Detection method of posaconazole impurities

Abstract

The invention provides a method for detecting impurities in a posaconazole injection, which adopts a liquid chromatograph-ultraviolet detector for detection, and adopts a principal component external standard method to calculate the content of the posaconazole impurities in the posaconazole injection according to the peak area of a chromatographic peak. The method is high in sensitivity, the detection limit of the posaconazole impurity is as low as 0.1085 [mu] g / mL, and the quantification limit is as low as 0.3617 [mu] g / mL; the method is good in specificity and free of interference of diluents and auxiliary materials, and posaconazole impurities and posaconazole can be rapidly and effectively separated; the method is simple in operation and good in reproducibility, in the range of 0.3617 mu g / mL to 3.6168 mu g / mL, the linear correlation coefficient R2 of the posaconazole impurity is larger than 0.999, and the recovery rate ranges from 101.2% to 109.8%.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical analysis, and particularly to a method for detecting posaconazole impurities in posaconazole injection by liquid chromatography. Background Art

[0002] The trade name of posaconazole injection is and its indication is to prevent invasive aspergillus and candida infections. Posaconazole (the structural formula is shown in Formula I) injection may degrade to produce impurities under the conditions of strong light, strong oxidation, and high heat. Among them, 2-((2S,3S)-2-hydroxypentan-3-yl)-4-(4-(piperazin-1-yl)phenyl)-2,4-dihydro-3H-1,2,4-triazol-3-one (the structural formula is shown in Formula II below, hereinafter referred to as Impurity II) is a photodegradation impurity of posaconazole injection. It has been confirmed by experiments that after posaconazole injection is irradiated with ultraviolet light (the total illuminance of the light source should not be less than 1.2×10 6 lux·hr, and the energy of the near-ultraviolet lamp is not less than 200W·hr / m 2 ) for 7 hours, the content of this impurity can be as high as more than 1.0%, which is far greater than the structural identification limit of 0.2% specified in ICH Q3B(R2). However, there is currently no reported detection method for Impurity II at home and abroad. Therefore, it is necessary to develop a detection method for Impurity II to strictly control the content of Impurity II in posaconazole injection to ensure the safety of clinical medication.

[0003] Summary of the Invention

[0004] To ensure the clinical medication safety of posaconazole injection and strengthen the monitoring of the content of posaconazole impurities, the present invention provides a method for detecting posaconazole impurities in posaconazole injection, which specifically includes the following steps:

[0005] Detect posaconazole impurities by liquid chromatography. The structure of the posaconazole impurity is shown in Formula II below.

[0006]

[0007] Further, the method includes the following steps:

[0008] (1) Dissolve the posaconazole sample in a diluent to obtain a test solution.

[0009] (2) Dissolve the posaconazole reference substance in acetonitrile and then dilute it with a diluent to obtain a reference solution.

[0010] (3) Use a liquid chromatograph - ultraviolet detector to detect the test solution in step (1) and the reference solution in step (2), and calculate the content of posaconazole impurities in the test solution by the external standard method of the main component.

[0011] Further, the chromatographic conditions of the liquid chromatograph are as follows:

[0012] Chromatographic column: octadecylsilane bonded silica gel chromatographic column;

[0013] Mobile phase: Mobile phase A is an aqueous solution of diammonium hydrogen phosphate and acetonitrile with a volume ratio of 80:20 to 95:5, preferably 85:15; Mobile phase B is an aqueous solution of diammonium hydrogen phosphate and acetonitrile with a volume ratio of 20:80 to 55:45, preferably 50:50; The mobile phase is gradient elution, and the gradient elution program is shown in the following table:

[0014]

[0015] Further, the concentration of the aqueous solution of diammonium hydrogen phosphate is 8 - 12 mmol / L, preferably 10 mmol / L.

[0016] Further, the flow rate of the mobile phase is 0.8 mL / min to 1.2 mL / min, preferably 1.0 mL / min.

[0017] Further, the chromatographic column is Agilent ZORBAX - Extend - C18, 4.6 mm × 150 mm × 3.5 μm.

[0018] Further, the column temperature of the chromatographic column is 25 - 35 °C, preferably 30 °C.

[0019] Further, the diluent is an aqueous acetonitrile solution, and the volume ratio of acetonitrile to water is 15% - 40%, preferably the volume ratio of acetonitrile to water is 30%.

[0020] Further, the absorption wavelength of the ultraviolet detector is 254 nm.

[0021] Further, the posaconazole sample is posaconazole raw material or posaconazole injection.

[0022] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0023] The present invention provides for the first time a method for detecting impurity II in posaconazole injection. This method has high sensitivity, with a detection limit as low as 0.1085 μg / mL (equivalent to 0.01% of the sample concentration) and a quantitation limit as low as 0.3617 μg / mL (equivalent to 0.02% of the sample concentration). The limit requirement for impurity II in posaconazole injection is 0.1%. The detection limit and quantitation limit of the present invention meet the requirements of pharmacopoeias such as the Chinese Pharmacopoeia and USP; it has good specificity, is not interfered by diluents and excipients, and can be rapidly and effectively separated from posaconazole; it has good reproducibility, and within the range of 0.3617 μg / mL - 3.6168 μg / mL, the linear correlation coefficient R 2 > 0.999, and the recovery rate is 101.2% - 109.8%. Description of the Drawings

[0024] Figure 1 It is the chromatogram of blank excipient 1 in Example 1.

[0025] Figure 2 It is the chromatogram of blank excipient 2 in Example 1.

[0026] Figure 3 It is the chromatogram of impurity stock solution 2 in Example 1.

[0027] Figure 4 It is the chromatogram of the test solution in Example 1.

[0028] Figure 5 It is the chromatogram of the spiked test solution in Example 1.

[0029] Figure 6 It is the linear graph of posaconazole reference substance in Example 3.

[0030] Figure 7 It is the linear graph of impurity II in Example 3.

[0031] Figure 8 It is the chromatogram of test solution 1 in Example 5. Detailed Implementation Modes

[0032] The following examples further illustrate the present invention. However, it is necessary to point out that the following examples are only used for the description of the invention content and do not constitute a limitation on the protection scope of the present invention. The protection scope of the present invention is subject to the claims.

[0033] In the following examples, posaconazole injection, blank excipients 1 and 2 were all produced by Zhejiang Hisun Pharmaceutical Co., Ltd.; unless otherwise specified, all temperatures are in degrees Celsius; all reagents are commercially available and used directly without further purification. Acetonitrile is commercially available chromatographic grade; diammonium hydrogen phosphate is commercially available analytical grade; water is self-made ultrapure water; impurity II was produced by Shenzhen Ruikai De Biotechnology Co., Ltd., batch number 23-04-200035, with a content of 98.56%; the posaconazole reference standard was provided by Zhejiang Hisun Pharmaceutical Co., Ltd., with a content of 99.8%.

[0034] Chromatographic conditions:

[0035] Chromatographic column: Agilent ZORBAX-Extend-C18, 150×4.6mm×3.5μm;

[0036] Flow rate: 1.0 mL / min;

[0037] Column temperature: 30°C;

[0038] Injection volume: 5 μl;

[0039] Detection wavelength: 254 nm;

[0040] Mobile phase A: 10 mmol / L ammonium dihydrogen phosphate aqueous solution: acetonitrile volume ratio = 85:15;

[0041] Mobile phase B: 10 mmol / L ammonium dihydrogen phosphate aqueous solution: acetonitrile volume ratio = 50:50;

[0042] The mobile phase was gradient elution, and the gradient elution program was as shown in the following table:

[0043]

[0044] Example 1

[0045] Specificity experiment

[0046] Diluent: 30% acetonitrile (i.e., the volume ratio of acetonitrile to water is 30%).

[0047] Reference standard stock solution 1: Weigh an appropriate amount of posaconazole reference standard and dissolve it in acetonitrile to obtain a reference standard stock solution with a concentration of 900 μg / mL.

[0048] Reference standard stock solution 2: Take an appropriate amount of reference standard stock solution 1 and dissolve it in acetonitrile to obtain a reference standard stock solution with a concentration of 90 μg / mL.

[0049] Reference standard solution: Transfer an appropriate amount of reference standard stock solution 2 and dissolve it in the diluent, and dilute it with the diluent to a reference standard solution with a concentration of 1.8 μg / mL.

[0050] Preparation of blank excipient 1: Precisely pipette 2.0 mL of blank excipient solution (without posaconazole raw material, and other excipients are the same as those in posaconazole injection) into a 20 mL volumetric flask, dilute to the mark with diluent, and shake well.

[0051] Preparation of blank excipient 2: Precisely pipette 6.0 mL of blank excipient solution (without posaconazole raw material, and other excipients are the same as those in posaconazole injection) into a 20 mL volumetric flask, dilute to the mark with diluent, and shake well.

[0052] Impurity stock solution 1: Weigh an appropriate amount of impurity II and dissolve it in 50% acetonitrile aqueous solution to obtain an impurity stock solution with a concentration of 450 μg / mL.

[0053] Impurity stock solution 2: Pipette an appropriate amount of impurity stock solution 1 and dilute it with diluent to obtain an impurity stock solution with a concentration of 90 μg / mL.

[0054] Impurity stock solution 3: Pipette an appropriate amount of impurity stock solution 1 and dilute it with diluent to obtain an impurity stock solution with a concentration of 18 μg / mL.

[0055] Preparation of test solution: Precisely pipette 2.0 mL of posaconazole injection into a 20 mL volumetric flask, dilute to the mark with diluent, and shake well.

[0056] Preparation of spiked test solution: Precisely pipette 2.0 mL of posaconazole injection and 2.0 mL of impurity stock solution 3 into a 20 mL volumetric flask respectively, dilute to the mark with diluent, and shake well.

[0057] Use the above liquid chromatography method to detect diluent, blank excipient 1, blank excipient 2, impurity stock solution 2, test solution and spiked test solution, and the test results are shown in Table 1. The chromatograms of blank excipient 1, blank excipient 2, impurity stock solution 2, test solution and spiked test solution are as Figure 1-5 shown.

[0058] Table 1. Determination results of blank excipient 1, blank excipient 2, impurity stock solution 2, test solution and spiked test solution

[0059] Name Retention time (min) Peak area Name Retention time (min) Peak area Diluent ND ND Impurity stock solution 2 4.850 1226.0 Blank excipient 1 ND ND Test solution ND ND Blank excipient 2 ND ND Spiked test solution 4.863 26.6

[0060] Conclusion: It can be seen from the experimental results that diluent, blank excipient 1 and blank excipient 2 have no interference at the peak position of impurity II. The method of the present invention has good specificity and can achieve effective separation from posaconazole within 35 minutes.

[0061] Example 2

[0062] Detection limit and quantitation limit experiments

[0063] Quantitation Limit Stock Solution 1: Pipette 2.0 mL of Impurity Stock Solution 1 and 1.0 mL of Reference Stock Solution 1 into a 50 mL volumetric flask, dilute to the mark with diluent, and mix well.

[0064] Quantitation Limit Stock Solution 2: Pipette 2.0 mL of Quantitation Limit Stock Solution 1 into a 20 mL volumetric flask, dilute to the mark with diluent, and mix well.

[0065] Quantitation Limit Solution: Pipette 4.0 mL of Quantitation Limit Stock Solution 2 into a 20 mL volumetric flask, dilute to the mark with diluent, and mix well.

[0066] Detection Limit Solution: Pipette 3.0 mL of Quantitation Limit Solution into a 10 mL volumetric flask, dilute to the mark with diluent, and mix well.

[0067] Detect the Quantitation Limit and Detection Limit Solutions by the above liquid chromatography method. It is required that the signal-to-noise ratio of the Quantitation Limit Solution is not less than 10, and the signal-to-noise ratio of the Detection Limit Solution is not less than 3. The test results are shown in Table 2 and Table 3 below.

[0068] Table 2. Test Results of Quantitation Limit

[0069]

[0070] Table 3. Test Results of Detection Limit

[0071]

[0072]

[0073] Conclusion: It can be seen from the experimental results that the quantitation limit of posaconazole is 0.3596 μg / mL (equivalent to 0.02% of the sample solution concentration), the signal-to-noise ratio of 6 injections of the quantitation limit is greater than 10, the detection limit is 0.1079 μg / mL (equivalent to 0.01% of the sample solution concentration), and the signal-to-noise ratio of 6 injections of the detection limit is greater than 3; the quantitation limit of impurity II is 0.3617 μg / mL (equivalent to 0.02% of the sample solution concentration), the signal-to-noise ratio of 6 injections of the quantitation limit is greater than 10, the detection limit is 0.1085 μg / mL (equivalent to 0.01% of the sample solution concentration), and the signal-to-noise ratio of 6 injections of the detection limit is greater than 3.

[0074] Example 3

[0075] Linearity and Correction Factor Experiment

[0076] Linear Solution: Pipette 0.4 mL, 1.0 mL, 2.0 mL, 3.0 mL, and 4.0 mL of Quantitation Limit Stock Solution 1 into 5 20 mL volumetric flasks respectively, dilute to the mark with diluent, and mix well.

[0077] The linear solution was detected by the above liquid chromatography method. Each linear solution was injected once, and its linear equation, slope and correlation coefficient are shown in Table 4. The linear graphs of posaconazole reference substance and impurity II are as Figure 6 , Figure 7 shown.

[0078] Table 4. Linear results of posaconazole reference substance and impurity II

[0079]

[0080] Conclusion:

[0081] 1) From the above experimental results, it can be seen that posaconazole has a good linear relationship in the range of 0.3596 μg / mL to 3.5964 μg / mL, and the linear correlation coefficient R 2 > 0.999; impurity II has a good linear relationship in the range of 0.3617 μg / mL to 3.6168 μg / mL, and the linear correlation coefficient R 2 > 0.999. The slope of posaconazole is 12.0377, the slope of impurity II is 13.2616, and the correction factor of the impurity is 0.91. According to the provisions of the Chinese Pharmacopoeia 2020 Edition, when the correction factor is between 0.90 and 1.10, it is rounded to 1.0. Therefore, when calculating the content of impurity II by the external standard method of the main component, the correction factor is calculated according to 1.0. Impurity II concentration = posaconazole reference substance concentration × impurity II peak area × correction factor / posaconazole reference substance peak area.

[0082] Example 4

[0083] Accuracy and precision experiments

[0084] Accuracy stock solution 1: That is, impurity stock solution 3.

[0085] Accuracy stock solution 2: Pipette 2.0 mL of impurity stock solution 3 into a 20 mL volumetric flask, dilute to the mark with the diluent, and shake well.

[0086] Test solution: Pipette 2.0 mL of posaconazole injection precisely into a 20 mL volumetric flask, dilute to the mark with the diluent, and shake well. Prepare 3 parallel portions.

[0087] Accuracy solution 1: Pipette 2.0 mL of posaconazole injection and 4.0 mL of accuracy stock solution 2 precisely into a 20 mL volumetric flask respectively, add the diluent to the mark, and shake well to obtain a posaconazole solution with an impurity II content of 0.02% (20% limit). Prepare 3 parallel portions.

[0088] Accuracy solution 2: Accurately pipette 2.0 mL of posaconazole injection and 1.0 mL of accuracy stock solution 1 into a 20 mL volumetric flask, add diluent to the mark, shake well, to obtain a posaconazole solution with an impurity II content of 0.05% (50% limit), and prepare 3 portions in parallel.

[0089] Accuracy solution 3: Accurately pipette 2.0 mL of posaconazole injection and 2.0 mL of accuracy stock solution 1 into a 20 mL volumetric flask, add diluent to the mark, shake well, to obtain a posaconazole solution with an impurity II content of 0.1% (100% limit), and prepare 6 portions in parallel.

[0090] Accuracy solution 4: Accurately pipette 2.0 mL of posaconazole injection and 4.0 mL of accuracy stock solution 1 into a 20 mL volumetric flask, add diluent to the mark, shake well, to obtain a posaconazole solution with an impurity II content of 0.2% (200% limit), and prepare 3 portions in parallel.

[0091] Use the above liquid chromatography method to detect the test solution and accuracy solutions 1 - 4. Take 3 portions of the test solution and accuracy solutions 1 - 4, inject one injection for each, and the recovery rate of each solution and the average recovery rate of each concentration level are shown in Table 5. Take 6 portions of accuracy solution 3 (i.e., precision solution), inject one injection for each portion, and the recovery rate of each solution and the average recovery rate of each concentration level are shown in Table 6.

[0092] Table 5. Results of accuracy experiment

[0093]

[0094] Table 6. Results of precision experiment

[0095] Accuracy solution 3 1 2 3 4 5 6 Average value % RSD % Impurity II content 0.11％ 0.11％ 0.11％ 0.11％ 0.11％ 0.11％ 0.11 0.0

[0096] Conclusion: From the above experimental results, it can be seen that the accuracy and precision of the detection method of the present invention are good. The recovery rate of the accuracy experiment is 101.2% - 109.8%, the average recovery rate is 102% - 106%, and the RSD of the precision experiment is 0.0%. The present invention can accurately detect the content of impurity II in posaconazole injection.

[0097] Example 5

[0098] Sample detection experiment

[0099] Preparation of test solution: Accurately pipette 2.0 mL of posaconazole injection into a 20 mL volumetric flask, dilute to the mark with diluent, shake well, and prepare 3 portions in parallel to obtain test solution 1, test solution 2, and test solution 3.

[0100] The above liquid chromatography method was used to detect 3 test solution samples, with each test solution sample injected once. The test results are shown in Table 7. The chromatogram of test solution 1 is as shown in Figure 8 shown.

[0101] Table 7. Test Results of Samples

[0102] Test solution 1 2 3 Average value % RSD % Impurity II content ND ND ND ND /

[0103] Conclusion: It can be seen from the above experimental results that impurity II was not detected in any of the 3 test solution samples. Combining the test results of the test solution samples in Example 1 and Example 4, the content of impurity II in posaconazole injection is lower than the quantification limit.

Claims

1. A method for detecting impurities in posaconazole, characterized in that: Liquid chromatography was used to detect the content of posaconazole impurities, and the structure of the posaconazole impurity was shown in the following formula II:

2. The method according to claim 1, characterized in that The method comprises the following steps: (1) dissolving a posaconazole sample in a diluent to obtain a test solution; (2) dissolving a posaconazole reference substance in acetonitrile, and then diluting with a diluent to obtain a reference substance solution; (3) The test solution of step (1) and the reference solution of step (2) are detected by liquid chromatography-ultraviolet detector, and the content of posaconazole impurities in the test solution is calculated by principal component external standard method.

3. The method according to claim 2, characterized in that The chromatographic conditions of the liquid chromatograph are: Chromatographic column: octadecylsilane bonded silica gel chromatographic column; Mobile phase: Mobile phase A is a diammonium phosphate aqueous solution and acetonitrile in a volume ratio of 80:20 to 95:5, preferably 85:15; Mobile phase B is a diammonium phosphate aqueous solution and acetonitrile in a volume ratio of 20:80 to 55:45, preferably 50:50; The mobile phase is gradient elution, and the gradient elution program is shown in the following table:

4. The method according to claim 3, characterized in that The concentration of the diammonium hydrogen phosphate aqueous solution is 8-12 mmol / L, preferably 10 mmol / L.

5. The method according to claim 3, characterized in that: The flow rate of the mobile phase is 0.8 mL / min to 1.2 mL / min, preferably 1.0 mL / min.

6. The method according to claim 3, characterized in that The chromatographic column is Agilent ZORBAX-Extend-C18, 4.6 mm×150 mm×3.5 μm.

7. The method according to claim 6, characterized in that The column temperature of the chromatographic column is 25-35°C, preferably 30°C.

8. The method according to claim 2, characterized in that: The diluent is an acetonitrile aqueous solution, the volume ratio of acetonitrile to water is 15% to 40%, and preferably the volume ratio of acetonitrile to water is 30%.

9. The method according to claim 2, characterized in that: The absorption wavelength of the ultraviolet detector is 254 nm.

10. The method according to any one of claims 2 to 9, characterized in that: The posaconazole sample is posaconazole bulk drug or posaconazole injection.

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