Detection method of pravastatin related substances

Abstract

The invention discloses a method for detecting related substances of pravastatin. The detection method comprises the following steps that a to-be-detected product is detected through a high performance liquid chromatography method, and a filler of a chromatographic column of the liquid chromatography method is ethylidene bridge hybrid particles; the to-be-detected product comprises one or more of pravastatin, a pravastatin salt and a pravastatin related substance. According to the detection method, effective separation of pravastatin and unknown degradation impurities adjacent to the pravastatin and effective separation of the impurity D and unknown impurities adjacent to the impurity D can be achieved at the same time, accurate detection of pravastatin and the impurity D can be achieved, operation and control are convenient, and the detection result is accurate; furthermore, the impurity B and the impurity E can be effectively separated, so that the accuracy of the content of the pravastatin, the impurity D, the impurity B and the impurity E is improved, and an effective detection method is provided for the content of pravastatin related substances.

Description

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 NMPA (formerly CFDA) and launched in China in January 2004 and February 2004, respectively. The dosage form is tablets. The 40mg 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, and the dosage forms included include pravastatin sodium tablets and pravastatin sodium capsules. The detection methods of 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 ChP2020.

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

[0004] Table 1. Impurities of pravastatin sodium API in various pharmacopoeias

[0005]

[0006]

[0007]

[0008] Note: The above impurity names refer to BP / EP regulations.

[0009] The detection capability of the impurities specified in the pharmacopoeias of various countries is qualified, but it still cannot meet the detection requirements of all the impurities mentioned above for this product. The types of impurities detected are relatively small, and there is still a poor separation between known impurities and unknown impurities, or between unknown impurities and pravastatin, resulting in inaccurate measurement results of the content of pravastatin and the above-mentioned known impurities. Therefore, how to accurately determine the content of related substances is a technical problem that needs to be solved urgently by those skilled in the art. Summary of the invention

[0010] The technical problem to be solved by the present invention is that when detecting pravastatin related substances, some impurity peaks are poorly separated from the main peak of pravastatin, and impurities cannot be separated from impurities, so that the content of pravastatin related substances cannot be accurately reflected; for this reason, the present invention provides a detection method for pravastatin related substances. The detection method can more effectively separate impurity B and impurity E, and further, pravastatin and its adjacent unknown impurities, impurity A and its adjacent unknown impurities, and impurity D and its adjacent unknown impurities can also be effectively separated, thereby improving the accuracy of the content of related substances such as pravastatin, impurity A, impurity D, impurity B and impurity E.

[0011] The present invention provides a method for detecting pravastatin-related substances, which comprises the following steps: using high performance liquid chromatography to detect a test product, wherein the filler of a chromatographic column of the liquid chromatography is ethylene bridged hybrid (BEH) particles;

[0012] The test products include one or more of pravastatin, pravastatin salts and pravastatin-related substances.

[0013] The column temperature of the liquid chromatography method is 25-35°C, preferably 28°C or 30°C, more preferably 28°C.

[0014] The mobile phase of the high performance liquid chromatography method may consist of a mobile phase A and a mobile phase B; the mobile phases A and B may be a mixture of water, phosphate buffer and acetonitrile.

[0015] In the mobile phases A and B, the pH value of the phosphate buffer may be 6.8-7.2, preferably 7.0.

[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] The high performance liquid chromatography method may adopt gradient elution, wherein the gradient elution may be a four-stage gradient elution or a five-stage gradient elution;

[0019] The four-stage gradient elution can be:

[0020]

[0021] Preferably:

[0022]

[0023] Or, the five-stage gradient elution can be

[0024]

[0025] Preferably

[0026]

[0027]

[0028] The elution duration is the difference between the elution end time and the elution start time of the elution stage.

[0029] The particle size of the filler of the chromatographic column may be 3.0-5.0 μm, preferably 3.5 μm.

[0030] The inner diameter of the chromatographic column may be 4.0-5.0 mm, preferably 4.6 mm.

[0031] The length of the chromatographic column may be 75-150 mm, preferably 150 mm.

[0032] The chromatography column is preferably XBridge Shield RP18 (eg Waters XBridge Shield RP18).

[0033] The test product is preferably pravastatin bulk drug and / or pravastatin preparation.

[0034] The pravastatin related substances may include one or more of the following compounds:

[0035] (Pravatin), (Impurity A), (Impurity B), (Impurity C),

[0036] (Impurity D), (Impurity E), (Impurity F), (Impurity G), (Impurity H) and (Impurity I).

[0037] The detection wavelength of the high performance liquid chromatography may be 236-240 nm, preferably 238 nm.

[0038] The detector of the HPLC method may be an ultraviolet visible light (UV / Vis) detector, preferably a 2489 ultraviolet visible light (UV / Vis) detector (eg, Waters 2489 ultraviolet visible light (UV / Vis) detector).

[0039] The injection volume of the high performance liquid chromatography method can be 8-12 μL, preferably 10 μL.

[0040] The flow rate of the HPLC method may be 0.8-1.2 mL / min, preferably 1.0 mL / min.

[0041] The injector temperature of the HPLC method may be 8-12°C, preferably 10°C.

[0042] The sample to be tested can be dissolved in a solvent before injection.

[0043] The solvent may be a mixture of methanol and water, or a mixture of water, phosphate buffer and acetonitrile.

[0044] The volume ratio of the water, phosphate buffer and acetonitrile can be (50-54):(28-32):(16-20), preferably 52:30:18.

[0045] The pH of the phosphate buffer may be 6.8-7.2, preferably 7.0.

[0046] The molar volume ratio of the test product to the solvent may be 0.8-1.2 mg / L, for example 1.0 mg / L.

[0047] In a preferred embodiment, the test products include pravastatin, (Impurity A), (Impurity B) and (impurity D);

[0048] The liquid chromatography method is: the chromatographic column is preferably XBridge Shield RP18 (e.g. Waters XBridge Shield RP18);

[0049] The column temperature of the liquid chromatography method is 25°C;

[0050] In the mobile phase A, the volume ratio of water, phosphate buffer and acetonitrile is 52:30:18;

[0051] In the mobile phase B, the volume ratio of water, phosphate buffer and acetonitrile is 10:30:60;

[0052] The gradient elution is:

[0053]

[0054] In a preferred embodiment,

[0055] The liquid chromatography method is as follows: the sample to be tested includes components of group (1) or group (2),

[0056] Group (1) pravastatin, (Impurity A) (Impurity B) and (impurity D);

[0057] Group (2) pravastatin, (Impurity A), (Impurity B), (Impurity C), (Impurity D), (Impurity E), (Impurity F), (Impurity G), (Impurity H) and (Impurity I);

[0058] The liquid chromatography method is: the chromatographic column is preferably XBridge Shield RP18 (e.g. Waters XBridge Shield RP18);

[0059] The column temperature of the liquid chromatography method is 28°C;

[0060] In the mobile phase A, the volume ratio of water, phosphate buffer and acetonitrile is 52:30:18;

[0061] In the mobile phase B, the volume ratio of water, phosphate buffer and acetonitrile is 10:30:60;

[0062] The gradient elution is:

[0063] Elution stage Elution time / min Mobile phase A (%) Mobile phase B (%) First elution stage 3 100％→100％ 0％→0％ Second elution stage 27 100％→0％ 0％→100％ The third elution stage 5 100％→0％ 0％→0％ Fourth elution stage 1 0％→100％ 100％→0％ Fifth elution stage 4 100％→100％ 0％→0％ .

[0064] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0065] The reagents and raw materials used in the present invention are commercially available.

[0066] The positive and progressive effects of the present invention are: the detection method can simultaneously realize the effective separation of pravastatin and its adjacent unknown degradation impurities, and impurity D and its adjacent unknown impurities, and can realize accurate detection of pravastatin and impurity D, is easy to operate and control, and the detection result is accurate; further, it can also realize the effective separation of impurity B and impurity E, thereby improving the accuracy of the content of pravastatin, impurity D, impurity B and impurity E, and provides an effective detection method for the content of pravastatin-related substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 HPLC spectrum of sample 1 in Example 1;

[0068] Figure 2 It is the HPLC spectrum of the sample 1 to be tested in Comparative Example 1. DETAILED DESCRIPTION

[0069] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0070] Example 1

[0071] 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 with 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.

[0072] 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 with 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.

[0073] Using a Waters XBridge Shield RP18 chromatographic column (4.6 mm × 150 mm, 3.5 μm), the sample 1 to be tested was injected under the chromatographic conditions shown in Table 2. The results showed that the main component Pravastatin and the adjacent unknown degradation impurities could be basically completely separated, as shown in FIG. Figure 1 As shown, the peak value, peak width / half peak width, separation degree and other data of the measured impurities are shown in Table 3 below.

[0074] Phosphate buffer: Weigh about 9.2 g of phosphoric acid, dilute to 1000 mL with water, and adjust the pH to 7.0 with triethylamine.

[0075] Detector model: Waters 2489 UV / Vis detector.

[0076] Table 2. Chromatographic conditions

[0077]

[0078] Table 3. Spectral data of API using Waters XBridge Shield RP18

[0079]

[0080]

[0081] From the above results, it can be seen that using the Waters XBridge Shield RP18 column, pravastatin and its adjacent unknown degradation impurities, impurity A and its adjacent unknown impurities, and impurity D and its adjacent unknown impurities can be completely separated.

[0082] Example 2

[0083] Optimization of column temperature and chromatographic gradient

[0084] The samples 1 and 2 to be tested were tested using a Waters XBridge Shield RP18 chromatographic column at different temperatures and under the chromatographic gradients shown in Table 4. Other conditions were consistent with those in Example 1. The results are shown in Table 5.

[0085] Table 4. Chromatographic gradient

[0086] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 3 100 0 30 0 100 35 0 100 36 100 0 40 100 0

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

[0088]

[0089]

[0090] The column temperature and chromatographic gradient were adjusted to ensure that all known impurities in the mixed solution and pravastatin could be completely separated, and that unknown degradation impurities that might exist in the raw material drug could also be completely separated from the known impurities.

[0091] From the above results, it can be seen that the higher the column temperature, the better the separation between impurity B and impurity E, the better the separation between the main peak and its adjacent unknown degradation impurities, but the worse the separation between the main peak and impurity A, between impurity A and its adjacent unknown impurities, and between impurity D and adjacent unknown impurities. When the column temperature is 28°C, the separation between impurities is greater than 1.5, and the separation between the main component and the adjacent impurities is greater than 2.0. Therefore, in the detection method of pravastatin related substances, the optimal column temperature is 28°C.

[0092] Comparative Example 1

[0093] The chromatographic column recommended by ChP 2020 is Aglient Zorbax SB-C18 column (4.6 mm × 75 mm, 3.5 μm). The sample 1 to be tested was injected under the chromatographic conditions shown in Table 6. The results showed that the main component Pravastatin and its adjacent unknown degradation impurities could not be separated, and impurity D and its adjacent unknown degradation impurities could not be effectively separated. Figure 2 The spectrum data are shown in Table 7 below.

[0094] Phosphate buffer: Weigh about 9.2 g of phosphoric acid, dilute to 1000 mL with water, and adjust the pH to 7.0 with triethylamine.

[0095] Detector model: Waters 2489 UV / Vis detector.

[0096] Table 6. Chromatographic conditions recommended by ChP 2020

[0097]

[0098]

[0099] Table 7. Spectral data of sample 1 using Aglient Zorbax SB-C18

[0100]

[0101] Note: “-” indicates that the pravastatin peak overlaps with the adjacent unknown degradation impurity peak 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 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 The column temperature of the liquid chromatography method is 25-35°C; The mobile phase of the high performance liquid chromatography method consists of mobile phase A and mobile phase B; the mobile phases A and B are a mixture of water, phosphate buffer and acetonitrile.

3. The detection method according to claim 2, 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, preferably 28°C; ② In the mobile phases A and B, the pH value of the phosphate buffer is 6.8-7.2, preferably 7.0; ③ In the mobile phase A, the volume ratio of water, phosphate buffer and acetonitrile is (50-54):(28-32):(16-20), preferably 52:30:18; In the mobile phase B, the volume ratio of water, phosphate buffer and acetonitrile is (8-12):(28-32):(58-62), preferably 10:30:

60.

4. The detection method according to claim 2 or 3, characterized in that: The high performance liquid chromatography method adopts gradient elution, wherein the gradient elution is a four-stage gradient elution or a five-stage gradient elution; The four-stage gradient elution is: The five-stage gradient elution is:

5. The detection method according to claim 4, characterized in that: The four-stage gradient elution is: The five-stage gradient elution 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 particle size of the filler of the chromatographic column is 3.0-5.0 μm; ② The inner diameter of the chromatographic column is 4.0-5.0 mm; ③The length of the chromatographic column is 75-150mm; ④The chromatographic column is XBridge Shield RP18; ⑤ The test product is pravastatin API and / or pravastatin preparation; ⑥ The pravastatin-related substances include one or more of the following compounds: ⑦ The sample to be tested is dissolved in a solvent before injection; ⑧ The detection wavelength of the high performance liquid chromatography is 236-240nm; ⑨ The detector of the high performance liquid chromatography is a UV-visible light detector; ⑩ The injection volume of the high performance liquid chromatography 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 particle size of the filler of the chromatographic column is 3.5 μm; ②The inner diameter of the chromatographic column is 4.6 mm; ③The length of the chromatographic column is 150 mm; ④ The detection wavelength of the high performance liquid chromatography is 238nm; ⑤ The detector of the high performance liquid chromatography is a 2489 ultraviolet visible light detector; ⑥ The injection volume of the high performance liquid chromatography method is 10 μL; ⑦ The flow rate of the high performance liquid chromatography is 1.0 mL / min; ⑧The injector temperature of the high performance liquid chromatography is 10°C.

8. The detection method according to claim 7, characterized in that The detection method meets one or both of the following conditions: ① The solvent is a mixture of methanol and water, or a mixture of water, phosphate buffer and acetonitrile; ② 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 more of the following conditions: ① In the solvent, the pH of the phosphate buffer is 6.8-7.2; preferably 7.0; ② The molar volume ratio of the test product to the solvent is 1.0 mg / L; ③ In the solvent, the volume ratio of water, phosphate buffer and acetonitrile is (50-54):(28-32):(16-20); preferably 52:30:

18.

10. The detection method according to claim 1, characterized in that: The detection method meets any one of the following conditions: ① The test products include pravastatin, The liquid chromatography method is as follows: the chromatographic column is XBridge Shield RP18; The column temperature was 25°C; In mobile phase A, the volume ratio of water, phosphate buffer, and acetonitrile was 52:30:18; In mobile phase B, the volume ratio of water, phosphate buffer, and acetonitrile was 10:30:60; Gradient elution: ② The test product includes components of group (1) or group (2), Group (1) pravastatin, Group (2) pravastatin, The liquid chromatography method is as follows: the chromatographic column is XBridge Shield RP18; The column temperature was 28°C; In mobile phase A, the volume ratio of water, phosphate buffer, and acetonitrile was 52:30:18; In mobile phase B, the volume ratio of water, phosphate buffer, and acetonitrile was 10:30:60; Gradient elution: 。

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