Measurement method for measuring pravastatin-related substance

Abstract

A measurement method for a pravastatin-related substance. The measurement method comprises the following steps: using a high-performance liquid chromatography method to measure a product to be measured, wherein a filler of a chromatography column of the liquid chromatography method is ethylene bridged hybrid particles, and said product comprises one or more of pravastatin, a pravastatin salt and a pravastatin-related substance. The measurement method can achieve effective separation of pravastatin and adjacent unknown degradation impurities, and impurities D and adjacent unknown impurities simultaneously, can achieve accurate measurement of the pravastatin and the impurities D, is easy to operate and control, has an accurate measurement result, and can further achieve effective separation of impurities B and impurities E, thereby improving the accuracy of measuring the amounts of the pravastatin, the impurities D, the impurities B and the impurities E, and providing an effective measurement method for the amount of the pravastatin-related substance.

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 limited, 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. Therefore, accurately determining the content of these substances remains 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 during the detection of pravastatin-related substances, some impurity peaks are poorly separated from the main pravastatin peak, and impurities cannot be separated from each other, resulting in an inability to accurately reflect the content of pravastatin-related substances. To this end, the present invention provides a method for detecting pravastatin-related substances. This detection method can more effectively separate impurities B and E. Furthermore, it can also effectively separate pravastatin and its adjacent unknown impurities, impurity A and its adjacent unknown impurities, and impurity D and its adjacent unknown impurities, thereby improving the accuracy of the content of related substances such as pravastatin, impurity A, impurity D, impurity B, and impurity E.

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

[0011] The column temperature of the liquid chromatography method is 25-35°C, preferably 28°C or 30°C, more preferably 28°C.

[0012] 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.

[0013] In the mobile phases A and B, the pH value of the phosphate buffer may be 6.8-7.2, preferably 7.0.

[0014] 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.

[0015] 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.

[0016] 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;

[0017] The four-stage gradient elution can be:

[0018] Preferably:

[0019] Or, the five-stage gradient elution can be

[0020] Preferably

[0021] The elution time is the difference between the elution end time and the elution start time of the elution stage.

[0022] The particle size of the filler of the chromatographic column may be 3.0-5.0 μm, preferably 3.5 μm.

[0023] The inner diameter of the chromatographic column may be 4.0-5.0 mm, preferably 4.6 mm.

[0024] The length of the chromatographic column may be 75-150 mm, preferably 150 mm.

[0025] The chromatography column is preferably XBridge Shield RP18 (eg Waters XBridge Shield RP18).

[0026] The test product is preferably pravastatin bulk drug and / or pravastatin preparation.

[0027] The pravastatin related substances may include one or more of the following compounds:

[0028] (pravastatin), (Impurity A), (impurity B), (impurity C), (impurity D), (impurity E), (impurity F), (impurity G), (Impurity H) and (Impurity I).

[0029] The detection wavelength of the high performance liquid chromatography method can be 236-240 nm, preferably 238 nm.

[0030] The detector of the HPLC method may be an ultraviolet-visible (UV / Vis) detector, preferably a 2489 ultraviolet-visible (UV / Vis) detector (eg, a Waters 2489 ultraviolet-visible (UV / Vis) detector).

[0031] The injection volume of the high performance liquid chromatography method can be 8-12 μL, preferably 10 μL.

[0032] The flow rate of the HPLC method may be 0.8-1.2 mL / min, preferably 1.0 mL / min.

[0033] The injector temperature of the HPLC method can be 8-12°C, preferably 10°C.

[0034] The sample to be tested can be dissolved in a solvent before injection.

[0035] The solvent can be a mixture of methanol and water, or a mixture of water, phosphate buffer and acetonitrile.

[0036] The volume ratio of the water, phosphate buffer and acetonitrile can be (50-54):(28-32):(16-20), preferably 52:30:18.

[0037] The pH of the phosphate buffer may be 6.8-7.2, preferably 7.0.

[0038] The molar volume ratio of the analyte to the solvent may be 0.8-1.2 mg / L, for example 1.0 mg / L.

[0039] In a preferred embodiment, the test products include pravastatin, (Impurity A), (Impurity B) and (impurity D);

[0040] The liquid chromatography method is as follows: the chromatographic column is preferably XBridge Shield RP18 (e.g. Waters XBridge Shield RP18);

[0041] The column temperature of the liquid chromatography method is 25°C;

[0042] In the mobile phase A, the volume ratio of water, phosphate buffer and acetonitrile is 52:30:18;

[0043] In the mobile phase B, the volume ratio of water, phosphate buffer and acetonitrile is 10:30:60;

[0044] The gradient elution is:

[0045] In a preferred embodiment,

[0046] The liquid chromatography method is as follows: the sample to be tested includes components of group (1) or group (2),

[0047] Group (1) is pravastatin, (Impurity A), (Impurity B) and (impurity D);

[0048] Group (2) is pravastatin, (Impurity A), (impurity B), (impurity C), (impurity D), (impurity E), (impurity F), (impurity G), (Impurity H) and (Impurity I);

[0049] The liquid chromatography method is as follows: the chromatographic column is preferably XBridge Shield RP18 (e.g. Waters XBridge Shield RP18);

[0050] The column temperature of the liquid chromatography method is 28°C;

[0051] In the mobile phase A, the volume ratio of water, phosphate buffer and acetonitrile is 52:30:18;

[0052] In the mobile phase B, the volume ratio of water, phosphate buffer and acetonitrile is 10:30:60;

[0053] The gradient elution is:

[0054] 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.

[0055] The reagents and raw materials used in the present invention are commercially available.

[0056] 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 effective separation of impurity B and impurity E, thereby improving the accuracy of the contents of pravastatin, impurity D, impurity B and impurity E, and providing an effective detection method for the content of pravastatin-related substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] FIG1 is a HPLC spectrogram of sample 1 in Example 1;

[0058] FIG2 is the HPLC spectrum of the sample 1 to be tested in Comparative Example 1. DETAILED DESCRIPTION

[0059] 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.

[0060] Example 1

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] Detector model: Waters 2489 ultraviolet-visible (UV / Vis) detector.

[0066] Table 2. Chromatographic conditions

[0067] Table 3. Spectral data of the drug substance using Waters XBridge Shield RP18

[0068] The above results show 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.

[0069] Example 2

[0070] Optimization of column temperature and chromatographic gradient

[0071] 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.

[0072] Table 4. Chromatographic gradient

[0073] Table 5. Test results of separation of pravastatin related substances at different column temperatures

[0074] 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.

[0075] 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, between impurity A and its adjacent unknown impurities, and between impurity D and its adjacent unknown impurities deteriorates. At a column temperature of 28°C, the resolution between impurities was greater than 1.5, and the resolution between the main component and its adjacent impurities was greater than 2.0. Therefore, the optimal column temperature for the detection of pravastatin-related substances is 28°C.

[0076] Comparative Example 1

[0077] 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 6. The results showed that the main component, pravastatin, could not be separated from its adjacent unknown degradation impurities, and impurity D could not be effectively separated from its adjacent unknown degradation impurities, as shown in Figure 2. The spectral data are shown in Table 7 below.

[0078] 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.

[0079] Detector model: Waters 2489 ultraviolet-visible (UV / Vis) detector.

[0080] Table 6. Chromatographic conditions recommended by ChP 2020

[0081] Table 7. Spectral data of sample 1 using Aglient Zorbax SB-C18

[0082] 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:

Images