Method for detecting pravastatin-related substance

Abstract

A method for detecting pravastatin-related substances. The detection method comprises the following steps: detecting a sample to be detected by means of using a high-performance liquid chromatography method, wherein the packing material of the chromatographic column for the liquid chromatography is ethylenebridge hybrid particles; a five-stage gradient elution is used in the high-performance liquid chromatography, and said sample comprises one or more of pravastatin, a salt of pravastatin and pravastatin-related substances. The detection method can simultaneously realize the effective separation of pravastatin from adjacent unknown degradation impurities thereof and impurity D from adjacent unknown impurities thereof, enabling accurate detection of pravastatin and impurity D, and it can also improve the resolution between impurity B and impurity E. Moreover, the detection method has passed methodological validation, thereby providing an effective method for detecting the content of pravastatin-related substances.

Description

Detection Methods for Pravastatin-Related Substances Technical Field

[0001] The invention belongs to the field of medicine, and particularly relates to a method for detecting pravastatin-related substances. Background Art

[0002] Pravastatin sodium is an HMG-CoA reductase inhibitor used to treat hyperlipidemia and familial hypercholesterolemia. Pravastatin sodium was developed by Daiichi Sankyo Co., Ltd. and has been launched in Japan, the United States, Europe and other countries. The 20mg and 10mg pravastatin sodium tablets produced by Daiichi Sankyo were approved by the NMPA (formerly CFDA) and launched in China in January 2004 and February 2004, respectively. The dosage form is tablets. The 40mg strength pravastatin sodium tablets produced by Daiichi Sankyo were launched in March 2006. In China, pravastatin sodium is included in the 2020 edition of the Chinese Pharmacopoeia, with the dosage forms included being pravastatin sodium tablets and pravastatin sodium capsules. The detection methods for related substances in pravastatin sodium tablets are included in the United States Pharmacopoeia USP 43, the British Pharmacopoeia BP 2021, the Japanese Pharmacopoeia JP 17, and the Chinese Pharmacopoeia ChP 2020.

[0003] The impurities listed in the pharmacopoeias of various countries for raw materials are shown in the table below.

[0004] Table 1. Impurities listed in the pharmacopoeias of various countries for pravastatin sodium API

[0005] Note: The above impurity names refer to BP / EP regulations.

[0006] While the detection capabilities of these impurities are acceptable in various pharmacopoeias, they still fall short of meeting the detection requirements for all of the aforementioned impurities in this product. The types of impurities detected are relatively few, and there is still poor resolution between known and unknown impurities, or between unknown impurities and pravastatin. This results in inaccurate measurements of pravastatin and the aforementioned known impurities. Accurately determining the content of these substances is a pressing technical challenge for those skilled in the art.

[0007] Summary of the Invention

[0008] The technical problem to be solved by the present invention is that when detecting pravastatin-related substances, some impurities are poorly separated from pravastatin and the impurities are difficult to separate from each other. A method for detecting pravastatin-related substances is provided. The method can simultaneously achieve effective separation of pravastatin and its adjacent unknown impurities, and impurity D and its adjacent unknown impurities. Furthermore, the separation degree of impurity B and impurity E can be improved, thereby improving the accuracy of the content of related substances such as pravastatin, impurity D, impurity B and impurity E. Furthermore, the detection method has passed methodological verification.

[0009] The present invention provides a method for detecting pravastatin-related substances, which comprises the following steps: using high performance liquid chromatography to detect a sample, wherein the filler of the chromatographic column of the liquid chromatography is ethylene bridged hybrid (BEH) particles;

[0010] The mobile phase of the high performance liquid chromatography method consists of mobile phase A and mobile phase B;

[0011] The mobile phases A and B are mixtures of water, phosphate buffer and acetonitrile;

[0012] In the mobile phases A and B, the pH value of the phosphate buffer is 6.8-7.2;

[0013] The high performance liquid chromatography method adopts a five-stage gradient elution:

[0014] The elution time is the difference between the elution end time and the elution start time of the elution stage;

[0015] The test products include one or more of pravastatin, pravastatin salts and pravastatin-related substances.

[0016] In the mobile phase A, the volume ratio of water, phosphate buffer and acetonitrile can be (50-54):(28-32):(16-20), preferably 52:30:18.

[0017] In the mobile phase B, the volume ratio of water, phosphate buffer and acetonitrile can be (8-12):(28-32):(58-62), preferably 10:30:60.

[0018] In the mobile phases A and B, the pH value of the phosphate buffer is 7.0.

[0019] The elution time of the first elution stage is preferably 3 minutes.

[0020] The elution time of the second elution stage is preferably 27 minutes.

[0021] The elution time of the third elution stage is preferably 5 minutes.

[0022] The elution time of the fourth elution stage is preferably 1 minute.

[0023] The elution time of the fifth elution stage is preferably 4 minutes.

[0024] In the first elution stage, the volume ratio of mobile phase A is preferably maintained at 100%, and the volume ratio of mobile phase B is preferably maintained at 0%.

[0025] In the second elution stage, the volume ratio of mobile phase A is preferably decreased from 100% to 0%, and the volume ratio of mobile phase B is preferably increased from 0% to 100%.

[0026] In the third elution stage, the volume ratio of mobile phase A is preferably maintained at 0%, and the volume ratio of mobile phase B is preferably maintained at 100%.

[0027] In the fourth elution stage, the volume ratio of mobile phase A is preferably increased from 0% to 100%, and the volume ratio of mobile phase B is preferably decreased from 100% to 0%.

[0028] In the fifth elution stage, the volume ratio of mobile phase A is preferably maintained at 100%, and the volume ratio of mobile phase B is preferably maintained at 0%.

[0029] The five-stage gradient elution of the high performance liquid chromatography is preferably:

[0030] The column temperature of the liquid chromatography method may be 25-35°C, preferably 28°C or 30°C, more preferably 28°C.

[0031] The detection wavelength of the high performance liquid chromatography method can be 236-240 nm and 218-222 nm, preferably 236 nm and 220 nm.

[0032] The detector of the high performance liquid chromatography method can be an ultraviolet-visible light (UV / Vis) detector or a photodiode array (PDA) detector; the ultraviolet-visible light (UV / Vis) detector is preferably a 2489 ultraviolet-visible light (UV / Vis) detector (for example, a Waters 2489 ultraviolet-visible light (UV / Vis) detector); the photodiode array (PDA) detector is preferably a 2998 photodiode array (PDA) detector (for example, a Waters 2998 photodiode array (PDA) detector).

[0033] The detector of the HPLC method is more preferably a photodiode array (PDA) detector (eg Waters 2998 photodiode array (PDA) detector).

[0034] The particle size of the filler of the chromatographic column may be 3.0-5.0 μm, preferably 3.5 μm.

[0035] The inner diameter of the chromatographic column may be 4.0-5.0 mm, preferably 4.6 mm.

[0036] The length of the chromatographic column may be 75-150 mm, preferably 150 mm.

[0037] The chromatography column is preferably XBridge Shield RP18 (eg Waters XBridge Shield RP18).

[0038] The test product is preferably pravastatin bulk drug and / or pravastatin preparation.

[0039] The pravastatin related substances may include one or more of the following compounds:

[0040] The injection volume of the high performance liquid chromatography method can be 8-12 μL, preferably 10 μL.

[0041] The flow rate of the HPLC method may be 0.8-1.2 mL / min, preferably 1.0 mL / min.

[0042] The injector temperature of the HPLC method can be 8-12°C, preferably 10°C.

[0043] The sample to be tested can be dissolved in a solvent before injection.

[0044] The solvent can be a mixture of methanol and water, or a mixture of water, phosphate buffer and acetonitrile.

[0045] In the solvent, the volume ratio of water, phosphate buffer and acetonitrile can be (50-54):(28-32):(16-20), preferably 52:30:18.

[0046] In the solvent, the pH of the phosphate buffer may be 6.8-7.2, preferably 7.0.

[0047] The molar volume ratio of the test substance to the solvent can be 0.8-1.2 mg / L, for example 1.0 mg / L. In a preferred embodiment,

[0048] The test product includes components of group (1) or group (2):

[0049] Group (1) is pravastatin,

[0050] Group (2) is pravastatin,

[0051] The chromatographic column is XBridge Shield RP18 (e.g., Waters XBridge Shield RP18);

[0052] The column temperature of the liquid chromatography method is 28°C;

[0053] In the mobile phase A, the volume ratio of water, phosphate buffer and acetonitrile is 52:30:18;

[0054] In the mobile phase B, the volume ratio of water, phosphate buffer and acetonitrile is 10:30:60;

[0055] The five-stage gradient elution is:

[0056] The detector of the high performance liquid chromatography method is a 2489 ultraviolet visible (UV / Vis) detector (for example, a Waters 2489 ultraviolet visible (UV / Vis) detector).

[0057] In a preferred embodiment,

[0058] The test products are pravastatin,

[0059] The chromatographic column is XBridge Shield RP18 (e.g., Waters XBridge Shield RP18);

[0060] The column temperature of the liquid chromatography method is 25°C;

[0061] In the mobile phase A, the volume ratio of water, phosphate buffer and acetonitrile is 52:30:18;

[0062] In the mobile phase B, the volume ratio of water, phosphate buffer and acetonitrile is 10:30:60;

[0063] The five-stage gradient elution is:

[0064] The detector of the HPLC method is a 2998 photodiode array (PDA) detector (eg, Waters 2998 photodiode array (PDA) detector).

[0065] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention.

[0066] The reagents and raw materials used in the present invention are commercially available.

[0067] The positive progress of the present invention is that: the detection method can simultaneously achieve effective separation of pravastatin and its adjacent unknown degradation impurities, and impurity D and its adjacent unknown impurities, and can achieve accurate detection of pravastatin and impurity D, is easy to operate and control, and has accurate detection results; further, it can also achieve improved separation of impurity B and impurity E, thereby improving the accuracy of the contents of pravastatin, impurity D, impurity B and impurity E; furthermore, the detection method has also passed methodological verification, providing an effective detection method for the content of pravastatin-related substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] FIG1 is a HPLC spectrum of the test sample 1 in Example 1;

[0069] FIG2 is an HPLC spectrum of the test sample 1 in Comparative Example 1;

[0070] FIG3 is the HPLC spectrum of the sample 3 to be tested in Example 3. DETAILED DESCRIPTION

[0071] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0072] Example 1

[0073] Test sample 1: The raw material drug (Shanghai Tianwei Biopharmaceutical Co., Ltd.) was treated with acid (1 mL of 0.5 mol / L hydrochloric acid solution, 20 minutes) and dissolved in water-phosphate buffer (pH 7.0)-acetonitrile (52:30:18) to obtain test sample 1; the specific components were pravastatin, impurity A, impurity B, impurity D, and various unknown impurities.

[0074] Test sample 2: Pravastatin sodium, impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H and impurity I were mixed and dissolved in water-phosphate buffer (pH 7.0)-acetonitrile (52:30:18), wherein pravastatin sodium was 1 mg / mL and the other impurities were all 2 μg / mL to obtain test sample 2.

[0075] Test sample 3: Pravastatin sodium, impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H and impurity I were mixed and dissolved in water-phosphate buffer (pH 7.0)-acetonitrile (52:30:18), wherein the concentration of pravastatin sodium and other impurities was 2 μg / mL to obtain test sample 3, i.e., the positioning solution of each component.

[0076] Using a Waters XBridge Shield RP18 column (4.6 mm × 150 mm, 3.5 μm), the test sample 1 was injected under the chromatographic conditions shown in Table 2. The results showed that the main component, pravastatin, and adjacent unknown degradation impurities could be almost completely separated, as shown in Figure 1. The peak value, peak width / half-peak width, and resolution of the measured impurities are shown in Table 3 below.

[0077] Phosphate buffer: Weigh approximately 9.2 g of phosphoric acid, dilute to 1000 mL with water, and adjust the pH to 7.0 with triethylamine.

[0078] Detector model: Waters 2489 ultraviolet-visible (UV / Vis) detector.

[0079] Table 2. Chromatographic conditions

[0080] Table 3. Spectral data of the drug substance using Waters XBridge Shield RP18

[0081] The above results show that using the Waters XBridge Shield RP18 column, pravastatin and its adjacent unknown degradation impurities, as well as impurity D and its adjacent unknown impurities can be completely separated.

[0082] Example 2

[0083] Optimization of column temperature and chromatographic gradient

[0084] Samples 1 and 2 were tested using Waters XBridge Shield RP18 columns, respectively, at different temperatures and under the chromatographic gradients shown in Table 4. Other conditions remained consistent with those of Example 1. The results are shown in Table 5.

[0085] Detector model: Waters 2489 ultraviolet-visible (UV / Vis) detector.

[0086] Table 4. Chromatographic gradient

[0087] Table 5. Test results of separation of pravastatin related substances at different column temperatures

[0088] The column temperature and chromatographic gradient were adjusted to ensure that all known impurities in the mixed solution and pravastatin were completely separated, and that any unknown degradation impurities that might exist in the API were also completely separated from the known impurities.

[0089] The above results show that higher column temperatures improve the resolution between impurities B and E, and between the main peak and its adjacent unknown degradation impurities. However, the resolution between the main peak and impurity A, and between impurity D and its adjacent unknown impurities, deteriorates. At a column temperature of 28°C, the resolution between impurities is greater than 1.5, and the resolution between the main component and its adjacent impurities is greater than 2.0. Therefore, the optimal column temperature for the detection of pravastatin-related substances is 28°C.

[0090] Example 3

[0091] Optimization of chromatographic gradients and detectors

[0092] The sample 3 to be tested was injected, and the chromatographic gradient was as shown in Table 4. The detector used was a Waters 2998 photodiode array (PDA) detector. Other test conditions were consistent with those of Example 1. The measured spectrum was shown in Figure 3, and the relevant data of the spectrum are shown in Table 6. Under this gradient, the separation degree of impurity B and impurity E was 1.81.

[0093] Table 4. Chromatographic gradient

[0094] Table 6. Spectral data corresponding to Figure 3 measured for each component positioning solution

[0095] Note: Resolution refers to the separation between a peak and its previous peak.

[0096] Example 4

[0097] Optimize the detection ability of impurities

[0098] Pharmacopoeias worldwide use 238 nm as the detection wavelength. Wavelength durability testing was conducted within the range of 238 nm ± 2 nm using an Agilent Zorbax SB-C18 column (4.6 × 75 μm, 3.5 μm) and mobile phase A. Other conditions remained consistent with those in Example 1. The results showed that the RSDs for impurities G and H were both greater than 10%, as shown in Table 7.

[0099] Table 7. Wavelength durability test results

[0100] The stock solutions of each impurity reference substance and the stock solution of pravastatin reference substance were injected into the liquid chromatograph, and full wavelength scanning was performed using a PDA detector. The results are shown in Table 8.

[0101] Table 8. Maximum absorption wavelength of each component

[0102] As can be seen from the above table, the maximum absorption wavelength of impurity G is 219.3nm, and the maximum absorption wavelength of impurity H is 233.4nm, which are inconsistent with the main component (maximum absorption wavelength 236.9nm), resulting in large differences in the measurement results of the two impurities within the range of 238nm±2nm. Therefore, the detection wavelength of impurity G is set to 220nm, and the detection wavelength of other impurities is set to 236nm. It has been verified that the selected wavelength has good durability in the range of ±2nm, and the RSD of the measurement results of each impurity is no more than 10%, indicating that the measurement results of each impurity are not affected by wavelength deviation. The results are shown in Table 9:

[0103] Table 9. Wavelength durability test results

[0104] The optimal detection wavelength of the detection method for pravastatin-related substances is 236nm+220nm.

[0105] Based on the above optimization conditions, the optimized measurement method is shown in Table 10.

[0106] Table 10. Optimal detection methods for pravastatin-related substances

[0107] This method has been validated for specificity, linearity, limit of quantification, limit of detection, accuracy, repeatability, solution stability, and robustness. This method is expected to be more applicable and robust for sample detection.

[0108] Comparative Example 1

[0109] The chromatographic column recommended by ChP 2020 is an Aglient Zorbax SB-C18 column (4.6 mm × 75 mm, 3.5 μm). Sample 1 was injected under the chromatographic conditions shown in Table 5. The results showed that the main component, pravastatin, and its adjacent unknown degradation impurities could not be separated. Impurity A and its adjacent unknown impurities, as well as impurity D and its adjacent unknown impurities, could not be effectively separated, as shown in Figure 2. The spectral data are shown in Table 6 below.

[0110] Phosphate buffer: Weigh approximately 9.2 g of phosphoric acid, dilute to 1000 mL with water, and adjust the pH to 7.0 with triethylamine.

[0111] Detector model: Waters 2489 ultraviolet-visible (UV / Vis) detector.

[0112] Table 11. Chromatographic conditions recommended by ChP 2020

[0113] Table 12. Spectral data of the drug substance using Aglient Zorbax SB-C18

[0114] Note: “-” indicates that pravastatin and the adjacent unknown degradation impurity peak of pravastatin overlap and cannot be completely separated, making it difficult to calculate the resolution.

Claims

1. A method for detecting pravastatin related substances, characterized in that: The method comprises the following steps: using high performance liquid chromatography to detect the sample to be tested, wherein the filler of the chromatographic column of the liquid chromatography is ethylene bridge hybrid particles; The mobile phase of the high performance liquid chromatography is composed of mobile phase A and mobile phase B; The mobile phases A and B are a mixture of water, phosphate buffer and acetonitrile; In the mobile phases A and B, the pH value of the phosphate buffer is 6.8-7.2; The high performance liquid chromatography method adopts a five-stage gradient elution: The test products include one or more of pravastatin, pravastatin salts and pravastatin-related substances.

2. The detection method according to claim 1, characterized in that In the mobile phase A, the volume ratio of water, phosphate buffer and acetonitrile is (50-54):(28-32):(16-20); In the mobile phase B, the volume ratio of water, phosphate buffer and acetonitrile is (8-12): (28-32): (58-62) And / or, in the mobile phases A and B, the pH value of the phosphate buffer is 7.

0.

3. The detection method according to claim 2, characterized in that In the mobile phase A, the volume ratio of water, phosphate buffer and acetonitrile is 52:30:18; In the mobile phase B, the volume ratio of water, phosphate buffer and acetonitrile is 10:30:

60.

4. The detection method according to claim 1, characterized in that The five-stage gradient elution satisfies one or more of the following conditions: ① The elution time of the first elution stage is 3 minutes; ② The elution time of the second elution stage is 27 minutes; ③ The elution time of the third elution stage is 5 minutes; ④ The elution time of the fourth elution stage is 1 min; ⑤ The elution time of the fifth elution stage is 4 minutes; ⑥ In the first elution stage, the volume ratio of mobile phase A is maintained at 100%, and the volume ratio of mobile phase B is maintained at 0%; ⑦ In the second elution stage, the volume ratio of mobile phase A is decreased from 100% to 0%, and the volume ratio of mobile phase B is increased from 0% to 100%; ⑧ In the third elution stage, the volume ratio of mobile phase A is maintained at 0%, and the volume ratio of mobile phase B is maintained at 100%; ⑨ In the fourth elution stage, the volume ratio of mobile phase A increases from 0% to 100%, and the volume ratio of mobile phase B decreases from 100% to 0%; ⑩ In the fifth elution stage, the volume ratio of mobile phase A is maintained at 100%, and the volume ratio of mobile phase B is maintained at 0%.

5. The detection method according to claim 4, characterized in that: The five-stage gradient elution of the high performance liquid chromatography is:

6. The detection method according to claim 1, characterized in that The detection method meets one or more of the following conditions: ① The column temperature of the liquid chromatography is 25-35°C; ② The detection wavelengths of the high performance liquid chromatography are 236-240nm and 218-222nm; ③ The detector of the high performance liquid chromatography is an ultraviolet visible light detector or a photodiode array detector; ④ The particle size of the filler of the chromatographic column is 3.0-5.0 μm; ⑤ The inner diameter of the chromatographic column is 4.0-5.0 mm; ⑥The length of the chromatographic column is 75-150mm; ⑦The chromatographic column is XBridge Shield RP18; ⑧ The test product is pravastatin bulk drug and / or pravastatin preparation; ⑨ The pravastatin related substances include one or more of the following compounds, ⑩ The sample to be tested is dissolved in a solvent before injection; The injection volume of the HPLC method is 8-12 μL; The flow rate of the HPLC method is 0.8-1.2 mL / min; The injector temperature of the HPLC method is 8-12°C.

7. The detection method according to claim 6, characterized in that The detection method meets one or more of the following conditions: ① The column temperature of the liquid chromatography is 28°C or 30°C; ② The detection wavelength of the high performance liquid chromatography is 236nm and 220nm; ③ The detector of the high performance liquid chromatography is a photodiode array detector; ④ The particle size of the filler of the chromatographic column is 3.5 μm; ⑤ The inner diameter of the chromatographic column is 4.6 mm; ⑥The length of the chromatographic column is 150 mm; ⑦ The injection volume of the high performance liquid chromatography method is 10 μL; ⑧ The flow rate of the high performance liquid chromatography is 1.0 mL / min; ⑨ The injector temperature of the HPLC method is 10°C; ⑩ The solvent is a mixture of methanol and water, or a mixture of water, phosphate buffer and acetonitrile.

8. The detection method according to claim 7, characterized in that The detection method meets one or more of the following conditions: ① The column temperature of the liquid chromatography is 28°C; ② The ultraviolet visible light detector is a 2489 ultraviolet visible light detector; the photodiode array detector is a 2998 photodiode array detector; ③ In the solvent, the volume ratio of water, phosphate buffer and acetonitrile is (50-54):(28-32):(16-20); ④ In the solvent, the pH of the phosphate buffer is 6.8-7.2; ⑤ The molar volume ratio of the test product to the solvent is 0.8-1.2 mg / L.

9. The detection method according to claim 8, characterized in that: The detection method meets one or both of the following conditions: ① In the solvent, the volume ratio of water, phosphate buffer and acetonitrile is 52:30:18; ② In the solvent, the pH of the phosphate buffer is 7.0; ③ The molar volume ratio of the test product to the solvent is 1.0 mg / L.

10. The detection method according to claim 1, characterized in that: The detection method is the following scheme ① or ②: Solution ①: The test product includes components of group (1) or group (2): Group (1) pravastatin, Group (2) pravastatin, The chromatographic column is XBridge Shield RP18; The column temperature of the liquid chromatography method is 28°C; In the mobile phase A, the volume ratio of water, phosphate buffer and acetonitrile is 52:30:18; In the mobile phase B, the volume ratio of water, phosphate buffer and acetonitrile is 10:30:60; The five-stage gradient elution is: The detector of the high performance liquid chromatography is a 2489 ultraviolet visible light detector; Scheme ②: The products to be tested include: Pravastatin, The chromatographic column is XBridge Shield RP18; The column temperature of the liquid chromatography method is 25°C; In the mobile phase A, the volume ratio of water, phosphate buffer and acetonitrile is 52:30:18; In the mobile phase B, the volume ratio of water, phosphate buffer and acetonitrile is 10:30:60; The five-stage gradient elution is: The detector of the high performance liquid chromatography is a 2998 photodiode array detector.

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