Method for separation and determination of posaconazole Z3 and its impurities and solvents by HPLC

Abstract

The present invention belongs to the technical field of chemical analysis, and specifically relates to a method for separating and determining posaconazole Z3 and its impurities and solvents by HPLC. The present invention adopts octadecylsilane bonded silica gel filler as the stationary phase, a mixed solution made of potassium hexafluorophosphate solution and methanol in a volume ratio of 90:10 as mobile phase A, and a mixed solution made of acetonitrile and methanol in a volume ratio of 90:10 as mobile phase B, and separates posaconazole Z3 and its related impurities and solvent toluene by gradient elution; after separation, a detector with a detection wavelength of 210nm is used for detection. The related impurities include impurities Z 3e and p-toluenesulfonic acid and / or other related impurities, other related impurities are impurities SM 3d 、Impurity SM3、Impurity SM 3i , impurity Z2, impurity Z 3f And / or methyl p-toluenesulfonate; the method of the invention has strong specificity, high sensitivity, good reproducibility, and the detection result is accurate and reliable.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical analysis, and in particular relates to a method for separating and determining posaconazole Z3 and its impurities and solvents by HPLC. Background Art

[0002] Posaconazole belongs to the second generation of triazole antifungal drugs and is a derivative of itraconazole. Posaconazole can be used for fungemia caused by Candida and Cryptococcus, respiratory, digestive tract, urinary tract fungal diseases, peritonitis, meningitis, etc. Posaconazole Z3 is an intermediate in the synthesis process of posaconazole, and its structural formula is shown in Formula I. According to the analysis of its synthetic process route, there may be impurities SM 3d 、Impurity SM3、Impurity SM 3i , impurity Z2, impurity Z 3e 、Impurity Z 3f , methyl p-toluenesulfonate, 8 impurities of p-toluenesulfonic acid and solvent toluene, among which impurity SM 3d 、Impurity SM3、Impurity SM 3i , impurity Z2, impurity Z 3e 、Impurity Z 3f In order to set the quality standard impurities, the quality requirements of posaconazole Z3 can be guaranteed. However, the crystallization solvent toluene used in the synthesis process may interfere with the detection of impurities. Therefore, it is necessary to establish a method that can simultaneously separate posaconazole Z3, toluene and the above 8 impurities, a total of 10 substances; and because most impurities have low limits, it is necessary to establish an HPLC method with a sensitivity that meets the requirements.

[0003]

[0004] In the existing data, the invention patent with application number 202211642366.X can realize impurity SM 3d 、Impurity SM3、Impurity SM 3i The invention patent with application number 202211655413.4 separated and determined the impurity Z2; the invention patent with application number 201610056478.5 disclosed a method for separating and determining methyl p-toluenesulfonate in posaconazole intermediate Z. However, none of the above inventions can achieve the separation and determination of posaconazole Z3 and impurity SM 3d 、Impurity SM3、Impurity SM 3i , impurity Z2, impurity Z 3e 、Impurity Z 3f , simultaneous separation and content detection of methyl p-toluenesulfonate, p-toluenesulfonic acid and solvent toluene.

[0005] Currently, there is no statutory analysis method or literature analysis method that can effectively separate all eight impurities and toluene that may be present in posaconazole Z3. Summary of the invention

[0006] In view of this, one of the objects of the present invention is to provide a method for separating posaconazole Z3 and its eight impurities and solvents based on high performance liquid chromatography. The method has high specificity and high sensitivity, and provides technical support for the qualitative and quantitative detection of posaconazole Z3 and its eight impurities and solvents in the later stage.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] A method for separating posaconazole Z3 and its related impurities and solvents based on high performance liquid chromatography, characterized in that the method comprises: using octadecylsilane bonded silica gel filler as the stationary phase, using a mixed solution of potassium hexafluorophosphate solution and methanol as mobile phase A, and a mixed solution of acetonitrile and methanol as mobile phase B for linear gradient elution, wherein the volume ratio of potassium hexafluorophosphate solution and methanol in the mobile phase A is 90:10; the volume ratio of acetonitrile and methanol in the mobile phase B is 90:10; and separating posaconazole Z3 and its related impurities and solvents by gradient elution; the structural formula of posaconazole Z3 is as shown in formula I; the related impurities include an impurity composition and / or other related impurities, and the impurity composition is an impurity Z as shown in formula VI 3e and p-toluenesulfonic acid as shown in formula IX, wherein the other related impurities are impurities SM as shown in formula II 3d , impurity SM3 as shown in formula III, impurity SM as shown in formula IV 3i , impurity Z2 as shown in formula V, impurity Z as shown in formula VII 3f and any one or more of methyl p-toluenesulfonate as shown in Formula VIII; the solvent is toluene;

[0009]

[0010]

[0011] Further, the method can sequentially convert p-toluenesulfonic acid, impurity SM 3d , methyl p-toluenesulfonate, impurity SM 3i , toluene, impurity SM3, impurity Z2, posaconazole Z3, impurity Z 3f and impurity Z 3e to separate.

[0012] Furthermore, the components can be characterized based on how quickly they are separated.

[0013] Further, the procedure of the gradient elution is:

[0014] At 0 minutes, the volume fraction of mobile phase A was set to 70%-74%, and the volume fraction of mobile phase B was set to 30%-26%;

[0015] At 5 minutes, the volume fraction of mobile phase A was set to 72%, and the volume fraction of mobile phase B was set to 28%;

[0016] At 30 minutes, the volume fraction of mobile phase A was set to 40%, and the volume fraction of mobile phase B was set to 60%;

[0017] At 50 minutes, the volume fraction of mobile phase A was set to 28%, and the volume fraction of mobile phase B was set to 72%;

[0018] At 55 minutes, the volume fraction of mobile phase A was set to 15%, and the volume fraction of mobile phase B was set to 85%;

[0019] At 65 minutes, the volume fraction of mobile phase A was set to 15%, and the volume fraction of mobile phase B was set to 85%;

[0020] At 65.1 minutes, the volume fraction of mobile phase A was set to 72%, and the volume fraction of mobile phase B was set to 28%;

[0021] At 75 minutes, the volume fraction of mobile phase A was set to 72% and the volume fraction of mobile phase B was set to 28%.

[0022] Preferably, the procedure of the gradient elution is:

[0023] At 0 minutes, the volume fraction of mobile phase A was set to 72% and the volume fraction of mobile phase B was set to 28%;

[0024] At 5 minutes, the volume fraction of mobile phase A was set to 72%, and the volume fraction of mobile phase B was set to 28%;

[0025] At 30 minutes, the volume fraction of mobile phase A was set to 40%, and the volume fraction of mobile phase B was set to 60%;

[0026] At 50 minutes, the volume fraction of mobile phase A was set to 28%, and the volume fraction of mobile phase B was set to 72%;

[0027] At 55 minutes, the volume fraction of mobile phase A was set to 15%, and the volume fraction of mobile phase B was set to 85%;

[0028] At 65 minutes, the volume fraction of mobile phase A was set to 15%, and the volume fraction of mobile phase B was set to 85%;

[0029] At 65.1 minutes, the volume fraction of mobile phase A was set to 72%, and the volume fraction of mobile phase B was set to 28%;

[0030] At 75 minutes, the volume fraction of mobile phase A was set to 72% and the volume fraction of mobile phase B was set to 28%.

[0031] Furthermore, the pH value of the mobile phase A is 4.8-5.2, preferably 5.0; in the mobile phase A, the concentration of the potassium hexafluorophosphate solution is 0.01 mol / L.

[0032] Furthermore, the injection flow rate is 1.4 ml / min-1.6 ml / min, preferably 1.5 ml / min; the column temperature of the chromatographic column is 33°C-37°C, preferably 35°C.

[0033] Furthermore, the specifications of the chromatographic column are 4.6 mm×250 mm, 5 μm.

[0034] Furthermore, the injection volume was 10 μl.

[0035] The second object of the present invention is to provide a method for simultaneously detecting the contents of posaconazole Z3 and its related impurities and solvents, wherein the method can achieve the content determination of posaconazole Z3 and its eight impurities and solvent toluene within 75 minutes or more.

[0036] To achieve the above object, the present invention adopts the following technical solutions:

[0037] A method for simultaneously detecting the contents of posaconazole Z3 and its related impurities and solvent toluene, comprising separating the posaconazole Z3 and its related impurities and solvent by the method described in the first objective, and then detecting them by using a detector; and setting the detection wavelength of the detector to 210 nm.

[0038] Furthermore, the retention times of the separated components from short to long are: p-toluenesulfonic acid, impurity SM 3d , methyl p-toluenesulfonate, impurity SM 3i , toluene, impurity SM3, impurity Z2, posaconazole Z3, impurity Z 3f and impurity Z 3e .

[0039] As a more preferred method, the volume ratio of potassium hexafluorophosphate solution and methanol in the mobile phase A is 90:10; the volume ratio of acetonitrile and methanol in the mobile phase B is 90:10; the pH value of the mobile phase A is 5.0, the injection flow rate is 1.5 ml / min; the column temperature of the chromatographic column is 35° C., the specifications of the chromatographic column are 4.6 mm×250 mm, 5 μm; the detection wavelength is 210 nm;

[0040] Perform linear gradient elution according to the gradient elution program and obtain a chromatogram; the gradient elution program is set as follows:

[0041] At 0 minutes, the volume fraction of mobile phase A was set to 72% and the volume fraction of mobile phase B was set to 28%;

[0042] At 5 minutes, the volume fraction of mobile phase A was set to 72%, and the volume fraction of mobile phase B was set to 28%;

[0043] At 30 minutes, the volume fraction of mobile phase A was set to 40%, and the volume fraction of mobile phase B was set to 60%;

[0044] At 50 minutes, the volume fraction of mobile phase A was set to 28%, and the volume fraction of mobile phase B was set to 72%;

[0045] At 55 minutes, the volume fraction of mobile phase A was set to 15%, and the volume fraction of mobile phase B was set to 85%;

[0046] At 65 minutes, the volume fraction of mobile phase A was set to 15%, and the volume fraction of mobile phase B was set to 85%;

[0047] At 65.1 minutes, the volume fraction of mobile phase A was set to 72%, and the volume fraction of mobile phase B was set to 28%;

[0048] At 75 minutes, the volume fraction of mobile phase A was set to 72% and the volume fraction of mobile phase B was set to 28%.

[0049] Further, the retention time of 1.7±0.5min was determined to be p-toluenesulfonic acid; the retention time of 6.0±0.5min was determined to be impurity SM 3d ; The retention time of 15.3±0.5min was determined to be methyl p-toluenesulfonate; the retention time of 18.9±0.5min was determined to be impurity SM 3i ; The retention time of 23.7±0.5min was determined to be toluene; the retention time of 28.4±0.5min was determined to be impurity SM3; the retention time of 31.1±0.5min was determined to be impurity Z2; the retention time of 49.4±0.5min was determined to be posaconazole Z3; the retention time of 61.7±0.5min was determined to be impurity Z 3f ; The retention time of 62.6±0.5min was determined to be impurity Z 3e .

[0050] Each component can be qualitatively detected based on the above retention time.

[0051] Furthermore, the sample to be tested is prepared with acetonitrile aqueous solution as solvent; in the solvent, the volume ratio of acetonitrile to water is 60:40.

[0052] Further, the p-toluenesulfonic acid, the impurity SM 3d, the methyl p-toluenesulfonate, the impurity SM 3i , the toluene, the impurity SM3, the impurity Z2, the posaconazole Z3, the impurity Z 3f and the impurity Z 3e The content.

[0053] Furthermore, standard substances are prepared to obtain a chromatogram of a standard sample; the content of the chromatogram obtained from the test sample is calculated by peak area according to the external standard method.

[0054] The content of the components of the standard is known, while the content of the components of the test sample is unknown.

[0055] The obtained standard sample chromatogram can be stored in the database as a chromatographic material for quality control in the smart pharmaceutical factory, to help the robot determine whether the products it produces are qualified and to provide quantitative improvement analysis suggestions for unqualified products.

[0056] The beneficial effects of the present invention are:

[0057] The present invention establishes a method for separating and determining posaconazole Z3 and its impurities and solvents based on HPLC by screening chromatographic columns and chromatographic conditions. The method can simultaneously separate p-toluenesulfonic acid, impurity SM 3d , methyl p-toluenesulfonate, impurity SM 3i , toluene, impurity SM3, impurity Z2, posaconazole Z3, impurity Z 3f and impurity Z 3e , and simultaneously determine its content; the method of the present invention has the characteristics of high sensitivity and high durability, and can separate the main peak of the test sample and 8 known impurities and solvents that may exist in the test sample within 75 minutes, and can achieve effective separation under the condition of fine-tuning the chromatographic conditions. The present invention solves the problem of separation and determination of posaconazole Z3 and possible impurities that cannot be solved by the prior art; the analytical method is short in time, high in sensitivity, strong in specificity, good in reproducibility, simple and feasible in operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 is the chromatogram of the test solution;

[0059] Figure 2 is the chromatogram of the mixed solution;

[0060] Figure 3 is the chromatogram of the limit of detection (LOD);

[0061] Figure 4 The chromatogram is for durability-flow rate 1.4 ml / min;

[0062] Figure 5 The chromatogram is for durability-flow rate 1.6 ml / min;

[0063] Figure 6 This is the chromatogram of durability-column temperature 33°C;

[0064] Figure 7 The chromatogram is for durability-column temperature 37°C;

[0065] Figure 8 The chromatogram is for durability-pH4.8;

[0066] Fig. 9 The chromatogram is for durability-pH5.2;

[0067] Fig.10 For durability - the chromatogram with a volume ratio of mobile phase A to mobile phase B of 70:30;

[0068] Fig.11 This is a chromatogram showing the robustness when the volume ratio of mobile phase A to mobile phase B is 74:26. DETAILED DESCRIPTION

[0069] The technical solution of the present invention will be further described clearly and completely in conjunction with specific embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention.

[0070] In the embodiments of the present invention, the determination was carried out strictly in accordance with the high performance liquid chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Rules 0512).

[0071] In the embodiment of the present invention, the solvent is an acetonitrile aqueous solution, and the volume ratio of acetonitrile to water in the solvent is 60:40.

[0072] In the embodiment of the present invention, the preparation method of the test solution is as follows: take an appropriate amount of the product, accurately weigh it, and quantitatively dilute it with a solvent to prepare a solution containing about 2 mg per 1 ml.

[0073] In the embodiment of the present invention, the control solution is prepared by accurately measuring 0.5 ml of the test solution, placing it in a 100 ml volumetric flask, adding a solvent to dilute to the mark, and shaking well.

[0074] In the embodiment of the present invention, the method for preparing the system suitability solution is as follows: weigh the posaconazole Z3 system suitability reference substance (containing posaconazole Z3, impurity SM 3i 、Impurity SM 3d , impurity SM3, impurity Z2, impurity Z 3f 、Impurity Z 3e), and measure an appropriate amount of toluene, add a solvent to dissolve and dilute to make a mixed solution containing about 0.1 μl of toluene and 2 mg of posaconazole Z3 system suitability reference substance per 1 ml.

[0075] In the embodiment of the present invention, the chromatographic conditions are: using octadecylsilane bonded silica gel as a filler (Shim-packVP-ODS 4.6mm×250mm, 5μm or a chromatographic column with equivalent performance), using mobile phase A and mobile phase B for linear gradient elution according to Table 1; the flow rate is 1.5ml per minute; the column temperature is 35°C; the detection wavelength is 210nm; the injection volume is 10μl; the mobile phase A is a mixed solution of potassium hexafluorophosphate solution and methanol, and the volume ratio of potassium hexafluorophosphate solution to methanol in the mobile phase A is 90:10; the concentration of the potassium hexafluorophosphate solution is 0.01mol / L; the mobile phase B is a mixed solution of acetonitrile and methanol, and the volume ratio of acetonitrile to methanol in the mobile phase B is 90:10.

[0076] Table 1. Gradient elution program

[0077]

[0078]

[0079] In the embodiment of the present invention, the preparation method of 0.01 mol / L potassium hexafluorophosphate solution is as follows: 1.84 g of potassium hexafluorophosphate is taken, 1000 ml of water is added to dissolve it, and the pH value is adjusted to 5.0 with phosphoric acid.

[0080] In the embodiment of the present invention, the system suitability requirement is: in the system suitability solution chromatogram, the separation degree between the posaconazole Z3 peak and the adjacent peaks should meet the requirements.

[0081] In the embodiment of the present invention, the determination method is: accurately measure the test solution and the control solution, inject them into the liquid chromatograph respectively, and record the chromatogram.

[0082] In the embodiment of the present invention, the limit is: in the chromatogram of the test solution, except for toluene, blank solvent peaks and gradient elution peaks, the content of each impurity shall comply with the provisions of Table 2 according to the principal component self-control method with correction factor, and the total amount of impurities shall not exceed 2.0%. The peak in the chromatogram of the test solution that is less than 0.1 times the main peak area of ​​the control solution can be ignored (0.05%).

[0083] Table 2

[0084] Main components and impurities Relative retention time Correction Factor Impurity Limit <![CDATA[Impurity SM 3d > 0.12 2.4 0.15％ <![CDATA[Impurity SM 3i > 0.38 1.7 0.15％ <![CDATA[Impurity SM3]]> 0.57 2.2 0.2％ <![CDATA[Impurity Z2]]> 0.63 0.85 1.3％ <![CDATA[Posaconazole Z3]]> 1.0 - - <![CDATA[Impurity Z 3f > 1.25 0.81 0.15％ <![CDATA[Impurity Z 3e > 1.27 0.86 0.4％ Other single impurities - 1.0 0.2％

[0085] Note: The relative retention times in Table 2 are for reference only. The peaks are located using the chromatogram of the system suitability solution.

[0086] In the embodiment of the present invention, the calculation formula of impurity content is as follows:

[0087]

[0088] Total impurities are the sum of all impurities.

[0089] A z : Peak area of ​​main component in reference solution;

[0090] A s : Impurity peak area in the test solution;

[0091] F: impurity correction factor (if there is no correction factor, F=1);

[0092] N: dilution factor for preparing control solution.

[0093] Example 1. Specificity

[0094] Impurities that may exist in the intermediate posaconazole Z3: Impurity SM 3d 、Impurity SM3、Impurity SM 3i , impurity Z2, impurity Z 3e 、Impurity Z 3f , methyl p-toluenesulfonate, p-toluenesulfonic acid and its solvate toluene, a total of 9 impurities, this method is for the main peak and impurity SM 3d 、Impurity SM3、Impurity SM 3i , impurity Z2, impurity Z 3e 、Impurity Z 3f , methyl p-toluenesulfonate, p-toluenesulfonic acid and toluene were investigated in specific tests.

[0095] Blank solution / solvent: acetonitrile aqueous solution, wherein the volume ratio of acetonitrile to water is 60:40.

[0096] Impurity SM3 stock solution: Accurately weigh 25.56 mg of impurity SM3, place it in a 50 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and obtain.

[0097] Impurity SM 3d Stock solution: Accurately weigh the impurity SM 3d 25.00 mg, put into a 50 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and you have it.

[0098] Impurity SM 3i Stock solution: Accurately weigh the impurity SM 3i 25.96 mg, put into a 50 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and you have it.

[0099] Impurity Z2 stock solution: Accurately weigh 49.19 mg of impurity Z2, place in a 50 ml volumetric flask, add about 5 ml of N, N-dimethylacetamide to dissolve, then dilute to the scale with solvent, shake well, and obtain.

[0100] Impurity Z 3e Stock solution: Accurately weigh impurity Z 3e 25.12 mg, put into a 50 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and you have it.

[0101] Impurity Z 3f Stock solution: Accurately weigh impurity Z 3e 24.97 mg, put in a 50 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and you have it.

[0102] Stock solution of methyl p-toluenesulfonate: Accurately weigh 24.36 mg of methyl p-toluenesulfonate, place in a 50 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and obtain.

[0103] p-Toluenesulfonic acid stock solution: Accurately weigh 52.04 mg of p-toluenesulfonic acid monohydrate, place in a 50 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and obtain.

[0104] Toluene fixation solution: Accurately weigh 24.66 mg of toluene, place in a 100 ml volumetric flask, add solvent to dissolve and dilute to the scale, and shake well.

[0105] Impurity localization solution: Accurately measure 0.5 ml of the impurity p-toluenesulfonic acid stock solution and 1.0 ml of the other known impurity stock solutions, place them in different 25 ml volumetric bottles, add solvent to dilute to the scale, shake well, and obtain.

[0106] Test sample solution: Accurately weigh 50.58 mg of the test sample, place it in a 25 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and obtain.

[0107] Mixed solution: Accurately weigh 48.99 mg of the test sample and place it in a 25 ml volumetric flask. Add an appropriate amount of solvent to dissolve it and then accurately measure 0.1 ml of methyl p-toluenesulfonate stock solution and impurity Z 3f 、Impurity SM 3d 、Impurity SM 3i 0.15 ml of each stock solution, 0.2 ml of impurity SM3 stock solution, 1.5 ml of impurity Z2 stock solution, and 1.5 ml of impurity Z 3e Place 0.5 ml of the stock solution in the same volumetric flask, dilute to the mark with solvent, and shake well.

[0108] Take 10 μl of each of the blank solution, each impurity location solution, toluene location solution, test solution, and mixed solution, inject according to the method, and record the chromatogram. The determination results are shown in Table 3 and Figure 1-2 As shown, Figure 1 and Figure 2 The corresponding integration results are shown in Table 4-5. The results show that the blank solution does not interfere with the determination of each impurity; the separation between the main peak and the adjacent impurity peak is not less than 1.5, and the separation between each known impurity peak and between the known impurity and the main peak is not less than 1.5, and the specificity meets the requirements.

[0109] Table 3. Specificity test results

[0110]

[0111] Note * :Toluene is the crystallization solvent for the synthesis of intermediate Z3 and actually exists in intermediate Z3, so it is located here to prove that it does not interfere with the determination of related substances of intermediate Z3.

[0112] Table 4. Figure 1 The integral result table

[0113]

[0114] Table 5. Figure 2 The integral result table

[0115]

[0116]

[0117] Example 2. Detection Limit

[0118] Quantitative limit solution: Accurately measure the impurity SM in Example 1 3d 、Impurity SM 3i 1.0 ml of each of impurity SM3 localization solution, 1.25 ml of methyl p-toluenesulfonate localization solution, 0.3 ml of impurity Z2 localization solution, and 0.3 ml of impurity Z 3e 、Impurity Z 3f Place 0.5 ml of the positioning solution and 1.5 ml of the test sample control solution in the same 50 ml volumetric flask, dilute to the scale with solvent, shake well, and obtain.

[0119] Detection limit solution: Accurately measure 3.0 ml of the quantitative limit solution, place it in a 10 ml volumetric flask, dilute to the scale with solvent, shake well, and obtain.

[0120] The detection limit solution was sampled three times continuously, and the ratio of the main peak height to the noise (signal-to-noise ratio) was calculated. The test results are shown in Table 6 and Figure 3 As shown, Figure 3 The corresponding integration results are shown in Table 7. The detection limit concentration of posaconazole Z3 is 0.091 μg / ml, and the mean signal-to-noise ratio is 4.2; the impurity SM 3dThe detection limit concentration was 0.118 μg / ml, and the mean signal-to-noise ratio was 6.5; the impurity SM 3i The detection limit concentration of impurity SM3 is 0.123 μg / ml, and the mean signal-to-noise ratio is 6.2; the detection limit concentration of impurity Z2 is 0.071 μg / ml, and the mean signal-to-noise ratio is 4.3; the detection limit concentration of impurity Z 3f The detection limit concentration was 0.058 μg / ml, and the mean signal-to-noise ratio was 8.3; impurity Z 3e The detection limit concentration was 0.060 μg / ml, and the average signal-to-noise ratio was 6.1, which met the requirement of signal-to-noise ratio S / N ≥ 3:1.

[0121] Table 6. Detection limit determination results

[0122]

[0123]

[0124] Table 7. Figure 3 The integral result table

[0125]

[0126] Example 3. Chromatographic Conditions Robustness

[0127] Take the mixed solution prepared in Example 1, use normal mobile phase, different mobile phase ratios, column temperatures, and column flow rates for the predetermined tests, and test them after the instrument system is stable, and record the separation degree, impurity content, and relative retention time between each peak. The test results are shown in Table 8. Figure 2 and Figures 4 to 11 As shown, Figures 4 to 11 The corresponding integration results are shown in Tables 9 to 16. When the flow rate, column temperature, initial ratio of mobile phase, and pH value of mobile phase A in the chromatographic conditions fluctuate slightly, the separation between the main peak and each known impurity peak and the adjacent peaks is greater than 1.5.

[0128] Table 8. Results of the durability test under chromatographic condition changes

[0129]

[0130]

[0131] Table 9. Figure 4 The integral result table

[0132]

[0133] Table 10. Figure 5 The integral result table

[0134]

[0135] Table 11. Figure 6 The integral result table

[0136]

[0137]

[0138] Table 12. Figure 7 The integral result table

[0139]

[0140] Table 13. Figure 8 The integral result table

[0141]

[0142]

[0143] Table 14. Fig. 9 The integral result table

[0144]

[0145] Table 15. Fig.10 The integral result table

[0146]

[0147] Table 16. Fig.11 The integral result table

[0148]

[0149] .

Claims

1. A method for separating posaconazole Z3 and its related impurities and solvents based on high performance liquid chromatography, characterized in that: The method comprises: using octadecylsilane bonded silica gel filler as the stationary phase, using a mixed solution of potassium hexafluorophosphate solution and methanol as mobile phase A, and a mixed solution of acetonitrile and methanol as mobile phase B for linear gradient elution, wherein the volume ratio of potassium hexafluorophosphate solution to methanol in the mobile phase A is 90:10; the volume ratio of acetonitrile to methanol in the mobile phase B is 90:10; separating posaconazole Z3 and its related impurities and solvents by gradient elution; and the gradient elution program is set as follows: At 0 minutes, the volume fraction of the mobile phase A is 70%-74%, and the volume fraction of the mobile phase B is 30%-26%; At 5 minutes, the volume fraction of the mobile phase A is 72%, and the volume fraction of the mobile phase B is 28%; At 30 minutes, the volume fraction of the mobile phase A is 40%, and the volume fraction of the mobile phase B is 60%; At 50 minutes, the volume fraction of the mobile phase A is 28%, and the volume fraction of the mobile phase B is 72%; At 55 minutes, the volume fraction of the mobile phase A is 15%, and the volume fraction of the mobile phase B is 85%; At 65 minutes, the volume fraction of the mobile phase A is 15%, and the volume fraction of the mobile phase B is 85%; At 65.1 minutes, the volume fraction of the mobile phase A was 72%, and the volume fraction of the mobile phase B was 28%; At 75 minutes, the volume fraction of the mobile phase A is 72%, and the volume fraction of the mobile phase B is 28%; The structural formula of posaconazole Z3 is shown in Formula I; the related impurities include the impurity SM shown in Formula II 3d , impurity SM3 shown in formula III, impurity SM shown in formula IV 3i , impurity Z2 represented by formula V, impurity Z represented by formula VI 3e , impurity Z represented by formula VII 3f , methyl p-toluenesulfonate represented by formula VIII and p-toluenesulfonic acid represented by formula IX; the solvent is toluene; 2. The method according to claim 1, characterized in that The pH value of the mobile phase A is 4.8-5.2; in the mobile phase A, the concentration of the potassium hexafluorophosphate solution is 0.01 mol / L.

3. The method according to claim 1, characterized in that The injection flow rate is 1.4ml / min-1.6ml / min; the column temperature is 33℃-37℃.

4. A method for simultaneously detecting the contents of posaconazole Z3 and its related impurities and solvents based on the method described in any one of claims 1 to 3, characterized in that: After separating the posaconazole Z3 and its related impurities and solvents by the method described in any one of claims 1 to 3, a detector is used for detection; the detection wavelength of the detector is set to 210 nm.

5. The method according to claim 4, characterized in that The retention times of the separated components are as follows: p-toluenesulfonic acid, impurity SM 3d , methyl p-toluenesulfonate, impurity SM 3i , toluene, impurity SM3, impurity Z2, posaconazole Z3, impurity Z 3f and impurity Z 3e .

6. The method according to claim 4, characterized in that The volume ratio of potassium hexafluorophosphate solution to methanol in the mobile phase A is 90:10; the volume ratio of acetonitrile to methanol in the mobile phase B is 90:10; the pH value of the mobile phase A is 5.0, and the injection flow rate is 1.5 ml / min; the column temperature of the chromatographic column is 35° C., the chromatographic column specifications are 4.6 mm×250 mm, 5 μm; and the detection wavelength is 210 nm; Perform linear gradient elution according to the gradient elution program and obtain a chromatogram; the gradient elution program is set as follows: At 0 minutes, the volume fraction of mobile phase A was set to 72% and the volume fraction of mobile phase B was set to 28%; At 5 minutes, the volume fraction of mobile phase A was set to 72%, and the volume fraction of mobile phase B was set to 28%; At 30 minutes, the volume fraction of mobile phase A was set to 40%, and the volume fraction of mobile phase B was set to 60%; At 50 minutes, the volume fraction of mobile phase A was set to 28%, and the volume fraction of mobile phase B was set to 72%; At 55 minutes, the volume fraction of mobile phase A was set to 15%, and the volume fraction of mobile phase B was set to 85%; At 65 minutes, the volume fraction of mobile phase A was set to 15%, and the volume fraction of mobile phase B was set to 85%; At 65.1 minutes, the volume fraction of mobile phase A was set to 72%, and the volume fraction of mobile phase B was set to 28%; At 75 minutes, the volume fraction of mobile phase A was set to 72% and the volume fraction of mobile phase B was set to 28%.

7. The method according to claim 6, characterized in that The retention time of 1.7±0.5min was determined to be p-toluenesulfonic acid; the retention time of 6.0±0.5min was determined to be impurity SM 3d ; The retention time of 15.3±0.5min was determined to be methyl p-toluenesulfonate; the retention time of 18.9±0.5min was determined to be impurity SM 3i ; The retention time of 23.7±0.5min was determined to be toluene; the retention time of 28.4±0.5min was determined to be impurity SM3; the retention time of 31.1±0.5min was determined to be impurity Z2; the retention time of 49.4±0.5min was determined to be posaconazole Z3; the retention time of 61.7±0.5min was determined to be impurity Z 3f ; The retention time of 62.6±0.5min was determined to be impurity Z 3e .

8. The method according to claim 4, characterized in that The p-toluenesulfonic acid, the impurity SM 3d , the methyl p-toluenesulfonate, the impurity SM 3i , the toluene, the impurity SM3, the impurity Z2, the posaconazole Z3, the impurity Z 3f and the impurity Z 3e The content.

9. The method according to claim 4, characterized in that Prepare standard products to obtain the chromatogram of standard samples; calculate the content of the chromatogram obtained from the test samples by using the peak area according to the external standard method.

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