Method for detecting chiral isomer of posaconazole

Abstract

The invention relates to a method for detecting a chiral isomer of posaconazole in a raw material medicine or a preparation, which comprises the following steps: (1) taking the raw material medicine or the preparation of posaconazole, dissolving with an organic solvent, and adding a drying agent to obtain a solution to be detected; and (2) determining the obtained to-be-detected solution by adopting high performance liquid chromatography, and calculating the content of the posaconazole isomer in the posaconazole raw material medicine or preparation. The sample pretreatment method of the detection method is simple, the durability of the chromatographic column can be ensured, the detection time is short, the effectiveness and accuracy of the detection result can be ensured, and the quality of posaconazole can be accurately and strictly controlled.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical analysis, in particular to a method for detecting chiral isomers of posaconazole in raw materials or preparations. BACKGROUND

[0002] Posaconazole is a triazole antifungal drug, which is used for the treatment of invasive aspergillosis, candida infection and fungal infection caused by hyalohyphomycetes and fusaria. Posaconazole is a lanosterol 14a-demethylase inhibitor. There are six new molecular entities for deep fungal infections on the market with the same target: fluconazole, itraconazole, voriconazole, posaconazole, and esproconazole. Posaconazole has the widest indication for prophylaxis and the second widest indication for treatment after voriconazole; its safety is only inferior to fluconazole. According to its position in the relevant treatment guidelines at home and abroad and its global sales data, posaconazole has a good clinical application prospect and great market value. At present, there are three dosage forms of posaconazole in China, including posaconazole enteric-coated tablets, posaconazole injection and posaconazole oral suspension, which can be used in clinical practice. However, there is no test analysis method for posaconazole in the current edition of Chinese Pharmacopoeia.

[0003] Isomer separation of chiral drugs is a difficulty in the treatment control of chiral drugs. Posaconazole has four chiral centers, and there are 15 potential stereoisomers in addition to posaconazole. According to the synthesis reaction route of posaconazole, six stereoisomers are likely to be introduced into the active pharmaceutical ingredient. Considering the safety and effectiveness of drug use, a simple and accurate detection method needs to be established to monitor the chiral isomers of posaconazole. SUMMARY

[0004] The present application aims to provide a detection method for chiral isomers of posaconazole, which solves the defects of the prior art in the detection method for isomers in posaconazole, can effectively detect and separate six chiral isomers in posaconazole oral suspension, ensures the effectiveness and accuracy of the detection results, and can accurately and strictly control the quality of posaconazole products.

[0005] The first aspect of the present application relates to a method for detecting chiral isomers of posaconazole in raw materials or preparations, comprising the following steps:

[0006] Step (1): taking posaconazole raw materials or posaconazole preparations, dissolving with an organic solvent, adding a drying agent to obtain a solution to be detected;

[0007] Step (2): determining the solution to be detected obtained in step (1) by high performance liquid chromatography to calculate the content of posaconazole isomers in posaconazole raw materials or preparations; the mobile phase used in the high performance liquid chromatography detection is dichloromethane and isopropyl alcohol.

[0008] In one or more embodiments of the present application, the compound of posaconazole has the following structure: with the chiral configuration of R,R,S,S.

[0009] In one or more embodiments of the present application, the chiral isomer of posaconazole is one or more of isomer I (R,R,S,R), isomer II (R,R,R,S), isomer III (R,R,R,R), isomer IV (S,R,S,S), isomer V (R,S,S,S), and isomer VI (S,S,S,S).

[0010] In one or more embodiments of the present application, the chiral isomer of posaconazole is a combination of isomer II (R,R,R,S) and isomer III (R,R,R,R).

[0011] In one or more embodiments of the present application, the chiral isomer of posaconazole is a combination of isomer I (R,R,S,R), isomer II (R,R,R,S), isomer III (R,R,R,R), isomer IV (S,R,S,S), isomer V (R,S,S,S), and isomer VI (S,S,S,S).

[0012] In one or more embodiments of the present application, the mobile phase is a mixture of dichloromethane and isopropanol in a volume ratio of 60-48:40-52. In one or more embodiments of the present application, the mobile phase is a mixture of dichloromethane and isopropanol in a volume ratio of 60-55:40-45. In one or more embodiments of the present application, the mobile phase is a mixture of dichloromethane and isopropanol in a volume ratio of 60:40.

[0013] In one or more embodiments of the present application, the mobile phase contains an organic amine in a volume percentage of 0.1-0.5%. In one or more embodiments of the present application, the mobile phase contains diethylamine in a volume percentage of 0.1-0.5%. In one or more embodiments of the present application, the mobile phase contains diethylamine in a volume percentage of 0.1-0.3%. In one or more embodiments of the present application, the mobile phase contains diethylamine in a volume percentage of 0.1%.

[0014] In one or more embodiments of the present application, the organic solvent in step (1) is an alcohol. In one or more embodiments of the present application, the organic solvent in step (1) is selected from one or more of methanol, ethanol, and isopropanol. In one or more embodiments of the present application, the organic solvent in step (1) is methanol.

[0015] In one or more embodiments of the present application, the drying agent in step (1) is a neutral drying agent. In one or more embodiments of the present application, the drying agent in step (1) is one of anhydrous sodium sulfate, anhydrous MgSO4, anhydrous CaCl2, or CaSO4. In one or more embodiments of the present application, the drying agent in step (1) is anhydrous sodium sulfate.

[0016] In one or more embodiments of the present application, the preparation in step (1) is posaconazole oral suspension or posaconazole enteric-coated tablet. In one or more embodiments of the present application, the preparation in step (1) is posaconazole oral suspension.

[0017] In one or more embodiments of the present application, the column used in the high performance liquid chromatography detection is a polysaccharide bonded chiral column.

[0018] In one or more embodiments of the present application, the column type is Chiralpak IA, Chiralpak IB, Chiralpak IC, Chiralpak ID3, Chiralpak IE3, Chiralpak IF3, or Chiralpak IG. In one or more embodiments of the present application, the column type is Chiralpak IB or Chiralpak IC, and the column parameters are 4.6 x 250 mm, 5 μm. In one or more embodiments of the present application, the column type is Chiralpak IC, and the column parameters are 4.6 x 250 mm, 5 μm.

[0019] In one or more embodiments of the present application, the column temperature used in the high performance liquid chromatography detection is 25-35°C. In one or more embodiments of the present application, the column temperature used in the high performance liquid chromatography detection is 30-35°C. In one or more embodiments of the present application, the column temperature used in the high performance liquid chromatography detection is 25°C.

[0020] In one or more embodiments of the present application, the detection wavelength used in the high performance liquid chromatography detection is 200 nm-262 nm. In one or more embodiments of the present application, the detection wavelength used in the high performance liquid chromatography detection is 200 nm-254 nm. In one or more embodiments of the present application, the detection wavelength used in the high performance liquid chromatography detection is 254 nm-262 nm. In one or more embodiments of the present application, the detection wavelength used in the high performance liquid chromatography detection is 260 nm.

[0021] In one or more embodiments of the present application, the detection time used in the high performance liquid chromatography detection is 30-35 min.

[0022] In one or more embodiments of the present application, the flow rate of the mobile phase used in the high performance liquid chromatography detection is 0.8-1.2 mL / min. In one or more embodiments of the present application, the flow rate of the mobile phase used in the high performance liquid chromatography detection is 1.0 mL / min.

[0023] In one or more embodiments of the present application, the sample injection amount used in the high performance liquid chromatography detection is 18-25 μL. In one or more embodiments of the present application, the sample injection amount used in the high performance liquid chromatography detection is 18-22 μL. In one or more embodiments of the present application, the sample injection amount used in the high performance liquid chromatography detection is 20 μL.

[0024] In one or more embodiments of the present application, the method for calculating the isomer content is self-control method or external standard method.

[0025] In one or more embodiments of the present application, the separation between isomer I, isomer IV, isomer V and isomer VI is greater than or equal to 1.5.

[0026] In one or more embodiments of the present application, the separation between isomer II and isomer III is greater than or equal to 1.

[0027] In the present application, the term "detection" refers to qualitative detection or quantitative detection if not otherwise specified.

[0028] In the present application, the term "chiral isomer" refers to the number and its corresponding compound as shown in Table 1 below if not otherwise specified.

[0029] Table 1 Compound number and structure

[0030]

[0031]

[0032] Advantages of the present application

[0033] The present application successfully obtains an analysis and detection method for simultaneously and accurately detecting multiple isomer components in posaconazole by optimizing the chromatographic conditions such as column temperature and mobile phase composition. The sample pretreatment method of the detection method of the present application is simple, and can ensure the durability of the chromatographic column, shorten the detection time, and ensure the effectiveness and accuracy of the detection results, which is conducive to accurately and strictly controlling the quality of posaconazole. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 Example 1 Chromatogram of the test sample

[0035] Figure 2Example 2 Detection of chromatographic profile.

[0036] Figure 3 Example 3 Detection of chromatographic profile.

[0037] Figure 4 Example 4 Detection of chromatographic profile DETAILED DESCRIPTION

[0038] Example 1 Analysis and detection of 6 chiral isomers in posaconazole oral suspension

[0039] 1. Experimental method

[0040] Preparation of reference solution: dissolve and dilute isomers I (R, R, S, R), II (R, R, R, S), III (R, R, R, R), IV (S, R, S, S), V (R, S, S, S), VI (S, S, S, S) with methanol to prepare 1 μg / ml of corresponding reference solution.

[0041] Preparation of test solution: take 0.7 g of posaconazole oral suspension, place in a 50 ml volumetric flask, add 1 ml of water, ultrasonicate for 10 min, dilute to the mark with methanol, shake well, add 14 g of anhydrous sodium sulfate to the solution, shake well, stand, filter the solution, and take the filtrate to obtain a 0.5 mg / ml test solution.

[0042] Preparation of system suitability solution: take the middle part of the well-shaken posaconazole oral suspension, accurately weigh about 0.7 g, place in a 50 ml volumetric flask, add 1 ml of water, add 2.5 ml of the above-mentioned reference solution of 20 μg / ml, respectively, ultrasonicate for 10 min, dilute to the mark with methanol, shake well, add 14 g of anhydrous sodium sulfate to the solution, shake well, stand, filter the solution, and take the filtrate to obtain a system suitability solution.

[0043] Preparation of mobile phase: measure 500 ml of isopropyl alcohol, add 500 ml of dichloromethane, and add 1 ml of diethylamine, mix well, and obtain the mobile phase.

[0044] High performance liquid chromatography detection: take 20 μl of the test solution and the reference solution, respectively, inject into the liquid chromatograph, and record the chromatogram. Instrument and chromatographic parameters: high performance liquid chromatograph (Agilent 1260 HPLC), chromatographic column: Daicel Chiralpak IC 4.6 x 250 mm, 5 μm; flow rate: 1.0 ml / min; detection wavelength: 260 nm; column temperature: 30 °C; injection volume: 20 μl; isocratic elution; running time: 35 min.

[0045] Calculation results:

[0046]

[0047] In the formula: M is the sample amount (mg), Ws is the reference amount (mg), At is the impurity peak area in the test solution, As is the impurity peak area in the reference solution, Dt is the dilution volume of the test solution (ml), Ds is the dilution volume of the reference solution (ml), P is the purity of the reference (%); 1.145 is the density of the sample (g / ml), and 40 is the sample specification (mg / ml).

[0048] 2. Experimental Results

[0049] like Figure 1 As shown, isomers I (R,R,S,R), IV (S,R,S,S), V (R,S,S,S), and VI (S,S,S,S) can be accurately and effectively detected in posaconazole oral suspension. However, the resolution between isomers III (R,R,R,R) and II (R,R,R,S) is 0.62, therefore, they need to be combined for control. If these six stereoisomers are introduced into the synthesis of posaconazole, the analytical method of this embodiment can accurately and effectively detect these six stereoisomers in posaconazole raw materials or preparations, ensuring product quality. Furthermore, the test sample used in this embodiment does not require extraction, the preparation method is simple, and the added desiccant can improve the lifespan of the chromatographic column.

[0050] Example 2 Investigation of mobile phase volume ratio

[0051] 1. Experimental Methods

[0052] The mobile phase was prepared as follows: 400 ml of isopropanol was added, along with 600 ml of dichloromethane and 1 ml of diethylamine. The mixture was then thoroughly mixed. The remaining detection methods and conditions were the same as in Example 1, yielding the liquid chromatogram of the posaconazole oral suspension.

[0053] 2. Experimental Results

[0054] like Figure 2 As shown, the resolution between isomer III (R,R,R,R) and isomer II (R,R,R,S) was 0.97, which is significantly higher than the resolution between isomers III and II in Example 1. This indicates that increasing the volume percentage of dichloromethane in the mobile phase can improve the resolution between posaconazole isomer III (R,R,R,R) and isomer II (R,R,R,S), and can also effectively detect the other four chiral isomers simultaneously.

[0055] Example 3: Column Temperature Investigation

[0056] 1. Experimental Methods

[0057] The mobile phase was prepared as follows: 500 ml of isopropanol was measured, 500 ml of dichloromethane was added, and 1 ml of diethylamine was added, and mixed. The column temperature was 25°C, and the rest of the detection method and conditions were the same as in Example 1, to obtain the liquid chromatogram of posaconazole oral suspension.

[0058] 2. Experimental results

[0059] As shown in Figure 3 , the separation degree of posaconazole isomer II (R, R, R, S) and isomer III (R, R, R, R) was 1.19, which was further increased compared to the separation degree of isomer III and isomer II in Example 2, indicating that reducing the column temperature can further improve the separation degree of posaconazole isomer III (R, R, R, R) and isomer II (R, R, R, S).

[0060] Experimental Example 4, column temperature investigation

[0061] 1. Experimental method

[0062] The mobile phase was prepared as follows: 500 ml of isopropanol was measured, 500 ml of dichloromethane was added, and 1 ml of diethylamine was added, and mixed. The column temperature was 35°C, and the rest of the detection method and conditions were the same as in Example 1, to obtain the liquid chromatogram of posaconazole oral suspension.

[0063] 2. Experimental results

[0064] As shown in Figure 4 , the separation degree of posaconazole isomer II (R, R, R, S) and isomer III (R, R, R, R) was only 0.78, indicating that the separation degree of posaconazole isomer II (R, R, R, S) and isomer III (R, R, R, R) was reduced after increasing the column temperature.

[0065] Example 5, methodological validation of the analysis method of the application

[0066] The same solution system and chromatographic conditions as in Example 1 were used to evaluate the specificity, sensitivity, and accuracy of the method described in the application.

[0067] 1. Specificity

[0068] The blank solvent and blank excipient did not interfere with the detection of posaconazole oral suspension, and the specificity met the requirements. The separation degree between posaconazole (R, R, S, S) and chiral isomer II (R, R, R, S) in the system suitability solution was greater than 1.5, and the system suitability met the requirements, indicating that the method described in the application had good specificity.

[0069] 2. Sensitivity

[0070] Isomer name 30% control solution S / N 30% spiked solution S / N Isomer IV 106.42 149.99 Isomer I 54.26 78.89 Isomer II 32.71 47.94 Isomer VI 15.54 21.74 Isomer V 137.08 154.00 Isomer III 78.26 88.29

[0071] Conclusion: The signal-to-noise ratio of each peak in the 30% control solution of the limit concentration and the 30% spiked solution of the limit concentration is greater than 10, and the sensitivity of the method meets the requirements.

[0072] 3. Accuracy (repeatability)

[0073]

[0074]

[0075] Conclusion: In the 6 100% recovery sample solutions, the individual recovery rate of each impurity is between 80% and 120%, the average recovery rate is between 90% and 108%, and RSD% < 3.0% (n = 6), which meets the requirements, indicating that the method described in the application can accurately detect the isomer impurities in the sample.

[0076] The above detailed description further describes the application. However, it should not be understood that the above-mentioned subject matter of the application is limited to only the listed examples, and any technical solutions implemented based on the content of the application fall within the scope of the application.

Claims

1. A method of detecting posaconazole chiral isomers in a bulk drug substance or a formulation, characterized in that, The method comprises the following steps: Step (1): taking posaconazole raw material or posaconazole preparation, dissolving with an organic solvent, adding a drying agent to obtain a solution to be detected; Step (2): determining the solution to be detected obtained in step (1) by high performance liquid chromatography to calculate the content of posaconazole isomers in the posaconazole raw material or preparation; the mobile phase used in the high performance liquid chromatography detection is a mixture of dichloromethane and isopropanol.

2. The detection method according to claim 1, characterized in that, The chiral isomers are one or more of isomer I (R, R, S, R), isomer II (R, R, R, S), isomer III (R, R, R, R), isomer IV (S, R, S, S), isomer V (R, S, S, S), and isomer VI (S, S, S, S).

3. The method of claim 1, wherein, The posaconazole chiral isomers are a combination of isomer I (R, R, S, R), isomer II (R, R, R, S), isomer III (R, R, R, R), isomer IV (S, R, S, S), isomer V (R, S, S, S), and isomer VI (S, S, S, S).

4. The assay of any one of claims 1-3, wherein, The mobile phase is a mixture of dichloromethane and isopropanol in a volume ratio of 60-48:40-52.

5. The detection method according to claim 4, characterized in that, The mobile phase contains an organic amine in a volume percentage of 0.1-0.5%.

6. The assay of any one of claims 1-3, wherein, The organic solvent in step (1) is an alcohol; preferably one or more of methanol, ethanol, and isopropanol.

7. The assay of any one of claims 1-3, wherein, The drying agent in step (1) is a neutral drying agent selected from one of anhydrous sodium sulfate, anhydrous MgSO4, anhydrous CaCl2, or CaSO4.

8. The assay of any one of claims 1-3, wherein, The chromatographic column used in the high performance liquid chromatography detection is a polysaccharide-bonded chiral column, and the preferred chromatographic column type is selected from Chiralpak IA, Chiralpak IB, Chiralpak IC, Chiralpak ID3, Chiralpak IE3, Chiralpak IF3, or Chiralpak IG.

9. The assay of any one of claims 1-3, wherein, The column temperature in the high performance liquid chromatography detection is 25-35℃.

10. The assay of any one of claims 1-3, wherein, The detection time in the high performance liquid chromatography detection is 30-35 min.

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