, acetate buffer and citrate buffer, and the buffer solution adjusts the pH value of the enzyme test solution to 7-9.

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

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

[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 the 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, and 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, and ethanol or its aqueous solution.

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

[0037] As a preferred technical solution, the volume proportion 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%, and 90%.

[0038] As a more preferred technical solution, the volume proportion 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, wherein the volume proportion 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, and 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 liquid chromatograph detection conditions are as follows:

[0043] Detector: UV detector;

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

[0045] Mobile phase A: ammonium acetate solution;

[0046] Mobile phase B: a mixture of ammonium acetate solution and acetonitrile;

[0047] Flow rate: 0.7ml / min;

[0048] Column temperature: 30°C;

[0049] Detection wavelength: 284nm;

[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 between 4.5 and 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 between 4.8 and 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 by glacial acetic acid.

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

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

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

[0062] Beneficial Effects

[0063] In the present invention, isavuconazole sulfate, a sample to be tested, is accelerated to hydrolyze under the action of an enzyme to obtain isavuconazole and its enantiomers. Since isavuconazole sulfate and its enantiomers have the same physicochemical properties, they are 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, thereby realizing the detection of the content of isavuconazole sulfate enantiomers, and then controlling the impurity content of enantiomers in isavuconazole sulfate bulk drug or its preparation, and ensuring that the quality of isavuconazole sulfate bulk drug and its preparation is controllable. The problem of difficulty in detecting isavuconazole sulfate enantiomers in the quality control of isavuconazole sulfate bulk drug and its preparation is solved. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 This is the liquid chromatogram of the solution for testing the system suitability in Example 5.

[0065] Figure 2 The liquid chromatogram of the test solution in Example 5 is shown in FIG.

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

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

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

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

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

[0071] Figure 8 is a linear diagram of the isavuconazole enantiomers in Example 11. DETAILED DESCRIPTION

[0072] To facilitate those skilled in the art to understand the content of the present invention, 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 cannot be understood as limiting the present invention.

[0073] Example 1 Selection of solvent

[0074] Appropriate amounts of isavuconazole sulfate and isavuconazole were taken respectively, and acetonitrile aqueous solutions with different volume proportions of acetonitrile were used to dissolve isavuconazole sulfate and isavuconazole respectively, and the dissolution was observed visually, 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, acetonitrile aqueous solution (the volume ratio of acetonitrile to water is 10:90) has poor solubility for isavuconazole, and the other solvents have good solubility for isavuconazole.

[0078] Example 2 Selection of chromatographic conditions

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

[0080] HPLC detection conditions:

[0081] Flow rate: 0.7 ml / min;

[0082] Column temperature: 30°C;

[0083] Detection wavelength: 284nm;

[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] The liquid chromatography conditions in test conditions 1-7 in Table 2 were screened respectively by the system suitability solution, and the separation between the two peaks of isavuconazole and isavuconazole enantiomers in the liquid chromatogram was recorded and observed. The specific chromatographic screening conditions and results 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 filler is cellulose-tris(3,5-dichlorophenylcarbamate) bonded on the surface of silica gel, the mobile phase A is 0.01 mol / L ammonium acetate solution (the pH value is adjusted to 5.0 with glacial acetic acid), the mobile phase B is 0.1 mol / L ammonium acetate solution (the pH value is adjusted to 5.0 with glacial acetic acid)-acetonitrile (the 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 enantiomers 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 hydrolases to explore the enzymatic hydrolysis conditions.

[0095] Pepsin, trypsin and butyrylcholinesterase were used to enzymolyze isavuconazole sulfate (the specific preparation method is: take an appropriate amount of isavuconazole sulfate, dissolve and dilute it with the corresponding enzyme solution to prepare a solution with a concentration of 4 mg / ml of isavuconazole sulfate, react according to the enzymolysis conditions in Table 3, accurately add an equal volume of acetonitrile to dissolve it, shake well, accurately measure 1 ml, and mix it with 1 ml of enzyme reaction termination solution (hydrochloric acid solution and acetonitrile aqueous solution (the volume ratio of acetonitrile to water is 50:50) to prepare an enzyme reaction termination solution with a hydrochloric acid concentration of 0.1 mol / L), mix well, and use the isavuconazole reference substance external standard method to detect the amount converted to isavuconazole (the chromatographic detection conditions use the test conditions 7 in Example 2, and compare it with the theoretical amount of isavuconazole sulfate converted to isavuconazole, and calculate the enzymolysis conversion rate (actual amount of isavuconazole sulfate converted to isavuconazole / theoretical amount of isavuconazole sulfate converted to isavuconazole = conversion rate), the results are shown in Table 3.

[0096] Table 3 - Enzymatic hydrolysis conditions and results

[0097]

[0098] Preparation of phosphate buffer: 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] The screening results of enzymatic hydrolysis conditions in Table 3 show that trypsin and butyrylcholinesterase have better enzymatic hydrolysis effects. Considering the comprehensive cost, 1wt% trypsin pH 8.0 phosphate buffer was selected as the enzyme test solution.

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

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

[0102] Example 4 Selection of enzyme reaction termination solution conditions

[0103] Preparation of enzyme reaction termination solution: prepared from hydrochloric acid solution and acetonitrile solution (the volume ratio between acetonitrile and water in acetonitrile solution is 50:50). Investigation of hydrochloric acid dosage: After isavuconazole sulfate is hydrolyzed in a water bath at 37°C, acid needs to be added to terminate the enzyme reaction activity, which is beneficial to the extraction of the target isavuconazole, so the amount of hydrochloric acid is investigated. Take 1 ml of the solution after isavuconazole sulfate hydrolysis obtained under test condition 4 in Example 3, mix it with 1 ml of hydrochloric acid extract of different concentrations, and then inject it into the liquid chromatography (liquid chromatography condition is 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 to isavuconazole / the amount of theoretical isavuconazole sulfate converted to isavuconazole = conversion rate), as shown in Table 4.

[0104] Table 4 - Experimental results

[0105]

[0106] The results showed that the conversion and extraction rate was over 95% when the enzyme reaction termination solution with a hydrochloric acid concentration of 0.1 mol / L was added. Therefore, it was beneficial to the conversion and extraction that the solution after enzymatic hydrolysis of the sample was mixed with the enzyme reaction termination solution with a hydrochloric acid concentration of 0.1 mol / L in equal proportions before sampling.

[0107] Example 5 Specificity Test

[0108] In this example, test condition 7 in Example 2 was used as the liquid chromatography detection condition.

[0109] Preparation of system suitability solution: Take appropriate amount of isavuconazole and its enantiomer, add acetonitrile-water solution (the volume ratio of acetonitrile to water is 50:50), dissolve and dilute to make 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, weigh accurately, dissolve it in acetonitrile-water solution (the volume ratio of acetonitrile to water is 50:50) and quantitatively dilute it to make a solution of about 0.3 μg per 1 ml.

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

[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 the test conditions 7 in Example 2. Record the chromatogram. The test results are shown in Tables 4 and Figure 1 In the system suitability solution chromatogram, isavuconazole enantiomers and isavuconazole eluted in sequence, and the separation degree between the two was greater than 1.5, indicating good separation; the theoretical plate number of the main peak of isavuconazole was greater than 5000, and the peak shape was good.

[0113] 10 μl of the sensitivity solution was accurately measured and injected into the liquid chromatograph. The chromatogram was recorded. The results are shown in Table 5. The signal-to-noise ratio of the peak height of the isavuconazole enantiomer peak was 175, which was greater than 10.

[0114] Accurately measure 10 μl of the test solution, inject it into the liquid chromatograph, and record the chromatogram. The results are shown in Table 5 and Figure 2 , enantiomers were not detected in the chromatogram of the test solution, which met the requirements.

[0115] Table 5 Specificity test results

[0116]

[0117] The above test results show that it has good sensitivity.

[0118] Example 6 Durability Test

[0119] In this example, test condition 7 in Example 2 was used as the liquid chromatography detection condition.

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

[0121] The durability of the HPLC detection method was investigated by changing the mobile phase ratio and the mobile phase pH value. Only one of the parameters was changed in each test, and the result was compared with the measurement result when the parameter was not changed. The chromatographic conditions in this embodiment are compared with the chromatographic conditions in the test condition 7 in Example 2, and the acetonitrile volume ratio in the mobile phase B is changed to 78% to 83%, and the pH value of the buffer in the mobile phase A and the mobile phase B is changed by ±0.2 at the same time. Take 10 μl of the system suitability solution respectively, inject it into the liquid chromatograph according to the standard chromatographic conditions, chromatographic conditions 1, chromatographic conditions 2, chromatographic conditions 3 and chromatographic conditions 4 in Table 5, and record the chromatogram. The chromatographic conditions and test results are shown in Table 6.

[0122] Table 6 Durability test results

[0123]

[0124] The test results show that under standard chromatographic conditions, chromatographic conditions 1, chromatographic conditions 2, chromatographic conditions 3 and chromatographic conditions 4, that is, when the pH of the ammonium acetate solution in mobile phase A and mobile phase B is between 4.8 and 5.2, or when the volume ratio of acetonitrile in mobile phase B is between 78% and 83%, the separation between the chromatographic peaks of isavuconazole and isavuconazole enantiomers is greater than 1.5, which meets all system suitability test requirements, indicating that the method has 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] Isavuconazole enantiomer reference solution was prepared (3 mg of isavuconazole enantiomer reference was dissolved in acetonitrile-water solution (the volume ratio of acetonitrile and water was 50:50) and quantitatively diluted to prepare a 0.6 μg / ml solution), placed at 5±3°C, and tested by liquid chromatography injection at 0, 16, and 24 hours, respectively. Compared with 0 hour, the change in peak area was calculated, and the change in peak area was no more 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 stability test of reference solution

[0129]

[0130] The test solution was prepared. The test solution in this example was prepared in the same manner as the test solution in Example 5. The prepared test solution was placed at 5±3°C, and samples were injected and tested at 0, 8, 12, and 24 hours, respectively. Compared with 0 hour, the change in the amount of enantiomers was calculated, and the peak area change values ​​were all less than 20%. The test solution was determined to have good stability within 24 hours. The results are shown in Table 8.

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

[0132]

[0133] Example 8 Quantitation Limit and Detection Limit Test

[0134] In this example, test condition 7 in Example 2 was used as the liquid chromatography detection condition.

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

[0136] Table 9 Quantitation limit and detection limit test results

[0137]

[0138] The test results showed that the limit of quantification of isavuconazole was 0.052µg / ml, the limit of quantification of isavuconazole enantiomer was 0.060µg / ml, the limit of detection of isavuconazole was 0.016µg / ml, and the limit of detection of isavuconazole enantiomer was 0.018µg / ml.

[0139] Example 9 Accuracy Test

[0140] In this example, test condition 7 in Example 2 was used as the liquid chromatography detection condition.

[0141] The accuracy of the method is achieved by examining the accuracy of the liquid chromatography test after adding different amounts of isavuconazole sulfate enantiomers to the sample. The accuracy of the liquid chromatography test was investigated when the amount of isavuconazole sulfate enantiomers added to the isavuconazole sulfate test sample was 0.01wt%, 0.1wt%, and 0.15wt% (the isavuconazole sulfate enantiomer reference substance was added with acetonitrile-water solution (wherein the volume ratio of acetonitrile and water was 50:50) to prepare a 0.44mg / ml solution as a stock solution, and 10μl, 100μl, and 150μl of the stock solution were added to the sample to be tested, respectively, and the other preparations were the same as the test solution preparation in Example 5). Using test condition 7 in Example 2 as the liquid chromatography detection condition, the recovery blank solution (prepared with the test solution in Example 5), 0.01wt% isavuconazole sulfate enantiomer solution (i.e., recovery limit solution), 0.1wt% isavuconazole sulfate enantiomer solution (i.e., recovery 100% solution) and 0.15wt% isavuconazole sulfate enantiomer solution (i.e., recovery 150% solution) were tested. Each concentration was repeated 3 times. The average recovery rate was consistent with 100±20%, the relative standard deviation was ≤10%, and the method had good accuracy. The results are shown in Table 10 and Figure 5 The accuracy is calculated by measured amount / added amount × 100%.

[0142] Table 10 Accuracy test results

[0143]

[0144] From the above test results, it can be seen that the accuracy of the isavuconazole enantiomers is within the range of 80% to 120%, and the liquid phase analysis method has good accuracy. From the high detection accuracy, it can be seen 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, test condition 7 in Example 2 was used as the liquid chromatography detection condition.

[0147] The precision test was conducted through method repeatability and intermediate precision. The method repeatability was tested by repeatedly preparing the same batch of test samples 6 times (the test sample solution was prepared in the same way as in Example 5, and 0.10wt% of isavuconazole enantiomers were added), and the amount of enantiomers detected in each test sample solution and the relative standard deviation of the detected amount were calculated, and the RSD did not exceed 10%; the intermediate precision was verified by repeating the "method repeatability" verification by different analysts, on different dates, and using different instruments, and the RSD did not exceed 10%; and the relative deviation of the results of method repeatability and intermediate precision did not exceed 20%, which met the requirements. The results are shown in Table 11 and Figure 6 .

[0148] Table 11 Precision test results

[0149]

[0150] Example 11 Linearity 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 of different concentrations (the isavuconazole reference substance was dissolved and diluted with acetonitrile-water solution (the volume ratio of acetonitrile and water was 50:50) to prepare a series of concentration solutions of 0.05, 0.52, 173.91, 347.81, 521.72, and 695.62 μg / ml; the isavuconazole enantiomer reference substance was dissolved and diluted with acetonitrile-water (the volume ratio of acetonitrile and water was 50:50) to prepare a series of concentration solutions of 0.06, 0.30, 0.60, 0.90, and 1.20 μg / ml). The measured peak area was plotted against the concentration, and the regression equation, correlation coefficient, and intercept were calculated using the least squares method. The method had good linearity, and the results are shown in Table 12 and Figure 7 .

[0153] Table 12 Linearity test results

[0154]

[0155] From the above results, it can be seen that the linear equations of isavuconazole reference substance and isavuconazole enantiomers 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: Take appropriate amount of isavuconazole and isavuconazole enantiomers, add the first solvent to dissolve and dilute, and prepare the 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 system suitability solution and 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 mixture of ammonium acetate solution and acetonitrile; 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: 。 2. The method for detecting isavuconazole sulfate enantiomers according to claim 1, wherein the enzyme test solution is prepared from a buffer solution and an enzyme, wherein the enzyme is selected from one of trypsin and butyrylcholinesterase.

3. The method for detecting isavuconazole sulfate enantiomers according to claim 2, 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.

4. The method for detecting isavuconazole sulfate enantiomers according to claim 1, wherein 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 ≥ 0.1 mol / L.

5. 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.

6. The method for detecting isavuconazole sulfate enantiomers according to claim 5, wherein the first solvent is an acetonitrile aqueous solution, and the volume proportion of acetonitrile in the acetonitrile aqueous solution is ≥30%.

7. 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.

8. The method for detecting isavuconazole sulfate enantiomers according to claim 1, wherein the volume ratio of ammonium acetate solution to acetonitrile in the mobile phase B is (15-25): (75-85).

9. 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.

10. The method for detecting isavuconazole sulfate enantiomers according to claim 9, 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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